patents.google.com

US20070299027A1 - Compositions and their uses directed to huntingtin - Google Patents

️Thu Dec 27 2007

US20070299027A1 - Compositions and their uses directed to huntingtin - Google Patents

Compositions and their uses directed to huntingtin Download PDF

Info

Publication number

US20070299027A1

US20070299027A1 US11/627,921 US62792107A US2007299027A1 US 20070299027 A1 US20070299027 A1 US 20070299027A1 US 62792107 A US62792107 A US 62792107A US 2007299027 A1 US2007299027 A1 US 2007299027A1 Authority

United States

Prior art keywords

huntingtin

antisense

nucleotides

oligonucleotide

seq

Prior art date

2006-01-26

Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)

Abandoned

Application number

US11/627,921

Inventor

Gene Hung

Janet Leeds

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Ionis Pharmaceuticals Inc

CHDI Foundation Inc

Original Assignee

Individual

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2006-01-26

Filing date

2007-01-26

Publication date

2007-12-27

2007-01-26 Application filed by Individual filed Critical Individual

2007-01-26 Priority to US11/627,921 priority Critical patent/US20070299027A1/en

2007-11-27 Assigned to ISIS PHARMACEUTICALS, INC. reassignment ISIS PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HUNG, GENE

2007-12-27 Publication of US20070299027A1 publication Critical patent/US20070299027A1/en

2009-02-18 Assigned to ISIS PHARMACEUTICALS, INC. reassignment ISIS PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BENNETT, C. FRANK, FREIER, SUSAN M.

2009-02-25 Assigned to CHDI FOUNDATION INC. reassignment CHDI FOUNDATION INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LEEDS, JANET M.

2009-11-13 Priority to US12/618,470 priority patent/US20100069472A1/en

2012-12-07 Priority to US13/708,839 priority patent/US9057066B2/en

2015-05-11 Priority to US14/708,511 priority patent/US20160053256A1/en

2017-10-05 Priority to US15/725,933 priority patent/US10738307B2/en

2020-08-10 Priority to US16/988,862 priority patent/US20210017522A1/en

Status Abandoned legal-status Critical Current

Links

239000000203 mixture Substances 0.000 title abstract description 34
150000001875 compounds Chemical class 0.000 claims abstract description 224
208000023105 Huntington disease Diseases 0.000 claims abstract description 141
230000000692 anti-sense effect Effects 0.000 claims abstract description 127
238000000034 method Methods 0.000 claims abstract description 81
238000011282 treatment Methods 0.000 claims abstract description 41
239000000074 antisense oligonucleotide Substances 0.000 claims description 170
238000012230 antisense oligonucleotides Methods 0.000 claims description 170
108091034117 Oligonucleotide Proteins 0.000 claims description 165
239000002773 nucleotide Substances 0.000 claims description 105
125000003729 nucleotide group Chemical group 0.000 claims description 97
108020004707 nucleic acids Proteins 0.000 claims description 87
102000039446 nucleic acids Human genes 0.000 claims description 87
235000000346 sugar Nutrition 0.000 claims description 30
239000008194 pharmaceutical composition Substances 0.000 claims description 26
-1 bicyclic nucleic acid Chemical class 0.000 claims description 21
210000001175 cerebrospinal fluid Anatomy 0.000 claims description 19
238000001802 infusion Methods 0.000 claims description 18
238000007913 intrathecal administration Methods 0.000 claims description 17
OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 16
238000000185 intracerebroventricular administration Methods 0.000 claims description 13
230000001965 increasing effect Effects 0.000 claims description 12
230000004083 survival effect Effects 0.000 claims description 11
229940104302 cytosine Drugs 0.000 claims description 8
RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 claims description 8
230000006872 improvement Effects 0.000 claims description 7
LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 claims description 6
125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 3
GHYOCDFICYLMRF-UTIIJYGPSA-N (2S,3R)-N-[(2S)-3-(cyclopenten-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]propanoyl]amino]propanamide Chemical compound C1(=CCCC1)C[C@@H](C(=O)[C@@]1(OC1)C)NC([C@H]([C@@H](C1=CC=C(C=C1)OC)O)NC([C@H](C)NC(CN1CCOCC1)=O)=O)=O GHYOCDFICYLMRF-UTIIJYGPSA-N 0.000 claims description 2
229940125797 compound 12 Drugs 0.000 claims description 2
230000014509 gene expression Effects 0.000 abstract description 63
208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 27
239000003814 drug Substances 0.000 abstract description 24
241001465754 Metazoa Species 0.000 abstract description 23
201000010099 disease Diseases 0.000 abstract description 21
230000005750 disease progression Effects 0.000 abstract description 8
238000004519 manufacturing process Methods 0.000 abstract description 5
208000037765 diseases and disorders Diseases 0.000 abstract description 2
108020000948 Antisense Oligonucleotides Proteins 0.000 description 106
150000007523 nucleic acids Chemical class 0.000 description 87
210000004027 cell Anatomy 0.000 description 76
230000000295 complement effect Effects 0.000 description 69
108091032973 (ribonucleotides)n+m Proteins 0.000 description 60
108090000623 proteins and genes Proteins 0.000 description 55
108020004999 messenger RNA Proteins 0.000 description 53
JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 48
230000005764 inhibitory process Effects 0.000 description 36
241000699666 Mus <mouse, genus> Species 0.000 description 28
101001030705 Homo sapiens Huntingtin Proteins 0.000 description 23
108020004459 Small interfering RNA Proteins 0.000 description 23
230000000694 effects Effects 0.000 description 22
230000006870 function Effects 0.000 description 22
210000001519 tissue Anatomy 0.000 description 22
108020004414 DNA Proteins 0.000 description 21
102000054185 human HTT Human genes 0.000 description 19
238000012986 modification Methods 0.000 description 19
238000012360 testing method Methods 0.000 description 19
101001030698 Mus musculus Huntingtin Proteins 0.000 description 18
230000004048 modification Effects 0.000 description 18
241000699670 Mus sp. Species 0.000 description 17
238000004458 analytical method Methods 0.000 description 17
210000004556 brain Anatomy 0.000 description 15
230000009467 reduction Effects 0.000 description 15
241000764238 Isis Species 0.000 description 14
210000003169 central nervous system Anatomy 0.000 description 12
239000002777 nucleoside Substances 0.000 description 12
230000015572 biosynthetic process Effects 0.000 description 11
238000003753 real-time PCR Methods 0.000 description 11
102100034343 Integrase Human genes 0.000 description 10
238000001727 in vivo Methods 0.000 description 10
230000035772 mutation Effects 0.000 description 10
102000004169 proteins and genes Human genes 0.000 description 10
230000002829 reductive effect Effects 0.000 description 10
150000003839 salts Chemical class 0.000 description 10
238000012228 RNA interference-mediated gene silencing Methods 0.000 description 9
230000009368 gene silencing by RNA Effects 0.000 description 9
238000009396 hybridization Methods 0.000 description 9
230000007246 mechanism Effects 0.000 description 9
241000894007 species Species 0.000 description 9
238000011830 transgenic mouse model Methods 0.000 description 9
102000016252 Huntingtin Human genes 0.000 description 8
108050004784 Huntingtin Proteins 0.000 description 8
108010092799 RNA-directed DNA polymerase Proteins 0.000 description 8
UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 8
238000009472 formulation Methods 0.000 description 8
230000002068 genetic effect Effects 0.000 description 8
125000000623 heterocyclic group Chemical group 0.000 description 8
230000002401 inhibitory effect Effects 0.000 description 8
125000003835 nucleoside group Chemical group 0.000 description 8
210000000056 organ Anatomy 0.000 description 8
238000003752 polymerase chain reaction Methods 0.000 description 8
239000000523 sample Substances 0.000 description 8
208000024891 symptom Diseases 0.000 description 8
238000003786 synthesis reaction Methods 0.000 description 8
206010061818 Disease progression Diseases 0.000 description 7
241000699660 Mus musculus Species 0.000 description 7
125000000217 alkyl group Chemical group 0.000 description 7
238000010171 animal model Methods 0.000 description 7
210000004369 blood Anatomy 0.000 description 7
239000008280 blood Substances 0.000 description 7
230000008859 change Effects 0.000 description 7
229940079593 drug Drugs 0.000 description 7
210000002950 fibroblast Anatomy 0.000 description 7
UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 7
125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 7
125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 7
238000002360 preparation method Methods 0.000 description 7
238000011160 research Methods 0.000 description 7
229940124597 therapeutic agent Drugs 0.000 description 7
230000014616 translation Effects 0.000 description 7
ABEXEQSGABRUHS-UHFFFAOYSA-N 16-methylheptadecyl 16-methylheptadecanoate Chemical compound CC(C)CCCCCCCCCCCCCCCOC(=O)CCCCCCCCCCCCCCC(C)C ABEXEQSGABRUHS-UHFFFAOYSA-N 0.000 description 6
238000011529 RT qPCR Methods 0.000 description 6
FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
238000003776 cleavage reaction Methods 0.000 description 6
208000035475 disorder Diseases 0.000 description 6
229910052739 hydrogen Inorganic materials 0.000 description 6
239000001257 hydrogen Substances 0.000 description 6
238000005417 image-selected in vivo spectroscopy Methods 0.000 description 6
238000012739 integrated shape imaging system Methods 0.000 description 6
150000003833 nucleoside derivatives Chemical class 0.000 description 6
230000007017 scission Effects 0.000 description 6
239000011780 sodium chloride Substances 0.000 description 6
238000010186 staining Methods 0.000 description 6
RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 6
238000013518 transcription Methods 0.000 description 6
230000035897 transcription Effects 0.000 description 6
238000013519 translation Methods 0.000 description 6
YIMATHOGWXZHFX-WCTZXXKLSA-N (2r,3r,4r,5r)-5-(hydroxymethyl)-3-(2-methoxyethoxy)oxolane-2,4-diol Chemical compound COCCO[C@H]1[C@H](O)O[C@H](CO)[C@H]1O YIMATHOGWXZHFX-WCTZXXKLSA-N 0.000 description 5
UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 5
208000035657 Abasia Diseases 0.000 description 5
UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 5
RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 5
ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 5
230000004075 alteration Effects 0.000 description 5
238000003556 assay Methods 0.000 description 5
230000027455 binding Effects 0.000 description 5
210000001124 body fluid Anatomy 0.000 description 5
230000001413 cellular effect Effects 0.000 description 5
230000007423 decrease Effects 0.000 description 5
238000009826 distribution Methods 0.000 description 5
238000005516 engineering process Methods 0.000 description 5
125000004437 phosphorous atom Chemical group 0.000 description 5
229910052698 phosphorus Inorganic materials 0.000 description 5
230000001225 therapeutic effect Effects 0.000 description 5
238000001890 transfection Methods 0.000 description 5
PEHVGBZKEYRQSX-UHFFFAOYSA-N 7-deaza-adenine Chemical compound NC1=NC=NC2=C1C=CN2 PEHVGBZKEYRQSX-UHFFFAOYSA-N 0.000 description 4
229930024421 Adenine Natural products 0.000 description 4
TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
101710163270 Nuclease Proteins 0.000 description 4
108091028043 Nucleic acid sequence Proteins 0.000 description 4
108700019146 Transgenes Proteins 0.000 description 4
HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 4
229960000643 adenine Drugs 0.000 description 4
125000000304 alkynyl group Chemical group 0.000 description 4
210000004748 cultured cell Anatomy 0.000 description 4
238000002405 diagnostic procedure Methods 0.000 description 4
239000003085 diluting agent Substances 0.000 description 4
NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 description 4
230000001976 improved effect Effects 0.000 description 4
238000010172 mouse model Methods 0.000 description 4
210000001577 neostriatum Anatomy 0.000 description 4
230000001537 neural effect Effects 0.000 description 4
230000004770 neurodegeneration Effects 0.000 description 4
210000002569 neuron Anatomy 0.000 description 4
125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 4
BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
239000000651 prodrug Substances 0.000 description 4
229940002612 prodrug Drugs 0.000 description 4
210000002966 serum Anatomy 0.000 description 4
238000006467 substitution reaction Methods 0.000 description 4
230000008685 targeting Effects 0.000 description 4
229940035893 uracil Drugs 0.000 description 4
238000001262 western blot Methods 0.000 description 4
102000040650 (ribonucleotides)n+m Human genes 0.000 description 3
KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 3
102000004190 Enzymes Human genes 0.000 description 3
108090000790 Enzymes Proteins 0.000 description 3
241000282412 Homo Species 0.000 description 3
238000000636 Northern blotting Methods 0.000 description 3
229910019142 PO4 Inorganic materials 0.000 description 3
230000005856 abnormality Effects 0.000 description 3
238000009825 accumulation Methods 0.000 description 3
230000009471 action Effects 0.000 description 3
125000003342 alkenyl group Chemical group 0.000 description 3
239000003963 antioxidant agent Substances 0.000 description 3
230000003078 antioxidant effect Effects 0.000 description 3
210000001106 artificial yeast chromosome Anatomy 0.000 description 3
125000002619 bicyclic group Chemical group 0.000 description 3
230000037396 body weight Effects 0.000 description 3
210000005013 brain tissue Anatomy 0.000 description 3
229910052799 carbon Inorganic materials 0.000 description 3
125000004432 carbon atom Chemical group C* 0.000 description 3
239000000969 carrier Substances 0.000 description 3
230000015556 catabolic process Effects 0.000 description 3
238000004113 cell culture Methods 0.000 description 3
238000007385 chemical modification Methods 0.000 description 3
239000003153 chemical reaction reagent Substances 0.000 description 3
230000003247 decreasing effect Effects 0.000 description 3
238000006731 degradation reaction Methods 0.000 description 3
231100000673 dose–response relationship Toxicity 0.000 description 3
239000000975 dye Substances 0.000 description 3
150000002243 furanoses Chemical group 0.000 description 3
125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
238000003119 immunoblot Methods 0.000 description 3
238000002955 isolation Methods 0.000 description 3
239000007788 liquid Substances 0.000 description 3
239000002609 medium Substances 0.000 description 3
238000012544 monitoring process Methods 0.000 description 3
239000000178 monomer Substances 0.000 description 3
208000015122 neurodegenerative disease Diseases 0.000 description 3
238000010984 neurological examination Methods 0.000 description 3
239000000546 pharmaceutical excipient Substances 0.000 description 3
230000003285 pharmacodynamic effect Effects 0.000 description 3
239000010452 phosphate Substances 0.000 description 3
230000008569 process Effects 0.000 description 3
230000002441 reversible effect Effects 0.000 description 3
238000012216 screening Methods 0.000 description 3
239000000243 solution Substances 0.000 description 3
239000000758 substrate Substances 0.000 description 3
229940113082 thymine Drugs 0.000 description 3
230000005945 translocation Effects 0.000 description 3
YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
MPCAJMNYNOGXPB-UHFFFAOYSA-N 1,5-anhydrohexitol Chemical class OCC1OCC(O)C(O)C1O MPCAJMNYNOGXPB-UHFFFAOYSA-N 0.000 description 2
FZWGECJQACGGTI-UHFFFAOYSA-N 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one Chemical compound NC1=NC(O)=C2N(C)C=NC2=N1 FZWGECJQACGGTI-UHFFFAOYSA-N 0.000 description 2
ICSNLGPSRYBMBD-UHFFFAOYSA-N 2-aminopyridine Chemical compound NC1=CC=CC=N1 ICSNLGPSRYBMBD-UHFFFAOYSA-N 0.000 description 2
125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 2
LZINOQJQXIEBNN-UHFFFAOYSA-N 4-hydroxybutyl dihydrogen phosphate Chemical compound OCCCCOP(O)(O)=O LZINOQJQXIEBNN-UHFFFAOYSA-N 0.000 description 2
OVONXEQGWXGFJD-UHFFFAOYSA-N 4-sulfanylidene-1h-pyrimidin-2-one Chemical compound SC=1C=CNC(=O)N=1 OVONXEQGWXGFJD-UHFFFAOYSA-N 0.000 description 2
108020003589 5' Untranslated Regions Proteins 0.000 description 2
RYVNIFSIEDRLSJ-UHFFFAOYSA-N 5-(hydroxymethyl)cytosine Chemical compound NC=1NC(=O)N=CC=1CO RYVNIFSIEDRLSJ-UHFFFAOYSA-N 0.000 description 2
ZAYHVCMSTBRABG-JXOAFFINSA-N 5-methylcytidine Chemical class O=C1N=C(N)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 ZAYHVCMSTBRABG-JXOAFFINSA-N 0.000 description 2
HCGHYQLFMPXSDU-UHFFFAOYSA-N 7-methyladenine Chemical compound C1=NC(N)=C2N(C)C=NC2=N1 HCGHYQLFMPXSDU-UHFFFAOYSA-N 0.000 description 2
UJOBWOGCFQCDNV-UHFFFAOYSA-N 9H-carbazole Chemical compound C1=CC=C2C3=CC=CC=C3NC2=C1 UJOBWOGCFQCDNV-UHFFFAOYSA-N 0.000 description 2
MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 2
LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
208000031648 Body Weight Changes Diseases 0.000 description 2
102000011727 Caspases Human genes 0.000 description 2
108010076667 Caspases Proteins 0.000 description 2
206010008748 Chorea Diseases 0.000 description 2
QCDFBFJGMNKBDO-UHFFFAOYSA-N Clioquinol Chemical compound C1=CN=C2C(O)=C(I)C=C(Cl)C2=C1 QCDFBFJGMNKBDO-UHFFFAOYSA-N 0.000 description 2
206010012289 Dementia Diseases 0.000 description 2
108700024394 Exon Proteins 0.000 description 2
102100039289 Glial fibrillary acidic protein Human genes 0.000 description 2
101710193519 Glial fibrillary acidic protein Proteins 0.000 description 2
102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
101710203526 Integrase Proteins 0.000 description 2
108091093037 Peptide nucleic acid Proteins 0.000 description 2
CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 2
241000283984 Rodentia Species 0.000 description 2
239000002253 acid Substances 0.000 description 2
DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
230000004913 activation Effects 0.000 description 2
239000004480 active ingredient Substances 0.000 description 2
125000002015 acyclic group Chemical group 0.000 description 2
238000007792 addition Methods 0.000 description 2
210000004227 basal ganglia Anatomy 0.000 description 2
230000003542 behavioural effect Effects 0.000 description 2
230000009286 beneficial effect Effects 0.000 description 2
239000000090 biomarker Substances 0.000 description 2
230000004579 body weight change Effects 0.000 description 2
125000002837 carbocyclic group Chemical group 0.000 description 2
230000003197 catalytic effect Effects 0.000 description 2
210000001159 caudate nucleus Anatomy 0.000 description 2
108091092328 cellular RNA Proteins 0.000 description 2
230000004700 cellular uptake Effects 0.000 description 2
210000001638 cerebellum Anatomy 0.000 description 2
238000006243 chemical reaction Methods 0.000 description 2
208000012601 choreatic disease Diseases 0.000 description 2
229960005228 clioquinol Drugs 0.000 description 2
230000001149 cognitive effect Effects 0.000 description 2
239000002299 complementary DNA Substances 0.000 description 2
238000002591 computed tomography Methods 0.000 description 2
CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
125000000753 cycloalkyl group Chemical group 0.000 description 2
RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 2
230000007850 degeneration Effects 0.000 description 2
230000003111 delayed effect Effects 0.000 description 2
230000001419 dependent effect Effects 0.000 description 2
238000001514 detection method Methods 0.000 description 2
238000011161 development Methods 0.000 description 2
238000003745 diagnosis Methods 0.000 description 2
239000000032 diagnostic agent Substances 0.000 description 2
229940039227 diagnostic agent Drugs 0.000 description 2
239000002552 dosage form Substances 0.000 description 2
239000003937 drug carrier Substances 0.000 description 2
230000002996 emotional effect Effects 0.000 description 2
YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 2
150000002148 esters Chemical class 0.000 description 2
238000002474 experimental method Methods 0.000 description 2
238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
230000005021 gait Effects 0.000 description 2
230000007277 glial cell activation Effects 0.000 description 2
210000005046 glial fibrillary acidic protein Anatomy 0.000 description 2
108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
125000001475 halogen functional group Chemical group 0.000 description 2
101150113725 hd gene Proteins 0.000 description 2
125000005842 heteroatom Chemical group 0.000 description 2
FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
238000000338 in vitro Methods 0.000 description 2
238000011534 incubation Methods 0.000 description 2
NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
239000007928 intraperitoneal injection Substances 0.000 description 2
210000003734 kidney Anatomy 0.000 description 2
210000004185 liver Anatomy 0.000 description 2
210000005228 liver tissue Anatomy 0.000 description 2
230000033001 locomotion Effects 0.000 description 2
229910001629 magnesium chloride Inorganic materials 0.000 description 2
239000003550 marker Substances 0.000 description 2
230000001404 mediated effect Effects 0.000 description 2
YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical group CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 2
239000003068 molecular probe Substances 0.000 description 2
125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 2
238000002610 neuroimaging Methods 0.000 description 2
230000002981 neuropathic effect Effects 0.000 description 2
238000006384 oligomerization reaction Methods 0.000 description 2
238000002515 oligonucleotide synthesis Methods 0.000 description 2
230000003204 osmotic effect Effects 0.000 description 2
238000007911 parenteral administration Methods 0.000 description 2
230000008506 pathogenesis Effects 0.000 description 2
RDOWQLZANAYVLL-UHFFFAOYSA-N phenanthridine Chemical compound C1=CC=C2C3=CC=CC=C3C=NC2=C1 RDOWQLZANAYVLL-UHFFFAOYSA-N 0.000 description 2
150000004713 phosphodiesters Chemical class 0.000 description 2
229910052697 platinum Inorganic materials 0.000 description 2
229920000642 polymer Polymers 0.000 description 2
230000002265 prevention Effects 0.000 description 2
238000012545 processing Methods 0.000 description 2
230000000750 progressive effect Effects 0.000 description 2
210000004129 prosencephalon Anatomy 0.000 description 2
230000006916 protein interaction Effects 0.000 description 2
150000003212 purines Chemical class 0.000 description 2
150000003230 pyrimidines Chemical class 0.000 description 2
230000010076 replication Effects 0.000 description 2
230000000717 retained effect Effects 0.000 description 2
159000000000 sodium salts Chemical class 0.000 description 2
239000007787 solid Substances 0.000 description 2
238000010532 solid phase synthesis reaction Methods 0.000 description 2
230000009870 specific binding Effects 0.000 description 2
230000000087 stabilizing effect Effects 0.000 description 2
239000000126 substance Substances 0.000 description 2
239000004094 surface-active agent Substances 0.000 description 2
230000009885 systemic effect Effects 0.000 description 2
ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 2
239000005451 thionucleotide Substances 0.000 description 2
230000000699 topical effect Effects 0.000 description 2
238000010361 transduction Methods 0.000 description 2
230000026683 transduction Effects 0.000 description 2
238000011144 upstream manufacturing Methods 0.000 description 2
230000004580 weight loss Effects 0.000 description 2
125000000008 (C1-C10) alkyl group Chemical group 0.000 description 1
UFSCXDAOCAIFOG-UHFFFAOYSA-N 1,10-dihydropyrimido[5,4-b][1,4]benzothiazin-2-one Chemical compound S1C2=CC=CC=C2N=C2C1=CNC(=O)N2 UFSCXDAOCAIFOG-UHFFFAOYSA-N 0.000 description 1
PTFYZDMJTFMPQW-UHFFFAOYSA-N 1,10-dihydropyrimido[5,4-b][1,4]benzoxazin-2-one Chemical compound O1C2=CC=CC=C2N=C2C1=CNC(=O)N2 PTFYZDMJTFMPQW-UHFFFAOYSA-N 0.000 description 1
FYADHXFMURLYQI-UHFFFAOYSA-N 1,2,4-triazine Chemical class C1=CN=NC=N1 FYADHXFMURLYQI-UHFFFAOYSA-N 0.000 description 1
FGODUFHTWYYOOB-UHFFFAOYSA-N 1,3-diaminopropan-2-yl dihydrogen phosphate Chemical compound NCC(CN)OP(O)(O)=O FGODUFHTWYYOOB-UHFFFAOYSA-N 0.000 description 1
WJFKNYWRSNBZNX-UHFFFAOYSA-N 10H-phenothiazine Chemical compound C1=CC=C2NC3=CC=CC=C3SC2=C1 WJFKNYWRSNBZNX-UHFFFAOYSA-N 0.000 description 1
TZMSYXZUNZXBOL-UHFFFAOYSA-N 10H-phenoxazine Chemical compound C1=CC=C2NC3=CC=CC=C3OC2=C1 TZMSYXZUNZXBOL-UHFFFAOYSA-N 0.000 description 1
UHUHBFMZVCOEOV-UHFFFAOYSA-N 1h-imidazo[4,5-c]pyridin-4-amine Chemical compound NC1=NC=CC2=C1N=CN2 UHUHBFMZVCOEOV-UHFFFAOYSA-N 0.000 description 1
101150028074 2 gene Proteins 0.000 description 1
VEPOHXYIFQMVHW-XOZOLZJESA-N 2,3-dihydroxybutanedioic acid (2S,3S)-3,4-dimethyl-2-phenylmorpholine Chemical compound OC(C(O)C(O)=O)C(O)=O.C[C@H]1[C@@H](OCCN1C)c1ccccc1 VEPOHXYIFQMVHW-XOZOLZJESA-N 0.000 description 1
QSHACTSJHMKXTE-UHFFFAOYSA-N 2-(2-aminopropyl)-7h-purin-6-amine Chemical compound CC(N)CC1=NC(N)=C2NC=NC2=N1 QSHACTSJHMKXTE-UHFFFAOYSA-N 0.000 description 1
JRYMOPZHXMVHTA-DAGMQNCNSA-N 2-amino-7-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1h-pyrrolo[2,3-d]pyrimidin-4-one Chemical compound C1=CC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O JRYMOPZHXMVHTA-DAGMQNCNSA-N 0.000 description 1
QNIZHKITBISILC-RPKMEZRRSA-N 2-amino-9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)-2-methyloxolan-2-yl]-3h-purin-6-one Chemical compound C1=NC(C(NC(N)=N2)=O)=C2N1[C@]1(C)O[C@H](CO)[C@@H](O)[C@H]1O QNIZHKITBISILC-RPKMEZRRSA-N 0.000 description 1
WKMPTBDYDNUJLF-UHFFFAOYSA-N 2-fluoroadenine Chemical compound NC1=NC(F)=NC2=C1N=CN2 WKMPTBDYDNUJLF-UHFFFAOYSA-N 0.000 description 1
125000004200 2-methoxyethyl group Chemical group [H]C([H])([H])OC([H])([H])C([H])([H])* 0.000 description 1
101150090724 3 gene Proteins 0.000 description 1
108020005345 3' Untranslated Regions Proteins 0.000 description 1
OALHHIHQOFIMEF-UHFFFAOYSA-N 3',6'-dihydroxy-2',4',5',7'-tetraiodo-3h-spiro[2-benzofuran-1,9'-xanthene]-3-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 OALHHIHQOFIMEF-UHFFFAOYSA-N 0.000 description 1
KUQZVISZELWDNZ-UHFFFAOYSA-N 3-aminopropyl dihydrogen phosphate Chemical compound NCCCOP(O)(O)=O KUQZVISZELWDNZ-UHFFFAOYSA-N 0.000 description 1
HYCSHFLKPSMPGO-UHFFFAOYSA-N 3-hydroxypropyl dihydrogen phosphate Chemical compound OCCCOP(O)(O)=O HYCSHFLKPSMPGO-UHFFFAOYSA-N 0.000 description 1
ZLAQATDNGLKIEV-UHFFFAOYSA-N 5-methyl-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CC1=CNC(=S)NC1=O ZLAQATDNGLKIEV-UHFFFAOYSA-N 0.000 description 1
UJBCLAXPPIDQEE-UHFFFAOYSA-N 5-prop-1-ynyl-1h-pyrimidine-2,4-dione Chemical compound CC#CC1=CNC(=O)NC1=O UJBCLAXPPIDQEE-UHFFFAOYSA-N 0.000 description 1
KXBCLNRMQPRVTP-UHFFFAOYSA-N 6-amino-1,5-dihydroimidazo[4,5-c]pyridin-4-one Chemical compound O=C1NC(N)=CC2=C1N=CN2 KXBCLNRMQPRVTP-UHFFFAOYSA-N 0.000 description 1
DCPSTSVLRXOYGS-UHFFFAOYSA-N 6-amino-1h-pyrimidine-2-thione Chemical compound NC1=CC=NC(S)=N1 DCPSTSVLRXOYGS-UHFFFAOYSA-N 0.000 description 1
QNNARSZPGNJZIX-UHFFFAOYSA-N 6-amino-5-prop-1-ynyl-1h-pyrimidin-2-one Chemical compound CC#CC1=CNC(=O)N=C1N QNNARSZPGNJZIX-UHFFFAOYSA-N 0.000 description 1
XYVLZAYJHCECPN-UHFFFAOYSA-N 6-aminohexyl phosphate Chemical compound NCCCCCCOP(O)(O)=O XYVLZAYJHCECPN-UHFFFAOYSA-N 0.000 description 1
XYVLZAYJHCECPN-UHFFFAOYSA-L 6-aminohexyl phosphate Chemical compound NCCCCCCOP([O-])([O-])=O XYVLZAYJHCECPN-UHFFFAOYSA-L 0.000 description 1
NJBMMMJOXRZENQ-UHFFFAOYSA-N 6H-pyrrolo[2,3-f]quinoline Chemical compound c1cc2ccc3[nH]cccc3c2n1 NJBMMMJOXRZENQ-UHFFFAOYSA-N 0.000 description 1
LOSIULRWFAEMFL-UHFFFAOYSA-N 7-deazaguanine Chemical compound O=C1NC(N)=NC2=C1CC=N2 LOSIULRWFAEMFL-UHFFFAOYSA-N 0.000 description 1
HRYKDUPGBWLLHO-UHFFFAOYSA-N 8-azaadenine Chemical compound NC1=NC=NC2=NNN=C12 HRYKDUPGBWLLHO-UHFFFAOYSA-N 0.000 description 1
LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 1
229960005508 8-azaguanine Drugs 0.000 description 1
102100027211 Albumin Human genes 0.000 description 1
108010088751 Albumins Proteins 0.000 description 1
108700028369 Alleles Proteins 0.000 description 1
206010003591 Ataxia Diseases 0.000 description 1
206010003694 Atrophy Diseases 0.000 description 1
206010006100 Bradykinesia Diseases 0.000 description 1
108010074051 C-Reactive Protein Proteins 0.000 description 1
102100032752 C-reactive protein Human genes 0.000 description 1
125000006519 CCH3 Chemical group 0.000 description 1
102000047934 Caspase-3/7 Human genes 0.000 description 1
108700037887 Caspase-3/7 Proteins 0.000 description 1
108020004394 Complementary RNA Proteins 0.000 description 1
206010010947 Coordination abnormal Diseases 0.000 description 1
125000000824 D-ribofuranosyl group Chemical group [H]OC([H])([H])[C@@]1([H])OC([H])(*)[C@]([H])(O[H])[C@]1([H])O[H] 0.000 description 1
208000019505 Deglutition disease Diseases 0.000 description 1
241000702421 Dependoparvovirus Species 0.000 description 1
102000012749 Dopamine and cAMP-Regulated Phosphoprotein 32 Human genes 0.000 description 1
108010090047 Dopamine and cAMP-Regulated Phosphoprotein 32 Proteins 0.000 description 1
239000006144 Dulbecco’s modiﬁed Eagle's medium Substances 0.000 description 1
208000012661 Dyskinesia Diseases 0.000 description 1
208000014094 Dystonic disease Diseases 0.000 description 1
238000002965 ELISA Methods 0.000 description 1
208000027534 Emotional disease Diseases 0.000 description 1
102100031780 Endonuclease Human genes 0.000 description 1
108010042407 Endonucleases Proteins 0.000 description 1
102000002494 Endoribonucleases Human genes 0.000 description 1
108010093099 Endoribonucleases Proteins 0.000 description 1
241000792859 Enema Species 0.000 description 1
108060002716 Exonuclease Proteins 0.000 description 1
108700039691 Genetic Promoter Regions Proteins 0.000 description 1
206010018341 Gliosis Diseases 0.000 description 1
WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical class C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 1
102000003964 Histone deacetylase Human genes 0.000 description 1
108090000353 Histone deacetylase Proteins 0.000 description 1
101000908713 Homo sapiens Dihydrofolate reductase Proteins 0.000 description 1
208000006083 Hypokinesia Diseases 0.000 description 1
UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
206010061218 Inflammation Diseases 0.000 description 1
108020005350 Initiator Codon Proteins 0.000 description 1
102000004877 Insulin Human genes 0.000 description 1
108090001061 Insulin Proteins 0.000 description 1
108010063738 Interleukins Proteins 0.000 description 1
102000015696 Interleukins Human genes 0.000 description 1
108091092195 Intron Proteins 0.000 description 1
208000015592 Involuntary movements Diseases 0.000 description 1
206010022998 Irritability Diseases 0.000 description 1
108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
208000019430 Motor disease Diseases 0.000 description 1
208000016285 Movement disease Diseases 0.000 description 1
101100198353 Mus musculus Rnasel gene Proteins 0.000 description 1
208000002740 Muscle Rigidity Diseases 0.000 description 1
108010021466 Mutant Proteins Proteins 0.000 description 1
102000008300 Mutant Proteins Human genes 0.000 description 1
208000009668 Neurobehavioral Manifestations Diseases 0.000 description 1
229910004679 ONO2 Inorganic materials 0.000 description 1
108700026244 Open Reading Frames Proteins 0.000 description 1
241000283973 Oryctolagus cuniculus Species 0.000 description 1
239000012807 PCR reagent Substances 0.000 description 1
PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Natural products C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 description 1
ABLZXFCXXLZCGV-UHFFFAOYSA-N Phosphorous acid Chemical class OP(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 description 1
239000004952 Polyamide Substances 0.000 description 1
108091034057 RNA (poly(A)) Proteins 0.000 description 1
238000002123 RNA extraction Methods 0.000 description 1
238000013381 RNA quantification Methods 0.000 description 1
230000006819 RNA synthesis Effects 0.000 description 1
108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
102000003661 Ribonuclease III Human genes 0.000 description 1
108010057163 Ribonuclease III Proteins 0.000 description 1
108091081021 Sense strand Proteins 0.000 description 1
UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
108020005038 Terminator Codon Proteins 0.000 description 1
108091036066 Three prime untranslated region Proteins 0.000 description 1
IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical class O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 1
102000003929 Transaminases Human genes 0.000 description 1
108090000340 Transaminases Proteins 0.000 description 1
206010044565 Tremor Diseases 0.000 description 1
108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
108020000999 Viral RNA Proteins 0.000 description 1
PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
238000010521 absorption reaction Methods 0.000 description 1
239000012190 activator Substances 0.000 description 1
230000002411 adverse Effects 0.000 description 1
239000000443 aerosol Substances 0.000 description 1
150000001336 alkenes Chemical class 0.000 description 1
125000005083 alkoxyalkoxy group Chemical group 0.000 description 1
125000002877 alkyl aryl group Chemical group 0.000 description 1
125000005600 alkyl phosphonate group Chemical group 0.000 description 1
VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 1
230000001668 ameliorated effect Effects 0.000 description 1
150000001408 amides Chemical group 0.000 description 1
125000005122 aminoalkylamino group Chemical group 0.000 description 1
238000000137 annealing Methods 0.000 description 1
PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 1
150000004056 anthraquinones Chemical class 0.000 description 1
230000002424 anti-apoptotic effect Effects 0.000 description 1
125000003710 aryl alkyl group Chemical group 0.000 description 1
230000037444 atrophy Effects 0.000 description 1
230000037429 base substitution Effects 0.000 description 1
230000006399 behavior Effects 0.000 description 1
230000008901 benefit Effects 0.000 description 1
ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 1
WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
239000003833 bile salt Substances 0.000 description 1
229940093761 bile salts Drugs 0.000 description 1
230000004071 biological effect Effects 0.000 description 1
230000008236 biological pathway Effects 0.000 description 1
229960002685 biotin Drugs 0.000 description 1
235000020958 biotin Nutrition 0.000 description 1
239000011616 biotin Substances 0.000 description 1
230000008499 blood brain barrier function Effects 0.000 description 1
238000004820 blood count Methods 0.000 description 1
210000001218 blood-brain barrier Anatomy 0.000 description 1
239000010839 body fluid Substances 0.000 description 1
101150006308 botA gene Proteins 0.000 description 1
230000005978 brain dysfunction Effects 0.000 description 1
239000000872 buffer Substances 0.000 description 1
238000004364 calculation method Methods 0.000 description 1
125000001369 canonical nucleoside group Chemical group 0.000 description 1
239000002775 capsule Substances 0.000 description 1
230000030570 cellular localization Effects 0.000 description 1
239000002738 chelating agent Substances 0.000 description 1
235000012000 cholesterol Nutrition 0.000 description 1
150000001841 cholesterols Chemical class 0.000 description 1
HGCIXCUEYOPUTN-UHFFFAOYSA-N cis-cyclohexene Natural products C1CCC=CC1 HGCIXCUEYOPUTN-UHFFFAOYSA-N 0.000 description 1
238000003340 combinatorial analysis Methods 0.000 description 1
230000002860 competitive effect Effects 0.000 description 1
239000003184 complementary RNA Substances 0.000 description 1
230000009918 complex formation Effects 0.000 description 1
239000008139 complexing agent Substances 0.000 description 1
239000002131 composite material Substances 0.000 description 1
238000007796 conventional method Methods 0.000 description 1
235000001671 coumarin Nutrition 0.000 description 1
125000000332 coumarinyl group Chemical class O1C(=O)C(=CC2=CC=CC=C12)* 0.000 description 1
229960003624 creatine Drugs 0.000 description 1
239000006046 creatine Substances 0.000 description 1
125000001995 cyclobutyl group Chemical group [H]C1([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
230000001934 delay Effects 0.000 description 1
238000004925 denaturation Methods 0.000 description 1
230000036425 denaturation Effects 0.000 description 1
230000003831 deregulation Effects 0.000 description 1
238000013461 design Methods 0.000 description 1
235000014113 dietary fatty acids Nutrition 0.000 description 1
LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical group P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
NAGJZTKCGNOGPW-UHFFFAOYSA-N dithiophosphoric acid Chemical class OP(O)(S)=S NAGJZTKCGNOGPW-UHFFFAOYSA-N 0.000 description 1
230000003828 downregulation Effects 0.000 description 1
230000004064 dysfunction Effects 0.000 description 1
208000010118 dystonia Diseases 0.000 description 1
238000004520 electroporation Methods 0.000 description 1
239000007920 enema Substances 0.000 description 1
229940079360 enema for constipation Drugs 0.000 description 1
239000003623 enhancer Substances 0.000 description 1
230000002708 enhancing effect Effects 0.000 description 1
125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
230000029142 excretion Effects 0.000 description 1
102000013165 exonuclease Human genes 0.000 description 1
230000004424 eye movement Effects 0.000 description 1
229930195729 fatty acid Natural products 0.000 description 1
239000000194 fatty acid Substances 0.000 description 1
150000004665 fatty acids Chemical class 0.000 description 1
239000012530 fluid Substances 0.000 description 1
239000007850 fluorescent dye Substances 0.000 description 1
229940014144 folate Drugs 0.000 description 1
OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 1
235000019152 folic acid Nutrition 0.000 description 1
239000011724 folic acid Substances 0.000 description 1
239000012634 fragment Substances 0.000 description 1
125000000524 functional group Chemical group 0.000 description 1
238000007306 functionalization reaction Methods 0.000 description 1
239000000499 gel Substances 0.000 description 1
238000001502 gel electrophoresis Methods 0.000 description 1
238000012224 gene deletion Methods 0.000 description 1
230000030279 gene silencing Effects 0.000 description 1
238000012226 gene silencing method Methods 0.000 description 1
230000007387 gliosis Effects 0.000 description 1
239000008103 glucose Substances 0.000 description 1
ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
244000144993 groups of animals Species 0.000 description 1
239000001963 growth medium Substances 0.000 description 1
230000036541 health Effects 0.000 description 1
150000002391 heterocyclic compounds Chemical class 0.000 description 1
125000000592 heterocycloalkyl group Chemical group 0.000 description 1
PHNWGDTYCJFUGZ-UHFFFAOYSA-L hexyl phosphate Chemical compound CCCCCCOP([O-])([O-])=O PHNWGDTYCJFUGZ-UHFFFAOYSA-L 0.000 description 1
230000001697 htt accumulation Effects 0.000 description 1
230000003463 hyperproliferative effect Effects 0.000 description 1
238000001114 immunoprecipitation Methods 0.000 description 1
238000000099 in vitro assay Methods 0.000 description 1
238000005462 in vivo assay Methods 0.000 description 1
208000016290 incoordination Diseases 0.000 description 1
238000010348 incorporation Methods 0.000 description 1
230000006698 induction Effects 0.000 description 1
239000012678 infectious agent Substances 0.000 description 1
230000004054 inflammatory process Effects 0.000 description 1
230000000977 initiatory effect Effects 0.000 description 1
239000007924 injection Substances 0.000 description 1
238000002347 injection Methods 0.000 description 1
229940125396 insulin Drugs 0.000 description 1
230000003993 interaction Effects 0.000 description 1
239000000138 intercalating agent Substances 0.000 description 1
229940047122 interleukins Drugs 0.000 description 1
238000001361 intraarterial administration Methods 0.000 description 1
230000003834 intracellular effect Effects 0.000 description 1
238000007917 intracranial administration Methods 0.000 description 1
238000010255 intramuscular injection Methods 0.000 description 1
239000007927 intramuscular injection Substances 0.000 description 1
210000001739 intranuclear inclusion body Anatomy 0.000 description 1
238000007912 intraperitoneal administration Methods 0.000 description 1
238000001990 intravenous administration Methods 0.000 description 1
238000007914 intraventricular administration Methods 0.000 description 1
125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
230000000366 juvenile effect Effects 0.000 description 1
230000003907 kidney function Effects 0.000 description 1
210000000265 leukocyte Anatomy 0.000 description 1
230000000670 limiting effect Effects 0.000 description 1
125000005647 linker group Chemical group 0.000 description 1
150000002632 lipids Chemical class 0.000 description 1
239000002502 liposome Substances 0.000 description 1
230000003908 liver function Effects 0.000 description 1
230000004807 localization Effects 0.000 description 1
238000002595 magnetic resonance imaging Methods 0.000 description 1
230000035800 maturation Effects 0.000 description 1
230000003340 mental effect Effects 0.000 description 1
230000004060 metabolic process Effects 0.000 description 1
239000002207 metabolite Substances 0.000 description 1
229910052751 metal Inorganic materials 0.000 description 1
239000002184 metal Substances 0.000 description 1
238000010208 microarray analysis Methods 0.000 description 1
239000011859 microparticle Substances 0.000 description 1
230000000897 modulatory effect Effects 0.000 description 1
230000007659 motor function Effects 0.000 description 1
210000004400 mucous membrane Anatomy 0.000 description 1
230000017311 musculoskeletal movement, spinal reflex action Effects 0.000 description 1
108700024542 myc Genes Proteins 0.000 description 1
210000004897 n-terminal region Anatomy 0.000 description 1
239000002105 nanoparticle Substances 0.000 description 1
239000006199 nebulizer Substances 0.000 description 1
125000001893 nitrooxy group Chemical group [O-][N+](=O)O* 0.000 description 1
230000009871 nonspecific binding Effects 0.000 description 1
238000010606 normalization Methods 0.000 description 1
238000007899 nucleic acid hybridization Methods 0.000 description 1
210000000287 oocyte Anatomy 0.000 description 1
125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 1
230000002018 overexpression Effects 0.000 description 1
229910052760 oxygen Inorganic materials 0.000 description 1
125000004430 oxygen atom Chemical group O* 0.000 description 1
210000000496 pancreas Anatomy 0.000 description 1
239000002245 particle Substances 0.000 description 1
230000008775 paternal effect Effects 0.000 description 1
230000001717 pathogenic effect Effects 0.000 description 1
230000001575 pathological effect Effects 0.000 description 1
230000007170 pathology Effects 0.000 description 1
230000035515 penetration Effects 0.000 description 1
230000035699 permeability Effects 0.000 description 1
208000022821 personality disease Diseases 0.000 description 1
230000003094 perturbing effect Effects 0.000 description 1
230000000144 pharmacologic effect Effects 0.000 description 1
239000012071 phase Substances 0.000 description 1
229950000688 phenothiazine Drugs 0.000 description 1
230000009120 phenotypic response Effects 0.000 description 1
150000002991 phenoxazines Chemical class 0.000 description 1
NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
239000002953 phosphate buffered saline Substances 0.000 description 1
150000003904 phospholipids Chemical class 0.000 description 1
UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 1
XUYJLQHKOGNDPB-UHFFFAOYSA-N phosphonoacetic acid Chemical compound OC(=O)CP(O)(O)=O XUYJLQHKOGNDPB-UHFFFAOYSA-N 0.000 description 1
150000008298 phosphoramidates Chemical class 0.000 description 1
150000008300 phosphoramidites Chemical class 0.000 description 1
230000004962 physiological condition Effects 0.000 description 1
239000013612 plasmid Substances 0.000 description 1
229920002647 polyamide Polymers 0.000 description 1
229920000768 polyamine Polymers 0.000 description 1
229920000570 polyether Polymers 0.000 description 1
229920001223 polyethylene glycol Polymers 0.000 description 1
108010040003 polyglutamine Proteins 0.000 description 1
230000003389 potentiating effect Effects 0.000 description 1
239000000843 powder Substances 0.000 description 1
230000002035 prolonged effect Effects 0.000 description 1
230000002685 pulmonary effect Effects 0.000 description 1
IGFXRKMLLMBKSA-UHFFFAOYSA-N purine Chemical compound N1=C[N]C2=NC=NC2=C1 IGFXRKMLLMBKSA-UHFFFAOYSA-N 0.000 description 1
UBQKCCHYAOITMY-UHFFFAOYSA-N pyridin-2-ol Chemical compound OC1=CC=CC=N1 UBQKCCHYAOITMY-UHFFFAOYSA-N 0.000 description 1
RXTQGIIIYVEHBN-UHFFFAOYSA-N pyrimido[4,5-b]indol-2-one Chemical compound C1=CC=CC2=NC3=NC(=O)N=CC3=C21 RXTQGIIIYVEHBN-UHFFFAOYSA-N 0.000 description 1
SRBUGYKMBLUTIS-UHFFFAOYSA-N pyrrolo[2,3-d]pyrimidin-2-one Chemical compound O=C1N=CC2=CC=NC2=N1 SRBUGYKMBLUTIS-UHFFFAOYSA-N 0.000 description 1
238000004445 quantitative analysis Methods 0.000 description 1
230000007115 recruitment Effects 0.000 description 1
230000011514 reflex Effects 0.000 description 1
230000014891 regulation of alternative nuclear mRNA splicing, via spliceosome Effects 0.000 description 1
125000006853 reporter group Chemical group 0.000 description 1
230000004044 response Effects 0.000 description 1
210000001995 reticulocyte Anatomy 0.000 description 1
239000003161 ribonuclease inhibitor Substances 0.000 description 1
238000010825 rotarod performance test Methods 0.000 description 1
101150116497 sacm1l gene Proteins 0.000 description 1
230000035807 sensation Effects 0.000 description 1
238000007493 shaping process Methods 0.000 description 1
230000019491 signal transduction Effects 0.000 description 1
239000002904 solvent Substances 0.000 description 1
210000000952 spleen Anatomy 0.000 description 1
230000002269 spontaneous effect Effects 0.000 description 1
230000000638 stimulation Effects 0.000 description 1
230000002739 subcortical effect Effects 0.000 description 1
238000007920 subcutaneous administration Methods 0.000 description 1
239000007929 subcutaneous injection Substances 0.000 description 1
125000001424 substituent group Chemical group 0.000 description 1
150000008163 sugars Chemical class 0.000 description 1
IIACRCGMVDHOTQ-UHFFFAOYSA-N sulfamic acid Chemical group NS(O)(=O)=O IIACRCGMVDHOTQ-UHFFFAOYSA-N 0.000 description 1
150000003456 sulfonamides Chemical group 0.000 description 1
BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
150000003457 sulfones Chemical group 0.000 description 1
150000003462 sulfoxides Chemical class 0.000 description 1
229910052717 sulfur Inorganic materials 0.000 description 1
230000008093 supporting effect Effects 0.000 description 1
239000000829 suppository Substances 0.000 description 1
239000000375 suspending agent Substances 0.000 description 1
235000020357 syrup Nutrition 0.000 description 1
239000006188 syrup Substances 0.000 description 1
239000003826 tablet Substances 0.000 description 1
238000011191 terminal modification Methods 0.000 description 1
230000002110 toxicologic effect Effects 0.000 description 1
231100000759 toxicological effect Toxicity 0.000 description 1
231100000041 toxicology testing Toxicity 0.000 description 1
108091006108 transcriptional coactivators Proteins 0.000 description 1
230000002103 transcriptional effect Effects 0.000 description 1
239000012096 transfection reagent Substances 0.000 description 1
238000012546 transfer Methods 0.000 description 1
230000009261 transgenic effect Effects 0.000 description 1
230000014621 translational initiation Effects 0.000 description 1
125000000876 trifluoromethoxy group Chemical group FC(F)(F)O* 0.000 description 1
125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 description 1
102000003390 tumor necrosis factor Human genes 0.000 description 1
230000003827 upregulation Effects 0.000 description 1
238000010200 validation analysis Methods 0.000 description 1
239000003981 vehicle Substances 0.000 description 1
230000003612 virological effect Effects 0.000 description 1
230000021542 voluntary musculoskeletal movement Effects 0.000 description 1
229940075420 xanthine Drugs 0.000 description 1

Classifications

- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
- C12N2310/111—Antisense spanning the whole gene, or a large part of it
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/322—2'-R Modification
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/323—Chemical structure of the sugar modified ring structure
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/323—Chemical structure of the sugar modified ring structure
- C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/33—Chemical structure of the base
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/33—Chemical structure of the base
- C12N2310/334—Modified C
- C12N2310/3341—5-Methylcytosine
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/33—Chemical structure of the base
- C12N2310/335—Modified T or U
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/34—Spatial arrangement of the modifications
- C12N2310/341—Gapmers, i.e. of the type ===---===
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/34—Spatial arrangement of the modifications
- C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications

Definitions

RTS0838US2SEQ.txt A computer-readable form of the sequence listing, named RTS0838US2SEQ.txt, which is 560 kb and was created on Jan. 26, 2007, is herein incorporated by reference. GENBANK® numbers and their submission dates are also incorporated herein by reference.
Huntington's disease is a neurodegenerative disorder caused by the mutation of the huntingtin gene. Alteration of this widely expressed single gene results in a progressive, neurodegenerative disorder with a large number of characteristic symptoms. Huntington's disease is an autosomal dominant disorder, with an onset generally in mid-life, although cases of onset from childhood to over 70 years of age have been documented. An earlier age of onset is associated with paternal inheritance, with 70% of juvenile cases being inherited through the father. Symptoms have an emotional, motor and cognitive component. Chorea is a characteristic feature of the motor disorder and is defined as excessive spontaneous movements which are irregularly timed, randomly distributed and abrupt. It can vary from being barely perceptible to severe.
⁇ disorders include dystonia, rigidity, bradykinesia, ocularmotor dysfunction and tremor.
Voluntary movement disorders include fine motor incoordination, dysathria, and dysphagia.
Emotional disorders commonly include depression and irritability, and cognitive component comprises subcortical dementia (Mangiarini et al., 1996 . Cell 87:493-506).
Changes in HD brains are widespread and include neuronal loss and gliosis, particularly in the cortex and striatum (Vonsattel and DiFiglia. 1998 . J. Neuropathol. Exp. Neurol., 57:369-384).
the HD mutation is a CAG expansion that results in the expansion of a poly-glutamine tract in the huntingtin protein, a 350 kDa protein of unknown function (Huntington Disease Collaborative Research Group, 1993 . Cell. 72:971-83).
the normal and expanded HD allele size have been found to be CAG 6-37 and CAG 35-121 repeats, respectively. Longer repeat sequences are associated with earlier disease onset. The mechanism by which the expansion results in pathology is unknown.
the absence of an HD phenotype in individuals deleted for one copy of huntingtin, or increased severtity of disease in those homozygous for the expansion suggests that the mutation does not result in a loss of function (Trottier et al., 1995, Nature Med., 10:104-110).
Models include mice carrying a fragment of the human gene, typically the first one or two exons, which contains the glutamine expansion, in addition to the undisrupted wild-type, endogenous, mouse gene; mice carrying the full length human huntingtin with an expanded glutamine repeat region, again with the endogenous mouse gene; and mice with pathogenic CAG repeats inserted into the CAG repeat region. All of the models have at least some shared features with the human disease. These mice have allowed for the testing of a number of different therapeutic agents for the prevention, amelioration and treatment of HD (see, e.g., Hersch and Ferrante, 2004 . NeuroRx.
the compounds are believed to function by a number of different mechanisms including transcription inhibition, caspace inhibition, histone deacetylase inhibition, antioxidant, huntingtin inhibition/antioxidant, biogenergetic/antioxidant, antiexcitotoxic, and antiapoptotic.
RNA interference RNA interference
RNAi directed to huntingtin reduced huntingtin mRNA and protein expression in cell culture and a HD mouse model. The authors report that huntingtin gene silencing improved behavioral and neuropathological abnormalities associated with HD.
Nguyen et al. (2005 . PNAS, 102: 11840-45; incorporated herein by reference), used the metal-binding compound clioquinol to treat PC12 cells expressing the mutant huntingtin gene and found reduced accumulation of mutant protein.
Treating the HD mouse model R6/2 with clioquinol resulted in improved behavioral and pathologic phenotypes, including decreased huntingtin aggregate accumulation, decreased striatal atrophy, improved rotarod performance, reduction of weight loss, normalization of blood glucose and insulin levels, and extension of lifespan, supporting the conclusion that reduction in mutant huntingtin protein is therapeutic for HD.
One embodiment of the invention is an antisense oligonucleotide 12 to 35 nucleotides in length comprising at least 12 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs 46-357.
the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 50, 93, 100, 105, 110, 125, 137, 345, 346, and 353.
the antisense oligonucleotide has at least 95% or 100% complementarity to SEQ ID NO: 4.
the antisense oligonucleotide has at least one modified internucleoside linkage, sugar moiety, or nucleobase.
the antisense oligonucleotide comprises a chimeric oligonucleotide having a gap segment positioned between 5′ and 3′ wing segments, and in some embodiments, the gap segment of the chimeric oligonucleotide is comprised of 2′-deoxynucleotides and the wing segments are comprised of nucleotides having modified sugar moieties.
the modified sugar moiety is 2′-OMe or a bicyclic nucleic acid.
the gap segment of the chimeric oligonucleotide consists of ten 2′-deoxynucleotides and each wing segment consists of five 2′-O-methoxyethyl-modified nucleotides, and in a more preferred embodiment said antisense oligonucleotide is 20 nucleotides in length.
each internucleoside linkage of the antisense oligonucleotide is a phosphorothioate internucleoside linkage.
each cytosine of the antisense oligonucleotide is a 5-methylcytosine.
the antisense oligonucleotide is 17 to 25 nucleotides in length. In another embodiment, the antisense oligonucleotide is 19 to 23 nucleotides in length. In another embodiment the antisense oligonucleotide is 20 nucleotides in length.
Another embodiment of the invention is a pharmaceutical composition
a pharmaceutical composition comprising any of the antisense oligonucleotide described herein and a pharmaceutically acceptable diluent.
Another embodiment is a method of treating an individual at risk of suffering from or currently suffering from Huntington's Disease (HD) comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an antisense compound 12 to 35 nucleobases in length having at least 90% complementarity to nucleotides 1650-1704, 1807-1874, 3183-3228, 4010-4087, 4265-4288, 4553-4608, 5781-5820, and 6793-6796 of SEQ ID NO: 4, where the administration treats the individual.
the administering comprises intrathecal delivery, intracerebroventricular delivery, or intraparenchymall delivery.
the administering comprises administration into the cerebrospinal fluid of the individual by intrathecal infusion.
the treatment comprises improvement in one or more indicators of HD.
the treatment comprises increasing the survival time of the individual.
the treatment comprises delaying the onset of HD.
the antisense compound has at least at least 95%, or 100%, complementarity to nucleotides 1650-1704, 1807-1874, 3183-3228, 4010-4087, 4265-4288, 4553-4608, 5781-5820, and 6793-6796 of SEQ ID NO: 4.
the antisense compound is an antisense oligonucleotide.
the antisense oligonucleotide has at least one modified internucleoside linkage, sugar moiety, or nucleobase.
the oligonucleotide comprises a chimeric oligonucleotide having a gap segment positioned between 5′ and 3′ wing segments.
the gap segment of the chimeric oligonucleotide is comprised of 2′-deoxynucleotides and the wing segments are comprised of nucleotides having modified sugar moieties.
the modified sugar moiety is 2′-OMe or a bicyclic nucleic acid.
the gap segment of the chimeric oligonucleotide consists of ten 2′-deoxynucleotides and each wing segment consists of five 2′-O-methoxyethyl-modified nucleotides.
each internucleoside linkage is a phosphorothioate internucleoside linkage.
each cytosine is a 5-methylcytosine.
the compound comprises 17 to 25 nucleotides, in others 19 to 23 nucleotides, in others, 20 nucleotides.
the method further comprises selecting an individual suffering from HD. In some embodiments, the method further comprises selecting an individual susceptible to HD.
Another embodiment is a method of treating an individual at risk of suffering from or currently suffering from Huntington's Disease (HD) comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an antisense oligonucleotide 12 to 35 nucleotides in length comprising at least 12 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs 46-357.
the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 50, 93, 100, 105, 110, 125, 137, 345, 346, and 353.
Another embodiment is the use of any of the antisense compounds or oligonucleotides disclosed herein in the manufacture of a medicament for treatment of HD.
One embodiment is use of an antisense oligonucleotide 12 to 35 nucleotides in length comprising at least 12 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs 46-357 in the preparation of a medicament for treating HD.
Another embodiment is the use of an antisense oligonucleotide 12 to 35 nucleotides in length comprising at least 12 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 50, 93, 100, 105, 110, 125, 137, 345, 346, and 353 in the preparation of a medicament for treating HD.
the treatment of HD is the slowing of HD progression in an individual suffering from HD.
the treatment of HD is preventing the onset of HD in an individual susceptible to HD.
the treatment of HD comprises increasing survival time of the individual.
Huntington's disease is a progressive, neurodegenerative disease caused by mutation of a widely expressed, single gene, huntingtin.
the mutation is an expansion of a CAG repeat region, wherein a larger expansion results in greater severity of the disease and an earlier age of onset.
the mutation results in a variety of motor, emotional and cognitive symptoms, and results in the formation of huntingtin aggregates in brain.
the absence of a phenotype for a single gene deletion, and an increase in disease severity in individuals carrying two mutated copies of the huntingtin gene suggests that the mutation does not result in a loss of function.
Antisense technology provides a mechanism for the development of therapeutic agents for a variety of diseases, including Huntington's Disease.
the principle behind antisense technology is that an antisense compound, which hybridizes to a target nucleic acid, modulates gene expression activities such as transcription or translation. This sequence specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in disease.
the present invention is directed to antisense compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding huntingtin, and which modulate the expression of huntingtin.
the antisense compound is targeted to human huntingtin (SEQ ID NOs 1-5 and 45).
Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of slowing HD progression, and methods of ameliorating or delaying the onset of HD symptoms. Such methods employ antisense compounds which modulate the expression of huntingtin.
Treatment encompasses slowing of disease progression in an individual suffering from Huntington's Disease (HD) as well as delaying the onset of HD in an individual susceptible to HD.
treatment methods comprise the administration to the cerebrospinal fluid of the individual a therapeutically effective amount of a pharmaceutical composition comprising an antisense compound or oligonucleotide targeted to huntingtin.
Such treatment methods further comprise increasing the survival time of an individual suffering from HD, or increasing the survival time of an individual susceptible to HD.
Slowing of disease progression is indicated by a lack of measurable change in, or an improvement of, one or more indicators of HD, including molecular markers or symptoms of the disease.
the delaying of the onset of HD is indicated by a lack of clinical presentation of indicators of HD.
the present invention employs antisense compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding huntingtin, ultimately modulating the amount of huntingtin protein produced.
a suitable form of modulation is inhibition of nucleic acid molecules encoding huntingtin, which is evidenced by a reduction in the levels of nucleic acids encoding huntingtin.
antisense compounds including antisense oligonucleotides, for use in inhibiting the expression of nucleic acid molecules encoding huntingtin, i.e. reducing the levels of nucleic acid molecules encoding huntingtin.
target nucleic acid and “nucleic acid molecule encoding huntingtin” have been used for convenience to encompass DNA encoding huntingtin, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA.
Antisense oligonucleotides which hybridize to and modulate the expression of one or more nucleic acids encoding huntingtin are considered to be “targeted to huntingtin.”
Antisense oligonucleotides of the present invention do not necessarily distinguish between wild-type huntingtin target nucleic acids and mutant huntingtin target nucleic acids. It is clinically desirable to reduce the levels of mutant huntingtin target nucleic acids, without introducing adverse effects due to reduction of the levels of wild-type huntingtin target nucleic acids.
antisense oligonucleotides at least 90% complementary to exon 30 of SEQ ID NO: 4, which encompasses nucleotides 4010-4087 of SEQ ID NO: 4.
antisense oligonucleotides are at least 90% complementary to nucleotides 4010-4087 of SEQ ID NO: 4.
This embodiment includes antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 99, 100, 101, or 102.
antisense oligonucleotides are at least 90% complementary to nucleotides 4028-4146 of SEQ ID NO: 4.
the antisense oligonucleotides include those comprising a sequence selected from SEQ ID NOs: 99, 100, 101, 102, or 103.
antisense oligonucleotides are at least 90% complementary to nucleotides 4538-4615 of SEQ ID NO: 4.
This embodiment includes antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 109, 110, 111, or 112.
antisense oligonucleotides are at least 90% complementary to exon 34 of SEQ ID NO: 4, which encompasses nucleotides 4553-4608 of SEQ ID NO: 4.
the antisense oligonucleotides are at least 90% complementary to nucleotides 4553-4608 of SEQ ID NO: 4.
This embodiment includes antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 110 or 112.
antisense oligonucleotides are at least 90% complementary to nucleotides 5781-5820 of SEQ ID NO: 4. In one embodiment, the antisense oligonucleotides comprise a sequence selected from SEQ ID NOs: 123, 124, or 125.
antisense oligonucleotides are at least 90% complementary to exon 42 of SEQ ID NO: 4, which encompasses nucleotides 5722-5863 of SEQ ID NO: 4.
antisense oligonucleotides are at least 90% complementary to nucleotides 5722-5863 of SEQ ID NO: 4.
antisense oligonucleotides comprise a sequence selected from SEQ ID NOs: 123, 124, or 125.
antisense oligonucleotides are at least 90% complementary to nucleotides 6763-6796 of SEQ ID NO: 4. In one embodiment, the antisense oligonucleotides comprise a sequence selected from SEQ ID NOs: 136, 137, or 138.
antisense oligonucleotides are at least 90% complementary to a region comprising both exon 48 of SEQ ID NO: 4, which encompasses nucleotides 6560-6773 of SEQ ID NO: 4, and exon 49 of SEQ ID NO: 4, which encompasses nucleotides 6774-6919 of SEQ ID NO: 4. Accordingly, antisense oligonucleotides are at least 90% complementary to nucleotides 6560-6919 of SEQ ID NO: 4.
antisense oligonucleotides comprise a sequence selected from SEQ ID NOs: 134, 135, 136, 137, 138, or 151.
antisense oligonucleotides are at least 90% complementary to nucleotides 3183-3253 of SEQ ID NO: 4.
antisense oligonucleotides comprise a sequence selected from SEQ ID NOs: 90, 91, 92, 93, and 94.
antisense oligonucleotides are at least 90% complementary to nucleotides 3183-3228 of SEQ ID NO: 4. This aspect encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 90, 91, 92, or 93.
antisense oligonucleotides are at least 90% complementary to a region comprising both exon 23 of SEQ ID NO: 4, which encompasses nucleotides 3019-3211 of SEQ ID NO:4, and exon 24 of SEQ ID NO: 4, which encompasses nucleotides 3212-3288 of SEQ ID NO: 4.
antisense oligonucleotides are at least 90% complementary to nucleotides 3091-3288 of SEQ ID NO: 4. This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 90, 91, 92, 93, or 94.
antisense oligonucleotides are at least 90% complementary to nucleotides 4265-4288 of SEQ ID NO: 4. This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 104 or 105.
antisense oligonucleotides are at least 90% complementary to exon 31 of SEQ ID NO: 4, which encompasses nucleotides 4088-4311 of SEQ ID NO: 4.
the antisense oligonucleotides are at least 90% complementary to nucleotides 4088-4311 of SEQ ID NO: 4.
This embodiment encompasses the antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 103, 104, or 105.
the antisense oligonucleotides are at least 90% complementary to nucleotides 1607-1704 of SEQ ID NO: 45.
This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 342, 343, 344, 345, 346, 347, 348, or 349.
antisense oligonucleotides are at least 90% complementary to nucleotides 1650-1704 of SEQ ID NO: 45.
This aspect encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 345, 346, 347, 348, or 349.
antisense oligonucleotides are at least 90% complementary to nucleotides 1807-1874 of SEQ ID NO: 45.
This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 351, 352, 353, 354, 355, 356, or 357.
antisense oligonucleotides are at least 90% complementary to nucleotides 985-1580 of SEQ ID NO: 45.
This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 47, 48, 49, 50, 51, 52, 53, or 54.
antisense oligonucleotides are at least 90% complementary to nucleotides 1079-1459 of SEQ ID NO: 45, which comprises a plurality of CAG repeats.
This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 48, 49, 50, 51, 52, or 53.
antisense oligonucleotides are at least 90% complementary to nucleotides 1055-1477 of SEQ ID NO: 45.
This region comprises a plurality of CAG repeats.
This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 338, 48, 49, 50, 51, 52, or 53.
antisense oligonucleotides are at least 90% complementary to nucleotides 1019-1542 of SEQ ID NO: 45.
This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 48, 49, 50, 51, 52, 53, or 54.
antisense oligonucleotides are at least 95% complementary to a nucleotide region recited herein. In additional embodiments, antisense oligonucleotides are at least 96%, 97%, 98%, 99% or 100% complementary to a nucleotide region recited herein.
an “individual suffering from Huntington's Disease (HD)” is an individual who has received from a health professional, such as a physician, a diagnosis of HD.
a health professional such as a physician
Relevant diagnostic tests are well known in the art and are understood to include, without limitation, genetic testing to determine the presence of a mutation in the huntingtin gene, neurological examination, and brain imaging. Genetic testing for mutations in the huntingtin gene is a particularly accurate diagnostic test for the presence of HD.
An “individual susceptible to Huntington's Disease (HD)” is understood to include an individual who, based on genetic testing and/or family history, is likely to develop HD. Genetic testing for mutations in the huntingtin gene is a particularly accurate diagnostic test for susceptibility to HD. Indicators of HD may also be employed in the identification of an individual susceptible to HD.
an antisense oligonucleotide targeted to huntingtin to the central nervous system (CNS) of an individual, and in particular to the regions of the CNS affected by HD.
CNS central nervous system
a preferred method of providing antisense oligonucleotides targeted to huntingtin to the tissues of the CNS is via administration of the antisense oligonucleotides directly into the cerebrospinal fluid (CSF).
Means of the delivery to the CSF and brain include intrathecal (IT), intracerebroventricular (ICV), and intraparenchymal administration.
IT or ICV administration may be achieved through the use of surgically implanted pumps that infuse a therapeutic agent into the cerebrospinal fluid. Intraparenchymal delivery may be achieved by the surgical placement of a catheter into the brain.
delivery to the CSF and “administration to the CSF” encompass the IT infusion or ICV infusion of antisense oligonucleotides targeted to huntingtin through the use of an infusion pump.
IT infusion is a suitable means for delivery to the CSF.
the antisense oligonucleotide is continuously infused into the CSF for the entire course of treatment; such administration is referred to as “continuous infusion” or, in the case of IT infusion, “continuous IT infusion.” Also contemplated is continuous intraparenchymal infusion using a pump.
an infusion pump such as, for example, the Medtronic SyncroMed® II pump, is employed to deliver antisense oligonucleotides targeted to huntingtin to the CNS.
the SyncroMed® II pump is surgically implanted according the procedures set forth by the manufacturer.
the pump contains a reservoir for retaining a drug solution, which is pumped at a programmed dose into a catheter that is surgically implanted.
the catheter is surgically intrathecally implanted.
the drug is the pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin.
a “pharmaceutical composition comprising an antisense oligonucleotide” refers to a composition comprising an antisense oligonucleotide targeted to huntingtin in a pharmaceutically acceptable diluent.
a suitable pharmaceutically acceptable diluent is phosphate-buffered saline.
an ISIS Number represents the nonadecasodium salt of the antisense oligonucleotide having the provided nucleobase sequence, where nucleosides 1 to 5 and 16 to 20 have 2′-O-methoxyethyl sugar moieties, nucleosides 6 to 15 are 2′-deoxynucleotides, each internucleoside linkage is a phosphorothioate linkage, and each cytosine is a 5-methylcytosine.
a “therapeutically effective amount” is an amount of a compound that provides a therapeutic benefit to an individual.
a therapeutically effective amount of an antisense compound targeted to huntingtin, such as an antisense oligonucleotide is an amount that slows, or prevents the progression of HD, or prevents or delays the onset of HD.
a therapeutically effective amount of an antisense oligonucleotide targeted to huntingtin ranges from 8 mg to 12 mg of antisense oligonucleotide. In other embodiments, a therapeutically effect amount of an antisense oligonucleotide targeted to huntingtin is 10 mg.
“treating” a patient with HD includes administering a therapeutically effective amount of a compound of the invention.
slowing disease progression means the prevention of, or delay in, a clinically undesirable change in one or more clinical parameters in an individual suffering from HD, such as those described herein. It is well within the abilities of a physician to identify a slowing of disease progression in an individual suffering from HD, using one or more of the disease assessment tests described herein. Additionally, it is understood that a physician may administer to the individual diagnostic tests other than those described herein to assess the rate of disease progression in an individual suffering from HD.
delaying the onset of HD means delaying undesirable changes in one or more indicators of HD that were previously negative for HD.
a physician may use family history of HD to determine an approximate age of HD onset in an individual susceptible to HD to determine if onset of HD is delayed.
indicators of HD are parameters employed by a medical professional, such as a physician, to diagnose or measure the progression of HD, and include, without limitation, genetic testing, hearing, eye movements, strength, coordination, chorea (rapid, jerky, involuntary movements), sensation, reflexes, balance, movement, mental status, dementia, personality disorder, family history, weight loss, and degeneration of the caudate nucleus. Degeneration of the caudate nucleus is assessed via brain imaging techniques such as magnetic resonance imaging (MRI) or computed tomography (CT) scan.
MRI magnetic resonance imaging
CT computed tomography
an “improvement in an indicator of HD” refers to the absence of an undesirable change, or the presence of a desirable change, in one or more indicators of HD.
an improvement in an indicator of HD is evidenced by the absence of a measureable change in one or more indicators of HD.
an improvement in an indicator of HD is evidenced by a desirable change in one or more indicators of HD.
a slowing of disease progression may further comprise an increase in survival time in an individual suffering from HD.
An “increase in survival time” is understood to mean increasing the survival of an individual suffering from HD, relative to an approximate survival time based upon HD progression and/or family history of HD.
a physician can use one or more of the disease assessment tests described herein to predict an approximate survival time of an individual suffering from HD.
a physician may additionally use the family history of an individual suffering from HD to predict survival time.
Antisense compounds targeted to huntingtin can be used to modulate the expression of huntingtin in an animal, such as a human, including humans suffering from, or susceptible to, HD.
the antisense compounds effectively inhibit the levels or function of huntingtin RNA. Because reduction in huntingtin mRNA levels can lead to alteration in huntingtin protein products of expression as well, such resultant alterations can also be measured.
Antisense compounds of the present invention that effectively inhibit the level or function of huntingtin RNA or protein products of expression are considered an active antisense compound.
the antisense compounds of the invention inhibit the expression of huntingtin causing a reduction of RNA by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%.
the reduction of the expression of huntingtin can be measured in a bodily fluid, tissue or organ of the animal.
Methods of obtaining samples for analysis, such as body fluids or tissues, and methods of preparation of the samples to allow for analysis are well known to those skilled in the art.
Methods for analysis of RNA and protein levels are discussed above and are well known to those skilled in the art.
the effects of treatment using the compounds of the invention can be assessed by measuring biomarkers associated with the target gene expression in the aforementioned fluids, tissues or organs, collected from an animal contacted with one or more compounds of the invention, by routine clinical methods known in the art.
Biomarkers of huntingtin include but are not limited to the accumulation of huntingtin positive neuronal inclusions, loss of certain neuronal tissue, etc.
a subject's systemic response to treatment can be assessed by monitoring clinically relevant measures that include but are not limited to: liver transaminases, bilirubin, albumin, blood urea nitrogen, creatine and other markers of kidney and liver function; interleukins, tumor necrosis factors, intracellular adhesion molecules, C-reactive protein and other markers of inflammation.
Bodily fluids, organs or tissues can be contacted with one or more of the compounds of the invention resulting in modulation of huntingtin expression in the cells of bodily fluids, organs or tissues.
An effective amount can be determined by monitoring the modulatory effect of the antisense compound or compounds or compositions on target nucleic acids or their products by methods routine to the skilled artisan.
ex vivo methods of treatment whereby cells or tissues are isolated from a subject, contacted with an effective amount of the antisense compound or compounds or compositions and reintroduced into the subject by routine methods known to those skilled in the art.
a compound of an isolated double stranded RNA oligonucleotide in the manufacture of a medicament for inhibiting huntingtin expression or overexpression.
Antisense compounds targeted to huntingtin can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier.
Acceptable carriers and dilutents are well known to those skilled in the art. Selection of a dilutent or carrier is based on a number of factors, including, but not limited to, the solubility of the compound and the route of administration. Such considerations are well understood by those skilled in the art.
the antisense compounds of the present invention inhibit the expression of huntingtin.
Antisense compounds targeted to huntingtin can also be used in the manufacture of a medicament for the treatment of diseases and disorders related to huntingtin expression.
the disease or disorder is Huntington's disease.
the antisense compounds of the present invention comprise any pharmaceutically acceptable salts, esters, or salts of such esters, or any other functional chemical equivalent which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the oligomeric compounds of the present invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
prodrug indicates a therapeutic agent that is prepared in an inactive or less active form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
prodrug versions of the oligonucleotides of the invention are prepared as SATE ((S-acetyl-2-thioethyl)phosphate) derivatives according to the methods disclosed in WO 93/24510 or WO 94/26764.
pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
Sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic administration to humans.
sodium salts of dsRNA compounds are also provided.
the oligomeric compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos.
the present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention.
the pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including but not limited to ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer (intratracheal, intranasal, epidermal and transdermal), oral or parenteral.
Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
Sites of administration are known to those skilled in the art. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be useful for oral administration.
the pharmaceutical formulations of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
compositions for non-parenteral administration include one or more modifications from naturally-occurring oligonucleotides (i.e. full-phosphodiester deoxyribosyl or full-phosphodiester ribosyl oligonucleotides). Such modifications may increase binding affinity, nuclease stability, cell or tissue permeability, tissue distribution, or other biological or pharmacokinetic property.
naturally-occurring oligonucleotides i.e. full-phosphodiester deoxyribosyl or full-phosphodiester ribosyl oligonucleotides.
modifications may increase binding affinity, nuclease stability, cell or tissue permeability, tissue distribution, or other biological or pharmacokinetic property.
Oral compositions for administration of non-parenteral oligomeric compounds can be formulated in various dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas.
the term “alimentary delivery” encompasses e.g. oral, rectal, endoscopic and sublingual/buccal administration.
Such oral oligomeric compound compositions can be referred to as “mucosal penetration enhancers.”
Oligomeric compounds such as oligonucleotides
Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860.
Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. Nos. 09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999) and 10/071,822, filed Feb. 8, 2002, which are herein incorporated by reference.
oral oligomeric compound compositions comprise at least one member of the group consisting of surfactants, fatty acids, bile salts, chelating agents, and non-chelating surfactants.
surfactants fatty acids, bile salts, chelating agents, and non-chelating surfactants.
fatty acids fatty acids
bile salts fatty acids
chelating agents chelating agents
non-chelating surfactants are well known to those skilled in the art.
a “pharmaceutical carrier” or “excipient” can be a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal and are known in the art.
the excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
Oral oligomeric compositions may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
compositions of the invention can contain two or more oligomeric compounds.
compositions of the present invention can contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target.
compositions of the present invention can contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Two or more combined compounds may be used together or sequentially.
oligomeric compound refers to a polymeric structure capable of hybridizing to a region of a nucleic acid molecule. This term includes oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics and chimeric combinations of these. Oligomeric compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular. Moreover, branched structures are known in the art.
An “antisense compound” or “antisense oligomeric compound” refers to an oligomeric compound that is at least partially complementary to the region of a nucleic acid molecule to which it hybridizes and which modulates (increases or decreases) its expression.
oligomeric compounds include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, and siRNAs.
EGS external guide sequence
these compounds can be introduced in the form of single-stranded, double-stranded, circular, branched or hairpins and can contain structural elements such as internal or terminal bulges or loops.
Oligomeric double-stranded compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
the compounds of the instant invention are non-catalytic.
an “antisense oligonucleotide” is an antisense compound that is a nucleic acid-based oligomer, but does not include siRNA duplexes.
the “antisense oligonucleotide” can be a single-stranded nucleic acid molecule.
An antisense oligonucleotide can be chemically modified.
Antisense compounds comprise from about 12 to about 35 linked nucleotides. This embodies antisense compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length.
the antisense compounds are 15 to 30 linked nucleotides in length, as exemplified above.
the antisense compounds are 17 to 25 linked nucleotides in length, as exemplified herein.
the antisense compounds are 19, 20, 21, 22, 23, or 24 linked nucleotides in length, or alternatively the oligomeric compounds range from 19 to 24 linked nucleotides in length.
the antisense compounds are 21, 22, 23, or 24 linked nucleotides in length, or alternatively the oligomeric compounds range from 21 to 24 linked nucleotides in length.
the antisense compounds are 20 linked nucleotides in length.
double-stranded antisense compounds encompass short interfering RNAs (siRNAs).
siRNA short interfering RNAs
the term “siRNA” is defined as a double-stranded compound having a first and second strand, each strand having a central portion and two independent terminal portions.
the central portion of the first strand is complementary to the central portion of the second strand, allowing hybridization of the strands.
the terminal portions are independently, optionally complementary.
the ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang.
the first strand of the siRNA is antisense to the target nucleic acid, while the second strand is complementary to the first strand.
the sense strand of the siRNA can then be designed and synthesized as the complement of the antisense strand and either strand may contain modifications or additions to either terminus.
both strands of the siRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini. It is possible for one end of a duplex to be blunt and the other to have overhanging nucleobases. In one embodiment, the number of overhanging nucleobases is from 1 to 6 on the 3′ end of each strand of the duplex.
the number of overhanging nucleobases is from 1 to 6 on the 3′ end of only one strand of the duplex. In a further embodiment, the number of overhanging nucleobases is from 1 to 6 on one or both 5′ ends of the duplexed strands. In another embodiment, the number of overhanging nucleobases is zero.
double-stranded antisense compounds are canonical siRNAs.
canonical siRNA is defined as a double-stranded oligomeric compound having a first strand and a second strand each strand being 21 nucleobases in length with the strands being complementary over 19 nucleobases and having on each 3′ termini of each strand a deoxy thymidine dimer (dTdT) which in the double-stranded compound acts as a 3′ overhang.
dTdT deoxy thymidine dimer
Each strand of the siRNA duplex may be from about 12 to about 35 nucleobases. In a preferred embodiment, each strand of the siRNA duplex is about 17 to about 25 nucleobases.
the central complementary portion may be from about 12 to about 35 nucleobases in length. In a preferred embodiment, the central complimentary portion is about 17 to about 25 nucleobases in length. It is understood that each the strand of the siRNA duplex and the central complementary portion may be about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases in length.
the terminal portions can be from 1 to 6 nucleobases. It is understood that the terminal portions can be about 1, 2, 3, 4, 5, or 6 nucleobases in length.
the siRNAs may also have no terminal portions.
the two strands of an siRNA can be linked internally leaving free 3′ or 5′ termini, or can be linked to form a continuous hairpin structure or loop.
the hairpin structure may contain an overhang on either the 5′ or 3′ terminus producing an extension of single-stranded character.
the double-stranded antisense compounds are blunt-ended siRNAs.
blunt-ended siRNA is defined as an siRNA having no terminal overhangs. That is, at least one end of the double-stranded compound is blunt.
siRNAs whether canonical or blunt, act to elicit dsRNAse enzymes and trigger the recruitment or activation of the RNAi antisense mechanism.
single-stranded RNAi (ssRNAi) compounds that act via the RNAi antisense mechanism are contemplated.
double-stranded compounds may include conjugate groups attached to one of the termini, selected nucleobase positions, sugar positions or to one of the internucleoside linkages.
the two strands can be linked via a non-nucleic acid moiety or linker group.
the compounds can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex.
the compounds can be fully or partially double-stranded.
duplex-forming regions of a single strand When formed from two strands, or a single strand that takes the form of a self-complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary when they base pair in Watson-Crick fashion.
the antisense compounds in accordance with this invention may comprise a complementary antisense compound from about 12 to about 35 nucleobases (i.e. from about 12 to about 35 linked nucleosides).
a single-stranded antisense compound of the invention comprises from about 12 to about 35 nucleobases
a double-stranded antisense compound of the invention (such as a siRNA, for example) comprises two strands, each of which is from about 12 to about 35 nucleobases.
Contained within the antisense compounds of the invention are antisense portions.
antisense portion is that part of the antisense compound that is designed to work by one of the aforementioned antisense mechanisms.
antisense portions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases.
the antisense compounds of the invention have antisense portions of 12 to 35 nucleobases. It is understood that the antisense portion may be about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases in length.
Antisense compounds 12 to 35 nucleobases in length comprising a stretch of at least 8, preferably at least 12, more preferably at least 17 consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well. Also contemplated are antisense compounds 12 to 35 nucleobases in length comprising a stretch of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucl nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.
Antisense compounds of the invention include antisense compound sequences that comprise at least the 8 (or 9-19) consecutive nucleobases from the 5′-terminus of one of the illustrative antisense compounds (the remaining nucleobases being a consecutive stretch of the same antisense beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the antisense contains about 12 to 35 nucleobases).
antisense compounds are represented by antisense compound sequences that comprise at least the 8 (or 9-19) consecutive nucleobases from the 3′-terminus of one of the illustrative antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the antisense compound contains about 12 to about 35 nucleobases).
antisense compounds may be represented by antisense compound sequences that comprise at least 8 (or 9-19) consecutive nucleobases from an internal portion of the sequence of an illustrative compound, and may extend in either or both directions until the antisense contains about 12 to about 35 nucleobases.
nucleoside is a base-sugar combination.
the base portion of the nucleoside is normally a heterocyclic base (sometimes referred to as a “nucleobase” or simply a “base”).
the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.
the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
the respective ends of this linear polymeric compound can be further joined to form a circular compound.
linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound.
the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
the normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.
Oligomeric compounds may comprise modified internucleoside linkages, e.g. non-naturally occurring internucleoside linkages.
oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom.
modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
Oligomeric compounds can have one or more modified internucleoside linkages.
Modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, phosphonoacetate and thiophosphonoacetate (see Sheehan et al., Nucleic Acids Research, 2003, 31(14), 4
Oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage, i.e., a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
Various salts, mixed salts and free acid forms are also included.
N3′-P5′-phosphoramidates have been reported to exhibit both a high affinity towards a complementary RNA strand and nuclease resistance (Gryaznov et al., J. Am. Chem. Soc., 1994, 116, 3143-3144). N3′-P5′-phosphoramidates have been studied with some success in vivo to specifically down regulate the expression of the c-myc gene (Skorski et al., Proc. Natl. Acad. Sci., 1997, 94, 3966-3971; and Faira et al., Nat. Biotechnol., 2001, 19, 40-44).
oligomeric compounds may have one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular —CH 2 —NH—O—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 — (known as a methylene (methylimino) or MMI backbone), —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —O—N(CH 3 )—CH 2 —CH 2 — (wherein the native phosphodiester internucleotide linkage is represented as —O—P( ⁇ O)(OH)—O—CH 2 —).
Some oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
morpholino linkages formed in part from the sugar portion of a nucleoside
siloxane backbones sulfide, sulfoxide and sulfone backbones
formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
riboacetyl backbones alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
Oligomeric compounds may also contain one or more substituted sugar moieties.
Suitable compounds can comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
oligonucleotides comprise one of the following at the 2′ position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
One modification includes 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group.
a further modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—(CH 2 ) 2 —O—(CH 2 ) 2 —N(CH 3 ) 2 , also described in examples hereinbelow.
2′-dimethylaminooxyethoxy i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group
2′-DMAOE also known as 2′-DMAOE
2′-dimethylaminoethoxyethoxy also known in the art as 2′-O-dimethyl-amino-ethoxy
modifications include 2′-methoxy (2′-O—CH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ), 2′-allyl (2′-CH 2 —CH ⁇ CH 2 ), 2′-O-allyl (2′-O—CH 2 —CH ⁇ CH 2 ) and 2′-fluoro (2′-F).
the 2′-modification may be in the arabino (up) position or ribo (down) position.
One 2′-arabino modification is 2′-F.
Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide.
Antisense compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.
a further modification includes bicyclic sugar moieties referred to as “bicyclic nucleic acids” or “BNAs” in which the 2′-hydroxyl group of the ribosyl sugar ring is linked to the 4′ carbon atom of the sugar ring to form the bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461).
BNAs bicyclic nucleic acids
the linkage can be a methylene (—CH 2 —) group bridging the 2′ oxygen atom and the 4′ carbon atom, or can be an ethylene group.
the alpha-L isomer of the bicyclic nucleic acid moiety wherein the linkage is a methylene group is an additional modified sugar moiety.
Another bicyclic sugar moiety that has been prepared and studied has the bridge going from the 3′-hydroxyl group via a single methylene group to the 4′ carbon atom of the sugar ring thereby forming a 3′-C,4′-C-oxymethylene linkage (see U.S. Pat. No. 6,043,060).
oligonucleotide mimetics Another group of oligomeric compounds includes oligonucleotide mimetics.
mimetics as it is applied to oligonucleotides includes oligomeric compounds wherein the furanose ring or the furanose ring and the internucleotide linkage are replaced with novel groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate.
the heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid.
Oligonucleotide mimetics include peptide nucleic acid (PNA) compounds (Nielsen et al., Science, 1991, 254, 1497-1500), morpholino-based compounds (see, for example, U.S. Pat. No. 5,034,506), cyclohexene nucleic acids (CeNA). In CeNA oligonucleotides (Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602), and phosphonomonoester nucleic acids.
PNA peptide nucleic acid
CeNA cyclohexene nucleic acids
Oligomeric compounds can also include nucleobase (often referred to in the art as heterocyclic base or simply as “base”) modifications or substitutions.
nucleobase often referred to in the art as heterocyclic base or simply as “base” modifications or substitutions.
“unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
a “substitution” is the replacement of an unmodified or natural base with another unmodified or natural base.
Modified nucleobases mean other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C ⁇ C—CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Certain nucleobase modifications increase the binding affinity of the compounds of the invention.
5-substituted pyrimidines include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently suitable base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications. It is understood in the art that modification of the base does not entail such chemical modifications as to produce substitutions in a nucleic acid sequence.
Oligomeric compounds of the present invention can also include polycyclic heterocyclic compounds in place of one or more of the naturally-occurring heterocyclic base moieties.
a number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G-clamps or cytidine analogs.
Oligomeric compounds may be chemically linked to one or more moieties or conjugates which enhance the oligomeric compound properties such as activity, cellular distribution or cellular uptake. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups.
Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligo-mers.
Additional conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention.
Oligomeric compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of an oligomeric compound to enhance properties such as for example nuclease stability. Included in stabilizing groups are cap structures. By “cap structure or terminal cap moiety” is meant chemical modifications, which have been incorporated at either terminus of oligonucleotides. These terminal modifications protect the oligomeric compounds having terminal nucleic acid molecules from exonuclease degradation, and can improve delivery and/or localization within a cell.
the cap can be present at either the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present on both termini of a single strand, or one or more termini of both strands of a double-stranded compound.
This cap structure is not to be confused with the inverted methylguanosine “5′ cap” present at the 5′ end of native mRNA molecules.
the 5′-cap includes inverted abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted nu
3′-cap structures include, for example 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxyp
the present invention also includes oligomeric compounds which are chimeric compounds.
“Chimeric” oligomeric compounds or “chimeras,” in the context of this invention are single- or double-stranded oligomeric compounds, such as oligonucleotides, which contain two or more chemically distinct regions, each comprising at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
Chimeric antisense oligonucleotides are one form of oligomeric compound.
oligonucleotides typically contain at least one region which is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, alteration of charge, increased stability and/or increased binding affinity for the target nucleic acid.
An additional region of the oligonucleotide may serve as a substrate for RNAses or other enzymes.
RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.
RNA target when bound by a DNA-like oligomeric compound, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression.
the cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNase III or RNAseL which cleaves both cellular and viral RNA. Cleavage products of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
Chimeric oligomeric compounds of the invention can be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, oligonucleotide mimetics, or regions or portions thereof. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922.
a “gapmer” is defined as an oligomeric compound, generally an oligonucleotide, having a 2′-deoxyoligonucleotide region flanked by non-deoxyoligonucleotide segments.
the central region is referred to as the “gap.”
the flanking segments are referred to as “wings.” While not wishing to be bound by theory, the gap of the gapmer presents a substrate recognizable by RNase H when bound to the RNA target whereas the wings do not provide such a substrate but can confer other properties such as contributing to duplex stability or advantageous pharmacokinetic effects.
Each wing can be one or more non-deoxyoligonucleotide monomers (if one of the wings has zero non-deoxyoligonucleotide monomers, a “emimer” is described).
the gapmer is a ten deoxynucleotide gap flanked by five non-deoxynucleotide wings. This is referred to as a 5-10-5 gapmer. Other configurations are readily recognized by those skilled in the art.
the wings comprise 2′-MOE modified nucleotides.
the gapmer has a phosphorothioate backbone.
the gapmer has 2′-MOE wings and a phosphorothioate backbone. Other suitable modifications are readily recognizable by those skilled in the art.
Oligomerization of modified and unmodified nucleosides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).
Oligomeric compounds can be conveniently and routinely made through the well-known technique of solid phase synthesis.
Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
Oligomeric compounds and phosphoramidites are made by methods well known to those skilled in the art. Oligomerization of modified and unmodified nucleosides is performed according to literature procedures for DNA like compounds (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA like compounds (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA:Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713) synthesis as appropriate. Alternatively, oligomers may be purchased from various oligonucleotide synthesis companies such as, for example, Dharmacon Research Inc., (Lafayette, Colo.).
the oligomeric compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed (including solution phase synthesis).
a 96-well plate format is particularly useful for the synthesis, isolation and analysis of oligonucleotides for small scale applications.
Hybridization means the pairing of complementary strands of oligomeric compounds. While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
An oligomeric compound is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
Stringent hybridization conditions or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.
“Complementarty,” as used herein, refers to the capacity for precise pairing between two nucleobases on one or two oligomeric compound strands. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position.
the oligomeric compound and the further DNA or RNA are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other.
“specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.
an antisense oligonucleotide is “fully complementary” to a target nucleic acid when each nucleobase of the antisense oligonucleotide is capable of undergoing precise base pairing with an equal number of nucleobases in the target nucleic acid. It is understood in the art that the sequence of the antisense oligonucleotide need not be fully complementary to that of its target nucleic acid to be active in inhibiting the activity of the target nucleic acid.
non-complementary positions also known as “mismatches”
mismatches there are “non-complementary” positions, also known as “mismatches”, between the antisense oligonucleotide and the target nucleic acid, and such non-complementary positions may be tolerated between an antisense oligonucleotide and the target nucleic acid provided that the antisense oligonucleotide remains specifically hybridizable to the target nucleic acid.
387916 having one non-complementary nucleobases with respect to mouse huntingtin, is capable of reducing mouse huntingtin mRNA levels in vitro and in vivo.
non-complementary nucleobase means a nucleobase of an antisense oligonucleotide that is unable to undergo precise base pairing with a nucleobase at a corresponding position in a target nucleic acid.
non-complementary and mismatch are interchangable.
antisense oligonucleotides having no more than three non-complementary nucleobases with respect to a nucleic acid encoding huntingtin are considered “complementary” to a nucleic acid encoding huntingtin.
antisense oligonucleotides contain no more than two non-complementary nucleobases with respect to a nucleic acid encoding huntingtin. In further embodiments, antisense oligonucleotides contain no more than one non-complementary nucleobase with respect to a nucleic acid encoding huntingtin.
non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense oligonucleotide.
the non-complementary nucleobase may be at an internal position in the antisense oligonucleotide.
two or more non-complementary nucleobases may be contiguous (i.e. linked) or non-embodiments contiguous.
the antisense oligonucleotides comprise at least 90% sequence complementarity to a huntingtin target nucleic acid. In further embodiments of the invention, the antisense oligonucleotides comprise at least 95% sequence complementarity to a huntingtin target nucleic acid. In further embodiments of the invention, the antisense oligonucleotides comprise at least 96%, 97%, 98% or 99% sequence complementarity to a huntingtin target nucleic acid.
Oligomeric compounds, or a portion thereof may have a defined percent identity to a SEQ ID NO, or a compound having a specific Isis number. This identity may be over the entire length of the oligomeric compound, or in a portion of the oligomeric compound (e.g., nucleobases 1-20 of a 27-mer may be compared to a 20-mer to determine percent identity of the oligomeric compound to the SEQ ID NO.). It is understood by those skilled in the art that an oligonucleotide need not have an identical sequence to those described herein to function similarly to the oligonucleotides described herein.
Shortened (i.e., deleted, and therefore non-identical) versions of oligonucleotides taught herein, or non-identical (i.e., one base replaced with another) versions of the oligonucleotides taught herein fall within the scope of the invention.
Percent identity is calculated according to the number of bases that are identical to the SEQ ID NO or compound to which it is being compared.
the non-identical bases may be adjacent to each other, dispersed through out the oligonucleotide, or both.
a 16-mer having the same sequence as nucleobases 2-17 of a 20-mer is 80% identical to the 20-mer.
a 20-mer containing four nucleobases not identical to the 20-mer is also 80% identical to the 20-mer.
a 14-mer having the same sequence as nucleobases 1-14 of an 18-mer is 78% identical to the 18-mer.
the percent identity is based on the percent of nucleobases in the original sequence present in a portion of the modified sequence. Therefore, a 30 nucleobase oligonucleotide comprising the full sequence of a 20 nucleobase SEQ ID NO would have a portion of 100% identity with the 20 nucleobase SEQ ID NO while further comprising an additional 10 nucleobase portion.
the full length of the modified sequence may constitute a single portion.
Oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the oligonucleotide were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the oligonucleotide that contained no mismatches. Similarly, target specific cleavage was achieved using a 13 nucleobase oligomer, including those with 1 or 3 mismatches. Maher and Dolnick (Nuc. Acid. Res.
tandem 14 nucleobase oligonucleotides 16:3341-3358.1988, incorporated herein by reference
28 and 42 nucleobase oligonucleotide comprised of the sequence of two or three of the tandem oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay.
Each of the three 14 nucleobase oligonucleotides alone were able to inhibit translation, albeit at a more modest level, than the 28 or 42 nucleobase oligonucleotide.
a mixture of the tandem 14 nucleobase oligonucleotides was as effective at inhibiting translation as the 28 nucleobase oligonucleotide targeted to the same region.
Targeting an oligomeric compound to a particular target nucleic acid molecule can be a multistep process. The process usually begins with the identification of a target nucleic acid whose expression is to be modulated.
target nucleic acid and “nucleic acid encoding huntingtin” encompass DNA encoding huntingtin, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA.
the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent.
the target nucleic acid encodes huntingtin.
the targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result.
“Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.
Target regions include, but are not limited to, contiguous nucleotide sequences, translation initiation and termination regions, coding regions, open reading frames, introns, exons, 3′-untranslated regions (3′-UTR), and 5′-untranslated regions (5′-UTR). Within regions of target nucleic acids are target segments.
a “target segment” means a sequence of a huntingtin target nucleic acid to which one or more antisense oligonucleotides are complementary.
the term “5′ target site” is defined as the 5′-most nucleobase of a target segment to which an antisense oligonucleotide is complementary.
a “3′ target site” is defined as the 3′-most nucleobase of a target segment to which an antisense oligonucleotide is complementary.
RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants.” More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence. Variants can result in mRNA variants including, but not limited to, those with alternate splice junctions, or alternate initiation and termination codons. Variants in genomic and mRNA sequences can result in disease. Oligonucleotides to such variants are within the scope of the instant invention.
compositions and methods for modulating the expression of genes which are presented in Table 2.
Table 2 Listed in Table 2 are the gene target names, as well as GENBANK® accession numbers used to design oligomeric compounds targeted to each gene.
TABLE 2 Gene Target Names and Sequences SEQ Species Genbank # ID NO Human AB209506.1 1 Human BE378835.1 2 Human L12392.1 3 Human NM_002111.5 4 Human nucleotides 462000 to 634000 of 5 NT_006081.17 Mouse AK042204.1 6 Mouse AK049546.1 7 Mouse L23312.1 8 Mouse L23313.1 9 Mouse NM_010414.1 10 Mouse nucleotides 2036000 to 2190000 of 11 NT_039302.4 Mouse NM_010414.1 (mouse short form) * 44 Human cut from genomic ad Sac1 and EcoR1 sites 45 surrounding exon 1, expanded CAG to results in 130 gln in this region * NM_010414.1 (mouse short form)
Modulation of expression of a target nucleic acid can be achieved through alteration of any number of nucleic acid (DNA or RNA) functions.
“Modulation” means a perturbation of function, for example, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in expression.
modulation of expression can include perturbing splice site selection of pre-mRNA processing.
“Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. These structures include the products of transcription and translation.
Modulation of expression means the perturbation of such functions.
the functions of DNA to be modulated can include replication and transcription.
RNA to be modulated can include translocation functions, which include, but are not limited to, translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, and translation of protein from the RNA.
RNA processing functions that can be modulated include, but are not limited to, splicing of the RNA to yield one or more RNA species, capping of the RNA, 3′ maturation of the RNA and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA.
Modulation of expression can result in the increased level of one or more nucleic acid species or the decreased level of one or more nucleic acid species, either temporally or by net steady state level.
modulation of the expression of HUNTINGTIN ⁇ can mean increase or decrease in target RNA or protein levels.
modulation of expression can mean an increase or decrease of one or more RNA splice products, or a change in the ratio of two or more splice products.
oligomeric compounds of the present invention can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels.
the effect of oligomeric compounds of the present invention on target nucleic acid expression can be routinely determined using, for example, PCR or Northern blot analysis.
Cell lines are derived from both normal tissues and cell types and from cells associated with various disorders (e.g. hyperproliferative disorders). Cell lines derived from multiple tissues and species can be obtained from American Type Culture Collection (ATCC, Manassas, Va.) and are well known to those skilled in the art.
Primary cells or those cells which are isolated from an animal and not subjected to continuous culture, can be prepared according to methods known in the art or obtained from various commercial suppliers. Additionally, primary cells include those obtained from donor human subjects in a clinical setting (i.e. blood donors, surgical patients).
Cells isolated from Huntington's disease patients are also used to test the effects of antisense compounds targeted to huntingtin.
the mutant huntingtin gene may be present in a heterozygous or homozygous form.
Such cells are available from National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository, examples of which include fibroblasts having repository number GM04281 or GM04478.
fibroblasts having repository number GM04281 or GM04478.
Cells from Huntington's disease patients are cultured according to procedures recommended by the supplier.
the pharmacological effects of antisense inhibition of huntingtin can be assessed in cell lines isolated from neuronal cells expressing either wild-type or mutant forms of the huntingtin gene.
the mutant forms of huntingtin are associated with particular phenotypes, and the effects on these phenotypes are evaluated following antisense inhibition of huntingtin.
An example of such cells are striatal cells established from Hdh Q111 knock-in mice, which bear 111 CAG repeats inserted into the mouse huntingtin locus. Establishment of striatal cell lines isolated from Hdh Q111 mice has been described by Trettel et al. (Human Mol. Genet., 2000, 9, 2799-2809). Striatal cell lines established from mice bearing a wild-type huntingtin gene are used for comparison studies.
Huntingtin mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR.
RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA by methods known in the art. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.
RNA blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology , Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.
Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISMTM 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
the method of analysis of modulation of RNA levels is not a limitation of the instant invention.
Levels of a protein encoded by huntingtin can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS).
Antibodies directed to a protein encoded by huntingtin can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.).
a validated target segment includes at least an 8-nucleobase portion of a target region. In another embodiment, a validated target segment includes at least a 12-nucleobase portion of a target region to which an active oligomeric compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.
Target segments can include DNA or RNA sequences that comprise at least the 8, or at least the 12, consecutive nucleobases from the 5′-terminus of a validated target segment (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA contains about 12 to about 35 nucleobases).
validated target segments are represented by DNA or RNA sequences that comprise at least the 8, or at least the 12 consecutive nucleobases from the 3′-terminus of a validated target segment (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA contains about 12 to about 35 nucleobases).
a validated oligomeric target segment can be represented by DNA or RNA sequences that comprise at least 8, or at least the 12, consecutive nucleobases from an internal portion of the sequence of a validated target segment, and can extend in either or both directions until the oligonucleotide contains about 12 to about 35 nucleobases.
the validated target segments identified herein can be employed in a screen for additional compounds that modulate the expression of huntingtin.
“Modulators” are those compounds that modulate the expression of huntingtin and which comprise at least an 8-nucleobase portion which is complementary to a validated target segment.
the screening method comprises the steps of contacting a validated target segment of a nucleic acid molecule encoding huntingtin with one or more candidate modulators, and selecting for one or more candidate modulators which perturb the expression of a nucleic acid molecule encoding huntingtin.
the candidate modulator or modulators are capable of modulating the expression of a nucleic acid molecule encoding huntingtin
the modulator can then be employed in further investigative studies of the function of huntingtin, or for use as a research, diagnostic, or therapeutic agent.
the validated target segments can also be combined with a second strand as disclosed herein to form stabilized double-stranded (duplexed) oligonucleotides for use as a research, diagnostic, or therapeutic agent.
Antisense compounds targeted to huntingtin are tested in experimental animal models.
the antisense compounds are targeted to the human huntingtin gene alone.
Such antisense compounds have, for example, less than four mismatches to human huntingtin and four or more mismatches to non-human huntingtin.
antisense compounds are targeted to both human and non-human huntingtin.
Such antisense compounds have, for example, less than four mismatches to human huntingtin and less than four mismatches to non-human huntingtin.
normal, wild-type animals may be used to perform toxicity studies of antisense oligonucleotides targeted to huntingtin.
the antisense compounds are administered systemically (e.g. via intraperitoneal injection) at doses of 25, 50, 75, or 100 mg/kg. Animals are monitored for any clinical changes, including changes in body weight. Serum is collected periodically, for example every week or every two weeks, during the dosing period and subjected to analysis using a clinical analyzer to detect any changes in serum chemistry profiles. At the end of the study, the animals are sacrificed. Blood is collected and analyzed for white blood cell count, platelet count, and serum chemistry. The weights of major organs are determined, and histological analyses are performed on spleen, liver, kidney and pancreas.
Antisense compounds targeted to huntingtin may be tested in experimental non-human models of Huntington's disease. Several non-human models have been developed and characterized.
the R6/2 transgenic mouse model has integrated into its genome 1 kilobase of the human huntingtin gene, including the 5′-UTR exon 1 and the first 262 basepairs of intron 1 (Mangiarin L. et al., Cell, 1996, 87, 493-506).
This transgene has 144 CAG repeats.
the transgene encodes for approximately 3% of the N-terminal region of the huntingtin protein, expression of which is driven by the human huntingtin promoter. Expression levels of this truncated version of human huntingtin protein are approximately 75% of the endogenous mouse huntingtin protein levels.
the R6/2 transgenic mice exhibit symptomos of human Huntington's disease and brain dysfunction.
the YAC128 transgenic mice harbor a yeast artificial chromosome (YAC) carrying the entire huntingtin gene, including the promoter region and 128 CAG repeats (Hodgson J. G. et al., Human Mol. Genet., 1998, 5, 1875). This YAC expresses all but exon 1 of the human gene. These transgenic mice do not express endogenouse mouse huntingtin.
YAC yeast artificial chromosome
the endogenous mouse huntingtin gene of the Q111 mice has 111 CAG repeats inserted into exon 1 of the gene (Wheeler V. C. et al., Human Mol. Genet, 8, 115-122).
the CAG repeat in exon 1 of the wild-type mouse huntingtin gene is replaced with 150 CAG repeats (Li C. H. et al., Human Mol. Genet, 2001, 10, 137).
Antisense compounds targeted to huntingtin are administered to the non-human experimental model, for example to transgenic mice that are used as models of Huntington's disease.
Antisense compounds may be administered directly into the central nervous system of the experimental animal, for example through intracerebroventricular (ICV), intrathecal (IT) or intraparenchymal administration. Dosages of antisense compounds administered may be 25, 50, 75, or 100 ug/day, and administration may be accomplished through continuous infusion using a surgically implanted osmotic pump (e.g. an Alzet mini-pump). 0.25, 0.5, or 1 uL/hour. Each dosage is administered to groups of 4 to 6 animals. Control groups of animals may receive saline infusion, or infusion of an antisense compound having a sequence not targeted to any known gene.
ICV intracerebroventricular
IT intrathecal
intraparenchymal administration Dosages of antisense compounds administered may be 25, 50, 75, or 100 ug/day, and administration may be accomplished through continuous infusion using a surgically implanted osmotic pump (e.g. an Alzet mini-pump). 0.25, 0.5,
Animals are treated for several weeks, for example 1, 2, 4, or 8 weeks. Animals are monitored for any clinical changes, including changes in body weight. At the end of the treatment period, animals are sacrificed. The brains are dissected into three regions: forebrain, basal ganglion, and cerebellum. Brain regions undergo histological assessment, including hemotoxylin/eosin staining, GFAP staining (to assess glial cell activation) and FluoroJ staining (to assess neurodegenerative changes). Huntingtin mRNA levels are measured by real-time PCR, and huntingtin protein levels are measured by immunoblotting (western blotting).
the duration of action of antisense compounds targeting huntingtin may also be evaluated.
animals are dosed for 2, 4, 6, or 8 weeks with antisense compounds targeting huntingtin.
the osmotic pumps are removed and animals are sacrificed 0, 1, 2, 4, 6, or 8 weeks following dosing termination.
the brains are dissected into three regions: forebrain, basal ganglion, and cerebellum. Brain regions undergo histological assessment, including hemotoxylin/eosin staining, GFAP staining (to assess glial cell activation) and FluoroJ staining (to assess neurodegenerative changes).
Huntingtin mRNA levels are measured by real-time PCR, and huntingtin protein levels are measured by immunoblotting (western blotting).
oligomeric compounds of the present invention can be utilized for diagnostics, research reagents and kits.
antisense compounds which are able to inhibit gene expression with specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
the oligomeric compounds of the present invention can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.
expression patterns within cells or tissues treated with one or more compounds or compositions of the present invention are compared to control cells or tissues not treated with compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.
gene expression patterns may be identified by microarray analysis.
oligomeric compounds on target nucleic acid expression was tested in cultured cells, for example A549 cells or HD patient fibroblasts for compounds targeted to human huntingtin, and in b.END cells for compounds targeted to mouse huntingtin.
the transfection reagent LIPOFECTIN® was used to introduce oligonucleotide into cells.
Other methods of transfection are well known to those skilled in the art. The method of screening is not a limitation of the instant invention.
Oligonucleotide was mixed with LIPOFECTIN® Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEM®-1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desired concentration of oligonucleotide and a LIPOFECTIN® concentration of 2.5 or 3 ⁇ g/mL per 100 nM oligonucleotide.
This transfection mixture was incubated at room temperature for approximately 0.5 hours.
For cells grown in 96-well plates, wells were washed once with 100 ⁇ L OPTI-MEM®-1 and then treated with 130 ⁇ L of the transfection mixture. Cells are treated and data were obtained in duplicate or triplicate. After approximately 4-7 hours of treatment at 37° C., the medium containing the transfection mixture was replaced with fresh culture medium. Cells were harvested 16-24 hours after oligonucleotide treatment.
the cells may be treated similarly, using appropriate volumes of medium and oligonucleotide.
Quantitation of huntingtin mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions.
RNA was subjected to sequential reverse transcriptase (RT) reaction and real-time PCR, both of which are performed in the same well.
RT and PCR reagents were obtained from Invitrogen Life Technologies (Carlsbad, Calif.).
RT real-time PCR was carried out in the same by adding 20 ⁇ L PCR cocktail (2.5 ⁇ PCR buffer minus MgCl 2 , 6.6 mM MgCl 2 , 375 ⁇ M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5 ⁇ ROX dye) to 96-well plates containing 30 ⁇ L total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).
PCR cocktail 2.5 ⁇ PCR buffer minus Mg
Gene target quantities obtained by RT, real-time PCR were normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RIBOGREEN® (Molecular Probes, Inc. Eugene, Oreg.).
GAPDH expression was quantified by RT, real-time PCR, by being run simultaneously with the target, multiplexing, or separately.
Total RNA was quantified using RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to the manufacturer's instructions.
Probes and primers for use in real-time PCR were designed to hybridize to target-specific sequences.
the primers and probes and the target nucleic acid sequences to which they hybridize are presented in Table 3.
the target-specific PCR probes have FAM covalently linked to the 5′ end and TAMRA or MGB covalently linked to the 3′ end, where FAM is the fluorescent dye and TAMRA or MGB is the quencher dye.
Antisense oligonucleotides were designed to target different regions of the human huntingtin gene, using published sequences cited in Table 2. The sequences and corresponding SEQ ID NOs are shown in Table 4. All compounds in Table 4 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of 10 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) by five-nucleotide “wings”. The wings are composed of 2′-O-(2-methoxyethyl) nucleotides, also known as 2′-MOE nucleotides. The internucleoside (backbone) linkages are phosphorothioate throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.
the antisense oligonucleotides were analyzed for their effect on huntingtin mRNA levels in A549 cells by quantitative real-time PCR as described in other examples herein.
the data presented in Table 4 represent percent inhibition of huntingtin mRNA levels relative to untreated cells. Data are averages from experiments in which cultured cells were treated with the disclosed antisense oligonucleotides.
Antisense oligonucleotides were designed to target different regions of the mouse huntingtin gene, using published sequences cited in Table 2. The sequences and corresponding SEQ ID NOs are shown in Table 5. All compounds in Table 5 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of 10 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) by five-nucleotide “wings”. The wings are composed of 2′-O-(2-methoxyethyl) nucleotides, also known as 2′-MOE nucleotides. The internucleoside (backbone) linkages are phosphorothioate throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.
the antisense oligonucleotides were analyzed for their effect on huntingtin mRNA levels in b.END cells by quantitative real-time PCR as described in other examples herein.
the data presented in Table 5 represent percent inhibition of huntingtin mRNA levels relative to untreated cells. Data are averages from experiments in which cultured cells were treated with the disclosed antisense oligonucleotides.
Isis numbers 387865-387942 are targeted to both human and mouse huntingtin.
antisense oligonucleotides were selected for additional testing in GMO4281 fibroblasts, which originated from an HD patient.
Control oligonucleotides having randomized nucleotide sequences not targeted to any known gene, were also tested.
GM04478 cells which are fibroblasts derived from and HD patient.
the testing was performed according to the procedure used for GM04281 cells. The results are shown in the following table as average percent inhibition, relative to untreated cells.
TABLE 8 Dose response inhibition of human huntingtin in GMO4478 fibroblasts Oligonucleotide Treatment Concentration 9.375 18.75 37.5 75.0 150.0 Isis No.
Each of the antisense oligonucleotides targeted to human huntingtin efficiently reduced huntingtin mRNA levels, in both GM04478 and GMO4281 fibroblasts.
the control oligonucleotides were not able to effectively inhibit huntingtin mRNA levels, particularly at lower doses.
the potency of antisense oligonucleotides targeted to huntingtin is summarized in Table 9.
the potency is illustrated as IC 50 , which is the concentration at which a 50% reduction in huntingtin mRNA levels is observed.
This table also indicates the huntingtin sequence to which the antisense oligonucleotides are complementary, as well as the corresponding 5′ target site. Particular features of the region of the huntingtin sequence to which the antisense oligonucleotides are complementary are also shown. Additionally indicated is the species of huntingtin gene to which the antisense oligonucleotides are targeted.
Target regions to which these inhibitory antisense oligonucleotides are complementary are herein referred to as “validated target segments.”
the antisense oligonucleotides are candidate therapeutic agents for the reduction of huntingtin mRNA levels in vivo.
the antisense oligonucleotides having demonstrated potency in vitro, are further tested in experimental animal models, including experimental models of Huntington's disease, to identify antisense oligonucleotides that may reduce huntingtin mRNA in humans.
the antisense oligonucleotides are administered at therapeutically effect amounts to a human, for the treatment or amelioration of Huntington's disease.
the antisense oligonucleotides are administered at therapeutically effective amounts, to delay the onset of Huntington's disease.
oligonucleotides targeted to huntingtin were selected for additional testing in huntingtin neuronal cell lines.
Mouse striatum cell lines with wild-type huntingtin, STHdhQ7/7 (Q7/7), and mutant huntingtin, STHdhQ111/111 (Q111/111) were transfected with various doses of oligos 387902 and 387916, ranging from approximately 0.05 ⁇ M, to 10 ⁇ M.
a 200V, 2 msec pulse in a 2 mm gap cuvette was used for electroploration transfection.
One million cells were electroporated in the presence of the indicated amount of oligonucleotide. Following electroporation, the cells were plated at a density of 5 ⁇ 10 4 cells per well.
cells are evaluated for phenotypic response by measuring caspase activity using the Promega Apo-ONE® Homogeneous Caspase-3/7 commercial assay. Briefly, cells are plated and Lipofectin® transfected the next day. After 48 hours the media is changed to serum-free DMEM for 24 h prior to the caspase assay.
antisense oligonucleotides In order to evaluate the effects of antisense inhibition of a gene in the central nervous system, it is beneficial to deliver antisense oligonucleotides directly to the central nervous system, for example, by intracerebroventricular (ICV), intrathecal (IT), or intraparenchymal administration.
ICV intracerebroventricular
ICV intrathecal
ICV intraparenchymal administration
ISIS 387902, 387916, 387918, 388249, 388503, 388509, and 388816 were selecting for in vivo testing.
Saline-treated mice were used as control animals. Each treatment or control group included four animals.
RNA was prepared from brain and liver tissues, and subjected to quantitative real-time PCR analysis to measure the reduction in mouse huntingtin mRNA levels.
Each antisense oligonucleotide targeted to huntingtin reduced huntingtin mRNA levels in house brain as shown in the following table.
the species of huntingtin nucleic acid to which antisense oligonucleotide is targeted is also shown.
Mouse huntingtin mRNA levels represent the average for each treatment group and are expressed as percentage of saline control (% saline control).
% saline control TABLE 11 In vivo antisense inhibition of mouse huntingtin Huntingtin Huntingtin mRNA levels, nucleic acid % of saline Isis No. SEQ ID NO: target species control 387902 human, mouse 37% 387916 human, rat 32% (single mismatch to mouse) 387918 human, mouse, 35% rat 388503 mouse 30% 388509 mouse 34%
Each of the antisense oligonucleotides shown in Table 11 reduced huntingtin mRNA levels in mouse brain following the ICV infusion period. Furthermore, ISIS 387916, which has one mismatch to mouse huntingtin, was able to reduce mouse huntingtin mRNA levels in vivo.
antisense oligonucleotides targeted to huntingtin are administered to R6/2 transgenic mice via ICV delivery.
ISIS 387902, 387916, 387918, 388249, 388503, 388509, and 388816 are selected for in vivo testing.
Saline-treated mice are used as control animals.
Each treatment or control group includes four animals.
Surgically implanted Alzet mini-pumps continuously infuse antisense oligonucleotides into mice at a dose of 100 ug/day over a two-week period. During the treatment period, mice are monitored for any clinical changes, such as body weight changes as well as phenotypical behaviors related to the huntingtin transgene.
mice are sacrificed and major organs are isolated.
RNA is prepared from brain and liver tissues, and is subjected to quantitative real-time PCR analysis to measure the reduction in mouse huntingtin mRNA levels. Huntingtin protein expression in the tissue is also measured using standard Western blotting techniques.
Such methods comprise the administration to the cerebrospinal fluid or brain tissue of the individual a pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin. Delivery of the pharmaceutical composition to the cerebrospinal fluid allows for contact of the antisense oligonucleotide with the cells of central nervous system tissues, including tissues affected by HD.
the physician's assessment includes the genetic testing of the HD gene, and a neurological examination.
a surgically implanted pump e.g, a Medtronic SyncroMed® II pump
a pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin to the cerebrospinal fluid or brain of an individual suffering from HD.
the pump is surgically implanted per the procedures outlined by the manufacturer. Drug is retained in the reservoir of the pump, and is pumped at a programmed dose preferably into a catheter that is surgically intrathecally implanted.
the reservoir is loaded with a pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin.
the pharmaceutical composition is administered at an amount that yields an infusion of 8 mg to 12 mg of antisense oligonucleotide into the cerebrospinal fluid.
the amount of antisense oligonucleotide infused is 10 mg.
Administration is for a period of at least 28 days. Individuals are monitored by a medical professional, who evaluates indicators of HD. It is clinically desirable for the administration to slow or halt the progression of HD, or prevent or slow the worsening of, or improve, a symptom or marker of HD.
Such methods comprise the administration to the cerebrospinal fluid or brain of the individual a pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin. Delivery of the pharmaceutical composition to the cerebrospinal fluid allows for contact of the antisense oligonucleotide with the cells of central nervous system tissues, including tissues affected by HD.
a surgically implanted pump e.g, a Medtronic SyncroMed® II pump
a pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin to the cerebrospinal fluid or brain of an individual susceptible to HD.
the pump is surgically implanted per the procedures outlined by the manufacturer. Drug is retained in the reservoir of the pump, and is pumped at a programmed dose preferably into a catheter that is surgically intrathecally implanted.
the reservoir is loaded with a pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin.
the pharmaceutical composition is administered at an amount that yields an infusion of 8 mg to 12 mg of antisense oligonucleotide into the cerebrospinal fluid.
the amount of antisense oligonucleotide infused is 10 mg.
Administration is for a period of at least 28 days. Individuals are monitored by a medical professional, who evaluates indicators of HD. It is clinically desirable for the administration to prevent or delay the onset of symptoms of HD.

Landscapes

Health & Medical Sciences (AREA)
Engineering & Computer Science (AREA)
Life Sciences & Earth Sciences (AREA)
Biomedical Technology (AREA)
Genetics & Genomics (AREA)
Bioinformatics & Cheminformatics (AREA)
Chemical & Material Sciences (AREA)
Organic Chemistry (AREA)
General Health & Medical Sciences (AREA)
Wood Science & Technology (AREA)
General Engineering & Computer Science (AREA)
Biotechnology (AREA)
Molecular Biology (AREA)
Zoology (AREA)
Neurosurgery (AREA)
Neurology (AREA)
Public Health (AREA)
General Chemical & Material Sciences (AREA)
Pharmacology & Pharmacy (AREA)
Veterinary Medicine (AREA)
Chemical Kinetics & Catalysis (AREA)
Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
Medicinal Chemistry (AREA)
Animal Behavior & Ethology (AREA)
Biophysics (AREA)
Physics & Mathematics (AREA)
Plant Pathology (AREA)
Microbiology (AREA)
Biochemistry (AREA)
Psychology (AREA)
Psychiatry (AREA)
Hospice & Palliative Care (AREA)
Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Disclosed herein are compounds, compositions and methods for modulating the expression of huntingtin in a cell, tissue or animal. Further provided are methods of slowing or preventing Huntington's disease progression using an antisense compound targeted to huntingtin. Additionally provided are methods of delaying or preventing the onset of Huntingtin's disease in an individual susceptible to Huntingtin's Disease. Also provided are uses of disclosed compounds and compositions in the manufacture of a medicament for treatment of diseases and disorders.

Description

This application claims priority under 35 U.S.C. 119 to U.S. Provisional Application Ser. No. 60/762,954, filed Jan. 26, 2006, titled “COMPOSITIONS AND THEIR USES DIRECTED TO HUNTINGTIN,” and to U.S. Provisional Application Ser. No. 60/836,290, filed Aug. 7, 2006, titled “COMPOSITIONS AND THEIR USES DIRECTED TO HUNTINGTIN.”
A computer-readable form of the sequence listing, named RTS0838US2SEQ.txt, which is 560 kb and was created on Jan. 26, 2007, is herein incorporated by reference. GENBANK® numbers and their submission dates are also incorporated herein by reference.
Huntington's disease (HD) is a neurodegenerative disorder caused by the mutation of the huntingtin gene. Alteration of this widely expressed single gene results in a progressive, neurodegenerative disorder with a large number of characteristic symptoms. Huntington's disease is an autosomal dominant disorder, with an onset generally in mid-life, although cases of onset from childhood to over 70 years of age have been documented. An earlier age of onset is associated with paternal inheritance, with 70% of juvenile cases being inherited through the father. Symptoms have an emotional, motor and cognitive component. Chorea is a characteristic feature of the motor disorder and is defined as excessive spontaneous movements which are irregularly timed, randomly distributed and abrupt. It can vary from being barely perceptible to severe. Other frequently observed abnormalities include dystonia, rigidity, bradykinesia, ocularmotor dysfunction and tremor. Voluntary movement disorders include fine motor incoordination, dysathria, and dysphagia. Emotional disorders commonly include depression and irritability, and cognitive component comprises subcortical dementia (Mangiarini et al., 1996. Cell 87:493-506). Changes in HD brains are widespread and include neuronal loss and gliosis, particularly in the cortex and striatum (Vonsattel and DiFiglia. 1998. J. Neuropathol. Exp. Neurol., 57:369-384).
The HD mutation is a CAG expansion that results in the expansion of a poly-glutamine tract in the huntingtin protein, a 350 kDa protein of unknown function (Huntington Disease Collaborative Research Group, 1993. Cell. 72:971-83). The normal and expanded HD allele size have been found to be CAG_6-37and CAG_35-121repeats, respectively. Longer repeat sequences are associated with earlier disease onset. The mechanism by which the expansion results in pathology is unknown. However, the absence of an HD phenotype in individuals deleted for one copy of huntingtin, or increased severtity of disease in those homozygous for the expansion suggests that the mutation does not result in a loss of function (Trottier et al., 1995, Nature Med., 10:104-110). Transcriptional deregulation and loss of function of transcriptional coactivator proteins have been implicated in HD pathogenesis. Mutant huntingtin has been shown specifically to disrupt activator-dependent transcription in the early stages of HD pathogenesis (Dunah et al., 2002. Science 296:2238-2243). Gene profiling of human blood has identified 322 mRNAs that show significantly altered expression in HD blood samples as compared to normal or presymptomatic individuals. Expression of marker genes was similarly substantially altered in post-mortem brain samples from HD caudate, suggesting that upregulation of genes in blood samples reflects disease mechanisms found in brain. Monitoring of gene expression may provide a sensitive and quantitative method to monitor disease progression, especially in the early stages of disease in both animal models and human patients (Borovecki et al., 2005, Proc. Natl. Acad. Sci. USA 102:11023-11028).
Identification of the gene has allowed for the development of animal models of the disease, including transgenic mice carrying mutated human or mouse forms of the gene. Models include mice carrying a fragment of the human gene, typically the first one or two exons, which contains the glutamine expansion, in addition to the undisrupted wild-type, endogenous, mouse gene; mice carrying the full length human huntingtin with an expanded glutamine repeat region, again with the endogenous mouse gene; and mice with pathogenic CAG repeats inserted into the CAG repeat region. All of the models have at least some shared features with the human disease. These mice have allowed for the testing of a number of different therapeutic agents for the prevention, amelioration and treatment of HD (see, e.g., Hersch and Ferrante, 2004. NeuroRx. 1:298-306) using a number of endpoints. The compounds are believed to function by a number of different mechanisms including transcription inhibition, caspace inhibition, histone deacetylase inhibition, antioxidant, huntingtin inhibition/antioxidant, biogenergetic/antioxidant, antiexcitotoxic, and antiapoptotic.
A number of authors have reported that the repression of the mutant huntingtin transgene in animal models of HD reduces the symptoms associated with the disease, (see e.g. Diaz-Hernandez et al., (2005. J. Neurosci. 25:9773-81; incorporated herein by reference). Wang et al., (2005. Neurosci. Res. 53:241-9; incorporated herein by reference) report that small interfering RNAs (siRNAs) directed against the huntingtin gene in the mouse model R6/2 inhibited transgenic huntingtin expression and significantly prolonged longevity, improved motor function and slowed loss of body weight.
Machida et al., (2006. Biochem. Biophys. Res. Commun. 343: 190-7; incorporated herein by reference), report that recombinant adeno-associated virus (rAAV)-mediated delivery of RNA interference (RNAi) into the striatum of a HD mouse model ameliorated neuropathological abnormalities associated with HD, such as insoluable protein accumulation and down-regulation of DARPP-32 expression. Importantly, the authors state that neuronal aggregates in the striatum were reduced after RNAi transduction in the animals compared to those at the time point of RNAi transduction.
Harper et al., (2005. PNAS 102:5820-25; incorporated herein by reference), found that RNAi directed to huntingtin reduced huntingtin mRNA and protein expression in cell culture and a HD mouse model. The authors report that huntingtin gene silencing improved behavioral and neuropathological abnormalities associated with HD.
Rodrigues-Lebron et al., (2005. Mol. Ther. 12:618-33; incorporated herein by reference), report that a recombinant adeno-associated viral serotype 5 (rAAV5) gene transfer of RNAi to suppress the levels of striatal mutant huntingtin in the R6/1 HD transgenic mouse resulted in reduced levels of huntingtin mRNA and protein. The reduction in huntingtin was concomitant with a reduction in the size and number of neuronal intranuclear inclusions and other markers of HD, and resulted in delayed onset of the rear paw clasping phenotype exhibited by the R6/1 mice.
Nguyen et al., (2005. PNAS, 102: 11840-45; incorporated herein by reference), used the metal-binding compound clioquinol to treat PC12 cells expressing the mutant huntingtin gene and found reduced accumulation of mutant protein. Treating the HD mouse model R6/2 with clioquinol resulted in improved behavioral and pathologic phenotypes, including decreased huntingtin aggregate accumulation, decreased striatal atrophy, improved rotarod performance, reduction of weight loss, normalization of blood glucose and insulin levels, and extension of lifespan, supporting the conclusion that reduction in mutant huntingtin protein is therapeutic for HD.
Based on these and other studies, one of skill in the art recognizes that reducing the expression of the mutant huntingtin gene will be therapeutic for HD.
One embodiment of the invention is an antisense oligonucleotide 12 to 35 nucleotides in length comprising at least 12 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs 46-357. In a preferred embodiment, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 50, 93, 100, 105, 110, 125, 137, 345, 346, and 353. In a further embodiment, the antisense oligonucleotide has at least 95% or 100% complementarity to SEQ ID NO: 4. In a further embodiment, the antisense oligonucleotide has at least one modified internucleoside linkage, sugar moiety, or nucleobase. In a further embodiment, the antisense oligonucleotide comprises a chimeric oligonucleotide having a gap segment positioned between 5′ and 3′ wing segments, and in some embodiments, the gap segment of the chimeric oligonucleotide is comprised of 2′-deoxynucleotides and the wing segments are comprised of nucleotides having modified sugar moieties. Is still other embodiments, the modified sugar moiety is 2′-OMe or a bicyclic nucleic acid. In a preferred embodiment, the gap segment of the chimeric oligonucleotide consists of ten 2′-deoxynucleotides and each wing segment consists of five 2′-O-methoxyethyl-modified nucleotides, and in a more preferred embodiment said antisense oligonucleotide is 20 nucleotides in length.
In another embodiment each internucleoside linkage of the antisense oligonucleotide is a phosphorothioate internucleoside linkage.
In another embodiment each cytosine of the antisense oligonucleotide is a 5-methylcytosine.
In another embodiment, the antisense oligonucleotide is 17 to 25 nucleotides in length. In another embodiment, the antisense oligonucleotide is 19 to 23 nucleotides in length. In another embodiment the antisense oligonucleotide is 20 nucleotides in length.
Another embodiment of the invention is a pharmaceutical composition comprising any of the antisense oligonucleotide described herein and a pharmaceutically acceptable diluent.
Another embodiment is a method of treating an individual at risk of suffering from or currently suffering from Huntington's Disease (HD) comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an antisense compound 12 to 35 nucleobases in length having at least 90% complementarity to nucleotides 1650-1704, 1807-1874, 3183-3228, 4010-4087, 4265-4288, 4553-4608, 5781-5820, and 6793-6796 of SEQ ID NO: 4, where the administration treats the individual. In a some embodiments, the administering comprises intrathecal delivery, intracerebroventricular delivery, or intraparenchymall delivery. In a some embodiments, the administering comprises administration into the cerebrospinal fluid of the individual by intrathecal infusion. In some embodiments, the treatment comprises improvement in one or more indicators of HD. In some embodiments, the treatment comprises increasing the survival time of the individual. In some embodiments, the treatment comprises delaying the onset of HD. In some embodiments, the antisense compound has at least at least 95%, or 100%, complementarity to nucleotides 1650-1704, 1807-1874, 3183-3228, 4010-4087, 4265-4288, 4553-4608, 5781-5820, and 6793-6796 of SEQ ID NO: 4.
In some embodiments, the antisense compound is an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide has at least one modified internucleoside linkage, sugar moiety, or nucleobase. In some embodiments, the oligonucleotide comprises a chimeric oligonucleotide having a gap segment positioned between 5′ and 3′ wing segments. In some embodiments, the gap segment of the chimeric oligonucleotide is comprised of 2′-deoxynucleotides and the wing segments are comprised of nucleotides having modified sugar moieties. In some embodiments, the modified sugar moiety is 2′-OMe or a bicyclic nucleic acid. In some embodiments, the gap segment of the chimeric oligonucleotide consists of ten 2′-deoxynucleotides and each wing segment consists of five 2′-O-methoxyethyl-modified nucleotides. In some embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage. In some embodiments, each cytosine is a 5-methylcytosine. In some embodiments, the compound comprises 17 to 25 nucleotides, in others 19 to 23 nucleotides, in others, 20 nucleotides.
In some embodiments, the method further comprises selecting an individual suffering from HD. In some embodiments, the method further comprises selecting an individual susceptible to HD.
Another embodiment is a method of treating an individual at risk of suffering from or currently suffering from Huntington's Disease (HD) comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an antisense oligonucleotide 12 to 35 nucleotides in length comprising at least 12 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs 46-357. In some embodiments, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 50, 93, 100, 105, 110, 125, 137, 345, 346, and 353.
Another embodiment is the use of any of the antisense compounds or oligonucleotides disclosed herein in the manufacture of a medicament for treatment of HD. One embodiment is use of an antisense oligonucleotide 12 to 35 nucleotides in length comprising at least 12 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs 46-357 in the preparation of a medicament for treating HD. Another embodiment is the use of an antisense oligonucleotide 12 to 35 nucleotides in length comprising at least 12 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 50, 93, 100, 105, 110, 125, 137, 345, 346, and 353 in the preparation of a medicament for treating HD. In a further embodiment, the treatment of HD is the slowing of HD progression in an individual suffering from HD. In a further embodiment, the treatment of HD is preventing the onset of HD in an individual susceptible to HD. In a further embodiment, the treatment of HD comprises increasing survival time of the individual.
Overview
Huntington's disease is a progressive, neurodegenerative disease caused by mutation of a widely expressed, single gene, huntingtin. The mutation is an expansion of a CAG repeat region, wherein a larger expansion results in greater severity of the disease and an earlier age of onset. The mutation results in a variety of motor, emotional and cognitive symptoms, and results in the formation of huntingtin aggregates in brain. The absence of a phenotype for a single gene deletion, and an increase in disease severity in individuals carrying two mutated copies of the huntingtin gene suggests that the mutation does not result in a loss of function.
Antisense technology provides a mechanism for the development of therapeutic agents for a variety of diseases, including Huntington's Disease. The principle behind antisense technology is that an antisense compound, which hybridizes to a target nucleic acid, modulates gene expression activities such as transcription or translation. This sequence specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in disease.
The present invention is directed to antisense compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding huntingtin, and which modulate the expression of huntingtin. In a preferred embodiment, the antisense compound is targeted to human huntingtin (SEQ ID NOs 1-5 and 45). Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of slowing HD progression, and methods of ameliorating or delaying the onset of HD symptoms. Such methods employ antisense compounds which modulate the expression of huntingtin.
Therapeutics
Provided herein are methods for treating an individual suffering from Huntington's Disease (HD). Treatment encompasses slowing of disease progression in an individual suffering from Huntington's Disease (HD) as well as delaying the onset of HD in an individual susceptible to HD. In some embodiments, such treatment methods comprise the administration to the cerebrospinal fluid of the individual a therapeutically effective amount of a pharmaceutical composition comprising an antisense compound or oligonucleotide targeted to huntingtin. Such treatment methods further comprise increasing the survival time of an individual suffering from HD, or increasing the survival time of an individual susceptible to HD. Slowing of disease progression is indicated by a lack of measurable change in, or an improvement of, one or more indicators of HD, including molecular markers or symptoms of the disease. The delaying of the onset of HD is indicated by a lack of clinical presentation of indicators of HD.
The present invention employs antisense compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding huntingtin, ultimately modulating the amount of huntingtin protein produced. A suitable form of modulation is inhibition of nucleic acid molecules encoding huntingtin, which is evidenced by a reduction in the levels of nucleic acids encoding huntingtin. Accordingly, disclosed herein are antisense compounds, including antisense oligonucleotides, for use in inhibiting the expression of nucleic acid molecules encoding huntingtin, i.e. reducing the levels of nucleic acid molecules encoding huntingtin. As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding huntingtin” have been used for convenience to encompass DNA encoding huntingtin, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. Antisense oligonucleotides which hybridize to and modulate the expression of one or more nucleic acids encoding huntingtin are considered to be “targeted to huntingtin.” Antisense oligonucleotides of the present invention do not necessarily distinguish between wild-type huntingtin target nucleic acids and mutant huntingtin target nucleic acids. It is clinically desirable to reduce the levels of mutant huntingtin target nucleic acids, without introducing adverse effects due to reduction of the levels of wild-type huntingtin target nucleic acids.
In one embodiment, antisense oligonucleotides at least 90% complementary to exon 30 of SEQ ID NO: 4, which encompasses nucleotides 4010-4087 of SEQ ID NO: 4. Thus, antisense oligonucleotides are at least 90% complementary to nucleotides 4010-4087 of SEQ ID NO: 4. This embodiment includes antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 99, 100, 101, or 102.
In another embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 4028-4146 of SEQ ID NO: 4. In one embodiment, the antisense oligonucleotides include those comprising a sequence selected from SEQ ID NOs: 99, 100, 101, 102, or 103.
In another embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 4538-4615 of SEQ ID NO: 4. This embodiment includes antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 109, 110, 111, or 112.
In another embodiment, antisense oligonucleotides are at least 90% complementary to exon 34 of SEQ ID NO: 4, which encompasses nucleotides 4553-4608 of SEQ ID NO: 4. Thus, in this embodiment, the antisense oligonucleotides are at least 90% complementary to nucleotides 4553-4608 of SEQ ID NO: 4. This embodiment includes antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 110 or 112.
In another embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 5781-5820 of SEQ ID NO: 4. In one embodiment, the antisense oligonucleotides comprise a sequence selected from SEQ ID NOs: 123, 124, or 125.
In another embodiment, antisense oligonucleotides are at least 90% complementary to exon 42 of SEQ ID NO: 4, which encompasses nucleotides 5722-5863 of SEQ ID NO: 4. Thus, in one embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 5722-5863 of SEQ ID NO: 4. In one embodiment, antisense oligonucleotides comprise a sequence selected from SEQ ID NOs: 123, 124, or 125.
In another embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 6763-6796 of SEQ ID NO: 4. In one embodiment, the antisense oligonucleotides comprise a sequence selected from SEQ ID NOs: 136, 137, or 138.
In another embodiment, antisense oligonucleotides are at least 90% complementary to a region comprising both exon 48 of SEQ ID NO: 4, which encompasses nucleotides 6560-6773 of SEQ ID NO: 4, and exon 49 of SEQ ID NO: 4, which encompasses nucleotides 6774-6919 of SEQ ID NO: 4. Accordingly, antisense oligonucleotides are at least 90% complementary to nucleotides 6560-6919 of SEQ ID NO: 4.
In one embodiment, antisense oligonucleotides comprise a sequence selected from SEQ ID NOs: 134, 135, 136, 137, 138, or 151.
In another embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 3183-3253 of SEQ ID NO: 4. In one embodiment, antisense oligonucleotides comprise a sequence selected from SEQ ID NOs: 90, 91, 92, 93, and 94. In a further embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 3183-3228 of SEQ ID NO: 4. This aspect encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 90, 91, 92, or 93.
In another embodiment, antisense oligonucleotides are at least 90% complementary to a region comprising both exon 23 of SEQ ID NO: 4, which encompasses nucleotides 3019-3211 of SEQ ID NO:4, and exon 24 of SEQ ID NO: 4, which encompasses nucleotides 3212-3288 of SEQ ID NO: 4. Thus, in one embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 3091-3288 of SEQ ID NO: 4. This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 90, 91, 92, 93, or 94.
In another embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 4265-4288 of SEQ ID NO: 4. This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 104 or 105.
In another embodiment, antisense oligonucleotides are at least 90% complementary to exon 31 of SEQ ID NO: 4, which encompasses nucleotides 4088-4311 of SEQ ID NO: 4. Thus, in this embodiment, the antisense oligonucleotides are at least 90% complementary to nucleotides 4088-4311 of SEQ ID NO: 4. This embodiment encompasses the antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 103, 104, or 105.
In another embodiment, the antisense oligonucleotides are at least 90% complementary to nucleotides 1607-1704 of SEQ ID NO: 45. This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 342, 343, 344, 345, 346, 347, 348, or 349. In one aspect, antisense oligonucleotides are at least 90% complementary to nucleotides 1650-1704 of SEQ ID NO: 45. This aspect encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 345, 346, 347, 348, or 349.
In another embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 1807-1874 of SEQ ID NO: 45. This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 351, 352, 353, 354, 355, 356, or 357.
In another embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 985-1580 of SEQ ID NO: 45. This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 47, 48, 49, 50, 51, 52, 53, or 54.
In another embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 1079-1459 of SEQ ID NO: 45, which comprises a plurality of CAG repeats. This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 48, 49, 50, 51, 52, or 53.
In another embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 1055-1477 of SEQ ID NO: 45. This region comprises a plurality of CAG repeats. This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 338, 48, 49, 50, 51, 52, or 53.
In another embodiment, antisense oligonucleotides are at least 90% complementary to nucleotides 1019-1542 of SEQ ID NO: 45. This embodiment encompasses antisense oligonucleotides comprising a sequence selected from SEQ ID NOs: 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 48, 49, 50, 51, 52, 53, or 54.
In further embodiments, antisense oligonucleotides are at least 95% complementary to a nucleotide region recited herein. In additional embodiments, antisense oligonucleotides are at least 96%, 97%, 98%, 99% or 100% complementary to a nucleotide region recited herein.
As used herein, an “individual suffering from Huntington's Disease (HD)” is an individual who has received from a health professional, such as a physician, a diagnosis of HD. Relevant diagnostic tests are well known in the art and are understood to include, without limitation, genetic testing to determine the presence of a mutation in the huntingtin gene, neurological examination, and brain imaging. Genetic testing for mutations in the huntingtin gene is a particularly accurate diagnostic test for the presence of HD.
An “individual susceptible to Huntington's Disease (HD)” is understood to include an individual who, based on genetic testing and/or family history, is likely to develop HD. Genetic testing for mutations in the huntingtin gene is a particularly accurate diagnostic test for susceptibility to HD. Indicators of HD may also be employed in the identification of an individual susceptible to HD.
In order for antisense inhibition of huntingtin to have a clinically desirable effect, it is beneficial to deliver an antisense oligonucleotide targeted to huntingtin to the central nervous system (CNS) of an individual, and in particular to the regions of the CNS affected by HD. As the blood-brain barrier is generally impermeable to antisense oligonucleotides administered systemically, a preferred method of providing antisense oligonucleotides targeted to huntingtin to the tissues of the CNS is via administration of the antisense oligonucleotides directly into the cerebrospinal fluid (CSF). Means of the delivery to the CSF and brain include intrathecal (IT), intracerebroventricular (ICV), and intraparenchymal administration. IT or ICV administration may be achieved through the use of surgically implanted pumps that infuse a therapeutic agent into the cerebrospinal fluid. Intraparenchymal delivery may be achieved by the surgical placement of a catheter into the brain. As used herein, “delivery to the CSF” and “administration to the CSF” encompass the IT infusion or ICV infusion of antisense oligonucleotides targeted to huntingtin through the use of an infusion pump. In some embodiments, IT infusion is a suitable means for delivery to the CSF. In other embodiments, the antisense oligonucleotide is continuously infused into the CSF for the entire course of treatment; such administration is referred to as “continuous infusion” or, in the case of IT infusion, “continuous IT infusion.” Also contemplated is continuous intraparenchymal infusion using a pump.
In some embodiments, an infusion pump such as, for example, the Medtronic SyncroMed® II pump, is employed to deliver antisense oligonucleotides targeted to huntingtin to the CNS. The SyncroMed® II pump is surgically implanted according the procedures set forth by the manufacturer. The pump contains a reservoir for retaining a drug solution, which is pumped at a programmed dose into a catheter that is surgically implanted. For intrathecal administration of a drug, the catheter is surgically intrathecally implanted. In the context of the methods provided herein, the drug is the pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin.
As used herein, a “pharmaceutical composition comprising an antisense oligonucleotide” refers to a composition comprising an antisense oligonucleotide targeted to huntingtin in a pharmaceutically acceptable diluent. By way of example, a suitable pharmaceutically acceptable diluent is phosphate-buffered saline. As provided herein, an ISIS Number represents the nonadecasodium salt of the antisense oligonucleotide having the provided nucleobase sequence, where nucleosides 1 to 5 and 16 to 20 have 2′-O-methoxyethyl sugar moieties, nucleosides 6 to 15 are 2′-deoxynucleotides, each internucleoside linkage is a phosphorothioate linkage, and each cytosine is a 5-methylcytosine.
As used herein, a “therapeutically effective amount” is an amount of a compound that provides a therapeutic benefit to an individual. For example, a therapeutically effective amount of an antisense compound targeted to huntingtin, such as an antisense oligonucleotide, is an amount that slows, or prevents the progression of HD, or prevents or delays the onset of HD. In one embodiment, a therapeutically effective amount of an antisense oligonucleotide that will result in an improvement to, or prevents or slows the worsening of, one or more indicators or symptoms of HD, such as those described herein. In some embodiments, a therapeutically effective amount of an antisense oligonucleotide targeted to huntingtin ranges from 8 mg to 12 mg of antisense oligonucleotide. In other embodiments, a therapeutically effect amount of an antisense oligonucleotide targeted to huntingtin is 10 mg. As used herein, “treating” a patient with HD includes administering a therapeutically effective amount of a compound of the invention.
As used herein, “slowing disease progression” means the prevention of, or delay in, a clinically undesirable change in one or more clinical parameters in an individual suffering from HD, such as those described herein. It is well within the abilities of a physician to identify a slowing of disease progression in an individual suffering from HD, using one or more of the disease assessment tests described herein. Additionally, it is understood that a physician may administer to the individual diagnostic tests other than those described herein to assess the rate of disease progression in an individual suffering from HD.
As used herein, “delaying the onset of HD” means delaying undesirable changes in one or more indicators of HD that were previously negative for HD. A physician may use family history of HD to determine an approximate age of HD onset in an individual susceptible to HD to determine if onset of HD is delayed.
As used herein, “indicators of HD,” are parameters employed by a medical professional, such as a physician, to diagnose or measure the progression of HD, and include, without limitation, genetic testing, hearing, eye movements, strength, coordination, chorea (rapid, jerky, involuntary movements), sensation, reflexes, balance, movement, mental status, dementia, personality disorder, family history, weight loss, and degeneration of the caudate nucleus. Degeneration of the caudate nucleus is assessed via brain imaging techniques such as magnetic resonance imaging (MRI) or computed tomography (CT) scan.
As used herein, an “improvement in an indicator of HD” refers to the absence of an undesirable change, or the presence of a desirable change, in one or more indicators of HD. In one embodiment, an improvement in an indicator of HD is evidenced by the absence of a measureable change in one or more indicators of HD. In another embodiment, an improvement in an indicator of HD is evidenced by a desirable change in one or more indicators of HD.
A slowing of disease progression may further comprise an increase in survival time in an individual suffering from HD. An “increase in survival time” is understood to mean increasing the survival of an individual suffering from HD, relative to an approximate survival time based upon HD progression and/or family history of HD. A physician can use one or more of the disease assessment tests described herein to predict an approximate survival time of an individual suffering from HD. A physician may additionally use the family history of an individual suffering from HD to predict survival time.
Antisense compounds targeted to huntingtin can be used to modulate the expression of huntingtin in an animal, such as a human, including humans suffering from, or susceptible to, HD. In one embodiment, the antisense compounds effectively inhibit the levels or function of huntingtin RNA. Because reduction in huntingtin mRNA levels can lead to alteration in huntingtin protein products of expression as well, such resultant alterations can also be measured. Antisense compounds of the present invention that effectively inhibit the level or function of huntingtin RNA or protein products of expression are considered an active antisense compound. In one embodiment, the antisense compounds of the invention inhibit the expression of huntingtin causing a reduction of RNA by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%.
The reduction of the expression of huntingtin can be measured in a bodily fluid, tissue or organ of the animal. Methods of obtaining samples for analysis, such as body fluids or tissues, and methods of preparation of the samples to allow for analysis are well known to those skilled in the art. Methods for analysis of RNA and protein levels are discussed above and are well known to those skilled in the art. The effects of treatment using the compounds of the invention can be assessed by measuring biomarkers associated with the target gene expression in the aforementioned fluids, tissues or organs, collected from an animal contacted with one or more compounds of the invention, by routine clinical methods known in the art. Biomarkers of huntingtin include but are not limited to the accumulation of huntingtin positive neuronal inclusions, loss of certain neuronal tissue, etc.
In addition, a subject's systemic response to treatment can be assessed by monitoring clinically relevant measures that include but are not limited to: liver transaminases, bilirubin, albumin, blood urea nitrogen, creatine and other markers of kidney and liver function; interleukins, tumor necrosis factors, intracellular adhesion molecules, C-reactive protein and other markers of inflammation.
Methods whereby bodily fluids, organs or tissues are contacted with an effective amount of one or more of the antisense compounds or compositions of the invention are also contemplated. Bodily fluids, organs or tissues can be contacted with one or more of the compounds of the invention resulting in modulation of huntingtin expression in the cells of bodily fluids, organs or tissues. An effective amount can be determined by monitoring the modulatory effect of the antisense compound or compounds or compositions on target nucleic acids or their products by methods routine to the skilled artisan. Further contemplated are ex vivo methods of treatment whereby cells or tissues are isolated from a subject, contacted with an effective amount of the antisense compound or compounds or compositions and reintroduced into the subject by routine methods known to those skilled in the art.
In one embodiment, provided are uses of a compound of an isolated double stranded RNA oligonucleotide in the manufacture of a medicament for inhibiting huntingtin expression or overexpression. Thus, provided herein is the use of an isolated double stranded RNA oligonucleotide targeted to huntingtin in the manufacture of a medicament for the treatment of a disease or disorder by means of the method described above.
Pharmaceutical Compositions
Antisense compounds targeted to huntingtin can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Acceptable carriers and dilutents are well known to those skilled in the art. Selection of a dilutent or carrier is based on a number of factors, including, but not limited to, the solubility of the compound and the route of administration. Such considerations are well understood by those skilled in the art. In one aspect, the antisense compounds of the present invention inhibit the expression of huntingtin.
Antisense compounds targeted to huntingtin can also be used in the manufacture of a medicament for the treatment of diseases and disorders related to huntingtin expression. In one embodiment, the disease or disorder is Huntington's disease.
The antisense compounds of the present invention comprise any pharmaceutically acceptable salts, esters, or salts of such esters, or any other functional chemical equivalent which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the oligomeric compounds of the present invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
The term “prodrug” indicates a therapeutic agent that is prepared in an inactive or less active form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE ((S-acetyl-2-thioethyl)phosphate) derivatives according to the methods disclosed in WO 93/24510 or WO 94/26764.
The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic administration to humans. In another embodiment, sodium salts of dsRNA compounds are also provided.
Formulations
The oligomeric compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756.
The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including but not limited to ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer (intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Sites of administration are known to those skilled in the art. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be useful for oral administration.
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
In some embodiments, compositions for non-parenteral administration include one or more modifications from naturally-occurring oligonucleotides (i.e. full-phosphodiester deoxyribosyl or full-phosphodiester ribosyl oligonucleotides). Such modifications may increase binding affinity, nuclease stability, cell or tissue permeability, tissue distribution, or other biological or pharmacokinetic property.
Oral compositions for administration of non-parenteral oligomeric compounds can be formulated in various dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The term “alimentary delivery” encompasses e.g. oral, rectal, endoscopic and sublingual/buccal administration. Such oral oligomeric compound compositions can be referred to as “mucosal penetration enhancers.”
Oligomeric compounds, such as oligonucleotides, may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. Nos. 09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999) and 10/071,822, filed Feb. 8, 2002, which are herein incorporated by reference.
In one embodiment, oral oligomeric compound compositions comprise at least one member of the group consisting of surfactants, fatty acids, bile salts, chelating agents, and non-chelating surfactants. Such formulations are well known to those skilled in the art.
A “pharmaceutical carrier” or “excipient” can be a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal and are known in the art. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
Oral oligomeric compositions may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
One of skill in the art will recognize that formulations are routinely designed according to their intended route of administration.
Combinations
Compositions of the invention can contain two or more oligomeric compounds. In another related embodiment, compositions of the present invention can contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the present invention can contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Two or more combined compounds may be used together or sequentially.
Compounds
The term “oligomeric compound” refers to a polymeric structure capable of hybridizing to a region of a nucleic acid molecule. This term includes oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics and chimeric combinations of these. Oligomeric compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular. Moreover, branched structures are known in the art. An “antisense compound” or “antisense oligomeric compound” refers to an oligomeric compound that is at least partially complementary to the region of a nucleic acid molecule to which it hybridizes and which modulates (increases or decreases) its expression. Consequently, while all antisense compounds can be said to be oligomeric compounds, not all oligomeric compounds are antisense compounds. Nonlimiting examples of oligomeric compounds include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, and siRNAs. As such, these compounds can be introduced in the form of single-stranded, double-stranded, circular, branched or hairpins and can contain structural elements such as internal or terminal bulges or loops. Oligomeric double-stranded compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound. The compounds of the instant invention are non-catalytic.
An “antisense oligonucleotide” is an antisense compound that is a nucleic acid-based oligomer, but does not include siRNA duplexes. In a preferred embodiment, and in any of the embodiments disclosed herein, the “antisense oligonucleotide” can be a single-stranded nucleic acid molecule. An antisense oligonucleotide can be chemically modified.
Antisense compounds comprise from about 12 to about 35 linked nucleotides. This embodies antisense compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length.
In one embodiment, the antisense compounds are 15 to 30 linked nucleotides in length, as exemplified above.
In one embodiment, the antisense compounds are 17 to 25 linked nucleotides in length, as exemplified herein.
In one embodiment, the antisense compounds are 19, 20, 21, 22, 23, or 24 linked nucleotides in length, or alternatively the oligomeric compounds range from 19 to 24 linked nucleotides in length.
In one embodiment, the antisense compounds are 21, 22, 23, or 24 linked nucleotides in length, or alternatively the oligomeric compounds range from 21 to 24 linked nucleotides in length.
In one embodiment, the antisense compounds are 20 linked nucleotides in length.
In one embodiment of the invention, double-stranded antisense compounds encompass short interfering RNAs (siRNAs). As used herein, the term “siRNA” is defined as a double-stranded compound having a first and second strand, each strand having a central portion and two independent terminal portions. The central portion of the first strand is complementary to the central portion of the second strand, allowing hybridization of the strands. The terminal portions are independently, optionally complementary. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. In one nonlimiting example, the first strand of the siRNA is antisense to the target nucleic acid, while the second strand is complementary to the first strand. Once the antisense strand is designed to target a particular nucleic acid target, the sense strand of the siRNA can then be designed and synthesized as the complement of the antisense strand and either strand may contain modifications or additions to either terminus. For example, in one embodiment, both strands of the siRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini. It is possible for one end of a duplex to be blunt and the other to have overhanging nucleobases. In one embodiment, the number of overhanging nucleobases is from 1 to 6 on the 3′ end of each strand of the duplex. In another embodiment, the number of overhanging nucleobases is from 1 to 6 on the 3′ end of only one strand of the duplex. In a further embodiment, the number of overhanging nucleobases is from 1 to 6 on one or both 5′ ends of the duplexed strands. In another embodiment, the number of overhanging nucleobases is zero.
In one embodiment of the invention, double-stranded antisense compounds are canonical siRNAs. As used herein, the term “canonical siRNA” is defined as a double-stranded oligomeric compound having a first strand and a second strand each strand being 21 nucleobases in length with the strands being complementary over 19 nucleobases and having on each 3′ termini of each strand a deoxy thymidine dimer (dTdT) which in the double-stranded compound acts as a 3′ overhang.
Each strand of the siRNA duplex may be from about 12 to about 35 nucleobases. In a preferred embodiment, each strand of the siRNA duplex is about 17 to about 25 nucleobases. The central complementary portion may be from about 12 to about 35 nucleobases in length. In a preferred embodiment, the central complimentary portion is about 17 to about 25 nucleobases in length. It is understood that each the strand of the siRNA duplex and the central complementary portion may be about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases in length. The terminal portions can be from 1 to 6 nucleobases. It is understood that the terminal portions can be about 1, 2, 3, 4, 5, or 6 nucleobases in length. The siRNAs may also have no terminal portions. The two strands of an siRNA can be linked internally leaving free 3′ or 5′ termini, or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5′ or 3′ terminus producing an extension of single-stranded character.
In another embodiment, the double-stranded antisense compounds are blunt-ended siRNAs. As used herein the term “blunt-ended siRNA” is defined as an siRNA having no terminal overhangs. That is, at least one end of the double-stranded compound is blunt. siRNAs, whether canonical or blunt, act to elicit dsRNAse enzymes and trigger the recruitment or activation of the RNAi antisense mechanism. In a further embodiment, single-stranded RNAi (ssRNAi) compounds that act via the RNAi antisense mechanism are contemplated.
Further modifications can be made to the double-stranded compounds and may include conjugate groups attached to one of the termini, selected nucleobase positions, sugar positions or to one of the internucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, the compounds can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the compounds can be fully or partially double-stranded. When formed from two strands, or a single strand that takes the form of a self-complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary when they base pair in Watson-Crick fashion.
The antisense compounds in accordance with this invention may comprise a complementary antisense compound from about 12 to about 35 nucleobases (i.e. from about 12 to about 35 linked nucleosides). In other words, a single-stranded antisense compound of the invention comprises from about 12 to about 35 nucleobases, and a double-stranded antisense compound of the invention (such as a siRNA, for example) comprises two strands, each of which is from about 12 to about 35 nucleobases. Contained within the antisense compounds of the invention (whether single or double stranded and on at least one strand) are antisense portions. The “antisense portion” is that part of the antisense compound that is designed to work by one of the aforementioned antisense mechanisms. One of ordinary skill in the art will appreciate that this comprehends antisense portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases.
In one embodiment, the antisense compounds of the invention have antisense portions of 12 to 35 nucleobases. It is understood that the antisense portion may be about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases in length.
Antisense compounds 12 to 35 nucleobases in length comprising a stretch of at least 8, preferably at least 12, more preferably at least 17 consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well. Also contemplated are antisense compounds 12 to 35 nucleobases in length comprising a stretch of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucl nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.
Antisense compounds of the invention include antisense compound sequences that comprise at least the 8 (or 9-19) consecutive nucleobases from the 5′-terminus of one of the illustrative antisense compounds (the remaining nucleobases being a consecutive stretch of the same antisense beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the antisense contains about 12 to 35 nucleobases). Other antisense compounds are represented by antisense compound sequences that comprise at least the 8 (or 9-19) consecutive nucleobases from the 3′-terminus of one of the illustrative antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the antisense compound contains about 12 to about 35 nucleobases). It is also understood that antisense compounds may be represented by antisense compound sequences that comprise at least 8 (or 9-19) consecutive nucleobases from an internal portion of the sequence of an illustrative compound, and may extend in either or both directions until the antisense contains about 12 to about 35 nucleobases.
One having skill in the art armed with the antisense compounds illustrated herein will be able, without undue experimentation, to identify further antisense compounds.
Chemical Modifications
As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base (sometimes referred to as a “nucleobase” or simply a “base”). The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.
Modified Internucleoside Linkages
Oligomeric compounds may comprise modified internucleoside linkages, e.g. non-naturally occurring internucleoside linkages. As defined in this specification, oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
Oligomeric compounds, including antisense compounds and antisense oligonucleotides, can have one or more modified internucleoside linkages. Modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, phosphonoacetate and thiophosphonoacetate (see Sheehan et al., Nucleic Acids Research, 2003, 31(14), 4109-4118 and Dellinger et al., J. Am. Chem. Soc., 2003, 125, 940-950), selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage, i.e., a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.
N3′-P5′-phosphoramidates have been reported to exhibit both a high affinity towards a complementary RNA strand and nuclease resistance (Gryaznov et al., J. Am. Chem. Soc., 1994, 116, 3143-3144). N3′-P5′-phosphoramidates have been studied with some success in vivo to specifically down regulate the expression of the c-myc gene (Skorski et al., Proc. Natl. Acad. Sci., 1997, 94, 3966-3971; and Faira et al., Nat. Biotechnol., 2001, 19, 40-44).
In some embodiments, oligomeric compounds may have one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene (methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the native phosphodiester internucleotide linkage is represented as —O—P(═O)(OH)—O—CH₂—).
Some oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂component parts.
Modified Sugars
Oligomeric compounds may also contain one or more substituted sugar moieties. Suitable compounds can comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Also suitable are O((CH₂)_nO)_mCH₃, O(CH₂)_nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON((CH₂)_nCH₃)₂, where n and m are from 1 to about 10. Other oligonucleotides comprise one of the following at the 2′ position: C₁to C₁₀lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. One modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group. A further modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—(CH₂)₂—O—(CH₂)₂—N(CH₃)₂, also described in examples hereinbelow.
Other modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. One 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Antisense compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; and, 6,147,200.
A further modification includes bicyclic sugar moieties referred to as “bicyclic nucleic acids” or “BNAs” in which the 2′-hydroxyl group of the ribosyl sugar ring is linked to the 4′ carbon atom of the sugar ring to form the bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene (—CH₂—) group bridging the 2′ oxygen atom and the 4′ carbon atom, or can be an ethylene group. The alpha-L isomer of the bicyclic nucleic acid moiety wherein the linkage is a methylene group is an additional modified sugar moiety. Another bicyclic sugar moiety that has been prepared and studied has the bridge going from the 3′-hydroxyl group via a single methylene group to the 4′ carbon atom of the sugar ring thereby forming a 3′-C,4′-C-oxymethylene linkage (see U.S. Pat. No. 6,043,060).
Oligonucleotide Mimetics
Another group of oligomeric compounds includes oligonucleotide mimetics. The term “mimetic” as it is applied to oligonucleotides includes oligomeric compounds wherein the furanose ring or the furanose ring and the internucleotide linkage are replaced with novel groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. Oligonucleotide mimetics include peptide nucleic acid (PNA) compounds (Nielsen et al., Science, 1991, 254, 1497-1500), morpholino-based compounds (see, for example, U.S. Pat. No. 5,034,506), cyclohexene nucleic acids (CeNA). In CeNA oligonucleotides (Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602), and phosphonomonoester nucleic acids.
Modified and Alternate Nucleobases
Oligomeric compounds can also include nucleobase (often referred to in the art as heterocyclic base or simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). A “substitution” is the replacement of an unmodified or natural base with another unmodified or natural base. “Modified” nucleobases mean other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3′,2′:4,5)pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Certain nucleobase modifications increase the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently suitable base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications. It is understood in the art that modification of the base does not entail such chemical modifications as to produce substitutions in a nucleic acid sequence.
Oligomeric compounds of the present invention can also include polycyclic heterocyclic compounds in place of one or more of the naturally-occurring heterocyclic base moieties. A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G-clamps or cytidine analogs.
Conjugates
Oligomeric compounds may be chemically linked to one or more moieties or conjugates which enhance the oligomeric compound properties such as activity, cellular distribution or cellular uptake. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligo-mers. Additional conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention.
Oligomeric compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of an oligomeric compound to enhance properties such as for example nuclease stability. Included in stabilizing groups are cap structures. By “cap structure or terminal cap moiety” is meant chemical modifications, which have been incorporated at either terminus of oligonucleotides. These terminal modifications protect the oligomeric compounds having terminal nucleic acid molecules from exonuclease degradation, and can improve delivery and/or localization within a cell. The cap can be present at either the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present on both termini of a single strand, or one or more termini of both strands of a double-stranded compound. This cap structure is not to be confused with the inverted methylguanosine “5′ cap” present at the 5′ end of native mRNA molecules.
In non-limiting examples, the 5′-cap includes inverted abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety. For siRNA constructs, the 5′ end (5′ cap) is commonly but not limited to 5′-hydroxyl or 5′-phosphate.
Particularly suitable 3′-cap structures include, for example 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramimdate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Tyer, 1993, Tetrahedron 49, 1925).
1. Chimeric compounds
It is not necessary for all positions in a given oligomeric compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even within a single nucleoside within an oligomeric compound.
The present invention also includes oligomeric compounds which are chimeric compounds. “Chimeric” oligomeric compounds or “chimeras,” in the context of this invention, are single- or double-stranded oligomeric compounds, such as oligonucleotides, which contain two or more chemically distinct regions, each comprising at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. Chimeric antisense oligonucleotides are one form of oligomeric compound. These oligonucleotides typically contain at least one region which is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, alteration of charge, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for RNAses or other enzymes. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target when bound by a DNA-like oligomeric compound, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNase III or RNAseL which cleaves both cellular and viral RNA. Cleavage products of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
Chimeric oligomeric compounds of the invention can be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, oligonucleotide mimetics, or regions or portions thereof. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922.
A “gapmer” is defined as an oligomeric compound, generally an oligonucleotide, having a 2′-deoxyoligonucleotide region flanked by non-deoxyoligonucleotide segments. The central region is referred to as the “gap.” The flanking segments are referred to as “wings.” While not wishing to be bound by theory, the gap of the gapmer presents a substrate recognizable by RNase H when bound to the RNA target whereas the wings do not provide such a substrate but can confer other properties such as contributing to duplex stability or advantageous pharmacokinetic effects. Each wing can be one or more non-deoxyoligonucleotide monomers (if one of the wings has zero non-deoxyoligonucleotide monomers, a “emimer” is described). In one embodiment, the gapmer is a ten deoxynucleotide gap flanked by five non-deoxynucleotide wings. This is referred to as a 5-10-5 gapmer. Other configurations are readily recognized by those skilled in the art. In one embodiment the wings comprise 2′-MOE modified nucleotides. In another embodiment the gapmer has a phosphorothioate backbone. In another embodiment the gapmer has 2′-MOE wings and a phosphorothioate backbone. Other suitable modifications are readily recognizable by those skilled in the art.
Oligomer Synthesis
Oligomerization of modified and unmodified nucleosides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).
Oligomeric compounds can be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
Oligonucleotide Synthesis
Oligomeric compounds and phosphoramidites are made by methods well known to those skilled in the art. Oligomerization of modified and unmodified nucleosides is performed according to literature procedures for DNA like compounds (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA like compounds (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA:Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713) synthesis as appropriate. Alternatively, oligomers may be purchased from various oligonucleotide synthesis companies such as, for example, Dharmacon Research Inc., (Lafayette, Colo.).
Irrespective of the particular protocol used, the oligomeric compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed (including solution phase synthesis).
Methods of isolation and analysis of oligonucleotides are well known in the art. A 96-well plate format is particularly useful for the synthesis, isolation and analysis of oligonucleotides for small scale applications.
Hybridization
“Hybridization” means the pairing of complementary strands of oligomeric compounds. While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
An oligomeric compound is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
“Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.
Complementarity
“Complementarty,” as used herein, refers to the capacity for precise pairing between two nucleobases on one or two oligomeric compound strands. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligomeric compound and the further DNA or RNA are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.
As used herein, an antisense oligonucleotide is “fully complementary” to a target nucleic acid when each nucleobase of the antisense oligonucleotide is capable of undergoing precise base pairing with an equal number of nucleobases in the target nucleic acid. It is understood in the art that the sequence of the antisense oligonucleotide need not be fully complementary to that of its target nucleic acid to be active in inhibiting the activity of the target nucleic acid. In some embodiments there are “non-complementary” positions, also known as “mismatches”, between the antisense oligonucleotide and the target nucleic acid, and such non-complementary positions may be tolerated between an antisense oligonucleotide and the target nucleic acid provided that the antisense oligonucleotide remains specifically hybridizable to the target nucleic acid. For example, as demonstrated herein, 387916, having one non-complementary nucleobases with respect to mouse huntingtin, is capable of reducing mouse huntingtin mRNA levels in vitro and in vivo. A “non-complementary nucleobase” means a nucleobase of an antisense oligonucleotide that is unable to undergo precise base pairing with a nucleobase at a corresponding position in a target nucleic acid. As used herein, the terms “non-complementary” and “mismatch” are interchangable. In some embodiments antisense oligonucleotides having no more than three non-complementary nucleobases with respect to a nucleic acid encoding huntingtin are considered “complementary” to a nucleic acid encoding huntingtin. In other embodiments, antisense oligonucleotides contain no more than two non-complementary nucleobases with respect to a nucleic acid encoding huntingtin. In further embodiments, antisense oligonucleotides contain no more than one non-complementary nucleobase with respect to a nucleic acid encoding huntingtin.
The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense oligonucleotide. Alternatively, the non-complementary nucleobase may be at an internal position in the antisense oligonucleotide. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-embodiments contiguous.
In other embodiments of the invention, the antisense oligonucleotides comprise at least 90% sequence complementarity to a huntingtin target nucleic acid. In further embodiments of the invention, the antisense oligonucleotides comprise at least 95% sequence complementarity to a huntingtin target nucleic acid. In further embodiments of the invention, the antisense oligonucleotides comprise at least 96%, 97%, 98% or 99% sequence complementarity to a huntingtin target nucleic acid.

Examples of oligonucleotides having mismatches or less than 100% sequence complementarity are shown in Table 1 below where the mismatch is designated by the letter X in the sequence.

	TABLE 1


	SEQ

Isis

	No.	NO:	Sequence (5′ to 3′)	is

387902	105	CGCCTGCACCATGTTCCTCA
	358	CGXCTGCACCATGTTCCTCA	A or T
	359	CGCCXGCACCATGTTCCTCA	C or G
	360	CGCCTGCACCAXGTTCCTCA	C or G
	361	CGCCTGCACCATGTTCXTCA	A or T

388816	345	GCCGTAGCCTGGGACCCGCC
	362	GCXGTAGCCTGGGACCCGCC	A or T
	363	GCCGTAGCXTGGGXCCCGCC	C or G
	364	GCCGTAGCCTGGGACCCXCC	A or T
	365	GCCGTAGCCTGGGACCCGCX	A or T

387916	125	TCTCTATTGCACATTCCAAG
	366	TCXCTATTGCACATTCCAAG	C or G
	367	TCTCTATXGCACATTCCAAG	C or G
	368	TCTCTATTGCAXATTCCAAG	A or T
	369	TCTCTATTGCACATTCXAAG	A or T

Identity

Oligomeric compounds, or a portion thereof, may have a defined percent identity to a SEQ ID NO, or a compound having a specific Isis number. This identity may be over the entire length of the oligomeric compound, or in a portion of the oligomeric compound (e.g., nucleobases 1-20 of a 27-mer may be compared to a 20-mer to determine percent identity of the oligomeric compound to the SEQ ID NO.). It is understood by those skilled in the art that an oligonucleotide need not have an identical sequence to those described herein to function similarly to the oligonucleotides described herein. Shortened (i.e., deleted, and therefore non-identical) versions of oligonucleotides taught herein, or non-identical (i.e., one base replaced with another) versions of the oligonucleotides taught herein fall within the scope of the invention. Percent identity is calculated according to the number of bases that are identical to the SEQ ID NO or compound to which it is being compared. The non-identical bases may be adjacent to each other, dispersed through out the oligonucleotide, or both.
For example, a 16-mer having the same sequence as nucleobases 2-17 of a 20-mer is 80% identical to the 20-mer. Alternatively, a 20-mer containing four nucleobases not identical to the 20-mer is also 80% identical to the 20-mer. A 14-mer having the same sequence as nucleobases 1-14 of an 18-mer is 78% identical to the 18-mer. Such calculations are well within the ability of those skilled in the art.
The percent identity is based on the percent of nucleobases in the original sequence present in a portion of the modified sequence. Therefore, a 30 nucleobase oligonucleotide comprising the full sequence of a 20 nucleobase SEQ ID NO would have a portion of 100% identity with the 20 nucleobase SEQ ID NO while further comprising an additional 10 nucleobase portion. In the context of the invention, the full length of the modified sequence may constitute a single portion.
It is well known by those skilled in the art that it is possible to increase or decrease the length of an antisense oligonucleotide and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309. 1992, incorporated herein by reference), a series of oligomers 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the oligonucleotide were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the oligonucleotide that contained no mismatches. Similarly, target specific cleavage was achieved using a 13 nucleobase oligomer, including those with 1 or 3 mismatches. Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358.1988, incorporated herein by reference) tested a series of tandem 14 nucleobase oligonucleotides, and a 28 and 42 nucleobase oligonucleotide comprised of the sequence of two or three of the tandem oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase oligonucleotides alone were able to inhibit translation, albeit at a more modest level, than the 28 or 42 nucleobase oligonucleotide. Interestingly, a mixture of the tandem 14 nucleobase oligonucleotides was as effective at inhibiting translation as the 28 nucleobase oligonucleotide targeted to the same region.
Target Nucleic Acids
“Targeting” an oligomeric compound to a particular target nucleic acid molecule can be a multistep process. The process usually begins with the identification of a target nucleic acid whose expression is to be modulated. As used herein, the terms “target nucleic acid” and “nucleic acid encoding huntingtin” encompass DNA encoding huntingtin, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. As disclosed herein, the target nucleic acid encodes huntingtin.
2. Target Regions, Segments, and Sites
The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. “Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Target regions include, but are not limited to, contiguous nucleotide sequences, translation initiation and termination regions, coding regions, open reading frames, introns, exons, 3′-untranslated regions (3′-UTR), and 5′-untranslated regions (5′-UTR). Within regions of target nucleic acids are target segments. As used herein, a “target segment” means a sequence of a huntingtin target nucleic acid to which one or more antisense oligonucleotides are complementary. The term “5′ target site” is defined as the 5′-most nucleobase of a target segment to which an antisense oligonucleotide is complementary. Likewise, a “3′ target site” is defined as the 3′-most nucleobase of a target segment to which an antisense oligonucleotide is complementary.
3. Variants
It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants.” More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence. Variants can result in mRNA variants including, but not limited to, those with alternate splice junctions, or alternate initiation and termination codons. Variants in genomic and mRNA sequences can result in disease. Oligonucleotides to such variants are within the scope of the instant invention.
4. Target Names and Synonyms

In accordance with the present invention are compositions and methods for modulating the expression of genes which are presented in Table 2. Listed in Table 2 are the gene target names, as well as GENBANK® accession numbers used to design oligomeric compounds targeted to each gene.

TABLE 2


Gene Target Names and Sequences

		SEQ
Species	Genbank #	ID NO

Human	AB209506.1	1
Human	BE378835.1	2
Human	L12392.1	3
Human	NM_002111.5	4
Human	nucleotides 462000 to 634000 of	5
	NT_006081.17
Mouse	AK042204.1	6
Mouse	AK049546.1	7
Mouse	L23312.1	8
Mouse	L23313.1	9
Mouse	NM_010414.1	10
Mouse	nucleotides 2036000 to 2190000 of	11
	NT_039302.4
Mouse	NM_010414.1 (mouse short form) *	44
Human	cut from genomic ad Sac1 and EcoR1 sites	45
	surrounding exon 1, expanded CAG to
	results in 130 gln in this region

* NM_010414.1 (mouse short form) extended with mouse genomic sequence to create transcript orthologous to human long form (NM_002111.5). Much of this extension is supported by mouse ESTs but the most 3′ end is supported only by homology to the human mRNA

Modulation of Target Expression

Modulation of expression of a target nucleic acid can be achieved through alteration of any number of nucleic acid (DNA or RNA) functions. “Modulation” means a perturbation of function, for example, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in expression. As another example, modulation of expression can include perturbing splice site selection of pre-mRNA processing. “Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. These structures include the products of transcription and translation. “Modulation of expression” means the perturbation of such functions. The functions of DNA to be modulated can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be modulated can include translocation functions, which include, but are not limited to, translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, and translation of protein from the RNA. RNA processing functions that can be modulated include, but are not limited to, splicing of the RNA to yield one or more RNA species, capping of the RNA, 3′ maturation of the RNA and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. Modulation of expression can result in the increased level of one or more nucleic acid species or the decreased level of one or more nucleic acid species, either temporally or by net steady state level. One result of such interference with target nucleic acid function is modulation of the expression of HUNTINGTINα. Thus, in one embodiment modulation of expression can mean increase or decrease in target RNA or protein levels. In another embodiment modulation of expression can mean an increase or decrease of one or more RNA splice products, or a change in the ratio of two or more splice products.
Cultured Cells
The effect of oligomeric compounds of the present invention on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. The effect of oligomeric compounds of the present invention on target nucleic acid expression can be routinely determined using, for example, PCR or Northern blot analysis. Cell lines are derived from both normal tissues and cell types and from cells associated with various disorders (e.g. hyperproliferative disorders). Cell lines derived from multiple tissues and species can be obtained from American Type Culture Collection (ATCC, Manassas, Va.) and are well known to those skilled in the art. Primary cells, or those cells which are isolated from an animal and not subjected to continuous culture, can be prepared according to methods known in the art or obtained from various commercial suppliers. Additionally, primary cells include those obtained from donor human subjects in a clinical setting (i.e. blood donors, surgical patients).
Cells isolated from Huntington's disease patients are also used to test the effects of antisense compounds targeted to huntingtin. In such cells, the mutant huntingtin gene may be present in a heterozygous or homozygous form. Such cells are available from National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository, examples of which include fibroblasts having repository number GM04281 or GM04478. Cells from Huntington's disease patients are cultured according to procedures recommended by the supplier.
The pharmacological effects of antisense inhibition of huntingtin can be assessed in cell lines isolated from neuronal cells expressing either wild-type or mutant forms of the huntingtin gene. The mutant forms of huntingtin are associated with particular phenotypes, and the effects on these phenotypes are evaluated following antisense inhibition of huntingtin. An example of such cells are striatal cells established from Hdh^Q111knock-in mice, which bear 111 CAG repeats inserted into the mouse huntingtin locus. Establishment of striatal cell lines isolated from Hdh^Q111mice has been described by Trettel et al. (Human Mol. Genet., 2000, 9, 2799-2809). Striatal cell lines established from mice bearing a wild-type huntingtin gene are used for comparison studies.
Assaying Modulation of Expression
Modulation of huntingtin expression can be assayed in a variety of ways known in the art. Huntingtin mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA by methods known in the art. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.
Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions. The method of analysis of modulation of RNA levels is not a limitation of the instant invention.
Levels of a protein encoded by huntingtin can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to a protein encoded by huntingtin can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.).
Validated Target Segments
The locations on the target nucleic acid to which active oligomeric compounds hybridize are hereinbelow referred to as “validated target segments.” In one embodiment, a validated target segment includes at least an 8-nucleobase portion of a target region. In another embodiment, a validated target segment includes at least a 12-nucleobase portion of a target region to which an active oligomeric compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.
Target segments can include DNA or RNA sequences that comprise at least the 8, or at least the 12, consecutive nucleobases from the 5′-terminus of a validated target segment (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA contains about 12 to about 35 nucleobases). Similarly validated target segments are represented by DNA or RNA sequences that comprise at least the 8, or at least the 12 consecutive nucleobases from the 3′-terminus of a validated target segment (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA contains about 12 to about 35 nucleobases). It is also understood that a validated oligomeric target segment can be represented by DNA or RNA sequences that comprise at least 8, or at least the 12, consecutive nucleobases from an internal portion of the sequence of a validated target segment, and can extend in either or both directions until the oligonucleotide contains about 12 to about 35 nucleobases.
Screening for Modulator Compounds
In another embodiment, the validated target segments identified herein can be employed in a screen for additional compounds that modulate the expression of huntingtin. “Modulators” are those compounds that modulate the expression of huntingtin and which comprise at least an 8-nucleobase portion which is complementary to a validated target segment. The screening method comprises the steps of contacting a validated target segment of a nucleic acid molecule encoding huntingtin with one or more candidate modulators, and selecting for one or more candidate modulators which perturb the expression of a nucleic acid molecule encoding huntingtin. Once it is shown that the candidate modulator or modulators are capable of modulating the expression of a nucleic acid molecule encoding huntingtin, the modulator can then be employed in further investigative studies of the function of huntingtin, or for use as a research, diagnostic, or therapeutic agent. The validated target segments can also be combined with a second strand as disclosed herein to form stabilized double-stranded (duplexed) oligonucleotides for use as a research, diagnostic, or therapeutic agent.
In Vivo Testing of Antisense Compounds Targeted to Huntingtin
Antisense compounds targeted to huntingtin are tested in experimental animal models. In one embodiment, the antisense compounds are targeted to the human huntingtin gene alone. Such antisense compounds have, for example, less than four mismatches to human huntingtin and four or more mismatches to non-human huntingtin. In another embodiment, antisense compounds are targeted to both human and non-human huntingtin. Such antisense compounds have, for example, less than four mismatches to human huntingtin and less than four mismatches to non-human huntingtin.
Normal Animals
Normal, wild-type animals may be used to perform toxicity studies of antisense oligonucleotides targeted to huntingtin. The antisense compounds are administered systemically (e.g. via intraperitoneal injection) at doses of 25, 50, 75, or 100 mg/kg. Animals are monitored for any clinical changes, including changes in body weight. Serum is collected periodically, for example every week or every two weeks, during the dosing period and subjected to analysis using a clinical analyzer to detect any changes in serum chemistry profiles. At the end of the study, the animals are sacrificed. Blood is collected and analyzed for white blood cell count, platelet count, and serum chemistry. The weights of major organs are determined, and histological analyses are performed on spleen, liver, kidney and pancreas.
Huntington's Disease Models
Antisense compounds targeted to huntingtin may be tested in experimental non-human models of Huntington's disease. Several non-human models have been developed and characterized.
The R6/2 transgenic mouse model has integrated into its genome 1 kilobase of the human huntingtin gene, including the 5′-UTR exon 1 and the first 262 basepairs of intron 1 (Mangiarin L. et al., Cell, 1996, 87, 493-506). This transgene has 144 CAG repeats. The transgene encodes for approximately 3% of the N-terminal region of the huntingtin protein, expression of which is driven by the human huntingtin promoter. Expression levels of this truncated version of human huntingtin protein are approximately 75% of the endogenous mouse huntingtin protein levels. The R6/2 transgenic mice exhibit symptomos of human Huntington's disease and brain dysfunction.
The YAC128 transgenic mice harbor a yeast artificial chromosome (YAC) carrying the entire huntingtin gene, including the promoter region and 128 CAG repeats (Hodgson J. G. et al., Human Mol. Genet., 1998, 5, 1875). This YAC expresses all but exon 1 of the human gene. These transgenic mice do not express endogenouse mouse huntingtin.
The endogenous mouse huntingtin gene of the Q111 mice has 111 CAG repeats inserted into exon 1 of the gene (Wheeler V. C. et al., Human Mol. Genet, 8, 115-122).
In the Q150 transgenic mice, the CAG repeat in exon 1 of the wild-type mouse huntingtin gene is replaced with 150 CAG repeats (Li C. H. et al., Human Mol. Genet, 2001, 10, 137).
Antisense compounds targeted to huntingtin are administered to the non-human experimental model, for example to transgenic mice that are used as models of Huntington's disease.
Antisense compounds may be administered directly into the central nervous system of the experimental animal, for example through intracerebroventricular (ICV), intrathecal (IT) or intraparenchymal administration. Dosages of antisense compounds administered may be 25, 50, 75, or 100 ug/day, and administration may be accomplished through continuous infusion using a surgically implanted osmotic pump (e.g. an Alzet mini-pump). 0.25, 0.5, or 1 uL/hour. Each dosage is administered to groups of 4 to 6 animals. Control groups of animals may receive saline infusion, or infusion of an antisense compound having a sequence not targeted to any known gene.
Animals are treated for several weeks, for example 1, 2, 4, or 8 weeks. Animals are monitored for any clinical changes, including changes in body weight. At the end of the treatment period, animals are sacrificed. The brains are dissected into three regions: forebrain, basal ganglion, and cerebellum. Brain regions undergo histological assessment, including hemotoxylin/eosin staining, GFAP staining (to assess glial cell activation) and FluoroJ staining (to assess neurodegenerative changes). Huntingtin mRNA levels are measured by real-time PCR, and huntingtin protein levels are measured by immunoblotting (western blotting).
The duration of action of antisense compounds targeting huntingtin may also be evaluated. For such analyses, animals are dosed for 2, 4, 6, or 8 weeks with antisense compounds targeting huntingtin. At the end of the dosing period, the osmotic pumps are removed and animals are sacrificed 0, 1, 2, 4, 6, or 8 weeks following dosing termination. The brains are dissected into three regions: forebrain, basal ganglion, and cerebellum. Brain regions undergo histological assessment, including hemotoxylin/eosin staining, GFAP staining (to assess glial cell activation) and FluoroJ staining (to assess neurodegenerative changes). Huntingtin mRNA levels are measured by real-time PCR, and huntingtin protein levels are measured by immunoblotting (western blotting).
Kits, Research Reagents, and Diagnostics
The oligomeric compounds of the present invention can be utilized for diagnostics, research reagents and kits. Furthermore, antisense compounds, which are able to inhibit gene expression with specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
For use in kits and diagnostics, the oligomeric compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.
As one nonlimiting example, expression patterns within cells or tissues treated with one or more compounds or compositions of the present invention are compared to control cells or tissues not treated with compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns. By way of example, gene expression patterns may be identified by microarray analysis.
Nonlimiting Disclosure and Incorporation by Reference
While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same. Each of the references, GENBANK® accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.
The effect of oligomeric compounds on target nucleic acid expression was tested in cultured cells, for example A549 cells or HD patient fibroblasts for compounds targeted to human huntingtin, and in b.END cells for compounds targeted to mouse huntingtin.
When cells reached 65-75% confluency, the transfection reagent LIPOFECTIN® was used to introduce oligonucleotide into cells. Other methods of transfection are well known to those skilled in the art. The method of screening is not a limitation of the instant invention.
Oligonucleotide was mixed with LIPOFECTIN® Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEM®-1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desired concentration of oligonucleotide and a LIPOFECTIN® concentration of 2.5 or 3 μg/mL per 100 nM oligonucleotide. This transfection mixture was incubated at room temperature for approximately 0.5 hours. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM®-1 and then treated with 130 μL of the transfection mixture. Cells are treated and data were obtained in duplicate or triplicate. After approximately 4-7 hours of treatment at 37° C., the medium containing the transfection mixture was replaced with fresh culture medium. Cells were harvested 16-24 hours after oligonucleotide treatment.
For culture chambers other than 96-well plates, the cells may be treated similarly, using appropriate volumes of medium and oligonucleotide.
Quantitation of huntingtin mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions.
After isolation from cells or tissues, RNA was subjected to sequential reverse transcriptase (RT) reaction and real-time PCR, both of which are performed in the same well. RT and PCR reagents were obtained from Invitrogen Life Technologies (Carlsbad, Calif.). RT, real-time PCR was carried out in the same by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).
Gene target quantities obtained by RT, real-time PCR were normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RIBOGREEN® (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression was quantified by RT, real-time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA was quantified using RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to the manufacturer's instructions.

Probes and primers for use in real-time PCR were designed to hybridize to target-specific sequences. The primers and probes and the target nucleic acid sequences to which they hybridize are presented in Table 3. The target-specific PCR probes have FAM covalently linked to the 5′ end and TAMRA or MGB covalently linked to the 3′ end, where FAM is the fluorescent dye and TAMRA or MGB is the quencher dye.

TABLE 3


Gene target-specific primers and probes
for use in real-time PCR

	SEQ		SEQ
	ID	Sequence	ID

Species	NO	Description	Sequence (5′ to 3′)	NO

Human	4	Forward	CTCCGTCCGGTAGACATGCT	38
		Primer

Human	4	Reverse	GGAAATCAGAACCCTCAAAAT	39
		Primer	GG

Human	4	Probe	TGAGCACTGTTCAACTGTGGA	40
			TATCGGGA

Mouse	10	Forward	CAGAGCTGGTCAACCGTATCC	41
		Primer

Mouse	10	Reverse	GGCTTAAACAGGGAGCCAAAA	42
		Primer

Mouse	10	Probe	ACTTCATGATGAGCTCGGAGT	43
			TCAAC

Human Huntingtin
Antisense oligonucleotides were designed to target different regions of the human huntingtin gene, using published sequences cited in Table 2. The sequences and corresponding SEQ ID NOs are shown in Table 4. All compounds in Table 4 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of 10 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) by five-nucleotide “wings”. The wings are composed of 2′-O-(2-methoxyethyl) nucleotides, also known as 2′-MOE nucleotides. The internucleoside (backbone) linkages are phosphorothioate throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.
The antisense oligonucleotides were analyzed for their effect on huntingtin mRNA levels in A549 cells by quantitative real-time PCR as described in other examples herein. The data presented in Table 4 represent percent inhibition of huntingtin mRNA levels relative to untreated cells. Data are averages from experiments in which cultured cells were treated with the disclosed antisense oligonucleotides.

If the huntingtin mRNA level in antisense oligonucleotide-treated cells was equal to or higher than in control cells, percent inhibition is expressed as zero inhibition. If present, “N.D.” indicates “not determined.” The target regions to which these antisense oligonucleotides are inhibitory are herein referred to as “validated target segments.”

TABLE 4


Inhibition of huntingtin mRNA levels by
chimeric oligonucleotides having 2′-MOE
wings and deoxy gap

SEQ

5′

SEQ

Target

ISIS #	NO	Site	Sequence (5′ to 3′)	Inhib	NO

388224	4	33	CAGGTAAAAGCAGAACCTGA	0	46

387865	4	155	GCCTTCATCAGCTTTTCCAG	65	47

388829	4	193	GCTGCTGCTGCTGCTGGAAG	46	48

388830	4	194	TGCTGCTGCTGCTGCTGCTG	46	49

388833	4	195	CTGCTGCTGTTGCTGCTGCT	62	50

388831	4	195	CTGCTGCTGCTGCTGCTGCT	56	51

388832	4	196	GCTGCTGCTGCTGCTGCTGC	36	52

388834	4	198	TGGCGGCTGCTGCTGCTGCT	62	53

388835	4	259	GCGGCGGCGGCGGTGGCGGC	52	54

387866	4	432	ATGATTCACACGGTCTTTCT	76	55

387867	4	489	AAATTCTGGAGAATTTCTGA	31	56

387868	4	497	AGTTTCTGAAATTCTGGAGA	58	57

387869	4	608	GAATCCATCAAAGCTTTGAT	53	58

387870	4	621	CCTTGGAAGATTAGAATCCA	45	59

387871	4	709	GAGCCAGCTCAGCAAACCTC	65	60

387872	4	718	GAACCAGGTGAGCCAGCTCA	37	61

387873	4	749	TTCACCAGGTAAGGCCTGCA	33	62

387874	4	821	ACAGCTGCAGCCAAGGTCTC	60	63

387875	4	845	CCAAAAGAAGCCATAATTTT	63	64

387876	4	876	AACCTTAATTTCATTGTCAT	75	65

388225	4	1000	GTAGCCAACTATAGAAATAT	53	66

388226	4	1005	ATTTAGTAGCCAACTATAGA	26	67

387877	4	1170	AGAGACTTCCATTTCTTTCC	81	68

387878	4	1176	AGAAGGAGAGACTTCCATTT	41	69

387879	4	1184	TGCTCTGCAGAAGGAGAGAC	46	70

387880	4	1201	CATAAACCTGGACAAGCTGC	79	71

387881	4	1208	GTCAGTTCATAAACCTGGAC	72	72

387882	4	1241	ACATTGTGGTCTTGGTGCTG	51	73

387883	4	1460	AAGAGCACTTTGCCTTTTTG	66	74

388227	4	1596	TGCTGACCCTGGAGTGGAAA	78	75

388228	4	1666	TGGCCAGATCCACTGAGTCC	30	76

387884	4	1775	TCATTCAGGTCCATGGCAGG	61	77

387885	4	1782	GGTCCCATCATTCAGGTCCA	68	78

387886	4	1876	CTAACACAATTTCAGAACTG	73	79

388229	4	1990	TGGAAGAGTTCCTGAAGGCC	29	80

388230	4	2022	GTTTTTCAATAAATGTGCCT	58	81

388231	4	2034	GCAGTGACTCATGTTTTTCA	60	82

388232	4	2039	TGCCTGCAGTGACTCATGTT	37	83

388233	4	2346	GTCAAGAGGAACTTTATAGA	55	84

387887	4	2400	ATCGATGTAGTTCAAGATGT	29	85

387888	4	2447	GTCCCACAGAGAATGGCAGT	73	86

388234	4	2677	TGATCAGCTGCAGTCCTAAC	1	87

387889	4	2820	TGTATAATGATGAGCCCCTC	76	88

387890	4	2971	GATCAGCTTGTCCTTGGTCA	81	89

388235	4	3183	TCTGGTGGTTGATGTGATTA	63	90

388236	4	3190	TGAGTGCTCTGGTGGTTGAT	26	91

387891	4	3203	CAGCATCCAAATGTGAGTGC	82	92

387892	4	3209	GCTTCACAGCATCCAAATGT	89	93

388237	4	3234	GAAGGCAGTGGAAAGAAGAC	62	94

387893	4	3641	AGAGAAGGCAAGGCTGCCTT	60	95

387894	4	3649	GGTTTGTTAGAGAAGGCAAG	63	96

387895	4	3851	ACATCATGCAGTTTGAGGTA	68	97

387896	4	3860	GCTTTCAGGACATCATGCAG	51	98

387897	4	4028	AAGCAGGATTTCAGGTATCC	78	99

387898	4	4036	CTCGACTAAAGCAGGATTTC	90	100

387899	4	4055	ACAGTTGCCATCATTGGTTC	67	101

388238	4	4069	ATTGTTGAACACAAACAGTT	50	102

387900	4	4127	TTGGAAGATAAGCCATCAAA	82	103

387901	4	4265	TGCACCATGTTCCTCAGGCT	79	104

387902	4	4269	CGCCTGCACCATGTTCCTCA	90	105

387903	4	4380	AATAGCATTCTTATCTGCAC	84	106

387904	4	4392	AATGTGATTATGAATAGCAT	64	107

388239	4	4458	TAACTGCACACATGTTGTAG	54	108

387905	4	4538	AACACCTGATCTGAATCCAG	78	109

388240	4	4558	GTTTCAATACAAAGCCAATA	78	110

387906	4	4586	AACTGGCCCACTTCAATGTA	78	111

387907	4	4596	TGATTCCCTGAACTGGCCCA	77	112

387908	4	4682	TTAGGAATTCCAATGATCTG	76	113

387909	4	4688	ATGATTTTAGGAATTCCAAT	77	114

387910	4	4715	CTGGCCATGATGCCATCACA	86	115

387911	4	4724	TTCCTTCCACTGGCCATGAT	77	116

387912	4	4805	GCATCAGCTTTATTTGTTCC	70	117

388241	4	4856	CTCAGTAACATTGACACCAC	71	118

388242	4	4868	TACTGGATGAGTCTCAGTAA	49	119

387913	4	4875	CTGATGGTACTGGATGAGTC	59	120

387914	4	4913	TGGCACTGCTGCAGGACAAG	71	121

387915	4	5219	TCCTGAATACGAGAAAGAAC	86	122

388243	4	5781	TTTGGCTGCCAAGTCAGAAT	52	123

388244	4	5787	TCCAAGTTTGGCTGCCAAGT	48	124

387916	4	5801	TCTCTATTGCACATTCCAAG	91	125

387917	4	5850	CTGACAGACATAATCACAGA	61	126

387918	4	5911	TGATCAGATCTTGAATGTGA	41	127

387919	4	6005	CGAGACTGAATTGCCTGGAT	59	128

387920	4	6296	GAATAGAGCCTTTGGTGTCT	56	129

388245	4	6333	GTCTTGCATGGTGGAGAGAC	39	130

387921	4	6466	AATCTGACCTGGTCCAACAC	65	131

387922	4	6476	AGCAGTGCAGAATCTGACCT	53	132

387923	4	6488	TCTGCACCTTCCAGCAGTGC	62	133

388246	4	6600	ACCAGAAATTTCACTCATCC	50	134

388247	4	6606	CTGGCCACCAGAAATTTCAC	21	135

388248	4	6763	CAGCATCCCCAAACAGATCA	65	136

388249	4	6769	ACAGTGCAGCATCCCCAAAC	72	137

388250	4	6777	GGACTGATACAGTGCAGCAT	65	138

387924	4	6860	TTCTCAGGAGGAAGGTGCAA	61	139

387925	4	6930	CTGCTCATGGATCAAATGCC	78	140

388251	4	7177	GTGTGTTTGGATCTACTTCC	67	141

388252	4	7199	GCAGTGATATACTTAGGATT	46	142

388253	4	7208	TCACAGGCTGCAGTGATATA	29	143

388254	4	7312	TGATGTTCCTGAGCAATGGC	51	144

388255	4	7383	TCCAAGCTTCCACACCAGTG	67	145

387926	4	7489	TGTTGATGCGGTAGATGAAC	29	146

387927	4	7556	GTCACCAGGACACCAAGGAG	70	147

387928	4	7709	TCCAAGCAGCTTACAGCTGG	69	148

388256	4	7816	TTGAAACCATTGCTTGAATC	64	149

388257	4	7855	ATGCCTGATATAAATGATGG	52	150

387942	4	7932	GTTGATCTGCAGCAGCAGCT	39	151

387929	4	7988	GAGTGTATGGACACCTGGCC	49	152

387930	4	8005	TGTTCCCCAGCCACACGGAG	85	153

387931	4	8363	GTGGCAGGCACCAGGTACTG	65	154

388258	4	8655	ATAGTTCTCAATGAGGTAAA	72	155

387932	4	8757	ACAGTGGTAAATGATGGAGG	41	156

387933	4	8903	ATGCAGGTGAGCATCAGGCC	29	157

387934	4	8910	TGTGTACATGCAGGTGAGCA	37	158

388259	4	9036	AGGAAAGCCTTTCCTGATCC	31	159

387935	4	9149	TATGGCTGCTGGTTGGACAG	57	160

387936	4	9240	CAGCATGACCCAGTCCCGGA	63	161

387937	4	9243	GGACAGCATGACCCAGTCCC	68	162

387938	4	9368	CCCATCCTGCTGATGACATG	69	163

387939	4	9407	ACCAGGCAGAAAAGGTTCAC	63	164

387940	4	9555	TCAGCAGGTGGTGACCTTGT	64	165

388260	4	9714	TCTGCCACATGGCAGAGACA	25	166

388261	4	9724	AAAGAGCACTTCTGCCACAT	56	167

388262	4	9735	GCCACTGCCACAAAGAGCAC	60	168

388263	4	9763	CACCAGGACTGCAGACACTC	65	169

388264	4	9785	TGGAAGGCCTCAGGCTCAGC	65	170

388265	4	9831	GGACCTGGTCACCCACATGG	22	171

388266	4	9863	GGCAACAACCAGCAGGTGAC	54	172

388267	4	9871	TGCAACCTGGCAACAACCAG	32	173

388268	4	9889	CCCAGATGCAAGAGCAGCTG	65	174

388269	4	9921	AACAGCCAGCCTGCAGGAGG	25	175

388270	4	9946	TCTACTGCAGGACAGCAGAG	20	176

388271	4	9973	TGTTCCCAAAGCCTGCTCAC	43	177

388272	4	9982	CCAGGCCAGTGTTCCCAAAG	41	178

388273	4	9988	GGAGACCCAGGCCAGTGTTC	44	179

388274	4	10047	AGCACAGGCCATGGCATCTG	43	180

388275	4	10054	CTGGCCCAGCACAGGCCATG	33	181

387941	4	10133	ACTGATATAATTAAATTTTA	0	182

388276	4	10274	GGCTATGCCAGTGGCTACAG	29	183

388277	4	10329	TGTGAATGCATAAACAGGAA	61	184

388278	4	10579	CTAGCAAGGAACAGGAGTGG	15	185

388279	4	10639	CCATGGAGCAGCAGGTCCCA	28	186

388280	4	10647	GCATGCATCCATGGAGCAGC	31	187

388281	4	10726	ACTAACAGTGCCAAGACACC	45	188

388282	4	10923	CCATTTTAATGACTTGGCTC	60	189

388283	4	11023	AGGAAGCAGAGCCCCTGCCT	48	190

388284	4	11150	GGCAGCACCTGCACAGAGTT	57	191

388285	4	11225	GCATACAAGTCCACATCTCA	54	192

388286	4	11293	CATACAGGCCTGGCAGAGGC	49	193

388287	4	11449	AAGAATGGTGATTTTCTTAC	46	194

388288	4	11637	TCTAGCCAGGAACAACATCT	47	195

388289	4	11646	ATGTAAACATCTAGCCAGGA	24	196

388290	4	11854	AATGAGCTCATATTCATCTC	20	197

388291	4	12076	GAATGAGCCCTGCCCTGACC	38	198

388292	4	12081	GCAATGAATGAGCCCTGCCC	57	199

388293	4	12122	AGCTGATATGGAGACCATCT	35	200

388294	4	12177	GGTGCTTGCCACAGATTTTT	65	201

388295	4	12324	TGCATTGCCAAACAATTCTA	57	202

388296	4	12409	TTGGCAGCTGGAAACATCAC	52	203

388297	4	12873	TCCAAGTCTACCCTGGCCAG	40	204

388298	4	13044	GTTGCCTTCAGTTGTCATGC	34	205

388299	4	13050	TTCCAGGTTGCCTTCAGTTG	59	206

388300	4	13167	CAGTTACCACCCAGATTGCA	46	207

388301	4	13251	GAGACCTGGACAAGGAGGCC	30	208

388842	5	3535	TGTAATTACAGAATTTGTAT	60	209

388852	5	16048	ACATTCCATGAATTCCATTT	43	210

388846	5	17007	GTTAATTTAGAGAAAATTCA	1	211

388845	5	24805	CAGAAGCATCCAAACCAGTA	40	212

388844	5	31595	CAAGAGGGTTGCATAGAAAC	17	213

388848	5	41489	CAAAGTATAAACAGTTTGAG	32	214

388839	5	41869	CCCAGTGCAGTTCACATTCA	54	215

388859	5	46461	TATTATAAAATACATGTTTC	26	216

388856	5	58668	ATTAGAGATTCATCATATTG	46	217

388857	5	59960	GGTATGGAAAGGTTCAACAT	58	218

388858	5	64678	TGGAAGGTGAGGGACAAAAA	57	219

388862	5	71659	AGCAGAAACAAGTATTCCAT	56	220

388853	5	86173	CAAATTCACATAGGGTTGGT	60	221

388860	5	97067	ACATGAGCAATGAAGGACAG	48	222

388840	5	98221	GCAATGTGTGATTTACCACA	67	223

388850	5	118154	ACCACATCATAATTTGTCAT	41	224

388855	5	120499	ATTATTTAAGAAGTACCCAC	36	225

388861	5	121068	TGCCCCAAAAAGTGGAACCA	55	226

388847	5	126660	ACATTTCCAAGAGGTTTTGA	48	227

388854	5	128596	TCAGCCCCAATTTGTAGCAG	59	228

388841	5	140692	GACATAAAGTTTAGAGGTAT	50	229

388843	5	142578	GAAGGACCCACAGAGGTTTG	53	230

388851	5	146457	TGAAAAGGAAGTGACATCAT	17	231

388849	5	165574	CAGTGTCAGGAGAAGCCCAG	46	232

388785	45	713	AGGTTCTGCCTCACACAGCA	57	311

388786	45	718	CCCGCAGGTTCTGCCTCACA	33	312

388787	45	740	AGGGAACCAGCCCGCCCCTG	56	313

388788	45	745	TGGCCAGGGAACCAGCCCGC	47	314

388789	45	750	ATGGCTGGCCAGGGAACCAG	25	315

388790	45	755	TGCCAATGGCTGGCCAGGGA	7	316

388791	45	777	GACAGCCCTAGCCTGCGGAC	19	317

388792	45	781	GATTGACAGCCCTAGCCTGC	0	318

388793	45	785	GCATGATTGACAGCCCTAGC	9	319

388794	45	885	ATCTTGGACCCGTCCCGGCA	63	320

388795	45	890	CGTCCATCTTGGACCCGTCC	53	321

388796	45	896	AGCGGCCGTCCATCTTGGAC	45	322

388797	45	902	AACCTGAGCGGCCGTCCATC	54	323

388798	45	906	GCAGAACCTGAGCGGCCGTC	62	324

388799	45	910	AAAAGCAGAACCTGAGCGGC	56	325

388800	45	913	GGTAAAAGCAGAACCTGAGC	36	326

388801	45	920	GGCCGCAGGTAAAAGCAGAA	65	327

388802	45	926	GCTCTGGGCCGCAGGTAAAA	64	328

388803	45	985	AGTCCCCGGAGGCCTCGGGC	56	329

388804	45	993	GGCACGGCAGTCCCCGGAGG	57	330

388805	45	1019	AGGGTCGCCATGGCGGTCTC	35	331

388806	45	1025	TTTTCCAGGGTCGCCATGGC	33	332

388807	45	1030	TCAGCTTTTCCAGGGTCGCC	63	333

388808	45	1034	TTCATCAGCTTTTCCAGGGT	54	334

388809	45	1040	AAGGCCTTCATCAGCTTTTC	48	335

388810	45	1045	ACTCGAAGGCCTTCATCAGC	57	336

388811	45	1050	GAGGGACTCGAAGGCCTTCA	51	337

388812	45	1056	GGACTTGAGGGACTCGAAGG	62	338

388836	45	1494	CTGAGGAAGCTGAGGAGGCG	45	339

388837	45	1511	TGTGCCTGCGGCGGCGGCTG	61	340

388838	45	1523	GGCAGCAGCGGCTGTGCCTG	53	341

388813	45	1607	CAAACTCACGGTCGGTGCAG	58	342

388814	45	1614	GCGGGCCCAAACTCACGGTC	51	343

388815	45	1623	GGAGCTGCAGCGGGCCCAAA	39	344

388816	45	1650	GCCGTAGCCTGGGACCCGCC	77	345

388817	45	1670	GCAGGGTTACCGCCATCCCC	70	346

388818	45	1675	AGGCTGCAGGGTTACCGCCA	66	347

388819	45	1680	CCCGCAGGCTGCAGGGTTAC	53	348

388820	45	1685	GCCGGCCCGCAGGCTGCAGG	49	349

388821	45	1773	AAGGCCTCGCCCCAGGAGGG	46	350

388822	45	1807	AGACCCAAGTGAGGGAGCGG	65	351

388823	45	1813	AAGGGAAGACCCAAGTGAGG	44	352

388824	45	1817	GGACAAGGGAAGACCCAAGT	68	353

388825	45	1825	TCGCGAGAGGACAAGGGAAG	24	354

388826	45	1830	TCCCCTCGCGAGAGGACAAG	59	355

388827	45	1850	GGCCCCAACAAGGCTCTGCC	58	356

388828	45	1855	GGACAGGCCCCAACAAGGCT	61	357

Mouse Huntingtin

Antisense oligonucleotides were designed to target different regions of the mouse huntingtin gene, using published sequences cited in Table 2. The sequences and corresponding SEQ ID NOs are shown in Table 5. All compounds in Table 5 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of 10 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) by five-nucleotide “wings”. The wings are composed of 2′-O-(2-methoxyethyl) nucleotides, also known as 2′-MOE nucleotides. The internucleoside (backbone) linkages are phosphorothioate throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.
The antisense oligonucleotides were analyzed for their effect on huntingtin mRNA levels in b.END cells by quantitative real-time PCR as described in other examples herein. The data presented in Table 5 represent percent inhibition of huntingtin mRNA levels relative to untreated cells. Data are averages from experiments in which cultured cells were treated with the disclosed antisense oligonucleotides.

TABLE 5


Inhibition of mouse huntingtin mRNA levels
by chimeric oligonucleotides having 2′-
MOE wings and deoxy gap

SEQ

Target

ISIS #	NO	Site	Sequence (5′ to 3′)	Inhib	NO

387869	8	517	GAATCCATCAAAGCTTTGAT	46	58

387884	8	1684	TCATTCAGGTCCATGGCAGG	54	77

387913	8	4787	CTGATGGTACTGGATGAGTC	32	120

387865	10	177	GCCTTCATCAGCTTTTCCAG	35	47

387866	10	394	ATGATTCACACGGTCTTTCT	38	55

387867	10	451	AAATTCTGGAGAATTTCTGA	22	56

387868	10	459	AGTTTCTGAAATTCTGGAGA	39	57

387870	10	583	CCTTGGAAGATTAGAATCCA	41	59

387871	10	671	GAGCCAGCTCAGCAAACCTC	34	60

387872	10	680	GAACCAGGTGAGCCAGCTCA	23	61

387874	10	783	ACAGCTGCAGCCAAGGTCTC	52	63

387875	10	807	CCAAAAGAAGCCATAATTTT	19	64

387876	10	838	AACCTTAATTTCATTGTCAT	42	65

387877	10	1132	AGAGACTTCCATTTCTTTCC	51	68

387878	10	1138	AGAAGGAGAGACTTCCATTT	24	69

387879	10	1146	TGCTCTGCAGAAGGAGAGAC	17	70

387880	10	1163	CATAAACCTGGACAAGCTGC	34	71

387882	10	1203	ACATTGTGGTCTTGGTGCTG	70	73

387883	10	1422	AAGAGCACTTTGCCTTTTTG	52	74

387885	10	1744	GGTCCCATCATTCAGGTCCA	44	78

387887	10	2365	ATCGATGTAGTTCAAGATGT	39	85

387888	10	2412	GTCCCACAGAGAATGGCAGT	31	86

387889	10	2785	TGTATAATGATGAGCCCCTC	48	88

387890	10	2936	GATCAGCTTGTCCTTGGTCA	55	89

387891	10	3168	CAGCATCCAAATGTGAGTGC	52	92

387892	10	3174	GCTTCACAGCATCCAAATGT	46	93

387893	10	3606	AGAGAAGGCAAGGCTGCCTT	46	95

387894	10	3614	GGTTTGTTAGAGAAGGCAAG	43	96

387895	10	3816	ACATCATGCAGTTTGAGGTA	57	97

387896	10	3825	GCTTTCAGGACATCATGCAG	38	98

387897	10	3993	AAGCAGGATTTCAGGTATCC	60	99

387898	10	4001	CTCGACTAAAGCAGGATTTC	48	100

387899	10	4020	ACAGTTGCCATCATTGGTTC	35	101

387900	10	4092	TTGGAAGATAAGCCATCAAA	41	103

387901	10	4230	TGCACCATGTTCCTCAGGCT	64	104

387902	10	4234	CGCCTGCACCATGTTCCTCA	47	105

387903	10	4345	AATAGCATTCTTATCTGCAC	46	106

387904	10	4357	AATGTGATTATGAATAGCAT	25	107

387905	10	4503	AACACCTGATCTGAATCCAG	29	109

387906	10	4551	AACTGGCCCACTTCAATGTA	64	111

387908	10	4647	TTAGGAATTCCAATGATCTG	74	113

387909	10	4653	ATGATTTTAGGAATTCCAAT	28	114

387910	10	4680	CTGGCCATGATGCCATCACA	27	115

387911	10	4689	TTCCTTCCACTGGCCATGAT	38	116

387912	10	4770	GCATCAGCTTTATTTGTTCC	45	117

387914	10	4878	TGGCACTGCTGCAGGACAAG	73	121

387915	10	5184	TCCTGAATACGAGAAAGAAC	8	122

387916	10	5763	TCTCTATTGCACATTCCAAG	59	125

387917	10	5812	CTGACAGACATAATCACAGA	55	126

387918	10	5873	TGATCAGATCTTGAATGTGA	69	127

387919	10	5967	CGAGACTGAATTGCCTGGAT	73	128

387920	10	6258	GAATAGAGCCTTTGGTGTCT	53	129

387921	10	6428	AATCTGACCTGGTCCAACAC	4	131

387922	10	6438	AGCAGTGCAGAATCTGACCT	16	132

387924	10	6822	TTCTCAGGAGGAAGGTGCAA	26	139

387925	10	6892	CTGCTCATGGATCAAATGCC	43	140

387926	10	7445	TGTTGATGCGGTAGATGAAC	8	146

387927	10	7512	GTCACCAGGACACCAAGGAG	47	147

387928	10	7665	TCCAAGCAGCTTACAGCTGG	31	148

387942	10	7888	GTTGATCTGCAGCAGCAGCT	54	151

387930	10	7961	TGTTCCCCAGCCACACGGAG	53	153

387931	10	8319	GTGGCAGGCACCAGGTACTG	62	154

387932	10	8713	ACAGTGGTAAATGATGGAGG	51	156

387933	10	8859	ATGCAGGTGAGCATCAGGCC	64	157

387934	10	8866	TGTGTACATGCAGGTGAGCA	45	158

387935	10	9105	TATGGCTGCTGGTTGGACAG	43	160

387936	10	9196	CAGCATGACCCAGTCCCGGA	53	161

387937	10	9199	GGACAGCATGACCCAGTCCC	34	162

387938	10	9324	CCCATCCTGCTGATGACATG	41	163

387939	10	9363	ACCAGGCAGAAAAGGTTCAC	28	164

387940	10	9511	TCAGCAGGTGGTGACCTTGT	54	165

387941	10	10042	ACTGATATAATTAAATTTTA	3	182

387873	11	39021	TTCACCAGGTAAGGCCTGCA	28	62

387881	11	46216	GTCAGTTCATAAACCTGGAC	57	72

387886	11	52829	CTAACACAATTTCAGAACTG	25	79

388535	11	64098	GATAAAACACCTTGTTAATG	0	233

388536	11	74028	GGAGCAGTACCTTATAGTTG	0	234

388467	11	85701	ATAGCTGCTGCACACAGACA	37	235

387907	11	90911	TGATTCCCTGAACTGGCCCA	77	112

388534	11	90914	GCATCAGTACCTGAACTGGC	18	236

388532	11	116664	GAGTGGTTGGCTAATGTTGA	26	237

387923	11	119259	TCTGCACCTTCCAGCAGTGC	25	133

387929	11	138172	GAGTGTATGGACACCTGGCC	64	152

388533	11	142848	CAGTTTTGTCCTGGATACAA	0	238

388459	44	962	GGAGCCAGTTGTAGAAGTAC	4	239

388460	44	1284	CCTGGTGTGGTCAGTGCTTG	39	240

388461	44	1306	CAGAGTGAGCTGCCCAAGCC	18	241

388462	44	1317	TCTTCTTGAACCAGAGTGAG	29	242

388463	44	1948	GTTTCTGAAAACATCTGAGA	13	243

388464	44	1998	CTATGGCCCATTCTTTCCAA	33	244

388465	44	2642	TAAGCAGTTGTAATCCCAAG	7	245

388466	44	3690	GGACTCATTGGAGTAGAAGC	34	246

388468	44	5944	AAGACCACTAGCTGCAGAAT	29	247

388469	44	6735	TGGTATGATGTGGTATCACC	53	248

388470	44	6855	GTCATTACCACAAACTTCAC	20	249

388471	44	7145	GACTGAGGTTTTGTATATCT	19	250

388472	44	7269	ACAATGTTCTTCAGCACAGC	24	251

388473	44	8515	CAGCAGATAGTCACTAACAA	20	252

388474	44	9228	ACTGGAGTTCTTTGTGTGAA	25	253

388475	44	9519	GGCACTACTCAGCAGGTGGT	49	254

388476	44	9532	CTTTTGTCCCACAGGCACTA	20	255

388477	44	9630	CTTGACACAAGTGGAAGCCT	15	256

388478	44	9676	GCATAGCCCTCATTGCAAAG	40	257

388479	44	9691	TAGTGCATGTTCCCTGCATA	45	258

388480	44	9701	AACCCCAACATAGTGCATGT	16	259

388481	44	9770	AAGACAAACACCTGGTCAAC	13	260

388482	44	9855	AACCATCTGGCAAGAGCTAG	23	261

388483	44	9924	TGTGGCAGGTATGCCTACTG	14	262

388484	44	9932	GACACTGGTGTGGCAGGTAT	36	263

388485	44	10102	CTTGCCAAGTCACACACTTT	19	264

388486	44	10135	ACTTCCATAAACTTTGTCAC	7	265

388487	44	10181	GACTGAGTAGCTACAGGAGA	40	266

388488	44	10275	TGCTGGCTTAATGGAATGCA	34	267

388489	44	10315	GGATTCTCACACAGGCAGTC	39	268

388490	44	10330	GTTAGGCCACAGGCAGGATT	30	269

388491	44	10348	CAGTTTTTCAGTTCCTCAGT	51	270

388492	44	10370	TTATAACTCTAACAGTGGAA	24	271

388493	44	10460	CTAGGAGAGTGCATCAACAC	38	272

388494	44	10480	TTTCTACCCAGGCTGAGAGA	30	273

388495	44	10550	CTACAGTGCAGGTCAGCCAC	42	274

388496	44	10582	CATCCACAATGGTCAGCTGG	30	275

388497	44	10616	CCCAACCATGCAGAAGATAC	17	276

388498	44	10634	GGTCAGCACTTCTCAGGTCC	50	277

388499	44	10950	TTAACATGACCTGGTTACTC	27	278

388500	44	10988	CCCAAACCAAGCCAGGAAAT	19	279

388501	44	11020	CTTGGTCATATAGTCAAACA	43	280

388502	44	11140	TAATCACAGGCTGCAAGCTC	28	281

388503	44	11170	AAGCAATCCATGGACTGAAG	52	282

388504	44	11211	GTCATGATGGAAAGATAGAG	35	283

388505	44	11240	AACCTTGCATCCCAGCAGCA	12	284

388506	44	11300	GGCAGATAGGAGGAGAGTCA	19	285

388507	44	11407	GGTGAATTTCTTTCATTAAA	53	286

388508	44	11525	TTGGACCAACCTCAGAGTGT	45	287

388509	44	11560	GTAATCAGGCCTGCACCATG	41	288

388510	44	11575	CATCTACCATGAGGAGTAAT	15	289

388511	44	11611	AATGGCTCTAGATTTTATAT	33	290

388512	44	11678	TTCTGATCACACTAAACAAG	31	291

388513	44	11750	CTAGGTTGTGGCACCCATGA	47	292

388514	44	11766	GTACCCAGGTGCATCTCTAG	52	293

388515	44	11890	TGTATGTGGCAGTTGCAAGA	51	294

388516	44	11940	ACTTTTAAAAATTGAGTCCC	17	295

388517	44	12054	TTAAATAAAGCTTGGAAATC	8	296

388518	44	12132	TGACAGTACCACCATGGAAA	27	297

388519	44	12176	GTGCATTGCCAAAAGTTCTA	41	298

388520	44	12248	AAGTCACCTACATGTCAAGG	22	299

388521	44	12262	ACTTGGCAGTGGCTAAGTCA	21	300

388522	44	12377	GTTAGGATTGGTCCCTTCCC	18	301

388523	44	12527	GACCAATTCTGCAGCCCCAC	28	302

388524	44	12648	CCATGATCCTAGTGCTCAAT	42	303

388525	44	12696	CCACATACCAATCCCTGGAG	38	304

388526	44	12726	CCAGCATCAGCAGCTCAGTG	40	305

388527	44	12756	TTTCCCAACCATGATATCCT	7	306

388528	44	12846	CCCTGAACCTTGATATCATC	2	307

388529	44	12971	TGCAGATAGGTCTCTGCCAC	16	308

388530	44	13020	TACAGCAGCAAGGCTTGGAC	29	309

388531	44	13100	GGAAATGGACAGCCAGGTCT	44	310

Isis numbers 387865-387942 are targeted to both human and mouse huntingtin.

Several antisense oligonucleotides were selected for additional testing in A549 cells. Cells in multi-well plates were treated with various amounts of the selected antisense oligonucleotides, as indicated in the following table (n=6 treatments per antisense oligonucleotide). Following the end of the treatment period, RNA was isolated from the cells, and human huntingtin mRNA levels in each individual cell culture well were measured by quantitative, real-time PCR. The data presented in the following table (Table 6) represent the average percent inhibition for each antisense oligonucleotide (n=6 treatments), relative to untreated cells. Control oligonucleotides, having randomized nucleotide sequences not targeted to any known gene, were also tested.

TABLE 6


Dose response inhibition of human huntingtin in A549 cells

	6.25	12.5	25	50	100	200
Isis No.	nM	nM	nM	nM	nM	nM

387892	97	91	77	68	45	28
387898	85	86	69	47	35	21
387902	91	104	67	47	23	9
387916	88	100	100	51	32	19
388227	86	92	114	80	69	58
388240	117	126	83	65	24	22
388249	101	100	106	54	35	24
388816	101	132	77	59	38	26
388817	92	97	84	69	50	30
388824	78	87	85	69	41	27
388833	81	82	68	65	47	41
Control #1	115	102	96	77	71	57

These results demonstrate that the antisense oligonucleotides targeted to huntingtin reduced huntingtin mRNA levels in A549 cells. The control oligonucleotides were not able to effectively inhibit huntingtin mRNA levels, particularly at lower doses. The target regions to which these inhibitory antisense oligonucleotides are complementary are herein referred to as “validated target segments.”
GMO4281
Several antisense oligonucleotides were selected for additional testing in GMO4281 fibroblasts, which originated from an HD patient. Cells in multi-well plates were treated with various amounts of the selected antisense oligonucleotides, as indicated in the following table (n=6 treatments per antisense oligonucleotide). Control oligonucleotides, having randomized nucleotide sequences not targeted to any known gene, were also tested.

Following the end of the treatment period, RNA was isolated from the cells, and human huntingtin mRNA levels in each individual cell culture well were measured by quantitative, real-time PCR. The data presented in the following table represent the average huntingtin mRNA level (n=6), relative to untreated cells, i.e. the data are expressed as percentage of control cell huntingtin mRNA levels. Percent control less than 100 indicates a reduction in huntingtin mRNA levels, whereas percent control greater than 100 indicates an increase in huntingtin mRNA levels. Percent inhibition can be calculated by subtracting the percentage of control from 100.

TABLE 7


Dose response inhibition of human
huntingtin in GMO4281 fibroblasts

Oligonucleotide Treatment Concentration

	9.375	18.75	37.5	75.0	150.0	300.0
Isis #	nM	nM	nM	nM	nM	nM

387892	77	55	47	33	22	21
387898	77	61	49	25	17	13
387902	87	58	52	27	17	13
387916	104	75	50	25	14	12
388240	81	74	57	26	17	16
388249	96	74	55	32	18	14
388816	86	61	48	26	14	12
388817	84	76	51	35	26	18
388824	86	78	59	38	24	20
388833	84	79	60	33	19	13
Control #1	99	95	106	67	63	48
Control #2	100	102	88	77	64	49

GMO4478 Cells

Several antisense oligonucleotides targeted to huntingtin were selected for additional testing in GM04478 cells, which are fibroblasts derived from and HD patient. The testing was performed according to the procedure used for GM04281 cells. The results are shown in the following table as average percent inhibition, relative to untreated cells.

TABLE 8


Dose response inhibition of human
huntingtin in GMO4478 fibroblasts

Oligonucleotide Treatment Concentration

	9.375	18.75	37.5	75.0	150.0
Isis No.	nM	nM	nM	nM	nM

387892	45	29	17	10	7
387898	50	27	9	2	2
387902	40	22	9	3	2
387916	60	39	18	6	3
388240	60	34	16	5	6
388249	78	56	34	13	7
388816	75	48	26	8	7
388817	70	52	37	38	32
388824	65	42	21	9	8
388833	43	31	16	7	3
Control #1	95	88	73	58	48
Control #2	101	94	90	64	56

Each of the antisense oligonucleotides targeted to human huntingtin efficiently reduced huntingtin mRNA levels, in both GM04478 and GMO4281 fibroblasts. The control oligonucleotides were not able to effectively inhibit huntingtin mRNA levels, particularly at lower doses.

The potency of antisense oligonucleotides targeted to huntingtin is summarized in Table 9. The potency is illustrated as IC₅₀, which is the concentration at which a 50% reduction in huntingtin mRNA levels is observed. This table also indicates the huntingtin sequence to which the antisense oligonucleotides are complementary, as well as the corresponding 5′ target site. Particular features of the region of the huntingtin sequence to which the antisense oligonucleotides are complementary are also shown. Additionally indicated is the species of huntingtin gene to which the antisense oligonucleotides are targeted. The target regions to which these inhibitory antisense oligonucleotides are complementary are herein referred to as “validated target segments.”

TABLE 9


Summary of potent antisense oligonucleotides targeted to huntingtin

				Target	5′		Target region
		IC₅₀		SEQ ID	Target	Target nucleic acid	within human
Isis No	A549	GM04281	GM04478	NO	Site	specifity	huntingin gene

387892	84	32	5	4	3209	Human-Mouse	exon 23:exon 24
387898	47	31	8	4	4036	Human-Mouse-Rat	exon 30
387902	39	35	5	4	4269	Human-Mouse	exon 31
387916	61	42	13	4	5801	Human-Rat	exon 42
388240	63	39	12	4	4558	Human; >4 mm to rodent	exon 34
388249	63	45	24	4	6769	human; >5 mm to rodent	exon 48:exon 49
388816	69	34	19	45	1650	targets R6/2 insert; >5	intron 1
						mm to mouse
388817	98	48	25	45	1670	targets R6/2 insert; >5	intron 1
						mm to mouse
388824	81	50	15	45	1817	targets R6/2 insert;	intron 1
						5 mm to mouse
388833	103	48	5	45	1128	targets CAG repeat	exon 1
						region

As the antisense oligonucleotides reduced huntingtin mRNA levels in cells isolated from HD patients, the antisense oligonucleotides are candidate therapeutic agents for the reduction of huntingtin mRNA levels in vivo. In one embodiment, the antisense oligonucleotides, having demonstrated potency in vitro, are further tested in experimental animal models, including experimental models of Huntington's disease, to identify antisense oligonucleotides that may reduce huntingtin mRNA in humans. Accordingly, in one embodiment, the antisense oligonucleotides are administered at therapeutically effect amounts to a human, for the treatment or amelioration of Huntington's disease. In another embodiment, the antisense oligonucleotides are administered at therapeutically effective amounts, to delay the onset of Huntington's disease.
Several antisense oligonucleotides targeted to huntingtin were selected for additional testing in huntingtin neuronal cell lines. Mouse striatum cell lines with wild-type huntingtin, STHdhQ7/7 (Q7/7), and mutant huntingtin, STHdhQ111/111 (Q111/111) were transfected with various doses of oligos 387902 and 387916, ranging from approximately 0.05 μM, to 10 μM. A 200V, 2 msec pulse in a 2 mm gap cuvette was used for electroploration transfection. One million cells were electroporated in the presence of the indicated amount of oligonucleotide. Following electroporation, the cells were plated at a density of 5×10⁴cells per well. The results are reported in the Table 10 as percent huntingtin mRNA as compared to no oligo control, with each concentration performed in triplicate.

TABLE 10

Inhibition of huntingtin in mouse neuronal cell lines

Q7/7 Q111/111

387902 387916 387902 387916

[Oligo]μM % mHtt % mHtt % mHtt % mHtt

0 100.0 100.0 100.0 100.0

0.675 85.9 36.5 156.4 59.3

1.25 50.2 34.5 103.4 31.4

3 31.4 11.0 38.9 18.8

5 12.2 1.6 16.5 6.4

10 5.5 2.7 6.8 3.1
In subsequent studies, cells are evaluated for phenotypic response by measuring caspase activity using the Promega Apo-ONE® Homogeneous Caspase-3/7 commercial assay. Briefly, cells are plated and Lipofectin® transfected the next day. After 48 hours the media is changed to serum-free DMEM for 24 h prior to the caspase assay.
In order to evaluate the effects of antisense inhibition of a gene in the central nervous system, it is beneficial to deliver antisense oligonucleotides directly to the central nervous system, for example, by intracerebroventricular (ICV), intrathecal (IT), or intraparenchymal administration. To evaluate the effects of antisense inhibition of huntingtin in the central nervous system of animals, antisense oligonucleotides targeted to huntingtin via ICV delivery.
ISIS 387902, 387916, 387918, 388249, 388503, 388509, and 388816 were selecting for in vivo testing. Saline-treated mice were used as control animals. Each treatment or control group included four animals. Surgically implanted Alzet mini-pumps continuously infused antisense oligonucleotides into mice at a dose of 100 ug/day over a two-week period. During the treatment period, mice were monitored for any clinical changes, such as body weight changes. At the end of the treatment period, mice were sacrificed and major organs were isolated. RNA was prepared from brain and liver tissues, and subjected to quantitative real-time PCR analysis to measure the reduction in mouse huntingtin mRNA levels.

TABLE 10


Inhibition of huntingtin in mouse neuronal cell lines

Each antisense oligonucleotide targeted to huntingtin reduced huntingtin mRNA levels in house brain, as shown in the following table. The species of huntingtin nucleic acid to which antisense oligonucleotide is targeted is also shown. Mouse huntingtin mRNA levels represent the average for each treatment group and are expressed as percentage of saline control (% saline control).

TABLE 11


In vivo antisense inhibition of mouse huntingtin

			Huntingtin
		Huntingtin	mRNA levels,
		nucleic acid	% of saline
Isis No.	SEQ ID NO:	target species	control

387902		human, mouse	37%
387916		human, rat	32%
		(single
		mismatch to
		mouse)
387918		human, mouse,	35%
		rat
388503		mouse	30%
388509		mouse	34%

Each of the antisense oligonucleotides shown in Table 11 reduced huntingtin mRNA levels in mouse brain following the ICV infusion period. Furthermore, ISIS 387916, which has one mismatch to mouse huntingtin, was able to reduce mouse huntingtin mRNA levels in vivo.
To evaluate the effects of antisense inhibition of huntingtin in the central nervous system of an animal model of HD, antisense oligonucleotides targeted to huntingtin are administered to R6/2 transgenic mice via ICV delivery.
ISIS 387902, 387916, 387918, 388249, 388503, 388509, and 388816 are selected for in vivo testing. Saline-treated mice are used as control animals. Each treatment or control group includes four animals. Surgically implanted Alzet mini-pumps continuously infuse antisense oligonucleotides into mice at a dose of 100 ug/day over a two-week period. During the treatment period, mice are monitored for any clinical changes, such as body weight changes as well as phenotypical behaviors related to the huntingtin transgene. At the end of the treatment period, mice are sacrificed and major organs are isolated. RNA is prepared from brain and liver tissues, and is subjected to quantitative real-time PCR analysis to measure the reduction in mouse huntingtin mRNA levels. Huntingtin protein expression in the tissue is also measured using standard Western blotting techniques.
Provided herein are methods of treating an individual suffering from Huntington's Disease (HD). Such methods comprise the administration to the cerebrospinal fluid or brain tissue of the individual a pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin. Delivery of the pharmaceutical composition to the cerebrospinal fluid allows for contact of the antisense oligonucleotide with the cells of central nervous system tissues, including tissues affected by HD.
Individuals suffering from HD receive a diagnosis of HD from a physician. The physician's assessment includes the genetic testing of the HD gene, and a neurological examination.
A surgically implanted pump (e.g, a Medtronic SyncroMed® II pump) is used to deliver a pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin to the cerebrospinal fluid or brain of an individual suffering from HD. The pump is surgically implanted per the procedures outlined by the manufacturer. Drug is retained in the reservoir of the pump, and is pumped at a programmed dose preferably into a catheter that is surgically intrathecally implanted.
The reservoir is loaded with a pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin. The pharmaceutical composition is administered at an amount that yields an infusion of 8 mg to 12 mg of antisense oligonucleotide into the cerebrospinal fluid. In some embodiments, the amount of antisense oligonucleotide infused is 10 mg. Administration is for a period of at least 28 days. Individuals are monitored by a medical professional, who evaluates indicators of HD. It is clinically desirable for the administration to slow or halt the progression of HD, or prevent or slow the worsening of, or improve, a symptom or marker of HD.
Provided herein are methods of preventing or delaying the onset of Huntington's Disease (HD) in individuals susceptible to HD. Such methods comprise the administration to the cerebrospinal fluid or brain of the individual a pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin. Delivery of the pharmaceutical composition to the cerebrospinal fluid allows for contact of the antisense oligonucleotide with the cells of central nervous system tissues, including tissues affected by HD.
Individuals susceptible to HD are identified by a physician following genetic testing of the HD gene, and a neurological examination.
A surgically implanted pump (e.g, a Medtronic SyncroMed® II pump) is used to deliver a pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin to the cerebrospinal fluid or brain of an individual susceptible to HD. The pump is surgically implanted per the procedures outlined by the manufacturer. Drug is retained in the reservoir of the pump, and is pumped at a programmed dose preferably into a catheter that is surgically intrathecally implanted.
The reservoir is loaded with a pharmaceutical composition comprising an antisense oligonucleotide targeted to huntingtin. The pharmaceutical composition is administered at an amount that yields an infusion of 8 mg to 12 mg of antisense oligonucleotide into the cerebrospinal fluid. In some embodiments, the amount of antisense oligonucleotide infused is 10 mg. Administration is for a period of at least 28 days. Individuals are monitored by a medical professional, who evaluates indicators of HD. It is clinically desirable for the administration to prevent or delay the onset of symptoms of HD.

Claims (23)

1. A method of treating an individual at risk of suffering from or currently suffering from Huntington's Disease (HD) comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an antisense compound 12 to 35 nucleobases in length having at least 90% complementarity to nucleotides 1650-1704, 1807-1874, 3183-3228, 4010-4087, 4265-4288, 4553-4608, 5781-5820, and 6793-6796 of SEQ ID NO: 4;

wherein said administration treats said individual.

2. The method of

claim 1

, wherein said administering comprises intrathecal delivery, intracerebroventricular delivery, or intraparenchymall delivery.

3. The method of

claim 2

, wherein the administering comprises administration into the cerebrospinal fluid of the individual by intrathecal infusion.

4. The method of

claim 1

, wherein said treatment comprises improvement in one or more indicators of HD.

5. The method of

claim 1

, wherein said treatment comprises increasing the survival time of the individual.

6. The method of

claim 1

, wherein said treatment comprises delaying the onset of HD.

7. The method of

claim 1

, wherein said antisense compound has at least at least 95% complementarity to nucleotides 1650-1704, 1807-1874, 3183-3228, 4010-4087, 4265-4288, 4553-4608, 5781-5820, and 6793-6796 of SEQ ID NO: 4.

8. The method of

claim 1

, wherein said antisense compound has 100% complementarity to nucleotides 1650-1704, 1807-1874, 3183-3228, 4010-4087, 4265-4288, 4553-4608, 5781-5820, and 6793-6796 of SEQ ID NO: 4.

9. The method of

claim 1

, wherein said antisense compound is an antisense oligonucleotide.

10. The method of

claim 9

, wherein said antisense oligonucleotide has at least one modified internucleoside linkage, sugar moiety, or nucleobase.

11. The method of

claim 10

, wherein said oligonucleotide comprises a chimeric oligonucleotide having a gap segment positioned between 5′ and 3′ wing segments.

12. The method of

claim 10

, wherein said gap segment of the chimeric oligonucleotide is comprised of 2′-deoxynucleotides and the wing segments are comprised of nucleotides having modified sugar moieties.

13. The method of

claim 12

, wherein the modified sugar moiety is 2′-OMe or a bicyclic nucleic acid.

14. The method of

claim 13

, wherein the gap segment of the chimeric oligonucleotide consists of ten 2′-deoxynucleotides and each wing segment consists of five 2′-O-methoxyethyl-modified nucleotides.

15. The method of

claim 9

, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

16. The method of

claim 9

, wherein each cytosine is a 5-methylcytosine.

17. The method of

claim 1

, comprising 17 to 25 nucleotides.

18. The method of

claim 1

, comprising 19 to 23 nucleotides.

19. The method of

claim 1

, comprising 20 nucleotides.

20. The method of

claim 1

, further comprising selecting an individual suffering from HD.

21. The method of

claim 1

, further comprising selecting an individual susceptible to HD.

22. A method of treating an individual at risk of suffering from or currently suffering from Huntington's Disease (HD) comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an antisense oligonucleotide 12 to 35 nucleotides in length comprising at least 12 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs 46-357.

23. The method of

claim 22

, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NOs: 50, 93, 100, 105, 110, 125, 137, 345, 346, and 353.

US11/627,921 2006-01-26 2007-01-26 Compositions and their uses directed to huntingtin Abandoned US20070299027A1 (en)

Priority Applications (6)

Application Number	Priority Date	Filing Date	Title
US11/627,921 US20070299027A1 (en)	2006-01-26	2007-01-26	Compositions and their uses directed to huntingtin
US12/618,470 US20100069472A1 (en)	2006-01-26	2009-11-13	Compositions and their uses directed to huntingtin
US13/708,839 US9057066B2 (en)	2006-01-26	2012-12-07	Compositions and their uses directed to huntingtin
US14/708,511 US20160053256A1 (en)	2006-01-26	2015-05-11	Compositions and their uses directed to huntingtin
US15/725,933 US10738307B2 (en)	2006-01-26	2017-10-05	Compositions and their uses directed to huntingtin
US16/988,862 US20210017522A1 (en)	2006-01-26	2020-08-10	Compositions and their uses directed to huntingtin

Applications Claiming Priority (3)

Application Number	Priority Date	Filing Date	Title
US76295406P	2006-01-26	2006-01-26
US83629006P	2006-08-07	2006-08-07
US11/627,921 US20070299027A1 (en)	2006-01-26	2007-01-26	Compositions and their uses directed to huntingtin

Related Child Applications (1)

Application Number	Title	Priority Date	Filing Date
US12/618,470 Continuation US20100069472A1 (en)	2006-01-26	2009-11-13	Compositions and their uses directed to huntingtin

Publications (1)

Publication Number	Publication Date
US20070299027A1 true US20070299027A1 (en)	2007-12-27

Family

ID=38164262

Family Applications (14)

Application Number	Title	Priority Date	Filing Date
US11/627,921 Abandoned US20070299027A1 (en)	2006-01-26	2007-01-26	Compositions and their uses directed to huntingtin
US11/627,916 Active US7951934B2 (en)	2006-01-26	2007-01-26	Compositions and their uses directed to huntingtin
US12/618,470 Abandoned US20100069472A1 (en)	2006-01-26	2009-11-13	Compositions and their uses directed to huntingtin
US13/090,146 Active US8415465B2 (en)	2006-01-26	2011-04-19	Compositions and their uses directed to huntingtin
US13/708,839 Active US9057066B2 (en)	2006-01-26	2012-12-07	Compositions and their uses directed to huntingtin
US13/789,368 Active US8952145B2 (en)	2006-01-26	2013-03-07	Compositions and their uses directed to huntingtin
US14/589,833 Active US9353372B2 (en)	2006-01-26	2015-01-05	Compositions and their uses directed to huntingtin
US14/708,511 Abandoned US20160053256A1 (en)	2006-01-26	2015-05-11	Compositions and their uses directed to huntingtin
US15/142,827 Active US9920321B2 (en)	2006-01-26	2016-04-29	Compositions and their uses directed to huntingtin
US15/725,933 Active US10738307B2 (en)	2006-01-26	2017-10-05	Compositions and their uses directed to huntingtin
US15/891,154 Active US10655129B2 (en)	2006-01-26	2018-02-07	Compositions and their uses directed to huntingtin
US16/848,968 Active US11072794B2 (en)	2006-01-26	2020-04-15	Compositions and their uses directed to huntingtin
US16/988,862 Pending US20210017522A1 (en)	2006-01-26	2020-08-10	Compositions and their uses directed to huntingtin
US17/352,806 Active 2027-04-08 US11761000B2 (en)	2006-01-26	2021-06-21	Compositions and their uses directed to huntingtin

Family Applications After (13)

Application Number	Title	Priority Date	Filing Date
US11/627,916 Active US7951934B2 (en)	2006-01-26	2007-01-26	Compositions and their uses directed to huntingtin
US12/618,470 Abandoned US20100069472A1 (en)	2006-01-26	2009-11-13	Compositions and their uses directed to huntingtin
US13/090,146 Active US8415465B2 (en)	2006-01-26	2011-04-19	Compositions and their uses directed to huntingtin
US13/708,839 Active US9057066B2 (en)	2006-01-26	2012-12-07	Compositions and their uses directed to huntingtin
US13/789,368 Active US8952145B2 (en)	2006-01-26	2013-03-07	Compositions and their uses directed to huntingtin
US14/589,833 Active US9353372B2 (en)	2006-01-26	2015-01-05	Compositions and their uses directed to huntingtin
US14/708,511 Abandoned US20160053256A1 (en)	2006-01-26	2015-05-11	Compositions and their uses directed to huntingtin
US15/142,827 Active US9920321B2 (en)	2006-01-26	2016-04-29	Compositions and their uses directed to huntingtin
US15/725,933 Active US10738307B2 (en)	2006-01-26	2017-10-05	Compositions and their uses directed to huntingtin
US15/891,154 Active US10655129B2 (en)	2006-01-26	2018-02-07	Compositions and their uses directed to huntingtin
US16/848,968 Active US11072794B2 (en)	2006-01-26	2020-04-15	Compositions and their uses directed to huntingtin
US16/988,862 Pending US20210017522A1 (en)	2006-01-26	2020-08-10	Compositions and their uses directed to huntingtin
US17/352,806 Active 2027-04-08 US11761000B2 (en)	2006-01-26	2021-06-21	Compositions and their uses directed to huntingtin

Country Status (12)

Country	Link
US (14)	US20070299027A1 (en)
EP (5)	EP3210633B1 (en)
JP (5)	JP5425474B2 (en)
AU (4)	AU2007210038B2 (en)
CA (1)	CA2638908C (en)
DK (1)	DK2161038T3 (en)
ES (2)	ES2744098T3 (en)
HK (1)	HK1141449A1 (en)
PL (2)	PL3210633T3 (en)
PT (1)	PT2161038E (en)
SI (1)	SI2161038T1 (en)
WO (2)	WO2007089584A2 (en)

Cited By (25)

* Cited by examiner, † Cited by third party

Publication number	Priority date	Publication date	Assignee	Title
WO2011032045A1 (en)	2009-09-11	2011-03-17	Isis Pharmaceuticals, Inc.	Modulation of huntingtin expression
US20110190222A1 (en) *	2008-07-29	2011-08-04	Corey David R	Selective Inhibition of Polyglutamine Protein Expression
WO2011139917A1 (en)	2010-04-29	2011-11-10	Isis Pharmaceuticals, Inc.	Modulation of transthyretin expression
US9605019B2 (en)	2011-07-19	2017-03-28	Wave Life Sciences Ltd.	Methods for the synthesis of functionalized nucleic acids
US9611471B2 (en)	2010-08-05	2017-04-04	Academisch Ziekenhuis Leiden	Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins
US9617547B2 (en)	2012-07-13	2017-04-11	Shin Nippon Biomedical Laboratories, Ltd.	Chiral nucleic acid adjuvant
WO2017062983A1 (en) *	2015-10-09	2017-04-13	The Children's Hospital Of Philadelphia	Compositions and methods for treating huntington's disease and related disorders
US9695211B2 (en)	2008-12-02	2017-07-04	Wave Life Sciences Japan, Inc.	Method for the synthesis of phosphorus atom modified nucleic acids
US9745576B2 (en)	2012-04-23	2017-08-29	Biomarin Technologies B.V.	RNA modulating oligonucleotides with improved characteristics for the treatment of neuromuscular disorders
US9890379B2 (en)	2006-08-11	2018-02-13	Biomarin Technologies B.V.	Treatment of genetic disorders associated with DNA repeat instability
EP3312189A1 (en)	2011-04-21	2018-04-25	Ionis Pharmaceuticals, Inc.	Modulation of hepatitis b virus (hbv) expression
US9982257B2 (en)	2012-07-13	2018-05-29	Wave Life Sciences Ltd.	Chiral control
US10144933B2 (en)	2014-01-15	2018-12-04	Shin Nippon Biomedical Laboratories, Ltd.	Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator
US10149905B2 (en)	2014-01-15	2018-12-11	Shin Nippon Biomedical Laboratories, Ltd.	Chiral nucleic acid adjuvant having antitumor effect and antitumor agent
US10160969B2 (en)	2014-01-16	2018-12-25	Wave Life Sciences Ltd.	Chiral design
US10167309B2 (en)	2012-07-13	2019-01-01	Wave Life Sciences Ltd.	Asymmetric auxiliary group
US10307434B2 (en)	2009-07-06	2019-06-04	Wave Life Sciences Ltd.	Nucleic acid prodrugs and methods of use thereof
US10322173B2 (en)	2014-01-15	2019-06-18	Shin Nippon Biomedical Laboratories, Ltd.	Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent
US10428019B2 (en)	2010-09-24	2019-10-01	Wave Life Sciences Ltd.	Chiral auxiliaries
US10479995B2 (en)	2015-07-22	2019-11-19	Wave Life Sciences Ltd.	Oligonucleotide compositions and methods thereof
US10724035B2 (en)	2016-05-04	2020-07-28	Wave Life Sciences Ltd.	Oligonucleotide compositions and methods thereof
US10876114B2 (en)	2007-10-26	2020-12-29	Biomarin Technologies B.V.	Methods and means for efficient skipping of at least one of the following exons of the human Duchenne muscular dystrophy gene: 43, 46, 50-53
US10913946B2 (en)	2012-01-27	2021-02-09	Biomarin Technologies B.V.	RNA modulating oligonucleotides with improved characteristics for the treatment of Duchenne and Becker muscular dystrophy
USRE48468E1 (en)	2007-10-26	2021-03-16	Biomarin Technologies B.V.	Means and methods for counteracting muscle disorders
US20220033814A1 (en) *	2018-12-03	2022-02-03	Triplet Therapeutics, Inc.	Methods for the treatment of trinucleotide repeat expansion disorders associated with mlh1 activity

Families Citing this family (71)

* Cited by examiner, † Cited by third party

Publication number	Priority date	Publication date	Assignee	Title
JP5425474B2 (en) *	2006-01-26	2014-02-26	アイシスファーマシューティカルズ，インコーポレーテッド	Composition and use thereof for huntingtin
ES2377327T5 (en)	2006-10-18	2020-04-28	Ionis Pharmaceuticals Inc	Antisense compounds
WO2008134646A2 (en) *	2007-04-26	2008-11-06	University Of Iowa Research Foundation	Rna interference suppression of neurodegenerative diseases and methods of use thereof
DK2164967T3 (en)	2007-05-31	2015-10-19	Univ Iowa Res Found	Reduction of off-target rna interferenstoksicitet
JP2010533170A (en)	2007-07-12	2010-10-21	プロセンサテクノロジーズビー．ブイ．	Molecules for targeting compounds to various selected organs, tissues or tumor cells
CN101980726A (en)	2008-02-08	2011-02-23	普罗森那控股公司	Methods and means for treating DNA repeat instability associated genetic disorders
US8679750B2 (en)	2008-05-09	2014-03-25	The University Of British Columbia	Methods and compositions for the treatment of Huntington'S disease
EP2119783A1 (en)	2008-05-14	2009-11-18	Prosensa Technologies B.V.	Method for efficient exon (44) skipping in Duchenne Muscular Dystrophy and associated means
WO2010017509A1 (en) *	2008-08-07	2010-02-11	Isis Pharmaceuticals, Inc.	Modulation of transthyretin expression for the treatment of cns related disorders
KR101773551B1 (en) *	2008-10-15	2017-08-31	아이오니스 파마수티컬즈, 인코포레이티드	Modulation of factor 11 expression
CN102459595A (en)	2009-04-24	2012-05-16	普罗森那技术公司	Inosine-containing oligonucleotides for the treatment of DMD
CN102575251B (en) *	2009-05-18	2018-12-04	库尔纳公司	The relevant disease of the reprogramming factor is treated by inhibiting the natural antisense transcript for the reprogramming factor
LT3305302T (en) *	2009-06-17	2018-12-10	Biogen Ma Inc.	Compositions and methods for modulation of smn2 splicing in a subject
JP6141018B2 (en)	2009-12-24	2017-06-07	バイオマリンテクノロジーズベー．フェー．	Molecules for treating inflammatory disorders
WO2011097614A1 (en) *	2010-02-08	2011-08-11	Isis Pharmaceuticals, Inc.	Mehods and compositions useful in diseases or conditions related to repeat expansion
CA2789005A1 (en) *	2010-02-08	2011-08-11	Isis Pharmaceuticals, Inc.	Selective reduction of allelic variants
US9006198B2 (en)	2010-02-08	2015-04-14	Isis Pharmaceuticals, Inc.	Selective reduction of allelic variants
EP2536738A4 (en) *	2010-02-08	2014-09-17	Isis Pharmaceuticals Inc	METHODS AND COMPOSITIONS SUITABLE FOR THE TREATMENT OF CONDITIONS OR CONDITIONS IN RELATION TO THE EXPANSION OF REPETITIONS
EP2380595A1 (en)	2010-04-19	2011-10-26	Nlife Therapeutics S.L.	Compositions and methods for selective delivery of oligonucleotide molecules to specific neuron types
EP2595663A4 (en) *	2010-07-19	2014-03-05	Isis Pharmaceuticals Inc	MODULATION OF THE EXPRESSION OF MYOTONIC DYSTROPHIC PROTEIN KINASE (DMPK)
AU2011329777B2 (en)	2010-11-17	2016-06-09	Ionis Pharmaceuticals, Inc.	Modulation of alpha synuclein expression
WO2012109395A1 (en)	2011-02-08	2012-08-16	Isis Pharmaceuticals, Inc.	Oligomeric compounds comprising bicyclic nucleotides and uses thereof
WO2012109667A1 (en) *	2011-02-12	2012-08-16	University Of Iowa Research Foundation	Therapeutic compounds
AU2012246822B2 (en)	2011-04-22	2017-05-25	Biomarin Technologies B.V.	New compounds for treating, delaying and/or preventing a human genetic disorder such as myotonic dystrophy type 1 (DM1)
US20150051389A1 (en) *	2011-08-11	2015-02-19	Isis Pharmaceuticals, Inc.	Selective antisense compounds and uses thereof
EP2751269B1 (en)	2011-08-29	2016-03-23	Ionis Pharmaceuticals, Inc.	Methods and compounds useful in conditions related to repeat expansion
EP3336189A1 (en)	2012-04-20	2018-06-20	Ionis Pharmaceuticals, Inc.	Oligomeric compounds comprising bicyclic nucleotides and uses thereof
WO2014059353A2 (en)	2012-10-11	2014-04-17	Isis Pharmaceuticals, Inc.	Oligomeric compounds comprising bicyclic nucleosides and uses thereof
EP4086347A3 (en)	2012-10-12	2023-01-11	Ionis Pharmaceuticals, Inc.	Selective antisense compounds and uses thereof
WO2014062736A1 (en)	2012-10-15	2014-04-24	Isis Pharmaceuticals, Inc.	Methods for monitoring c9orf72 expression
CN104968783B (en)	2012-10-15	2019-12-10	Ionis制药公司	Compositions for modulating expression of C9ORF72
BR112015027369B1 (en)	2013-05-01	2021-06-08	Ionis Pharmaceuticals, Inc	compounds comprising a modified oligonucleotide and a conjugate group, composition comprising said compounds and uses thereof
GB201308259D0 (en) *	2013-05-08	2013-06-12	Iles Raymond K	Cancer diagnosis and monitoring
US10435430B2 (en)	2013-07-31	2019-10-08	Ionis Pharmaceuticals, Inc.	Methods and compounds useful in conditions related to repeat expansion
TW201536329A (en)	2013-08-09	2015-10-01	Isis Pharmaceuticals Inc	Compound and method for regulating the manifestation of dystrophic myotonic protein kinase (DMPK)
MX2016004651A (en)	2013-10-11	2016-08-05	Ionis Pharmaceuticals Inc	Compositions for modulating c9orf72 expression.
CN107075514A (en)	2014-05-20	2017-08-18	衣阿华大学研究基金会	The therapeutic compounds of Huntington's disease
GB201410693D0 (en)	2014-06-16	2014-07-30	Univ Southampton	Splicing modulation
US10436802B2 (en)	2014-09-12	2019-10-08	Biogen Ma Inc.	Methods for treating spinal muscular atrophy
AU2015327836B2 (en)	2014-10-03	2021-07-01	Cold Spring Harbor Laboratory	Targeted augmentation of nuclear gene output
GB201504124D0 (en)	2015-03-11	2015-04-22	Proqr Therapeutics B V	Oligonucleotides
HUE049233T2 (en)	2015-04-16	2020-09-28	Ionis Pharmaceuticals Inc	Compositions for modifying C9ORF72 expression
KR102422625B1 (en)	2015-10-09	2022-07-20	유니버시티 오브 사우스앰톤	Regulation of gene expression and screening of deregulated protein expression
WO2017079291A1 (en)	2015-11-02	2017-05-11	Ionis Pharmaceuticals, Inc.	Compounds and methods for modulating c90rf72
US10874672B2 (en)	2015-12-10	2020-12-29	Ptc Therapeutics, Inc.	Methods for treating Huntington's disease
ES2882500T3 (en)	2015-12-14	2021-12-02	Cold Spring Harbor Laboratory	Antisense oligomers for the treatment of Dravet syndrome
US11096956B2 (en)	2015-12-14	2021-08-24	Stoke Therapeutics, Inc.	Antisense oligomers and uses thereof
US20180193270A1 (en)	2016-11-29	2018-07-12	PureTech Health LLC	Exosomes for delivery of therapeutic agents
KR20200033249A (en)	2017-06-05	2020-03-27	피티씨 테라퓨틱스, 인크.	Huntington's Disease Treatment Compound
CN111182898B (en)	2017-06-28	2024-04-16	Ptc医疗公司	Methods for treating huntington's disease
CA3067592A1 (en)	2017-06-28	2019-01-03	Ptc Therapeutics, Inc.	Methods for treating huntington's disease
AU2018322319B2 (en)	2017-08-25	2021-08-05	Stoke Therapeutics, Inc.	Antisense oligomers for treatment of conditions and diseases
TWI809004B (en)	2017-11-09	2023-07-21	美商Ionis製藥公司	Compounds and methods for reducing snca expression
US10668247B2 (en)	2017-11-15	2020-06-02	Alcyone Lifesciences, Inc.	Therapy specific, pre-programmed auto injection device
SG11202006526YA (en)	2018-01-12	2020-08-28	Bristol Myers Squibb Co	Antisense oligonucleotides targeting alpha-synuclein and uses thereof
EA202091693A1 (en)	2018-01-12	2021-04-14	Бристол-Маерс Сквибб Компани	ANTI-SENSE OLIGONUCLEOTIDES TARGETINGLY AFFECTING ALPHA-SYNUCLEINE AND THEIR APPLICATIONS
CA3094703A1 (en)	2018-03-27	2019-10-03	Ptc Therapeutics, Inc.	Compounds for treating huntington's disease
CA3099280A1 (en)	2018-05-04	2019-11-07	Stoke Therapeutics, Inc.	Methods and compositions for treatment of cholesteryl ester storage disease
EP3797160A1 (en)	2018-05-23	2021-03-31	The Broad Institute Inc.	Base editors and uses thereof
SG11202012674PA (en)	2018-06-27	2021-01-28	Ptc Therapeutics Inc	Heterocyclic and heteroaryl compounds for treating huntington's disease
KR20210038845A (en)	2018-06-27	2021-04-08	피티씨 테라퓨틱스, 인크.	Heteroaryl compounds for the treatment of Huntington's disease
JP2023500251A (en)	2019-11-01	2023-01-05	ノバルティスアーゲー	Use of splicing modulators for treatment to slow the progression of Huntington's disease
JP2023507741A (en) *	2019-12-20	2023-02-27	アスクレピオスバイオファーマシューティカル，インコーポレイテッド	Method for treating Huntington's disease
EP4146804A1 (en)	2020-05-08	2023-03-15	The Broad Institute Inc.	Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
IL298063A (en)	2020-05-11	2023-01-01	Stoke Therapeutics Inc	opa1 antisense oligomers for the treatment of conditions and diseases
TW202304446A (en)	2021-03-29	2023-02-01	瑞士商諾華公司	The use of a splicing modulator for a treatment slowing progression of huntington’s disease
EP4314296A2 (en)	2021-03-29	2024-02-07	Alnylam Pharmaceuticals, Inc.	Huntingtin (htt) irna agent compositions and methods of use thereof
WO2023034870A2 (en)	2021-09-01	2023-03-09	Ionis Pharmaceuticals, Inc.	Compounds and methods for reducing dmpk expression
EP4423272A2 (en) *	2021-10-29	2024-09-04	Alnylam Pharmaceuticals, Inc.	Huntingtin (htt) irna agent compositions and methods of use thereof
WO2023168304A2 (en) *	2022-03-02	2023-09-07	Triplet Therapeutics, Inc.	Methods for the treatment of nucleotide repeat expansion disorders associated with msh3 activity
WO2024077267A1 (en) *	2022-10-07	2024-04-11	The Broad Institute, Inc.	Prime editing methods and compositions for treating triplet repeat disorders

Citations (14)

* Cited by examiner, † Cited by third party

Publication number	Priority date	Publication date	Assignee	Title
US5595756A (en) *	1993-12-22	1997-01-21	Inex Pharmaceuticals Corporation	Liposomal compositions for enhanced retention of bioactive agents
US5700922A (en) *	1991-12-24	1997-12-23	Isis Pharmaceuticals, Inc.	PNA-DNA-PNA chimeric macromolecules
US5998148A (en) *	1999-04-08	1999-12-07	Isis Pharmaceuticals Inc.	Antisense modulation of microtubule-associated protein 4 expression
US6043060A (en) *	1996-11-18	2000-03-28	Imanishi; Takeshi	Nucleotide analogues
US6147200A (en) *	1999-08-19	2000-11-14	Isis Pharmaceuticals, Inc.	2'-O-acetamido modified monomers and oligomers
US20030109476A1 (en) *	2001-08-07	2003-06-12	Kmiec Eric B.	Compositions and methods for the prevention and treatment of Huntington's disease
US6670461B1 (en) *	1997-09-12	2003-12-30	Exiqon A/S	Oligonucleotide analogues
US20040096880A1 (en) *	2001-08-07	2004-05-20	Kmiec Eric B.	Compositions and methods for the treatment of diseases exhibiting protein misassembly and aggregation
US20050042646A1 (en) *	2002-08-05	2005-02-24	Davidson Beverly L.	RNA interference suppresion of neurodegenerative diseases and methods of use thereof
US20050096284A1 (en) *	2002-02-20	2005-05-05	Sirna Therapeutics, Inc.	RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US20050191638A1 (en) *	2002-02-20	2005-09-01	Sirna Therapeutics, Inc.	RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US20050255086A1 (en) *	2002-08-05	2005-11-17	Davidson Beverly L	Nucleic acid silencing of Huntington's Disease gene
US20050255487A1 (en) *	2002-11-14	2005-11-17	Dharmacon, Inc.	Methods and compositions for selecting siRNA of improved functionality
US20070099860A1 (en) *	2005-10-28	2007-05-03	Sah Dinah W	Compositions and methods for inhibiting expression of huntingtin gene

Family Cites Families (109)

* Cited by examiner, † Cited by third party

Publication number	Priority date	Publication date	Assignee	Title
US667046A (en)	1899-06-23	1901-01-29	John W Weeks	Pressure-reducing valve.
US2526893A (en)	1946-08-28	1950-10-24	Cutler Hammer Inc	Dynamic braking control
US4534899A (en)	1981-07-20	1985-08-13	Lipid Specialties, Inc.	Synthetic phospholipid compounds
US4426330A (en)	1981-07-20	1984-01-17	Lipid Specialties, Inc.	Synthetic phospholipid compounds
US5118800A (en)	1983-12-20	1992-06-02	California Institute Of Technology	Oligonucleotides possessing a primary amino group in the terminal nucleotide
FR2567892B1 (en)	1984-07-19	1989-02-17	Centre Nat Rech Scient	NOVEL OLIGONUCLEOTIDES, THEIR PREPARATION PROCESS AND THEIR APPLICATIONS AS MEDIATORS IN DEVELOPING THE EFFECTS OF INTERFERONS
US5034506A (en)	1985-03-15	1991-07-23	Anti-Gene Development Group	Uncharged morpholino-based polymers having achiral intersubunit linkages
EP0260032B1 (en)	1986-09-08	1994-01-26	Ajinomoto Co., Inc.	Compounds for the cleavage at a specific position of RNA, oligomers employed for the formation of said compounds, and starting materials for the synthesis of said oligomers
DE3855864T2 (en)	1987-11-30	1997-09-25	Univ Iowa Res Found	DNA MOLECULES STABILIZED BY MODIFICATIONS ON THE 3'-TERMINAL PHOSPHODIESTERBINDING, THEIR USE AS NUCLEIC ACID PROBE AND AS A THERAPEUTIC AGENT FOR INHIBITING THE EXPRESSION OF SPECIFIC TARGET GENES
US5403711A (en)	1987-11-30	1995-04-04	University Of Iowa Research Foundation	Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is cleaved
US5354844A (en)	1989-03-16	1994-10-11	Boehringer Ingelheim International Gmbh	Protein-polycation conjugates
US5108921A (en)	1989-04-03	1992-04-28	Purdue Research Foundation	Method for enhanced transmembrane transport of exogenous molecules
US5256775A (en)	1989-06-05	1993-10-26	Gilead Sciences, Inc.	Exonuclease-resistant oligonucleotides
US5227170A (en)	1989-06-22	1993-07-13	Vestar, Inc.	Encapsulation process
US5591722A (en)	1989-09-15	1997-01-07	Southern Research Institute	2'-deoxy-4'-thioribonucleosides and their antiviral activity
US5013556A (en)	1989-10-20	1991-05-07	Liposome Technology, Inc.	Liposomes with enhanced circulation time
US5356633A (en)	1989-10-20	1994-10-18	Liposome Technology, Inc.	Method of treatment of inflamed tissues
US5527528A (en)	1989-10-20	1996-06-18	Sequus Pharmaceuticals, Inc.	Solid-tumor treatment method
JPH05504552A (en)	1989-10-24	1993-07-15	ギリアド　サイエンシズ，インコーポレイテッド	Oligonucleotide modified at the 2' position
US5580575A (en)	1989-12-22	1996-12-03	Imarx Pharmaceutical Corp.	Therapeutic drug delivery systems
US5469854A (en)	1989-12-22	1995-11-28	Imarx Pharmaceutical Corp.	Methods of preparing gas-filled liposomes
US5646265A (en)	1990-01-11	1997-07-08	Isis Pharmceuticals, Inc.	Process for the preparation of 2'-O-alkyl purine phosphoramidites
US5670633A (en)	1990-01-11	1997-09-23	Isis Pharmaceuticals, Inc.	Sugar modified oligonucleotides that detect and modulate gene expression
US5623065A (en)	1990-08-13	1997-04-22	Isis Pharmaceuticals, Inc.	Gapped 2' modified oligonucleotides
US5220007A (en)	1990-02-15	1993-06-15	The Worcester Foundation For Experimental Biology	Method of site-specific alteration of RNA and production of encoded polypeptides
US5149797A (en)	1990-02-15	1992-09-22	The Worcester Foundation For Experimental Biology	Method of site-specific alteration of rna and production of encoded polypeptides
US5264618A (en)	1990-04-19	1993-11-23	Vical, Inc.	Cationic lipids for intracellular delivery of biologically active molecules
GB9009980D0 (en)	1990-05-03	1990-06-27	Amersham Int Plc	Phosphoramidite derivatives,their preparation and the use thereof in the incorporation of reporter groups on synthetic oligonucleotides
ES2116977T3 (en)	1990-05-11	1998-08-01	Microprobe Corp	SOLID SUPPORTS FOR NUCLEIC ACID HYBRIDIZATION TESTS AND METHODS TO IMMOBILIZE OLIGONUCLEOTIDES IN A COVALENT WAY.
US6582908B2 (en)	1990-12-06	2003-06-24	Affymetrix, Inc.	Oligonucleotides
JP3220180B2 (en)	1991-05-23	2001-10-22	三菱化学株式会社	Drug-containing protein-bound liposomes
US5521291A (en)	1991-09-30	1996-05-28	Boehringer Ingelheim International, Gmbh	Conjugates for introducing nucleic acid into higher eucaryotic cells
NZ244306A (en)	1991-09-30	1995-07-26	Boehringer Ingelheim Int	Composition for introducing nucleic acid complexes into eucaryotic cells, complex containing nucleic acid and endosomolytic agent, peptide with endosomolytic domain and nucleic acid binding domain and preparation
EP0538194B1 (en)	1991-10-17	1997-06-04	Novartis AG	Bicyclic nucleosides, oligonucleotides, their method of preparation and intermediates therein
NZ244820A (en) *	1991-10-25	1994-01-26	Isis Pharmaceuticals Inc	Oligonucleotide inhibitor of epstein-barr virus.
US5359044A (en)	1991-12-13	1994-10-25	Isis Pharmaceuticals	Cyclobutyl oligonucleotide surrogates
FR2687679B1 (en)	1992-02-05	1994-10-28	Centre Nat Rech Scient	OLIGOTHIONUCLEOTIDES.
FR2692265B1 (en)	1992-05-25	1996-11-08	Centre Nat Rech Scient	BIOLOGICALLY ACTIVE COMPOUNDS OF THE PHOSPHOTRIESTER TYPE.
EP0577558A2 (en)	1992-07-01	1994-01-05	Ciba-Geigy Ag	Carbocyclic nucleosides having bicyclic rings, oligonucleotides therefrom, process for their preparation, their use and intermediates
US5652355A (en)	1992-07-23	1997-07-29	Worcester Foundation For Experimental Biology	Hybrid oligonucleotide phosphorothioates
US6180403B1 (en) *	1999-10-28	2001-01-30	Isis Pharmaceuticals Inc.	Antisense inhibition of tumor necrosis factor alpha converting enzyme (TACE) expression
US5583020A (en)	1992-11-24	1996-12-10	Ribozyme Pharmaceuticals, Inc.	Permeability enhancers for negatively charged polynucleotides
JP3351476B2 (en)	1993-01-22	2002-11-25	三菱化学株式会社	Phospholipid derivatives and liposomes containing the same
US5395619A (en)	1993-03-03	1995-03-07	Liposome Technology, Inc.	Lipid-polymer conjugates and liposomes
EP0691968B1 (en)	1993-03-30	1997-07-16	Sanofi	Acyclic nucleoside analogs and oligonucleotide sequences containing them
DE4311944A1 (en)	1993-04-10	1994-10-13	Degussa	Coated sodium percarbonate particles, process for their preparation and detergent, cleaning and bleaching compositions containing them
US5462854A (en)	1993-04-19	1995-10-31	Beckman Instruments, Inc.	Inverse linkage oligonucleotides for chemical and enzymatic processes
FR2705099B1 (en)	1993-05-12	1995-08-04	Centre Nat Rech Scient	Phosphorothioate triester oligonucleotides and process for their preparation.
US5534259A (en)	1993-07-08	1996-07-09	Liposome Technology, Inc.	Polymer compound and coated particle composition
US5543158A (en)	1993-07-23	1996-08-06	Massachusetts Institute Of Technology	Biodegradable injectable nanoparticles
US5417978A (en)	1993-07-29	1995-05-23	Board Of Regents, The University Of Texas System	Liposomal antisense methyl phosphonate oligonucleotides and methods for their preparation and use
US5801154A (en)	1993-10-18	1998-09-01	Isis Pharmaceuticals, Inc.	Antisense oligonucleotide modulation of multidrug resistance-associated protein
US5446137B1 (en)	1993-12-09	1998-10-06	Behringwerke Ag	Oligonucleotides containing 4'-substituted nucleotides
KR100386337B1 (en)	1993-12-09	2004-03-24	토마스 제퍼슨 대학교	Compounds and Method for Site-Specific Mutations in Eukaryotic Cells
US5519134A (en)	1994-01-11	1996-05-21	Isis Pharmaceuticals, Inc.	Pyrrolidine-containing monomers and oligomers
US5627053A (en)	1994-03-29	1997-05-06	Ribozyme Pharmaceuticals, Inc.	2'deoxy-2'-alkylnucleotide containing nucleic acid
US5543152A (en)	1994-06-20	1996-08-06	Inex Pharmaceuticals Corporation	Sphingosomes for enhanced drug delivery
US5597909A (en)	1994-08-25	1997-01-28	Chiron Corporation	Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use
US5591721A (en)	1994-10-25	1997-01-07	Hybridon, Inc.	Method of down-regulating gene expression
US5512295A (en)	1994-11-10	1996-04-30	The Board Of Trustees Of The Leland Stanford Junior University	Synthetic liposomes for enhanced uptake and delivery
US5792747A (en)	1995-01-24	1998-08-11	The Administrators Of The Tulane Educational Fund	Highly potent agonists of growth hormone releasing hormone
US5652356A (en)	1995-08-17	1997-07-29	Hybridon, Inc.	Inverted chimeric and hybrid oligonucleotides
US20040266706A1 (en) *	2002-11-05	2004-12-30	Muthiah Manoharan	Cross-linked oligomeric compounds and their use in gene modulation
US6368855B1 (en) *	1996-06-11	2002-04-09	Antigen Express, Inc.	MHC class II antigen presenting cells containing oligonucleotides which inhibit Ii protein expression
JP3756313B2 (en)	1997-03-07	2006-03-15	武今西	Novel bicyclonucleosides and oligonucleotide analogues
US20030228597A1 (en)	1998-04-13	2003-12-11	Cowsert Lex M.	Identification of genetic targets for modulation by oligonucleotides and generation of oligonucleotides for gene modulation
US20030096775A1 (en) *	2001-10-23	2003-05-22	Isis Pharmaceuticals Inc.	Antisense modulation of complement component C3 expression
US6287860B1 (en)	2000-01-20	2001-09-11	Isis Pharmaceuticals, Inc.	Antisense inhibition of MEKK2 expression
US20020187931A1 (en) *	2000-04-13	2002-12-12	Michael Hayden	Modulating cell survival by modulating huntingtin function
AU2001296412A1 (en) *	2000-09-29	2002-04-08	Isis Pharmaceuticals, Inc.	Antisense modulation of mekk4 expression
US7176303B2 (en)	2003-11-06	2007-02-13	Isis Pharmaceuticals, Inc.	Modulation of STAT5 expression
EP3415625A1 (en)	2002-02-01	2018-12-19	Life Technologies Corporation	Double-stranded oligonucleotides
US20030232443A1 (en) *	2002-06-18	2003-12-18	Isis Pharmaceuticals Inc.	Antisense modulation of centromere protein B expression
US20040092465A1 (en) *	2002-11-11	2004-05-13	Isis Pharmaceuticals Inc.	Modulation of huntingtin interacting protein 1 expression
US20040102398A1 (en) *	2002-11-23	2004-05-27	Isis Pharmaceuticals Inc.	Modulation of B7H expression
US20050106731A1 (en)	2002-08-05	2005-05-19	Davidson Beverly L.	siRNA-mediated gene silencing with viral vectors
US20080274989A1 (en) *	2002-08-05	2008-11-06	University Of Iowa Research Foundation	Rna Interference Suppression of Neurodegenerative Diseases and Methods of Use Thereof
WO2004027030A2 (en)	2002-09-18	2004-04-01	Isis Pharmaceuticals, Inc.	Efficient reduction of target rna’s by single- and double-stranded oligomeric compounds
US7250496B2 (en) *	2002-11-14	2007-07-31	Rosetta Genomics Ltd.	Bioinformatically detectable group of novel regulatory genes and uses thereof
US7250289B2 (en) *	2002-11-20	2007-07-31	Affymetrix, Inc.	Methods of genetic analysis of mouse
US7790867B2 (en) *	2002-12-05	2010-09-07	Rosetta Genomics Inc.	Vaccinia virus-related nucleic acids and microRNA
WO2004076639A2 (en) *	2003-02-26	2004-09-10	Wyeth	Use of gene expression profiling in the diagnosis and treatment of lupus nephritis and systemic lupus erythematosus
EP1605978B1 (en)	2003-03-07	2010-09-01	Alnylam Pharmaceuticals Inc.	Therapeutic compositions
JP4179562B2 (en) *	2003-05-14	2008-11-12	独立行政法人科学技術振興機構	Suppression of huntingtin gene expression
MXPA06000514A (en) *	2003-07-14	2006-06-20	Decode Genetics Ehf	Method of diagnosis and treatment for asthma based on haplotype association.
CA2533701A1 (en) *	2003-07-31	2005-02-17	Isis Pharmaceuticals, Inc.	Oligomeric compounds and compositions for use in modulation of small non-coding rnas
EP2821085B1 (en) *	2003-09-12	2020-04-29	University of Massachusetts	Rna interference for the treatment of gain-of-function disorders
WO2005045032A2 (en)	2003-10-20	2005-05-19	Sima Therapeutics, Inc.	RNA INTERFERENCE MEDIATED INHIBITION OF EARLY GROWTH RESPONSE GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US7341835B2 (en) *	2004-01-13	2008-03-11	Affymetrix, Inc.	Methods of analysis of alternative splicing in mouse
WO2005083436A1 (en) *	2004-02-26	2005-09-09	Bayer Healthcare Ag	Diagnostics and therapeutics for diseases associated with integrin-linked kinase 1 (ilk1)
WO2005105995A2 (en)	2004-04-14	2005-11-10	Sirna Therapeutics, Inc.	RNA INTERFERENCE MEDIATED TREATMENT OF POLYGLUTAMINE (POLYQ) REPEAT EXPANSION DISEASES USING SHORT INTERFERING NUCLEIC ACID (siNA)
US7374927B2 (en) *	2004-05-03	2008-05-20	Affymetrix, Inc.	Methods of analysis of degraded nucleic acid samples
AU2005248147A1 (en) *	2004-05-11	2005-12-08	Alphagen Co., Ltd.	Polynucleotides for causing RNA interference and method for inhibiting gene expression using the same
US7323308B2 (en) *	2004-09-03	2008-01-29	Affymetrix, Inc.	Methods of genetic analysis of E. coli
WO2006128141A2 (en)	2005-05-27	2006-11-30	Sirna Therapeutics, Inc.	Rna interference mediated inhibition of stromal cell-derived factor-1 (sdf-1) gene expression using short interfering nucleic acid (sina)
DE202005008426U1 (en)	2005-05-31	2005-09-15	Ricon Sieb Und Foerdertechnik	Conveyor unit has conveyor belts provided with recesses on inner facing sides for fitting of side edges of conveying apron, whereby conveying apron is connected via edges to conveyor belts in region of recesses
CA2619534A1 (en)	2005-08-18	2007-02-22	Alnylam Pharmaceuticals, Inc.	Methods and compositions for treating neurological disease
US8658608B2 (en) *	2005-11-23	2014-02-25	Yale University	Modified triple-helix forming oligonucleotides for targeted mutagenesis
JP5425474B2 (en)	2006-01-26	2014-02-26	アイシスファーマシューティカルズ，インコーポレーテッド	Composition and use thereof for huntingtin
US7901882B2 (en) *	2006-03-31	2011-03-08	Affymetrix, Inc.	Analysis of methylation using nucleic acid arrays
US8076306B2 (en)	2006-04-12	2011-12-13	Isis Pharmaceuticals, Inc.	Compositions and their uses directed to hepcidin
EP2046993A4 (en)	2006-07-07	2010-11-17	Univ Massachusetts	RNA INTERFERENCE COMPOSITIONS AND METHOD FOR TREATING MORBUS HUNTINGTON
US20100184833A1 (en)	2006-08-11	2010-07-22	Prosenta Technologies B.V.	Methods and means for treating dna repeat instability associated genetic disorders
US20080039415A1 (en)	2006-08-11	2008-02-14	Gregory Robert Stewart	Retrograde transport of sirna and therapeutic uses to treat neurologic disorders
WO2009023855A2 (en) *	2007-08-15	2009-02-19	Isis Pharmaceuticals, Inc.	Tetrahydropyran nucleic acid analogs
WO2010111522A2 (en)	2009-03-26	2010-09-30	The Regents Of The University Of California	Mesenchymal stem cells producing inhibitory rna for disease modification
US8957038B2 (en)	2009-07-15	2015-02-17	Medtronic, Inc.	Treatment of neurological disorders
WO2011097388A1 (en)	2010-02-03	2011-08-11	Alnylam Pharmaceuticals, Inc.	Selective inhibition of polyglutamine protein expression
US10867398B2 (en)	2017-11-21	2020-12-15	Reliance Core Consulting LLC	Methods, systems, apparatuses and devices for facilitating motion analysis in an environment

2007
- 2007-01-26 JP JP2008552447A patent/JP5425474B2/en active Active
- 2007-01-26 SI SI200731421T patent/SI2161038T1/en unknown
- 2007-01-26 PL PL17151157T patent/PL3210633T3/en unknown
- 2007-01-26 EP EP17151157.9A patent/EP3210633B1/en active Active
- 2007-01-26 EP EP07717061A patent/EP1991677A2/en not_active Withdrawn
- 2007-01-26 EP EP09075505.9A patent/EP2161038B1/en active Active
- 2007-01-26 DK DK09075505.9T patent/DK2161038T3/en active
- 2007-01-26 EP EP10193685.4A patent/EP2428227B1/en active Active
- 2007-01-26 PT PT90755059T patent/PT2161038E/en unknown
- 2007-01-26 ES ES17151157T patent/ES2744098T3/en active Active
- 2007-01-26 ES ES09075505.9T patent/ES2453380T3/en active Active
- 2007-01-26 AU AU2007210038A patent/AU2007210038B2/en active Active
- 2007-01-26 US US11/627,921 patent/US20070299027A1/en not_active Abandoned
- 2007-01-26 PL PL09075505T patent/PL2161038T3/en unknown
- 2007-01-26 EP EP10193707.6A patent/EP2422819B1/en active Active
- 2007-01-26 WO PCT/US2007/002171 patent/WO2007089584A2/en active Application Filing
- 2007-01-26 CA CA2638908A patent/CA2638908C/en active Active
- 2007-01-26 US US11/627,916 patent/US7951934B2/en active Active
- 2007-01-26 WO PCT/US2007/002215 patent/WO2007089611A2/en active Application Filing
2009
- 2009-11-13 US US12/618,470 patent/US20100069472A1/en not_active Abandoned
2010
- 2010-08-18 HK HK10107888.8A patent/HK1141449A1/en unknown
2011
- 2011-04-19 US US13/090,146 patent/US8415465B2/en active Active
2012
- 2012-12-07 US US13/708,839 patent/US9057066B2/en active Active
2013
- 2013-03-07 US US13/789,368 patent/US8952145B2/en active Active
- 2013-04-10 AU AU2013203297A patent/AU2013203297B2/en active Active
- 2013-05-31 JP JP2013115737A patent/JP5916660B2/en active Active
2015
- 2015-01-05 US US14/589,833 patent/US9353372B2/en active Active
- 2015-05-11 US US14/708,511 patent/US20160053256A1/en not_active Abandoned
- 2015-12-25 JP JP2015252875A patent/JP6395058B2/en active Active
2016
- 2016-04-29 US US15/142,827 patent/US9920321B2/en active Active
2017
- 2017-07-12 AU AU2017204772A patent/AU2017204772B2/en active Active
- 2017-10-05 US US15/725,933 patent/US10738307B2/en active Active
2018
- 2018-02-07 US US15/891,154 patent/US10655129B2/en active Active
- 2018-03-28 JP JP2018061748A patent/JP2018117635A/en active Pending
2019
- 2019-09-30 AU AU2019237389A patent/AU2019237389A1/en not_active Abandoned
2020
- 2020-02-14 JP JP2020023793A patent/JP2020096624A/en active Pending
- 2020-04-15 US US16/848,968 patent/US11072794B2/en active Active
- 2020-08-10 US US16/988,862 patent/US20210017522A1/en active Pending
2021
- 2021-06-21 US US17/352,806 patent/US11761000B2/en active Active

Patent Citations (14)

* Cited by examiner, † Cited by third party

Publication number	Priority date	Publication date	Assignee	Title
US5700922A (en) *	1991-12-24	1997-12-23	Isis Pharmaceuticals, Inc.	PNA-DNA-PNA chimeric macromolecules
US5595756A (en) *	1993-12-22	1997-01-21	Inex Pharmaceuticals Corporation	Liposomal compositions for enhanced retention of bioactive agents
US6043060A (en) *	1996-11-18	2000-03-28	Imanishi; Takeshi	Nucleotide analogues
US6670461B1 (en) *	1997-09-12	2003-12-30	Exiqon A/S	Oligonucleotide analogues
US5998148A (en) *	1999-04-08	1999-12-07	Isis Pharmaceuticals Inc.	Antisense modulation of microtubule-associated protein 4 expression
US6147200A (en) *	1999-08-19	2000-11-14	Isis Pharmaceuticals, Inc.	2'-O-acetamido modified monomers and oligomers
US20030109476A1 (en) *	2001-08-07	2003-06-12	Kmiec Eric B.	Compositions and methods for the prevention and treatment of Huntington's disease
US20040096880A1 (en) *	2001-08-07	2004-05-20	Kmiec Eric B.	Compositions and methods for the treatment of diseases exhibiting protein misassembly and aggregation
US20050096284A1 (en) *	2002-02-20	2005-05-05	Sirna Therapeutics, Inc.	RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US20050191638A1 (en) *	2002-02-20	2005-09-01	Sirna Therapeutics, Inc.	RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US20050042646A1 (en) *	2002-08-05	2005-02-24	Davidson Beverly L.	RNA interference suppresion of neurodegenerative diseases and methods of use thereof
US20050255086A1 (en) *	2002-08-05	2005-11-17	Davidson Beverly L	Nucleic acid silencing of Huntington's Disease gene
US20050255487A1 (en) *	2002-11-14	2005-11-17	Dharmacon, Inc.	Methods and compositions for selecting siRNA of improved functionality
US20070099860A1 (en) *	2005-10-28	2007-05-03	Sah Dinah W	Compositions and methods for inhibiting expression of huntingtin gene

Cited By (55)

* Cited by examiner, † Cited by third party

Publication number	Priority date	Publication date	Assignee	Title
US11274299B2 (en)	2006-08-11	2022-03-15	Vico Therapeutics B.V.	Methods and means for treating DNA repeat instability associated genetic disorders
US10689646B2 (en)	2006-08-11	2020-06-23	Biomarin Technologies B.V.	Treatment of genetic disorders associated with DNA repeat instability
US9890379B2 (en)	2006-08-11	2018-02-13	Biomarin Technologies B.V.	Treatment of genetic disorders associated with DNA repeat instability
US11427820B2 (en)	2007-10-26	2022-08-30	Biomarin Technologies B.V.	Methods and means for efficient skipping of exon 45 in Duchenne muscular dystrophy pre-mRNA
USRE48468E1 (en)	2007-10-26	2021-03-16	Biomarin Technologies B.V.	Means and methods for counteracting muscle disorders
US10876114B2 (en)	2007-10-26	2020-12-29	Biomarin Technologies B.V.	Methods and means for efficient skipping of at least one of the following exons of the human Duchenne muscular dystrophy gene: 43, 46, 50-53
US20110190222A1 (en) *	2008-07-29	2011-08-04	Corey David R	Selective Inhibition of Polyglutamine Protein Expression
US8901095B2 (en)	2008-07-29	2014-12-02	The Board Of Regents Of The University Of Texas System	Selective inhibition of polyglutamine protein expression
US9340785B2 (en)	2008-07-29	2016-05-17	The Board Of Regents Of The University Of Texas System	Selective inhibition of polyglutamine protein expression
US9695211B2 (en)	2008-12-02	2017-07-04	Wave Life Sciences Japan, Inc.	Method for the synthesis of phosphorus atom modified nucleic acids
US10329318B2 (en)	2008-12-02	2019-06-25	Wave Life Sciences Ltd.	Method for the synthesis of phosphorus atom modified nucleic acids
US10307434B2 (en)	2009-07-06	2019-06-04	Wave Life Sciences Ltd.	Nucleic acid prodrugs and methods of use thereof
US10202603B2 (en)	2009-09-11	2019-02-12	Ionis Pharmaceuticals, Inc.	Modulation of huntingtin expression
KR102279458B1 (en) *	2009-09-11	2021-07-21	아이오니스 파마수티컬즈, 인코포레이티드	Modulation of huntingtin expression
EP3178933A1 (en)	2009-09-11	2017-06-14	Ionis Pharmaceuticals, Inc.	Modulation of huntingtin expression
KR102175230B1 (en) *	2009-09-11	2020-11-06	아이오니스 파마수티컬즈, 인코포레이티드	Modulation of huntingtin expression
KR101774526B1 (en) *	2009-09-11	2017-09-04	아이오니스 파마수티컬즈, 인코포레이티드	Modulation of huntingtin expression
KR20200124782A (en) *	2009-09-11	2020-11-03	아이오니스 파마수티컬즈, 인코포레이티드	Modulation of huntingtin expression
US10619158B2 (en)	2009-09-11	2020-04-14	Ionis Pharmaceuticals, Inc.	Modulation of huntingtin expression
EP3626823A1 (en)	2009-09-11	2020-03-25	Ionis Pharmaceuticals, Inc.	Modulation of huntingtin expression
US11421231B2 (en)	2009-09-11	2022-08-23	Ionis Pharmaceuticals, Inc.	Modulation of Huntington expression
US10837016B2 (en)	2009-09-11	2020-11-17	Ionis Pharmaceuticals, Inc.	Modulation of huntingtin expression
WO2011032045A1 (en)	2009-09-11	2011-03-17	Isis Pharmaceuticals, Inc.	Modulation of huntingtin expression
US9683236B2 (en)	2009-09-11	2017-06-20	Ionis Pharmaceuticals, Inc.	Modulation of huntingtin expression
KR20190000924A (en) *	2009-09-11	2019-01-03	아이오니스 파마수티컬즈, 인코포레이티드	Modulation of huntingtin expression
US9061044B2 (en)	2010-04-29	2015-06-23	Isis Pharmaceuticals, Inc.	Modulation of transthyretin expression
US11535849B2 (en)	2010-04-29	2022-12-27	Ionis Pharmaceuticals, Inc.	Modulation of transthyretin expression
US9399774B2 (en)	2010-04-29	2016-07-26	Ionis Pharmaceuticals, Inc.	Modulation of transthyretin expression
WO2011139917A1 (en)	2010-04-29	2011-11-10	Isis Pharmaceuticals, Inc.	Modulation of transthyretin expression
US8697860B1 (en)	2010-04-29	2014-04-15	Isis Pharmaceuticals, Inc.	Diagnosis and treatment of disease
US9611471B2 (en)	2010-08-05	2017-04-04	Academisch Ziekenhuis Leiden	Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins
US10364432B2 (en)	2010-08-05	2019-07-30	Academisch Ziekenhuis Leiden H.O.D.N. Lumc	Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins
US10590421B2 (en)	2010-08-05	2020-03-17	Academisch Ziekenhuis Leiden H.O.D.N. Lumc	Antisense oligonucleotide directed removal of proteolytic cleavage sites, the HCHWA-D mutation, and trinucleotide repeat expansions
US10428019B2 (en)	2010-09-24	2019-10-01	Wave Life Sciences Ltd.	Chiral auxiliaries
EP3505528A1 (en)	2011-04-21	2019-07-03	Ionis Pharmaceuticals, Inc.	Modulation of hepatitis b virus (hbv) expression
EP3816288A2 (en)	2011-04-21	2021-05-05	Glaxo Group Limited	Modulation of hepatitis b virus (hbv) expression
EP3312189A1 (en)	2011-04-21	2018-04-25	Ionis Pharmaceuticals, Inc.	Modulation of hepatitis b virus (hbv) expression
US9605019B2 (en)	2011-07-19	2017-03-28	Wave Life Sciences Ltd.	Methods for the synthesis of functionalized nucleic acids
US10280192B2 (en)	2011-07-19	2019-05-07	Wave Life Sciences Ltd.	Methods for the synthesis of functionalized nucleic acids
US10913946B2 (en)	2012-01-27	2021-02-09	Biomarin Technologies B.V.	RNA modulating oligonucleotides with improved characteristics for the treatment of Duchenne and Becker muscular dystrophy
US11345915B2 (en)	2012-04-23	2022-05-31	Vico Therapeutics B.V.	RNA modulating oligonucleotides with improved characteristics for the treatment of neuromuscular disorders
US9745576B2 (en)	2012-04-23	2017-08-29	Biomarin Technologies B.V.	RNA modulating oligonucleotides with improved characteristics for the treatment of neuromuscular disorders
US10167309B2 (en)	2012-07-13	2019-01-01	Wave Life Sciences Ltd.	Asymmetric auxiliary group
US10590413B2 (en)	2012-07-13	2020-03-17	Wave Life Sciences Ltd.	Chiral control
US9617547B2 (en)	2012-07-13	2017-04-11	Shin Nippon Biomedical Laboratories, Ltd.	Chiral nucleic acid adjuvant
US9982257B2 (en)	2012-07-13	2018-05-29	Wave Life Sciences Ltd.	Chiral control
US10322173B2 (en)	2014-01-15	2019-06-18	Shin Nippon Biomedical Laboratories, Ltd.	Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent
US10149905B2 (en)	2014-01-15	2018-12-11	Shin Nippon Biomedical Laboratories, Ltd.	Chiral nucleic acid adjuvant having antitumor effect and antitumor agent
US10144933B2 (en)	2014-01-15	2018-12-04	Shin Nippon Biomedical Laboratories, Ltd.	Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator
US10160969B2 (en)	2014-01-16	2018-12-25	Wave Life Sciences Ltd.	Chiral design
US10479995B2 (en)	2015-07-22	2019-11-19	Wave Life Sciences Ltd.	Oligonucleotide compositions and methods thereof
US11473084B2 (en) *	2015-10-09	2022-10-18	The Children's Hospital Of Philadelphia	Compositions and methods for treating Huntington's disease and related disorders
WO2017062983A1 (en) *	2015-10-09	2017-04-13	The Children's Hospital Of Philadelphia	Compositions and methods for treating huntington's disease and related disorders
US10724035B2 (en)	2016-05-04	2020-07-28	Wave Life Sciences Ltd.	Oligonucleotide compositions and methods thereof
US20220033814A1 (en) *	2018-12-03	2022-02-03	Triplet Therapeutics, Inc.	Methods for the treatment of trinucleotide repeat expansion disorders associated with mlh1 activity

Also Published As

Publication number	Publication date
EP3210633A2 (en)	2017-08-30
DK2161038T3 (en)	2014-03-03
WO2007089584A3 (en)	2007-11-29
AU2017204772B2 (en)	2019-07-11
ES2453380T3 (en)	2014-04-07
US20160053256A1 (en)	2016-02-25
AU2017204772A1 (en)	2017-07-27
EP3210633B1 (en)	2019-06-19
PL2161038T3 (en)	2014-06-30
PT2161038E (en)	2014-03-10
JP5425474B2 (en)	2014-02-26
JP2020096624A (en)	2020-06-25
HK1141449A1 (en)	2010-11-12
AU2013203297A1 (en)	2013-05-09
US20080039418A1 (en)	2008-02-14
US20110306652A1 (en)	2011-12-15
US9057066B2 (en)	2015-06-16
US8415465B2 (en)	2013-04-09
US20130281684A1 (en)	2013-10-24
US11761000B2 (en)	2023-09-19
US7951934B2 (en)	2011-05-31
US10655129B2 (en)	2020-05-19
US20180340178A1 (en)	2018-11-29
EP1991677A2 (en)	2008-11-19
US20210017522A1 (en)	2021-01-21
JP2016116520A (en)	2016-06-30
JP6395058B2 (en)	2018-09-26
PL3210633T3 (en)	2019-12-31
CA2638908A1 (en)	2007-08-09
AU2007210038A1 (en)	2007-08-09
JP5916660B2 (en)	2016-05-11
WO2007089611A2 (en)	2007-08-09
JP2009524431A (en)	2009-07-02
SI2161038T1 (en)	2014-05-30
JP2013226147A (en)	2013-11-07
US20160369273A1 (en)	2016-12-22
US20200318116A1 (en)	2020-10-08
US20100069472A1 (en)	2010-03-18
EP3210633A3 (en)	2017-11-01
ES2744098T3 (en)	2020-02-21
EP2422819A2 (en)	2012-02-29
EP2428227B1 (en)	2016-06-22
US8952145B2 (en)	2015-02-10
AU2007210038B2 (en)	2013-01-10
AU2013203297B2 (en)	2017-04-13
EP2428227A1 (en)	2012-03-14
US20150307877A1 (en)	2015-10-29
EP2422819B1 (en)	2017-03-01
US9353372B2 (en)	2016-05-31
EP2161038A1 (en)	2010-03-10
EP2161038B1 (en)	2013-12-25
JP2018117635A (en)	2018-08-02
US10738307B2 (en)	2020-08-11
CA2638908C (en)	2021-04-27
US20220042013A1 (en)	2022-02-10
US20180273945A1 (en)	2018-09-27
US20130189782A1 (en)	2013-07-25
WO2007089584A2 (en)	2007-08-09
US9920321B2 (en)	2018-03-20
US11072794B2 (en)	2021-07-27
WO2007089611A3 (en)	2008-01-10
EP2422819A3 (en)	2012-06-27
AU2019237389A1 (en)	2019-10-17

Legal Events

Date	Code	Title	Description
2007-11-27	AS	Assignment	Owner name: ISIS PHARMACEUTICALS, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:HUNG, GENE;REEL/FRAME:020158/0962 Effective date: 20070905
2009-02-18	AS	Assignment	Owner name: ISIS PHARMACEUTICALS, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BENNETT, C. FRANK;FREIER, SUSAN M.;REEL/FRAME:022279/0437;SIGNING DATES FROM 20090217 TO 20090218 Owner name: ISIS PHARMACEUTICALS, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BENNETT, C. FRANK;FREIER, SUSAN M.;SIGNING DATES FROM 20090217 TO 20090218;REEL/FRAME:022279/0437
2009-02-25	AS	Assignment	Owner name: CHDI FOUNDATION INC., NEW YORK Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:LEEDS, JANET M.;REEL/FRAME:022308/0314 Effective date: 20090212
2009-12-22	STCB	Information on status: application discontinuation	Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

0	100.0	100.0	100.0	100.0
0.675	85.9	36.5	156.4	59.3
1.25	50.2	34.5	103.4	31.4
3	31.4	11.0	38.9	18.8
5	12.2	1.6	16.5	6.4
10	5.5	2.7	6.8	3.1

	387902	387916	387902	387916
[Oligo]μM	% mHtt	% mHtt	% mHtt	% mHtt

US20070299027A1 - Compositions and their uses directed to huntingtin - Google Patents