CN115702940A - Use of formulations comprising cyclic RNA molecules for the preparation of a medicament for the treatment of tumors - Google Patents

Disclosure of Invention

Problems to be solved by the invention

In view of the problems in the prior art, there is a need to develop more drugs suitable for intratumoral immunotherapy, for example. To this end, the present disclosure provides the use of a formulation comprising a circular RNA molecule in the manufacture of a medicament for the prevention or treatment of a tumour. The invention discovers for the first time that the circular RNA molecule is suitable for being used as a tumor immunotherapy medicament, can effectively slow down the growth rate of tumors, realizes the in-vivo immunotherapy effect of inhibiting and killing the tumors, and has wide application prospect in the clinical therapy of the tumors.

Means for solving the problems

In a first aspect, the present disclosure provides the use of a formulation comprising a cyclic RNA molecule encoding a polypeptide having tumor immunotherapeutic activity in the manufacture of a medicament for the prevention or treatment of a tumor.

In some embodiments, the use according to the present disclosure, wherein the circular RNA molecule is an unencapsulated circular RNA molecule.

In some embodiments, the use according to the present disclosure, wherein the formulation comprising a circular RNA molecule is an injectable formulation; preferably, the injection further comprises a solvent for dissolving the circular RNA molecule; more preferably, the injection is composed of a circular RNA molecule and a solvent dissolving the circular RNA molecule.

In some embodiments, the use according to the present disclosure, wherein the circular RNA molecule comprises a coding region encoding the polypeptide having tumor immunotherapeutic activity, and an IRES element operably linked to the coding region;

preferably, the IRES element comprises any one of the following groups (i) - (iv):

(i) Comprises the amino acid sequence shown as SEQ ID NO:11-14, or a combination thereof;

(ii) Comprises the amino acid sequence shown as SEQ ID NO:11-14, or a reverse complement thereof;

(iii) (iii) a reverse complement of a sequence that is capable of hybridizing to the nucleotide sequence set forth in (i) or (ii) under high stringency hybridization conditions or very high stringency hybridization conditions;

(iv) (iii) a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the nucleotide sequence set forth in (i) or (ii);

preferably, the IRES element is selected from the following (a) ₁ )-(a ₇ ) Any one of (1):

(a ₁ ) Comprises the amino acid sequence shown as SEQ ID NO: 11;

(a ₂ ) Comprises a nucleotide sequence as set forth in SEQ ID NO: 12;

(a ₃ ) Comprises a nucleotide sequence as set forth in SEQ ID NO: 13;

(a ₄ ) Comprises a nucleotide sequence as set forth in SEQ ID NO: 14;

(a ₅ ) Comprises the amino acid sequence shown as SEQ ID NO:15, and the nucleotide sequence of the sequence shown in the sequence；

(a ₆ ) Comprises a nucleotide sequence as set forth in SEQ ID NO: 16;

(a ₇ ) Comprises the amino acid sequence shown as SEQ ID NO:17, or a nucleotide sequence of the sequence shown in 17.

In some embodiments, the use according to the present disclosure, wherein the circular RNA molecule further comprises a5 'spacer upstream of the IRES element, and a 3' spacer downstream of the coding region;

preferably, the 5' spacer comprises the following (b) ₁ )-(b ₂ ) Any one of the sequences set forth in:

(b ₁ ) As shown in SEQ ID NO:20-21, or a pharmaceutically acceptable salt thereof;

(b ₂ ) And (b) ₁ ) A sequence wherein the nucleotide sequence shown has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity;

preferably, the 3' spacer comprises (c) ₁ )-(c ₂ ) Any one of the sequences set forth in:

(c ₁ ) As shown in SEQ ID NO: 22-23;

(c ₂ ) And (c) ₁ ) A sequence wherein the nucleotide sequence is at least 90%, alternatively at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity.

In some embodiments, the use according to the present disclosure, wherein the circular RNA molecule further comprises a second exon upstream of the 5 'spacer, and a first exon downstream of the 3' spacer;

preferably, the second exon comprises the following (d) ₁ )-(d ₂ ) Any one of the sequences set forth in:

(d ₁ ) As shown in SEQ ID NO: 19;

(d ₂ ) And (d) ₁ ) The nucleotide sequence shown has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 9%Sequences with 8%, most preferably at least 99% sequence identity;

preferably, said first exon comprises the following (e) ₁ )-(e ₂ ) Any one of the sequences set forth in:

(e ₁ ) As shown in SEQ ID NO: 18;

(e ₂ ) And (e) ₁ ) A sequence whose nucleotide sequence as set forth has at least 90%, alternatively at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity.

In some embodiments, the use according to the present disclosure, wherein the formulation comprising a circular RNA molecule is administered to a subject by intratumoral administration; preferably, the formulation comprising the circular RNA molecule is administered to the subject by intratumoral administration.

In some embodiments, the use according to the present disclosure, wherein the circular RNA molecule comprises a coding region encoding the polypeptide having tumor immunotherapeutic activity, wherein the polypeptide having tumor immunotherapeutic activity is selected from one or more of: cytokines, antigen binding fragments, immune checkpoint inhibitors;

optionally, the cytokine is selected from one or more of IL, IFN, TNF, GM-CSF, M-CSF;

optionally, the antigen-binding fragment specifically binds to one or more of the following tumor antigens: CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, AFP, CEA, PSCA, GD2, NKG2D, BCMA, EGFR, her2, EGFRv iii, CD171, FAP, IL13 ra 2, VEGFR1, VEGFR2, GPC-3, mesothelin, claudin 18.2, epCAM, MUC1, MUC16, EPHA2, EPHA3, CD133, PSMA;

optionally, the immune checkpoint inhibitor is one or more of the following immune checkpoint protein inhibitors: PD-1, PD-L1, PDL2, CTLA4, LAG3, TIM3, TIGIT and CD103.

In some embodiments, the use according to the present disclosure, wherein the coding region encodes a cytokine selected from one or more of: IL-15, IL-12, IFN alpha-2b, GM-CSF;

preferably, said IL-15 comprises the following (j) ₁ )-(j ₂ ) Any one of the sequences set forth in:

(j ₁ ) As shown in SEQ ID NO:1 (c) or a pharmaceutically acceptable salt thereof,

(j ₂ ) As shown in SEQ ID NO:1, and has the cytokine activity of IL-15 through one or more amino acid substitution, repetition, deletion or addition;

preferably, the IL-12 comprises the following (k) ₁ )-(k ₂ ) Any one of the sequences set forth in seq id no:

(k ₁ ) As shown in SEQ ID NO:2, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is shown in 2,

(k ₂ ) As shown in SEQ ID NO:2 through substitution, repetition, deletion or addition of one or more amino acids, and has the cytokine activity of IL-12;

preferably, the IFN alpha-2b comprises the following (l) ₁ )-(l ₂ ) Any one of the sequences set forth in:

(l ₁ ) As shown in SEQ ID NO:3, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is shown in the specification,

(l ₂ ) As shown in SEQ ID NO:3 by substituting, repeating, deleting or adding one or more amino acids, and has the cytokine activity of IFN alpha-2 b;

preferably, the GM-CSF comprises the following (m) ₁ )-(m ₂ ) Any one of the sequences set forth in:

(m ₁ ) As shown in SEQ ID NO:4, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is shown as 4,

(m ₂ ) As shown in SEQ ID NO:4 by substitution, repetition, deletion or addition of one or more amino acids, and has the cytokine activity of GM-CSF.

In some embodiments, the use according to the present disclosure, wherein the formulation comprises (g) as follows ₁ )-(g ₄ ) One or more of the cyclic RNA molecules of (a):

(g ₁ ) And SEQ ID NO:7 has at least 90%, optionallyA circular RNA molecule with at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity;

(g ₂ ) And SEQ ID NO:8, or a variant thereof, wherein the nucleotide sequence set forth in seq id No. 8 comprises a circular RNA molecule having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity;

(g ₃ ) And SEQ ID NO:8, or a variant thereof, wherein the nucleotide sequence set forth in seq id No. 8 comprises a circular RNA molecule having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity;

(g ₄ ) And SEQ ID NO:8, or a variant thereof, and a circular RNA molecule having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the nucleotide sequence set forth in seq id no.

In some embodiments, the use according to the present disclosure, wherein the preparation comprising a circular RNA molecule is prepared as follows (f) ₁ )-(f ₃ ) Use in at least one medicament:

(f ₁ ) A medicament for inhibiting or killing tumor cells,

(f ₂ ) A medicament for inducing an anti-tumor immune response in a subject,

(f ₃ ) A medicament for reducing or eliminating tumor immunosuppressive microenvironment.

In some embodiments, the use according to the present disclosure, wherein the formulation comprising a circular RNA molecule further comprises a tumor therapeutic agent, wherein the tumor therapeutic agent is selected from one or more of a radiotherapeutic agent, a chemotherapeutic agent, an immunomodulator, a cytotoxic agent, an antibody, a vaccine;

preferably, the tumor therapeutic agent is an immunomodulator; more preferably, the immune modulator is an immune checkpoint inhibitor.

In a second aspect, the present disclosure provides a preparation comprising a cyclic RNA molecule for use in the prevention or treatment of a tumour, the preparation comprising a cyclic RNA molecule as defined in the first aspect; preferably, the formulation comprising the circular RNA molecule is an injection; preferably, the formulation comprising the cyclic RNA molecule is administered to the subject by intratumoral administration; more preferably, the preparation comprising the circular RNA molecule is an injection to be administered intratumorally.

In a third aspect, the present disclosure provides a pharmaceutical composition for preventing or treating a tumor, wherein the pharmaceutical composition comprises:

(h ₁ ) A preparation comprising a circular RNA molecule, the preparation comprising a circular RNA molecule being as defined in the first aspect;

(h ₂ ) A tumour therapeutic agent as defined in the first aspect.

In a fourth aspect, the present disclosure provides a method of preventing or treating a tumour, wherein the method comprises administering to a subject a preparation comprising a circular RNA molecule, wherein the preparation comprising a circular RNA molecule is as defined in the first aspect;

optionally, the mode of administration is selected from oral, intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, transdermal, nasal, rectal, intratumoral injection, intrathecal injection, subarachnoid injection, or systemic administration;

preferably, the mode of administration is intratumoral injection.

ADVANTAGEOUS EFFECTS OF INVENTION

In some embodiments, the present disclosure finds for the first time that a cyclic RNA molecule encoding a polypeptide having tumor immunotherapeutic activity is effective in inhibiting tumor growth, exerting an in vivo therapeutic effect of inhibiting, killing, or otherwise inhibiting tumor. The preparation containing the circular RNA molecule provided by the disclosure is suitable for being used as a tumor immunotherapy medicament and has wide application prospect in clinical treatment of tumors.

In some embodiments, the preparations of the present disclosure comprising a cyclic RNA molecule are administered to a subject by intratumoral administration, which enables efficient expression of the cyclic RNA molecule and thus efficient tumor killing and inhibition effects.

In some embodiments, the preparation comprising the cyclic RNA molecule of the present disclosure is an injection, and can be administered into a subject by intratumoral injection, so as to achieve efficient delivery into a tumor patient, and maintain the expression stability, durability and efficiency of the cyclic RNA molecule, thereby fully exerting the immunotherapy effect on a living tumor.

In some embodiments, the present disclosure demonstrates for the first time that the injection of cyclic mRNA into a tumor for immunotherapy can transform the tumor into a vaccine, promote the body to generate a specific immune response against tumor cells, generate a distant effect, and effectively inhibit tumor metastasis.

In some embodiments, the preparations of the present disclosure containing the cyclic RNA molecule do not need to be encapsulated by liposome material, i.e., have high stability, and can achieve efficient delivery into tumor, thereby effectively exerting the tumor killing and inhibiting effects.

In some embodiments, the present disclosure provides circular RNA molecules with better protein translation levels, protein expression durability, and superior tumor killing, inhibition effects compared to linear mRNA molecules; and the steps of capping, poly (A) tail addition, nucleotide modification and the like are not required to be carried out on the circular RNA molecules, the production process is simple, and the production cost is reduced.

In some embodiments, the present disclosure can effectively activate immune response in vivo, promote tumor infiltration by immune cells, effectively activate immune response against tumors in the body, and improve tumor treatment effect by administering the cyclic RNA molecule and the immune checkpoint inhibitor in combination, which act synergistically.

Detailed Description

Definition of

The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification can mean "one," but can also mean "one or more," at least one, "and" one or more than one.

As used in the claims and specification, the words "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as merely an alternative and "and/or," the term "or" in the claims means "and/or" unless it is explicitly stated that only alternatives or mutual exclusions between alternatives are mutually exclusive.

As used in this disclosure, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and are polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component). The polypeptides may be isolated from natural sources, may be produced by recombinant techniques from eukaryotic or prokaryotic hosts, and may be the product of synthetic methods.

As used in this disclosure, "recombinant polypeptide" also referred to as "engineered polypeptide" refers to polypeptides, proteins having peptide sequences that are not linked in nature. The recombinant polypeptide can be a target polypeptide obtained in prokaryotic or eukaryotic cells by using a genetic engineering technology; the target polypeptide may be a polypeptide obtained by a synthetic technique.

As used in this disclosure, the term "antigen-binding fragment" includes an intact antibody, or a portion or fragment of an intact or complete antibody having fewer amino acid residues than the intact or complete antibody, which is capable of binding to an antigen or competing with an intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) for binding to an antigen. Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, fab ', F (ab') ₂ Fv, single-chain Fv, diabodies (diabodies), single-domain antibodies (sdabs). The Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains, and can be obtained, for example, by papain digestion of whole antibodies. In addition, complete antibody production F (ab') by pepsin digestion below the disulfide bond in the hinge region ₂ Which is a dimer of Fab' and is a bivalent antibody fragment. F (ab') ₂ Can be reduced under neutral conditions by disrupting the disulfide bond in the hinge region, thereby converting F (ab') ₂ The dimer is converted to Fab' monomer. The Fab' monomers are essentially Fab fragments with a hinge region (see: basic Immunology, editor W.E.Paul, raven Press, N.Y. (1993) for a more detailed description of other antibody fragments). The Fv plateThe fragments consist of VL and VH domains of a single arm of the antibody. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, using recombinant methods, they can be joined by a synthetic linker peptide that enables the two domains to be produced as a single protein chain in which the VL and VH regions pair to form a single chain Fv. The antibody fragment may be obtained by a chemical method, a recombinant DNA method, or a protease digestion method.

As used in this disclosure, the term "circular RNA" is a RNA molecule in the form of a closed loop consisting essentially of exons, IRES elements, protein coding regions, and spacers. In some preferred embodiments, the circular RNA has the structure: "second exon E2-spacer-IRES element-coding region-spacer-first exon E1". The circular RNA used in the present disclosure has protein translation activity and may also be referred to as "circular mRNA".

"sequence identity" and "percent identity" in the present disclosure refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions in the aligned polynucleotides or polypeptides containing the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotides or polypeptides containing different nucleotide or amino acid residues. Polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or deleted nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or both polynucleotides). Polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or deleting amino acids (i.e., amino acid insertions or amino acid deletions in one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

Illustratively, in the present disclosure, two or more sequences or subsequences have "sequence identity" or "percent identity" of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residues when compared and aligned for maximum correspondence as measured using a sequence comparison algorithm or by visual inspection. The determination/calculation of "sequence identity" or "percent identity" may be based on any suitable region of the sequence. For example, a region of at least about 50 residues, a region of at least about 100 residues, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues in length. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the biopolymers (i.e., nucleic acids or polypeptides) to be compared.

As used in this disclosure, "treating" means: after suffering from a disease, contacting (e.g., administering) a subject with a cyclic RNA, a circularized precursor RNA, a recombinant nucleic acid molecule, a recombinant expression vector, a composition of the invention, thereby alleviating a symptom of the disease compared to when not contacting, does not imply that a symptom of the disease must be completely inhibited. The suffering of a disease is: the body develops symptoms of the disease.

As used in this disclosure, "preventing" refers to: before the onset of a disease, the subject is contacted (e.g., administered) with the cyclic RNA, the circularized precursor RNA, the recombinant nucleic acid molecule, the recombinant expression vector, the composition, and the like of the present invention, whereby the symptoms after the onset of the disease are alleviated as compared with the case of no contact, and it is not necessarily required to completely suppress the onset of the disease.

As used in this disclosure, the term "effective amount" refers to an amount or dose of a recombinant nucleic acid molecule, recombinant expression vector, circularized precursor RNA, circular RNA, vaccine or composition of the invention that produces the desired effect in a patient in need of treatment or prevention following administration of the patient in a single or multiple doses. An effective amount can be readily determined by the attending physician, as one skilled in the art, by considering a number of factors including: species such as mammals; its size, age and general health; the specific diseases involved; the degree or severity of the disease; the response of the individual patient; the specific antibody administered; a mode of administration; bioavailability characteristics of the administered formulation; a selected dosing regimen; and the use of any concomitant therapies.

As used in this disclosure, the term "individual", "patient" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

As used in this disclosure, the term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous (pre-cancer) and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

As used in this disclosure, the terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial diffuse melanoma, lentigo nevus melanoma, acromelanoma, melanoma, multiple myeloma and B-cell lymphoma, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (ptphald), as well as associated with scarring (koospes), edema (such as associated with brain tumors) and proliferative brain (Meigs) cancer, and head and neck cancer. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, or colon cancer, including metastatic forms of those cancers.

As used in this disclosure, the term "radiotherapeutic agent" in this disclosure includes the use of drugs that cause DNA damage. Radiotherapy has been widely used in cancer and disease treatment and includes those commonly referred to as gamma rays, X-rays and/or the targeted delivery of radioisotopes to tumor cells.

The term "chemotherapeutic agent" in this disclosure is a chemical compound useful for the treatment of cancer. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, photosensitizers, anti-estrogen and selective estrogen receptor modulators, anti-progestins, estrogen receptor downregulators, estrogen receptor antagonists, luteinizing hormone-releasing hormone agonists, anti-androgens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth. Chemotherapeutic agents useful in the treatment methods of the present disclosure include cytostatic and/or cytotoxic agents.

Unless defined otherwise or clearly indicated by the background, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Technical scheme

SEQ ID NO:1 is the amino acid sequence of a cytokine IL-15;

the amino acid sequence of SEQ ID NO:2 is the amino acid sequence of the cytokine IL-12;

SEQ ID NO:3 is the amino acid sequence of the cytokine IFN alpha-2 b;

SEQ ID NO:4 is the amino acid sequence of a cell factor GM-CSF;

SEQ ID NO:5 is the amino acid sequence of the fluorescent protein Luciferse;

the amino acid sequence of SEQ ID NO:6 is a nucleotide sequence of a circular RNA molecule encoding Luciferase;

the amino acid sequence of SEQ ID NO:7 is a nucleotide sequence of a circular RNA molecule encoding IL-15;

the amino acid sequence of SEQ ID NO:8 is the nucleotide sequence of a circular RNA molecule encoding IL-12;

the amino acid sequence of SEQ ID NO:9 is the nucleotide sequence of the circular RNA molecule encoding IFN alpha-2 b;

SEQ ID NO:10 is a nucleotide sequence of a circular RNA molecule encoding GM-CSF;

the amino acid sequence of SEQ ID NO:11 is the nucleotide sequence of CVB3 IRES;

SEQ ID NO:12 is the nucleotide sequence of EV24 IRES;

SEQ ID NO:13 is the nucleotide sequence of EV29 IRES;

SEQ ID NO:14 is the nucleotide sequence of EV33 IRES;

SEQ ID NO:15 is the nucleotide sequence of EV24 and CVB3v chimeric IRES;

SEQ ID NO:16 is the nucleotide sequence of EV29 and CVB3v chimeric IRES;

SEQ ID NO:17 is the nucleotide sequence of EV33 chimeric IRES with CVB3 v;

SEQ ID NO:18 is the nucleotide sequence of the first exon (E1);

SEQ ID NO:19 is the nucleotide sequence of the second exon (E2);

SEQ ID NO:21 is the nucleotide sequence of 5' spacer sequence 2;

SEQ ID NO:23 is the nucleotide sequence of 3' spacer sequence 2;

SEQ ID NO:24 is a nucleotide sequence of the 5' intron in the DNA vector for constructing the circular mRNA;

SEQ ID NO:25 is a nucleotide sequence of a 3' intron in a DNA vector for constructing a circular mRNA;

SEQ ID NO:26 is a nucleotide sequence of 5' homologous arm sequence 1 (H1) in a DNA vector for constructing circular mRNA;

SEQ ID NO:27 is a nucleotide sequence of 5' homology arm sequence 2 (H2) in a DNA vector for constructing a circular mRNA;

SEQ ID NO:29 is the nucleotide sequence of 3' homology arm sequence 2 in the DNA vector for constructing the circular mRNA.

Formulations comprising circular RNA molecules

The present disclosure provides formulations comprising a cyclic RNA molecule, wherein the cyclic RNA molecule encodes a polypeptide having tumor immunotherapeutic activity. The invention discovers for the first time that the preparation containing the cyclic RNA molecules can obviously inhibit the growth of tumors in tumor patients, plays an obvious tumor treatment effect in tumor mouse models, and provides a novel treatment medicament with a wide application prospect for tumor immunotherapy.

In some embodiments, the circular RNA molecule is a wrapped circular RNA molecule. For example, a formulation comprising a cyclic RNA molecule is an encapsulate formed by encapsulating a cyclic RNA molecule with materials such as Liposome Polymer (LPR), liposome complex (LP), liposome Nanoparticle (LNP), and the like. In some embodiments, the circular RNA molecule is an unencapsulated circular RNA molecule. The present disclosure surprisingly found that the cyclic RNA molecules in the present disclosure can achieve high in vivo delivery efficiency and high in vivo expression efficiency without being encapsulated by liposome materials, and the naked cyclic RNA molecules can exert an obvious in vivo tumor treatment effect, and are suitable for large-scale popularization and use.

In some embodiments, the formulation comprising a circular RNA molecule in the present disclosure is an injection. As generally known to those skilled in the art, the dosage form of the drug has an important influence on the activity of the drug molecule, and the present disclosure finds that when the preparation containing the cyclic RNA molecule is an injection, the preparation is suitable for in vivo delivery, and after being delivered in vivo, the preparation can exert high tumor killing and inhibiting effects, and can specifically stimulate the anti-tumor immune response of the organism, so as to realize the immunotherapy of the tumor.

In some preferred embodiments, the injection further comprises a solvent for dissolving the circular RNA molecule. Illustratively, the solvent that solubilizes the circular RNA molecule is water, a chloride solution, TE buffer, PBS buffer, or any other solvent suitable for solubilizing the circular RNA molecule. Illustratively, in some embodiments, the solvent that dissolves the circular RNA molecules is a TE buffer, e.g., a 1 × TE buffer, a2 × TE buffer, or the like. In some embodiments, the solvent that dissolves the circular RNA molecule is PBS buffer, e.g., 1 x PBS buffer, 2 x PBS buffer, and the like. In some embodiments, the solvent that dissolves the circular RNA molecule is a chloride solution, e.g., a 0.9wt% sodium chloride solution, and the like.

Further, the amount of the circular RNA molecule in the injectable preparation may vary depending on various factors such as the disease state, the age, sex and weight of the individual and the ability of the circular RNA molecule to elicit a desired response in the individual.

In some more preferred embodiments, the injection consists of the circular RNA molecule and a solvent that dissolves the circular RNA molecule. The injection disclosed by the disclosure has the advantages of simple components, no containing of biological toxic substances, high biological safety and suitability for administration to animals. In some embodiments, the injection only consisting of the cyclic RNA molecule and the solvent for dissolving the cyclic RNA molecule in the present disclosure can achieve high in vivo delivery efficiency and in vivo expression efficiency, effectively inhibit the growth of in vivo tumor, and has the advantages of high safety, good stability, significant effect, and the like.

In some embodiments, the formulations of the present disclosure comprising a cyclic RNA molecule are administered to a subject by intratumoral administration, which can greatly reduce the likelihood of causing immune-related side effects (irAEs), improve the safety of drug delivery, achieve high local drug concentrations in tumors, improve the bioavailability of the drug,

in some embodiments, the formulations of the present disclosure comprising a circular RNA molecule are administered to a subject by intratumoral administration. Compared with subcutaneous injection, intramuscular injection, intradermal injection, intravenous injection, intraperitoneal injection and intratumoral injection, the cyclic RNA molecule has high in-vivo expression efficiency under the administration mode of intratumoral administration, and can realize obvious inhibition on tumor growth in a mouse tumor model through intratumoral injection administration, which shows that the cyclic RNA molecule contained in the disclosure can exert a tumor treatment effect which is remarkably superior to that of other administration modes when being administered by intratumoral injection.

Circular RNA molecules

The circular RNA molecules in the present disclosure comprise a coding region encoding a polypeptide having tumor immunotherapeutic activity, and an IRES element operably linked to the coding region.

In the present disclosure, the polypeptide having tumor immunotherapeutic activity may be any type of polypeptide having tumor immunotherapeutic activity, and the sequence of the polypeptide having tumor immunotherapeutic activity is not particularly limited in the present disclosure.

In some embodiments, the polypeptide having tumor immunotherapeutic activity is a cytokine. Exemplary cytokines include, but are not limited to, interleukins (IL), interferons (IFN), tumor Necrosis Factor (TNF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF). The circular RNA molecule can effectively activate the immune system of an organism, kill tumor cells and realize the treatment of tumors by expressing cell factors.

In some embodiments, the polypeptide having tumor immunotherapeutic activity is an antigen binding fragment. More specifically, the polypeptide having tumor immunotherapeutic activity is an antigen-binding fragment that specifically binds to a tumor antigen. Exemplary, tumor antigens include, but are not limited to, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, AFP, CEA, PSCA, GD2, NKG2D, BCMA, EGFR, her2, EGFRv iii, CD171, FAP, IL13 ra 2, VEGFR1, VEGFR2, GPC-3, mesothelin, claudin 18.2, epCAM, MUC1, MUC16, EPHA2, EPHA3, CD133, PSMA. The circular RNA molecule can block the signal path of the tumor growth factor by targeting the surface antigen or the specific receptor of the tumor cell, thereby playing a role in killing the tumor cell.

In the present disclosure, an antigen-binding fragment includes an antibody that specifically binds to a tumor antigen, or comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds.

Further, antigen-binding fragments include natural and artificial antibodies that encompass a variety of structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies (e.g., scFv), intact antibodies, fv, fab '-SH, F (ab') 2; a linear antibody; a single domain antibody; a bivalent or bispecific antibody or fragment thereof; camelid antibodies; and bispecific or multispecific antibodies formed from antibody fragments.

In some alternative embodiments, the polypeptide having tumor immunotherapeutic activity is IL-15 having the amino acid sequence set forth in SEQ ID NO:1, or the amino acid sequence as shown in SEQ ID NO:1 is substituted, repeated, deleted or added with one or more amino acids, and has the amino acid sequence shown in SEQ ID NO:1, or a mutant of the activity of the polypeptide as shown in the figure.

In some alternative embodiments, the circular RNA molecule comprises a nucleotide sequence encoding a nucleotide sequence set forth as SEQ ID NO:1, the coding region for IL-15. The present disclosure achieves effective inhibition of mouse tumors by intratumoral injection of a circular RNA molecule expressing IL-15 into a mouse tumor model.

In some alternative embodiments, the polypeptide having tumor immunotherapeutic activity is IL-12, having the amino acid sequence set forth in SEQ ID NO:2, or the amino acid sequence as shown in SEQ ID NO:2 is substituted, repeated, deleted or added with one or more amino acids, and has the amino acid sequence shown in SEQ ID NO: 2.

In some alternative embodiments, the circular RNA molecule comprises a nucleotide sequence encoding a nucleotide sequence set forth as SEQ ID NO:2, the present disclosure realizes the effective inhibition of mouse tumor by injecting a circular RNA molecule expressing IL-12 into the tumor of a mouse tumor model.

In some alternative embodiments, the polypeptide having tumor immunotherapeutic activity is IFN alpha-2b having the amino acid sequence set forth in SEQ ID NO:3, or the amino acid sequence as set forth in SEQ ID NO:3 is substituted, repeated, deleted or added with one or more amino acids, and has the amino acid sequence shown in SEQ ID NO: 3.

In some alternative embodiments, the circular RNA molecule comprises a nucleotide sequence encoding a nucleotide sequence set forth as SEQ ID NO:3, the present disclosure realizes effective inhibition of mouse tumor by injecting a circular RNA molecule expressing IFN alpha-2b into tumor of mouse tumor model.

In some alternative embodiments, the polypeptide having tumor immunotherapeutic activity is GM-CSF, which has the amino acid sequence as set forth in SEQ ID NO:4, or the amino acid sequence as shown in SEQ ID NO:4 is substituted, repeated, deleted or added with one or more amino acids, and has the amino acid sequence shown in SEQ ID NO: 4.

In some alternative embodiments, the circular RNA molecule comprises a nucleotide sequence encoding a nucleotide sequence set forth as SEQ ID NO:4, the present disclosure realizes the effective inhibition of mouse tumor by injecting a circular RNA molecule expressing GM-CSF intratumorally into a mouse tumor model.

In some preferred embodiments, the IRES sequence comprises a sequence as set forth in SEQ ID NO:11-17, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the nucleotide sequence of one or more sequences in the group consisting of the sequences of any of claims 11-17. In some embodiments, the IRES sequence is SEQ ID NO:11, CVB3 IRES, SEQ ID NO:12, EV24 IRES, SEQ ID NO:13, EV29IRES, SEQ ID NO:14, EV33 IRES of the nucleotide sequence shown in fig. 14. In some embodiments, the IRES sequence comprises a chimeric sequence of a CVB3v IRES and any one of an EV24 IRES, an EV29IRES and an EV33 IRES. In some embodiments, the IRES sequence comprises a chimeric sequence of CVB3v and EV24 IRES, having a nucleotide sequence as set forth in SEQ ID NO:15, respectively. In some embodiments, the IRES sequence comprises a chimeric sequence of CVB3v and EV29IRES, having a nucleotide sequence as set forth in SEQ ID NO: shown at 16. In some embodiments, the IRES sequence comprises a chimera sequence of CVB3v and EV33 IRES, having a nucleotide sequence as set forth in SEQ ID NO: shown at 17.

The disclosure unexpectedly finds that the circular RNA molecule containing the IRES sequence is suitable for being applied as an injection for in vivo tumor injection, can be efficiently delivered to the body in an unencapsulated naked molecule state, and has obvious immunotherapy effects of activating the anti-tumor immune response of the body, inhibiting and killing tumors.

In some preferred embodiments, the circular RNA molecule further comprises a5 'spacer located upstream of the IRES element, and a 3' spacer located downstream of the coding region. Specifically, the 5' spacer sequence is identical to SEQ ID NO:20-21, and a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the nucleotide sequence set forth in any one of sequences 20-21. The 3' spacer sequence is identical to SEQ ID NO:22-23, and a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the nucleotide sequence set forth in any one of sequences 22-23.

In some preferred embodiments, the circular RNA molecule further comprises a second exon located upstream of said 5 'spacer region, and a first exon located downstream of said 3' spacer region. Specifically, the second exon sequence is a sequence identical to SEQ ID NO:19, or a variant thereof, and a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the nucleotide sequence set forth in 19. The first exon sequence is identical to SEQ ID NO:18, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity compared to the nucleotide sequence set forth in seq id No. 18.

In some preferred embodiments, the circular RNA molecule comprises the following elements connected in sequence: a second exon, a5 'spacer, an IRES element, a coding region, a 3' spacer, and a first exon.

According to the method, the 5 'spacer region, the 3' spacer region, the second exon and the first exon with specific sequences are selected, so that the efficient delivery of the circular RNA molecules into the tumor of an organism can be realized, the circular RNA molecules can be effectively delivered into the tumor only by dissolving, the growth of the tumor can be effectively inhibited, and the immunotherapy of the tumor can be realized.

In some preferred embodiments, the circular RNA molecule comprises a nucleotide sequence as set forth in SEQ ID NO:7, or a nucleotide sequence substantially identical to SEQ ID NO:7, has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity compared to the nucleotide sequence set forth in seq id no.

In some preferred embodiments, the circular RNA molecule comprises a nucleotide sequence as set forth in SEQ ID NO:8, or a nucleotide sequence corresponding to SEQ ID NO:8, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity compared to the nucleotide sequence set forth in seq id No. 8.

In some preferred embodiments, the circular RNA molecule comprises a nucleotide sequence as set forth in SEQ ID NO:9, or a nucleotide sequence corresponding to SEQ ID NO:9, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity compared to the nucleotide sequence set forth in seq id No. 9.

In some preferred embodiments, the circular RNA molecule comprises a nucleotide sequence as set forth in SEQ ID NO:10, or a nucleotide sequence identical to SEQ ID NO:7, has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity compared to the nucleotide sequence set forth in seq id no.

The cyclic RNA molecules are injected singly or in a combined mode in the tumor, so that the growth inhibition of the tumor is realized, the fact that the cyclic RNA molecules injected in the tumor can be used for tumor immunotherapy is proved for the first time, and an effective immunotherapy medicine is provided for clinical therapy of the tumor. In some preferred embodiments, the polypeptide is produced by co-administration of a polypeptide as set forth in SEQ ID NO:7-10, and the tumor growth speed in a tumor mouse model is remarkably inhibited.

In some embodiments, the injection containing the circular RNA molecule in the present disclosure can achieve efficient in vivo delivery and in vivo expression without encapsulating the circular RNA molecule, and exert tumor killing and inhibiting effects.

In some embodiments, the circular RNA molecules of the present disclosure, when administered to a subject by intratumoral injection, can transform a tumor into a vaccine, promote the body to generate a specific immune response against tumor cells, generate a distancing effect, and effectively inhibit tumor metastasis.

Pharmaceutical composition

Pharmaceutical compositions of the disclosure include a formulation of the disclosure comprising a cyclic RNA molecule, and a tumor therapeutic. By combined administration of the preparation containing the circular RNA molecules and the tumor therapeutic agent, the curative effect and specificity of tumor therapy are improved, and the clinical application range of the tumor therapeutic agent is widened.

In some alternative embodiments, the tumor therapeutic agent is selected from one or more of a radiotherapeutic agent, a chemotherapeutic agent, an immunomodulator, a cytotoxic agent, an antibody, a vaccine. In some preferred embodiments, the tumor therapeutic is an immune modulator "comprising immune checkpoint modulators, such as immune checkpoint protein receptors and their ligands, that mediate inhibition of T cell-mediated cytotoxicity and are typically expressed by tumors or on anergic T cells in the tumor microenvironment and allow the tumors to evade immune attack. Inhibitors of the activity of immunosuppressive checkpoint protein receptors and their ligands can overcome the immunosuppressive tumor environment to allow cytotoxic T cell attack of the tumor. Examples of immune checkpoint proteins include, but are not limited to, PD-1, PD-L1, PDL2, CTLA4, LAG3, TIM3, TIGIT, and CD103. Modulation (including inhibition) of the activity of such proteins can be accomplished by immune checkpoint modulators, which can include, for example, antibodies, aptamers, small molecules, and soluble forms of checkpoint receptor proteins, among others, that target checkpoint proteins. In addition, the "immunomodulator" also includes cytokines such as Interleukin (IL), interferon (IFN), tumor Necrosis Factor (TNF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), etc., which can achieve the effect of enhancing the immune response and immunoregulation of the organism of a tumor patient or a pathogen infected person.

In some preferred embodiments, the immune modulator is an immune checkpoint inhibitor. The invention jointly applies the circular RNA molecule and the immune check point inhibitor to a mouse tumor model by an intratumoral injection mode, finds that the circular RNA molecule and the immune check point inhibitor can act synergistically, improves the number of tumor-infiltrating immune cells, and particularly can specifically activate tumor-infiltrating CD8 ⁺ T cells have important significance for stimulating the specific immune response of organisms and playing the roles of killing and inhibiting tumors.

Medical application

The preparation containing the circular RNA molecule or the pharmaceutical composition containing the preparation is suitable for being used as a tumor immunotherapy medicament and used for preventing or treating tumors.

In some embodiments, the formulations comprising a circular RNA molecule in the present disclosure are used to treat solid tumors, exemplary, including but not limited to: lung cancer, liver cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, renal cancer, lymphoma, ovarian cancer, gastric cancer, pancreatic cancer, multiple myeloma, glioma, prostate cancer, cervical cancer, cholangiocarcinoma, esophageal cancer, or melanoma.

The preparation containing the circular RNA molecule in the disclosure can be prepared into an unencapsulated injection, and is applied to a subject suffering from a solid tumor in an intratumoral injection mode, the circular RNA molecule is efficiently and stably expressed in the subject, the anti-tumor immune response of an organism is effectively activated, tumor cells are inhibited and killed, and the immunotherapy effect on the tumor is fully exerted.

Examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

The experimental techniques and experimental procedures used in this example are, unless otherwise specified, conventional techniques, e.g., those in the following examples, in which specific conditions are not specified, and generally according to conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The materials, reagents and the like used in the examples are commercially available from normal sources unless otherwise specified.

The following examples relate to experimental animals and experimental methods as follows:

B16F10 tumor model

Experimental animals: female C57BL/6 (Beijing Wittingle laboratory animal technology Co., ltd.), 6-8 weeks of age, body weight between 18 and 22 grams, was acclimatized in the rearing room for at least three days prior to the study. Mice had free access to food and sterile water.

A breeding environment: maintaining 12 hours of light and 12 hours of dark day and night alternation, maintaining the humidity at 40-70% and the difference of the daily humidity less than 5%; the temperature is maintained at 20-25 ℃. Human care was given according to the 3R principle used for experimental animals.

Intratumoral mRNA injection was started around 5 days after subcutaneous tumor inoculation. Tumor growth was assessed every 2-3 days by caliper measurements and tumor volume size was calculated using the following formula: a b ² /2 wherein b<a。

MC38 tumor model

Experimental animals: female C57BL/6 (Beijing Wittingle laboratory animal technology Co., ltd.), 6-8 weeks of age, body weight between 18 and 22 grams, was acclimatized in the rearing room for at least three days prior to the study. Mice had free access to food and sterile water.

A breeding environment: maintaining 12 hours of bright light and 12 hours of dark day and night, maintaining the humidity at 40-70% and the difference of the day humidity less than 5%; the temperature is maintained at 20-25 ℃. The humane care was given according to the 3R principle used in experimental animals.

A549 tumor model

Experimental animals: female CD-1Nude (Beijing Wittingle laboratory animal technology Co., ltd.), 6-8 weeks of age, body weight between 18 and 22 grams, was acclimatized in the rearing room for at least three days prior to the study. Mice had free access to food and sterile water.

A breeding environment: maintaining 12 hours of bright light and 12 hours of dark day and night, maintaining the humidity at 40-70% and the difference of the day humidity less than 5%; the temperature is maintained at 20-25 ℃.

NCI-H358 tumor model

Experimental animals: female CD-1Nude (Beijing Wittingle laboratory animal technology Co., ltd.), 6-8 weeks of age, body weight between 18 and 22 grams, was acclimatized in the rearing room for at least three days prior to the study. Mice had free access to food and sterile water.

A breeding environment: maintaining 12 hours of light and 12 hours of dark day and night alternation, maintaining the humidity at 40-70% and the difference of the daily humidity less than 5%; the temperature is maintained at 20-25 ℃.

Preparation of mRNA for in vivo studies

Preparation of circular mRNA:

a DNA plasmid vector for producing circular mRNA containing a gene of interest to be expressed is synthesized and constructed by committing Suzhou Jinzhi Biotech, ltd to gene synthesis and cloning. As used herein, a DNA vector for constructing a circular mRNA comprises a T7 promoter, a 5' homology arm, a 3' intron, a second exon E2,5' spacer, an IRES element, a luciferase sequence coding region, a downstream spacer, a 5' intron, a first exon E1,3' homology arm, and an enzyme cleavage site XbaI useful for plasmid linearization. Plasmids were obtained by a plasmid extraction kit (Tiangen endotoxin-free small-volume medium extraction kit), and linearized by restriction enzyme XbaI. Circular mRNA precursor RNA is synthesized by in vitro transcription. The transcription system is as follows:

TABLE 1

The T7 RNA polymerase adopts PureScribe ^TM T7 High Yield RNA Synthesis Kit (Jiangsu Purikang biomedicine Co., ltd.). The resulting transcription product was centrifuged using silica membraneColumn Purification (Thermo, geneJET RNA Purification Kit). Circularization of RNA to give circular mRNA under the following conditions:

TABLE 2

The above solution was heated at 55 ℃ for 15min, and the circularized RNA product was purified by silica-membrane centrifugal column method (Thermo, geneJET RNA Purification Kit) to obtain circular mRNA, and the OD value was measured. Dissolving the circular mRNA in water, adding 2 XPBS buffer solution and water to prepare an injection in which the circular mRNA is dissolved by 1 XPBS buffer solution,

the linear mRNA was synthesized as follows:

the method for enzyme digestion linearization and template purification of the pUC57-TRNP-EGFP-DASP plasmid is the same as (2) -2). Linear mRNA with a Cap1 structure is synthesized by in vitro transcription by adopting an APExBio Kit HyperScribeTMAllin One mRNA Synthesis Kit II, and nucleoside modification is carried out by adopting pseudouracil. Finally obtaining the Cap1 structure, linear mRNA with a polyA tail and pseudouridine modification.

The circular mRNA is subpackaged and placed in a refrigerator at 90 ℃ below zero, unfrozen on the day of injection, the corresponding 2X injection solution is prepared, the 2X injection solution, enzyme-free water and the circular mRNA are used for preparing the corresponding 1X injection solution, and the subpackaged circular mRNA is used for intratumoral injection.

The linear mRNA is subpackaged and put in a refrigerator at 90 ℃ below zero, unfreezes on the day of injection, prepares a corresponding 2X injection solution, prepares a corresponding 1X injection solution by using the 2X solution, enzyme-free water and the linear mRNA, and is subpackaged for intratumoral injection.

Mouse in vivo fluorescence imaging

After intratumoral injection of nucleotide-modified linear or circular mRNA encoding Luciferase into mice for a period of time, the mice were anesthetized by intraperitoneal injection of Luciferase substrate fluorescein aqueous solution (300 μ L,4.5mg promega). Fluorescence of live animal images was quantified 10min after intraperitoneal injection of fluorescein. The region of interest (ROI) is quantized to mean radiance (photons) ^-1 cm ^-2 sr ^-1 Represented by a color scaled Image superimposed on a mouse grayscale photograph using the Living Image software from Caliper Life Sciences). For absolute quantification, bioluminescent signals were blotted as total flux (photons s) ^-1 )。

Tumor monitoring

Tumor size was measured twice weekly with a vernier caliper until the final euthanasia. When the tumor size reaches about 2000mm ³ Or when there was a health problem in the animal (20% area ulceration of the tumor), the animal was euthanized. The following cases were defined as tumor regression: tumor volume at study termination<20mm ³ ；TF/T0<1, where TF is the final tumor volume and T0 is the tumor volume on the day of the first intratumoral mRNA injection. The data were collated using GraphPad Prism 8.0.2 and a tumor growth plot and a survival plot were plotted.

Tumor infiltrating immune cell assay

After euthanasia, the mice were dissected and the tumor draining lymph nodes, spleens and tumor bodies were removed. Lymph nodes and spleen were ground and passed through a 70 μm sieve, and cells were collected by centrifugation for flow antibody staining. After the tumor was minced, the cells were digested with collagen Type IV (Collagenase IV, from Gibco) at 37 ℃ for 30min, filtered through a 70 μm filter, and centrifuged to collect cells for flow antibody staining.

Flow antibody staining: firstly, cells are stained with eFluor780 cell viatility dye for death and viability, the cells are stained for 20min at 4 ℃ in the dark, then the cells are centrifuged, and the cells are respectively stained with antibodies after being washed once by PBS. (1) Lymph node: detecting immune cell group, T cell group and CD4 by staining CD45, CD3, CD4, CD8 and CD69 respectively ⁺ T cell clustering, CD8 ⁺ T cell clustering, activated T cell clustering; (2) spleen: detection of immune cell, T cell and CD4 by staining CD45, CD3, CD4, CD8 and CD69 ⁺ T cell clustering, CD8 ⁺ T cell clustering, activated T cell clustering; (3) tumor tissue: the detection is divided into two groups, wherein one group is stained with CD45, CD3, CD4, CD8 and CD69 to detect immune cell subgroup, T cell subgroup and CD4 respectively ⁺ T cell clustering, CD8 ⁺ T cell subpopulations, activated T cell subpopulations, and another group stained CD45, CD11b, F4/80 to detect macrophage subpopulations. After staining at 4 ℃ in the dark for 20min, centrifugation was carried out, washed once with PBS, resuspended in 200. Mu.L of PBS, and examined by a sonic focusing flow cytometer (Life, atture NXT). All flow-through antibodies were purchased from Biolegend.

Example 1 Effect of different routes of administration on expression of circular mRNA

1.1 Experimental methods

For B1F10 tumor model mice, intratumoral, peritumoral subcutaneous and intradermal injections of 10. Mu.g of circular mRNA encoding Fireflyfluenase were performed, respectively; for normal wild-type mice, 10. Mu.g of mRNA for circular Luciferase was injected intramuscularly, intraperitoneally, and caudally, respectively. After 6h, the mice were injected with fluorescein water solution in the abdominal cavity and subjected to fluorescence imaging of the living bodies. Effect of different routes of administration on expression of circular mRNA by fluorescence intensity analysis.

1.2 results of the experiment

Figure 1 shows the effect of different routes of administration on the expression of circular mRNA, as shown by B and C in figure 1, with no apparent expression seen by peri-tumoral subcutaneous and intradermal injection of circular mRNA. No fluorescence was detected in any of the other modes of administration, such as intramuscular (D), intraperitoneal (E), and intravenous (F). In contrast, in the mice injected with (A) intratumorally, the entire tumor body showed significant fluorescence. Therefore, the administration mode of the cyclic mRNA can significantly influence the expression of the cyclic mRNA without a delivery system, and the method of intratumoral injection of the cyclic mRNA is feasible.

Example 2 validation of expression of circular mRNA in various tumor models

2.1 Experimental methods

For A549, NCI-H358, B16F10 and MC38 tumor model mice, 10. Mu.g of circular mRNA encoding Fireflyfluenase was intratumorally injected, and 6H later, the mice were intraperitoneally injected with a fluorescein aqueous solution for in vivo fluorescence imaging, respectively. The expression of the circular mRNA in the tumors of different tumor model mice is analyzed through fluorescence intensity.

2.2 results of the experiment

FIG. 2 shows the results of verifying the expression of circular mRNA on various tumor models, wherein A-D are, in order, an A549 tumor model mouse, an H358 tumor model mouse, a B16F10 tumor model mouse, and an MC38 tumor model mouse. As can be seen from FIG. 2, the intratumoral injection of circular mRNA can be expressed in all A549, NCI-H358, B16F10 and MC38 tumor models. Intratumoral injection of circular mRNA has the potential for widespread use in a variety of solid tumors.

Example 3 comparison of expression of circular mRNA in tumors with Linear mRNA

3.1 Experimental methods

For B16F10 tumor model mice, 10 μ g of nucleotide-modified linear mRNA and cyclic mRNA encoding firefly luciferase were intratumorally injected, and 6h, 24h, 48h, 72h and 96h later, the mice were intraperitoneally injected with fluorescein aqueous solution for in vivo fluorescence imaging, respectively. The expression of linear and circular Luciferase mRNA in the tumor was analyzed by fluorescence intensity.

3.2 results of the experiment

FIG. 3-1 is a comparison of circular versus linear mRNA expression in B16F10 tumor model mouse tumors. (A-E) in vivo fluorescence imaging plots of intratumoral injection of linear mice; (F-J) is a live fluorescence imaging image of the intratumoral injection ring-shaped mouse. As can be seen from FIG. 3-1, the intratumoral fluorescence was observed after 96h in mice injected with the circular Firefly Luciferase mRNA, while the intratumoral fluorescence was very weak or disappeared in mice injected with the linear Luciferase mRNA. Indicating that the circular mRNA is more stable and expressed for a longer time in the tumor relative to the linear mRNA.

FIG. 3-2 is a comparison of circular versus linear mRNA expression in MC38 tumor model mouse tumors. (A-E) shows in vivo fluorescence imaging plots of intratumorally injected linear mice; (F-J) is a graph of in vivo fluorescence imaging of intratumoral injected ring mice. As can be seen from FIG. 3-2, the circular mRNA was more stable in the tumor and expressed for a longer period of time than the linear mRNA.

Example 4 verification of the efficacy of the circular mRNA encoding cytokine mix for intratumoral injection

4.1 Experimental methods

TABLE 3

4.2 results of the experiment

FIG. 4 shows the B16F10 model tumor growth curves of the PBS Control injection group, the Luciferase Control injection group, the Cytokine mix group and the PD-1 group. As shown in fig. 4, the intratumoral injection of cyclic mRNA groups encoding various cytokines (cytokine mix group) showed the least tumor volume in all groups and significantly slower tumor growth rate.

Example 5 verification of the drug efficacy of the cyclic mRNA encoding cytokine mix administered intratumorally in combination with the PD-1 antibody System

5.1 Experimental methods

Based on the above example 4, the cyclic mRNA encoding cytokine mix was treated with PD-1 antibody to establish MC-38 colorectal cancer model and B16F10 melanoma model for experiments.

TABLE 4

5.2 results of the experiment

FIG. 5 shows graphs of MC38 model tumor growth in injection Control group, mRNA Control group, cytokine mix group, PD-1 group, combination group. From the results in FIG. 5, the MC38 colon cancer mouse model itself has a response rate to PD-1 antibody therapy, and the mean tumor volume is smaller and the tumor growth rate is significantly slowed down after the combined intratumoral injection of the cyclic mRNA encoding the cytokine. FIG. 6 shows the graphs of the tumor growth of the B16F10 model in the Control group, mRNA Control group, cytokine mix group, PD-1 group, combination group. According to the results of fig. 6, the B16F10 mouse melanoma model responded little to the PD-1 antibody treatment, and the tumor growth curve thereof was not significantly different from that of the control group. After the cyclic mRNA for coding the cytokine is injected into the tumor and is combined with the PD-1 antibody for administration, the B16F10 melanoma responds to the PD-1 antibody, and the two treatment means generate synergistic effect, so that the growth speed of the tumor is remarkably slowed down.

Example 6 Effect of intratumoral injection of circular mRNA on tumor immune infiltrating cells

6.1 Experimental methods

On the basis of the B16F10 model of examples 4 and 5 above, 3 mice were taken from each group, euthanized 24 hours after the second administration, and tumors, tumor-draining lymph nodes, and spleens were taken, prepared as single cell suspensions, and divided into two parts for cell death and viability staining and antibody labeling, respectively. First part marking: CD45, CD3, CD4, CD8, CD69. Second part marking: CD45, F4/80, CD11b. Analysis of CD45 Positive cell Rate after flow assay, and CD4 ⁺ T cells and CD8 ⁺ Activation of T cells.

6.2 results of the experiment

FIG. 7 shows the effect of intratumoral injection of circular mRNA encoding various cytokines (mix group) and combination treatment with PD-1 antibody (combination group) on immune cell infiltration. Wherein the group of cyclic mRNAs encoding various cytokines (mix group) and the group of combination therapy with PD-1 antibody (combination group) were intratumorally injected without causing lymph nodes and spleen CD8 at drainage sites as shown in A and B ⁺ Massive activation of T cells. However, at the tumor site, as shown by C, intratumoral injection of various cytokines resulted in more immune cell infiltration, and the immune cell proportion in the tumor was more significantly increased after treatment with the PD-1 antibody. In addition to the increase in the proportion of immune cells, the status of immune cells also appears to vary greatly. As shown in D and E, the cyclic mRNA group (mix) encoding various cytokines and the combination treatment group (combination group) with PD-1 antibody were intratumorally injected with tumor-infiltrated CD4 ⁺ T cells and CD8 ⁺ T cell activation levels were as high as 80%.

Sequence listing

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Lys His Tyr Ser Cys Thr Ala Glu Asp Ile Asp His Glu Asp Ile Thr

385 390 395 400

Arg Asp Gln Thr Ser Thr Leu Lys Thr Cys Leu Pro Leu Glu Leu His

405 410 415

Lys Asn Glu Ser Cys Leu Ala Thr Arg Glu Thr Ser Ser Thr Thr Arg

420 425 430

Gly Ser Cys Leu Pro Pro Gln Lys Thr Ser Leu Met Met Thr Leu Cys

435 440 445

Leu Gly Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Thr Glu Phe Gln

450 455 460

Ala Ile Asn Ala Ala Leu Gln Asn His Asn His Gln Gln Ile Ile Leu

465 470 475 480

Asp Lys Gly Met Leu Val Ala Ile Asp Glu Leu Met Gln Ser Leu Asn

485 490 495

His Asn Gly Glu Thr Leu Arg Gln Lys Pro Pro Val Gly Glu Ala Asp

500 505 510

Pro Tyr Arg Val Lys Met Lys Leu Cys Ile Leu Leu His Ala Phe Ser

515 520 525

Thr Arg Val Val Thr Ile Asn Arg Val Met Gly Tyr Leu Ser Ser Ala

530 535 540

<210> 3

<211> 190

<212> PRT

<213> Artificial Sequence

<220>

<223> polypeptide sequence

<400> 3

Met Arg Val Thr Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Gly Ser Cys Asp Leu Pro

20 25 30

His Thr Tyr Asn Leu Gly Asn Lys Arg Ala Leu Thr Val Leu Glu Glu

35 40 45

Met Arg Arg Leu Pro Pro Leu Ser Cys Leu Lys Asp Arg Lys Asp Phe

50 55 60

Gly Phe Pro Leu Glu Lys Val Asp Asn Gln Gln Ile Gln Lys Ala Gln

65 70 75 80

Ala Ile Leu Val Leu Arg Asp Leu Thr Gln Gln Ile Leu Asn Leu Phe

85 90 95

Thr Ser Lys Asp Leu Ser Ala Thr Trp Asn Ala Thr Leu Leu Asp Ser

100 105 110

Phe Cys Asn Asp Leu His Gln Gln Leu Asn Asp Leu Lys Ala Cys Val

115 120 125

Met Gln Glu Pro Pro Leu Thr Gln Glu Asp Ser Leu Leu Ala Val Arg

130 135 140

Thr Tyr Phe His Arg Ile Thr Val Tyr Leu Arg Lys Lys Lys His Ser

145 150 155 160

Leu Cys Ala Trp Glu Val Ile Arg Ala Glu Val Trp Arg Ala Leu Ser

165 170 175

Ser Ser Thr Asn Leu Leu Ala Arg Leu Ser Glu Glu Lys Glu

180 185 190

<210> 4

<211> 141

<212> PRT

<213> Artificial Sequence

<220>

<223> polypeptide sequence

<400> 4

Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val Tyr Ser Leu

1 5 10 15

Ser Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His

20 25 30

Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val

35 40 45

Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys

50 55 60

Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu

65 70 75 80

Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser

85 90 95

Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr

100 105 110

Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu

115 120 125

Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro Gly Gln Lys

130 135 140

<210> 5

<211> 550

<212> PRT

<213> Artificial Sequence

<220>

<223> polypeptide sequence

<400> 5

Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro

1 5 10 15

Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg

20 25 30

Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu

35 40 45

Val Asp Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala

50 55 60

Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val

65 70 75 80

Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu

85 90 95

Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg

100 105 110

Glu Leu Leu Asn Ser Met Gly Ile Ser Gln Pro Thr Val Val Phe Val

115 120 125

Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro

130 135 140

Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly

145 150 155 160

Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe

165 170 175

Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile

180 185 190

Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val

195 200 205

Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp

210 215 220

Pro Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser Val

225 230 235 240

Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu

245 250 255

Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu

260 265 270

Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val

275 280 285

Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr

290 295 300

Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser

305 310 315 320

Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile

325 330 335

Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr

340 345 350

Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe

355 360 365

Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val

370 375 380

Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly

385 390 395 400

Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly

405 410 415

Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His Phe

420 425 430

Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Tyr Gln

435 440 445

Val Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro Asn Ile

450 455 460

Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu

465 470 475 480

Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys

485 490 495

Glu Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu

500 505 510

Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly

515 520 525

Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys

530 535 540

Gly Gly Lys Ile Ala Val

545 550

<210> 6

<211> 2572

<212> RNA

<213> Artificial Sequence

<220>

<223> sequence of circular RNA molecule

<400> 6

aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60

cgcaauagcc gaaaaaacaa aaacaaaaaa aacaaaaaaa caaaaaaaaa accaaaacac 120

auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180

aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240

cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300

accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360

ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420

cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480

ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540

cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600

gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660

accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720

auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780

auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840

auuuuaauac ugaacaccgc aaaauggagg augccaagaa caucaagaag ggcccugccc 900

cuuucuaccc ucuggaggau ggcacagcug gagagcagcu gcacaaagcc augaagaggu 960

augcccuggu gccuggcacc auugccuuca cagaugccca cauugaggug gacaucaccu 1020

augcugagua cuuugagaug ucugugaggc uggcugaggc caugaagagg uauggccuga 1080

acaccaacca uaggauugug gugugcucug agaacagccu gcaguucuuc augccugugc 1140

ugggagcccu guucauugga guggcugugg ccccugccaa ugacaucuac aaugagaggg 1200

agcugcugaa cagcaugggc aucucucagc cuacaguggu cuuugugagu aaaaagggcc 1260

ugcagaagau ccugaaugug cagaagaagc ugccuaucau ucagaagauc aucaucaugg 1320

acagcaagac agacuaccaa ggcuuucaga gcauguacac cuuugugaca agccaccugc 1380

cuccuggcuu caaugaguau gacuuugugc cugagagcuu ugauagggac aagaccauug 1440

cccugaucau gaauagcucu ggcagcacug gccugccuaa gggaguggcc cugccucaua 1500

ggacagccug ugugagguuc agccaugcua gggacccuau cuuuggcaau cagaucaucc 1560

cugacacagc cauccugucu guggugcccu uccaucaugg cuuuggcaug uucaccaccc 1620

ugggcuaccu gaucuguggc uuuagggugg ugcugaugua uagguuugag gaggagcugu 1680

uccugaggag ccugcaagac uacaagauuc agucugcccu gcuggugccu acccuguuca 1740

gcuucuuugc caagagcacc cugauugaca aguaugaccu gagcaaccug caugagauug 1800

ccucuggagg agccccucuc agcaaagagg ugggagaggc uguggccaag agguuccacc 1860

ugccuggcau uaggcaaggc uauggccuga cagagaccac cucugccauc cugaucaccc 1920

cugagggaga ugacaagccu ggagcugugg gcaagguggu accauucuuu gaggccaagg 1980

ugguggaccu ggacacuggc aagacccugg gagugaauca gaggggagag cuguguguga 2040

ggggcccuau gaucaugucu ggcuauguga acaacccuga ggccaccaau gcccucauug 2100

acaaggaugg auggcugcac ucuggagaca uugccuacug ggaugaggau gagcacuucu 2160

ucauugugga uaggcugaag agccugauca aguacaaggg cuaccaagug gccccugcug 2220

agcuugagag cauccugcug cagcacccua acaucuuuga ugcuggagug gcuggcuugc 2280

cugaugauga ugcuggagag cugccugcug cugugguggu gcuggagcau ggcaagacca 2340

ugacagagaa ggagauugug gacuaugugg cuagccaagu gaccacagcc aagaagcuga 2400

ggggaggagu gguguuugug gaugaggugc cuaagggccu gacuggcaag cuggaugcua 2460

ggaagauuag ggagauccug aucaaggcca agaagggagg caagauugcu gugugaaaaa 2520

aacaaaaaac aaaacggcua uuaugcguua ccggcgagac gcuacggacu ua 2572

<210> 7

<211> 1681

<212> RNA

<213> Artificial Sequence

<220>

<223> sequence of circular RNA molecule

<400> 7

aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60

cgcaauagcc gaaaaaacaa aaacaaaaaa aacaaaaaaa caaaaaaaaa accaaaacac 120

auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180

aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240

cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300

accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360

ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420

cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480

ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540

cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600

gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660

accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720

auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780

auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840

auuuuaauac ugaacaccgc aaaauggccc cuaggagggc caggggcugc aggacccugg 900

gccugcccgc ccugcuucug cugcugcucc ugaggccccc ugccaccagg ggcgacuaca 960

aggacgacga cgauaagauc gagggcagga ucaccugccc ucccccuaug agcguggagc 1020

acgccgacau cugggugaag agcuacagcc uguacagcag ggagagguac aucugcaaca 1080

gcggcuucaa gaggaaggcc ggcaccagca gccugaccga gugcgugcug aacaaggcca 1140

ccaacguggc ccacuggacc acccccagcc ugaagugcau cagagacccc gcccuggugc 1200

accagaggcc cgccccuccc agcggcggca gcggcggcgg aggcagcggc ggcggcagcg 1260

gcggaggcgg cagccugcag aacuggguga acgugaucag cgaccugaag aagaucgagg 1320

accugaucca gagcaugcau auugaugcua cauuauauac cgaaagcgac gugcacccca 1380

gcugcaaggu gaccgccaug aagugcuucc ugcuggagcu gcaggugauc agccuggaga 1440

gcggcgacgc cagcauccac gacaccgugg agaaccugau cauccuggcc aacaacagcc 1500

ugagcagcaa cggcaaugug accgagagcg gcugcaagga gugcgaggag cuggaggaga 1560

agaacaucaa ggaguuccug cagagcuucg ugcacaucgu gcagauguuc aucaacacca 1620

gcugaaaaaa acaaaaaaca aaacggcuau uaugcguuac cggcgagacg cuacggacuu 1680

a 1681

<210> 8

<211> 2539

<212> RNA

<213> Artificial Sequence

<220>

<223> sequence of circular RNA molecule

<400> 8

aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60

cgcaauagcc gaaaaaacaa aaacaaaaaa aacaaaaaaa caaaaaaaaa accaaaacac 120

auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180

aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240

cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300

accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360

ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420

cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480

ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540

cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600

gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660

accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720

auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780

auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840

auuuuaauac ugaacaccgc aaaaugugcc accagcagcu ggugaucagc ugguucagcc 900

ugguguuccu ggccagcccc cugguggcca ucugggagcu gaagaaggac guguacgugg 960

uggagcugga cugguacccc gacgcccccg gcgagauggu ggugcugacc ugcgacaccc 1020

ccgaggagga cggcaucacc uggacccugg accagagcag cgaggugcug ggcagcggca 1080

agacccugac cauccaggug aaggaguucg gcgacgccgg ccaguacacc ugccacaagg 1140

gcggcgaggu gcugagccac agccugcugc ugcuccacaa gaaggaggac ggcaucugga 1200

gcaccgacau ccugaaggac cagaaggagc ccaagaacaa gaccuuccug aggugcgagg 1260

ccaagaacua cagcggcagg uucaccugcu gguggcugac caccaucagc accgaccuga 1320

ccuucagcgu gaagagcagc aggggcagca gcgaccccca gggcgugacc ugcggcgcug 1380

ccacauuguc ugcugaaagg guuagaggcg acaacaagga guacgaauac agcguggagu 1440

gccaggagga cagcgccugc cccgccgccg aggagagccu gcccaucgag gugauggugg 1500

acgccgugca caagcugaag uacgagaacu acaccagcag cuucuucauc agggacauca 1560

ucaagcccga cccucccaag aaccugcagc ugaagccccu gaagaacagc aggcaggugg 1620

aggugagcug ggaguacccc gacaccugga gcaccccuca cagcuacuuc agccugaccu 1680

ucugcgugca gguccagggc aagagcaagc gggagaagaa ggacagggug uucaccgaca 1740

agaccagcgc caccgugauc ugcaggaaga acgccagcau cagcgugagg gcccaggaca 1800

gguacuacag cagcagcugg agcgaguggg ccagcgugcc cugcagcggc agcagcggcg 1860

gcgggggcag ccccggcggc ggcagcagca ggaaccugcc cguggccacc ccugaucccg 1920

gaauguuccc uugccugcac cacagccaga accugcugag ggccgugagc aacaugcugc 1980

agaaggccag gcagacccug gaguucuacc ccugcaccag cgaggagauc gaccacgagg 2040

acaucaccaa ggacaagacc agcaccgugg aggccugucu gcccuuggag cugaccaaga 2100

acgagagcug ccugaacagc agggagacca gcuucaucac caacggcagc ugccuggcca 2160

gcaggaagac cagcuucaug auggcccugu gccugagcag caucuacgag gaccugaaga 2220

uguaccaggu ggaguucaag accaugaacg ccaagcugcu gauggacccc aagaggcaga 2280

ucuuccugga ccagaacaug cuggccguga ucgacgagcu gaugcaggcc cugaacuuca 2340

acagcgagac cgugccccag aagagcagcc uggaggagcc cgacuucuac aagaccaaga 2400

ucaagcugug cauccugcug cacgccuuca ggaucagagc cgugaccauc gacaggguga 2460

ugagcuaccu gaacgccagc ugaaaaaaac aaaaaacaaa acggcuauua ugcguuaccg 2520

gcgagacgcu acggacuua 2539

<210> 9

<211> 1486

<212> RNA

<213> Artificial Sequence

<220>

<223> sequence of circular RNA molecule

<400> 9

aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60

cgcaauagcc gaaaaaacaa aaacaaaaaa aacaaaaaaa caaaaaaaaa accaaaacac 120

auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180

aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240

cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300

accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360

ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420

cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480

ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540

cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600

gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660

accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720

auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780

auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840

auuuuaauac ugaacaccgc aaaauggccc ugaccuucgc ccugcuggug gcccugcugg 900

ugcugagcug caagagcagc ugcagcgugg gcugcgaccu gccccagacc cacagccugg 960

gcagcaggag gacccugaug cugcuggccc agaugaggag gaucagccug uucagcugcc 1020

ugaaggacag gcacgacuuc ggcuuccccc aggaggaguu cggcaaccag uuccagaagg 1080

ccgagaccau ccccgugcug cacgagauga uccagcagau cuucaaccug uucagcacca 1140

aggacagcag cgccgccugg gacgagaccc ugcuggacaa guucuacacc gagcuguacc 1200

agcagcugaa cgaccuggag gccugcguga uccagggcgu gggggugacc gagaccccuc 1260

ugaugaagga ggacagcauc cuggccguga ggaaguacuu ccagaggauc acccuguacc 1320

ugaaggagaa gaaguacagc cccugcgccu gggagguggu gagggccgag aucaugagga 1380

gcuucagccu gagcaccaac cugcaggaga gccugaggag caaggaguga aaaaaacaaa 1440

aaacaaaacg gcuauuaugc guuaccggcg agacgcuacg gacuua 1486

<210> 10

<211> 1354

<212> RNA

<213> Artificial Sequence

<220>

<223> sequence of circular RNA molecule

<400> 10

aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60

cgcaauagcc gaaaaaacaa aaacaaaaaa aacaaaaaaa caaaaaaaaa accaaaacac 120

auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180

aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240

cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300

accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360

ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420

cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480

ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540

cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600

gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660

accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720

auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780

auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840

auuuuaauac ugaacaccgc aaaauguggc ugcagagccu gcugcugcuc ggcaccgugg 900

ccugcagcau cagcgccccc gccagaagcc ccagcccuag cacccagccc ugggagcacg 960

ugaacgccau ccaggaggcc aggaggcugc ugaaccugag cagggacacc gccgccgaga 1020

ugaacgagac cguggaggug aucagcgaga uguucgaccu gcaggagccc accugccugc 1080

agaccaggcu ggagcuguac aagcagggcc ugaggggcag ccugaccaag cugaagggcc 1140

cccugaccau gauggccagc cacuacaagc agcacugccc ucccaccccc gagaccagcu 1200

gcgccaccca gaucaucacc uucgagagcu ucaaggagaa ccugaaggac uuccugcugg 1260

ugauccccuu cgacugcugg gagcccgugc aggagugaaa aaaacaaaaa acaaaacggc 1320

uauuaugcgu uaccggcgag acgcuacgga cuua 1354

<210> 11

<211> 741

<212> RNA

<213> Artificial Sequence

<220>

<223> IRES sequence

<400> 11

uuaaaacagc cuguggguug aucccaccca caggcccauu gggcgcuagc acucugguau 60

cacgguaccu uugugcgccu guuuuauacc cccuccccca acuguaacuu agaaguaaca 120

cacaccgauc aacagucagc guggcacacc agccacguuu ugaucaagca cuucuguuac 180

cccggacuga guaucaauag acugcucacg cgguugaagg agaaagcguu cguuauccgg 240

ccaacuacuu cgaaaaaccu aguaacaccg uggaaguugc agaguguuuc gcucagcacu 300

accccagugu agaucagguc gaugagucac cgcauucccc acgggcgacc guggcggugg 360

cugcguuggc ggccugccca uggggaaacc caugggacgc ucuaauacag acauggugcg 420

aagagucuau ugagcuaguu gguaguccuc cggccccuga augcggcuaa uccuaacugc 480

ggagcacaca cccucaagcc agagggcagu gugucguaac gggcaacucu gcagcggaac 540

cgacuacuuu ggguguccgu guuucauuuu auuccuauac uggcugcuua uggugacaau 600

ugagagaucg uuaccauaua gcuauuggau uggccauccg gugacuaaua gagcuauuau 660

auaucccuuu guuggguuua uaccacuuag cuugaaagag guuaaaacau uacaauucau 720

uguuaaguug aauacagcaa a 741

<210> 12

<211> 682

<212> RNA

<213> Artificial Sequence

<220>

<223> IRES sequence

<400> 12

uuaaaacagc cuguggguug cacccaccca cagggcccac agggcgcuag cacucuggua 60

ucacgguacc uuugugcgcc uguuuuauua ccccuucccc aauugaaaau uagaagcaau 120

gcacaccgau caacagcagg cguggcgcac cagucacguc ucgaucaagc acuucuguuu 180

ccccggaccg aguaucaaua gacugcucac gcgguugaag gagaaagugu ucguuauccg 240

gcuaaccacu ucgagaaacc caguaacacc augaaaguug caggguguuu cgcucagcac 300

uuccccagug uagaucaggu cgaugaguca ccgcguuccc cacgggcgac cguggcggug 360

gcugcguugg cggccugccu auggguuaac ccauaggacg cucuaauaca gacauggugc 420

gaagaguuua uugagcuggu uaguaucccu ccggccccug aaugcggcua auccuaacug 480

cggagcacgu gccuccaauc caggggguug caugucguaa cggguaacuc ugcagcggaa 540

ccgacuacuu uggguguccg uguuuccuuu uauucuuaua cuggcugcuu auggugacaa 600

ucgaggaauu guuaccauau agcuauugga uuggccaucc ggugucuaac agagcgauua 660

uauaccucuu uguuggauuu au 682

<210> 13

<211> 742

<212> RNA

<213> Artificial Sequence

<220>

<223> IRES sequence

<400> 13

uuaaaacagc cuguggguug aucccaccca cagggcccac ugggcgcuag cacucuggua 60

ucacgguacc uuugugcgcc uguuuuauac uuccuccccc aacugcaacu uagaaguaac 120

acaaaccgau caacagucag cguggcacac cagccacguu uugaucaaac acuucuguua 180

ccccggacug aguaucaaua gacugcucac gcgguugaag gagaaaacgu ucguuauccg 240

gccaacuacu ucgagaaacc uaguaacgcc auggaaguug uggaguguuu cgcucagcac 300

uaccccagug uagaucaggu ugaugaguca ccgcauuccc cacgggugac cguggcggug 360

gcugcguugg cggccugccc auggggaaac ccaugggacg cucuuauaca gacauggugc 420

gaagagucua uugagcuagu ugguaguccu ccggccccug aaugcggcua aucccaacug 480

cggagcauac acucucaagc cagaggguag ugugucguaa ugggcaacuc ugcagcggaa 540

ccgacuacuu uggguguccg uguuucauuu uauuccuaua cuggcugcuu auggugacaa 600

uugagagauu guuaccauau agcuauugga uuggccaucc ggugacuaac agagcuauua 660

uauaucuuuu uguuggguuu auaccacuua gcuugaaaga gguuaaaacu cuacauuaca 720

uuuuaauacu gaacaccgca aa 742

<210> 14

<211> 737

<212> RNA

<213> Artificial Sequence

<220>

<223> IRES sequence

<400> 14

uuaaaacagc cuguggguug aucccaccca cagggcccau ugggcgcuag cacucuggua 60

ucacgguacc cuugugcgcc uguuuuaugu cccuucccuc aacuguaacu uagaaguaac 120

gcacaccgau caacagucag cguggcacac cagccauguu uugaucaagc acuucuguua 180

ccccggaccg aguaucaaca gacugcucac gcgguugaag gagaaagugu ucguuauccg 240

gccaacuacu ucgaaaaacc uaguaacacc auggaaguug cagaguguuu cgcucagcac 300

uaccccagug uagaucaggu cgaugaguca ccgcaucccc cacgggcgac cguggcggug 360

gcugcguugg cggccugccu augggggaac ccauaggacg cucuaauaca gacauggugc 420

gaagagucca uugagcuagu ugguaguccu ccggccccug aaugcggcua auccuaacug 480

cggagcacac accuucaagc cagagggcag ugugucguaa cgggcaacuc ugcagcggaa 540

ccgacuacuu uggguguccg uguuucauuu uauucuuaua cuggcugcuu auggugacaa 600

uugagagauu guuaccauau agcuauugga uuggccaucc agugacuagc agagcuauua 660

uauaccucuu uguuggguuu auaccaccua auuugaaaga aguuaaaaca uuagaauuca 720

uuauuaaauu gaauaca 737

<210> 15

<211> 683

<212> RNA

<213> Artificial Sequence

<220>

<223> IRES sequence

<400> 15

uuaaaacagc cuguggguug cacccaccca cagggcccac agggcgcuag cacucuggua 60

ucacgguacc uuugugcgcc uguuuuauua ccccuucccc aauugaaaau uagaagcaau 120

gcacaccgau caacagcagg cguggcgcac cagucacguc ucgaucaagc acuucuguuu 180

ccccggaccg aguaucaaua gacugcucac gcgguugaag gagaaagugu ucguuauccg 240

gcuaaccacu ucgagaaacc caguaacacc augaaaguug caggguguuu cgcucagcac 300

uuccccagug uagaucaggu cgaugaguca ccgcguuccc cacgggcgac cguggcggug 360

gcugcguugg cggccugccu auggguuaac ccauaggacg cucuaauaca gacauggugc 420

gaagaguuua uugagcuggu uaguaucucc uccggccccu gaaugcggcu aauccuaacu 480

gcggagcaca cacccucaag ccagagggca gugugucgua acgggcaacu cugcagcgga 540

accgacuacu uugggugucc guguuuccuu uuauucuuau acuggcugcu uauggugaca 600

aucgaggaau uguuaccaua uagcuauugg auuggccauc cggugucuaa cagagcgauu 660

auauaccucu uuguuggauu uau 683

<210> 16

<211> 742

<212> RNA

<213> Artificial Sequence

<220>

<223> IRES sequence

<400> 16

uuaaaacagc cuguggguug aucccaccca cagggcccac ugggcgcuag cacucuggua 60

ucacgguacc uuugugcgcc uguuuuauac uuccuccccc aacugcaacu uagaaguaac 120

acaaaccgau caacagucag cguggcacac cagccacguu uugaucaaac acuucuguua 180

ccccggacug aguaucaaua gacugcucac gcgguugaag gagaaaacgu ucguuauccg 240

gccaacuacu ucgagaaacc uaguaacgcc auggaaguug uggaguguuu cgcucagcac 300

uaccccagug uagaucaggu ugaugaguca ccgcauuccc cacgggugac cguggcggug 360

gcugcguugg cggccugccc auggggaaac ccaugggacg cucuuauaca gacauggugc 420

gaagagucua uugagcuagu ugguaguccu ccggccccug aaugcggcua auccuaacug 480

cggagcacac acccucaagc cagagggcag ugugucguaa cgggcaacuc ugcagcggaa 540

ccgacuacuu uggguguccg uguuucauuu uauuccuaua cuggcugcuu auggugacaa 600

uugagagauu guuaccauau agcuauugga uuggccaucc ggugacuaac agagcuauua 660

uauaucuuuu uguuggguuu auaccacuua gcuugaaaga gguuaaaacu cuacauuaca 720

uuuuaauacu gaacaccgca aa 742

<210> 17

<211> 737

<212> RNA

<213> Artificial Sequence

<220>

<223> IRES sequence

<400> 17

uuaaaacagc cuguggguug aucccaccca cagggcccau ugggcgcuag cacucuggua 60

ucacgguacc cuugugcgcc uguuuuaugu cccuucccuc aacuguaacu uagaaguaac 120

gcacaccgau caacagucag cguggcacac cagccauguu uugaucaagc acuucuguua 180

ccccggaccg aguaucaaca gacugcucac gcgguugaag gagaaagugu ucguuauccg 240

gccaacuacu ucgaaaaacc uaguaacacc auggaaguug cagaguguuu cgcucagcac 300

uaccccagug uagaucaggu cgaugaguca ccgcaucccc cacgggcgac cguggcggug 360

gcugcguugg cggccugccu augggggaac ccauaggacg cucuaauaca gacauggugc 420

gaagagucca uugagcuagu ugguaguccu ccggccccug aaugcggcua auccuaacug 480

cggagcacac acccucaagc cagagggcag ugugucguaa cgggcaacuc ugcagcggaa 540

ccgacuacuu uggguguccg uguuucauuu uauucuuaua cuggcugcuu auggugacaa 600

uugagagauu guuaccauau agcuauugga uuggccaucc agugacuagc agagcuauua 660

uauaccucuu uguuggguuu auaccaccua auuugaaaga aguuaaaaca uuagaauuca 720

uuauuaaauu gaauaca 737

<210> 18

<211> 16

<212> RNA

<213> Artificial Sequence

<220>

<223> first exon sequence

<400> 18

agacgcuacg gacuua 16

<210> 19

<211> 51

<212> RNA

<213> Artificial Sequence

<220>

<223> second exon sequence

<400> 19

aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc a 51

<210> 20

<211> 50

<212> RNA

<213> Artificial Sequence

<220>

<223> 5' spacer sequence

<400> 20

aaaaaacaaa aacaaaaaaa acaaaaaaac aaaaaaaaaa ccaaaacaca 50

<210> 21

<211> 50

<212> RNA

<213> Artificial Sequence

<220>

<223> 5' spacer sequence

<400> 21

aaaaacaaaa aacaaaaaaa aaaccaaaaa aacaaaaaaa acaaaacaca 50

<210> 22

<211> 25

<212> RNA

<213> Artificial Sequence

<220>

<223> 3' spacer sequence

<400> 22

aaaaaaacaa aaaaacaaaa caaac 25

<210> 23

<211> 22

<212> RNA

<213> Artificial Sequence

<220>

<223> 3' spacer sequence

<400> 23

aaaaacaaaa aacaaaacaa ac 22

<210> 24

<211> 114

<212> DNA

<213> Artificial Sequence

<220>

<223> 5' Intron sequence

<400> 24

aataattgag ccttaaagaa gaaattcttt aagtggatgc tctcaaactc agggaaacct 60

aaatctagtt atagacaagg caatcctgag ccaagccgaa gtagtaatta gtaa 114

<210> 25

<211> 131

<212> DNA

<213> Artificial Sequence

<220>

<223> 3' Intron sequence

<400> 25

aacaatagat gacttacaac taatcggaag gtgcagagac tcgacgggag ctaccctaac 60

gtcaagacga gggtaaagag agagtccaat tctcaaagcc aataggcagt agcgaaagct 120

gcaagagaat g 131

<210> 26

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> 5' homology arm sequence

<400> 26

accgtcagtt gctcactgtg c 21

<210> 27

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> 5' homology arm sequence

<400> 27

accgtgctat gtccacgtgt c 21

<210> 28

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> 3' homology arm sequence

<400> 28

gcacagtgag caactgacgg a 21

<210> 29

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> 3' homology arm sequence

<400> 29

gacacgtgga catagcacgg a 21