WO2023165477A1 - Expression cassette of interleukin 1 receptor antagonist protein and aav-based gene delivery system - Google Patents

Abstract

The present invention relates to a nucleic acid molecule encoding an interleukin 1 receptor antagonist protein, a transgenic expression cassette comprising the nucleic acid molecule, a gene delivery system comprising the transgenic expression cassette and use thereof. The transgenic expression cassette and the gene delivery system of the present invention can be used to treat various diseases that can be improved by blocking the IL-1 signaling pathway, including malignant tumors and inflammatory diseases, such as arthritis, particularly osteoarthritis.

Description

Expression cassette of interleukin-1 receptor antagonist protein and AAV-based gene delivery system technical field

The present disclosure relates to a nucleic acid molecule encoding an interleukin-1 receptor antagonist protein, a transgene expression cassette comprising the nucleic acid molecule, a gene delivery system comprising the transgene expression cassette and applications thereof.

Background technique

Interleukin-1 (IL-1) is a pleiotropic cytokine that mainly affects inflammation and immune response, and also regulates other physiological functions of the body, which has an important impact on the pathogenesis of diseases. IL-1 levels are elevated in acute inflammatory diseases (such as septic shock), chronic inflammatory diseases (such as rheumatoid arthritis) and malignant tumors. IL-1 is secreted by macrophages and initiates the inflammatory response. In addition, studies have shown that IL-1 plays an important role in tumorigenesis (Krelin Y et al., Cancer Res, 2007;67(3):1062-1071). The IL-1 family mainly includes IL-1α and IL-1β. IL-1β is not produced under normal physiological conditions, and is only secreted under inflammatory signals; while IL-1α exists in the cytoplasm and on the cell membrane under normal physiological conditions, and its expression level increases when inflammation occurs (WU X et al., Chinese journal of lung cancer, 2010; 13(12):1145-1148).

Interleukin 1 receptor antagonist protein (IL-1Ra or IL1RA) is a naturally occurring IL-1β inhibitory protein molecule, which can bind to the IL1 receptor on the cell surface without initiating signal transduction, thereby competitively blocking the IL1 signaling pathway . The cDNA encoding IL-1Ra is introduced into the cells of the target tissue of the patient to achieve the expression of the target protein IL-1Ra, thereby playing a therapeutic role. Studies have shown that recombinant IL-1Ra can reduce the volume of mouse tumors and inhibit tumor-mediated neovascularization (Bar D et al., FASEB J., 2004; 18(1):161-163). Anakinra is a recombinant IL-1Ra that has been marketed for the treatment of osteoarthritis (OA), but due to the short half-life of the drug, it is difficult to maintain an effective therapeutic concentration, so its therapeutic effect on OA is not ideal. In addition, repeated injections will also bring a lot of inconvenience and pain to patients.

Osteoarthritis (OA) is a common joint disease. According to statistics, males account for about 10% of the elderly population over 60 years old, while females account for about 18%. Clinically, the main manifestations are joint pain, stiffness, severe and even complete loss of joint function, which brings a heavy burden to the patient's family and society. human and financial burden. The pathogenesis of osteoarthritis is complex and diverse. Studies have shown that the disease is caused by multiple factors such as genetics, biology (including aging, inflammation, etc.) and biomechanics. So far, there is no effective treatment .

The most common symptom in the pathogenesis of osteoarthritis is synovial inflammation. Joint inflammation can cause the production of proinflammatory factors such as interleukin 1 (IL-1) and tumor necrosis factor (TNFα), which in turn aggravate the occurrence and development of OA. Although the pathogenesis of OA has not been elucidated, interleukin-1 plays a key role in the development of osteoarthritis as a pro-inflammatory factor. in the joint, IL-1β is synthesized by chondrocytes, osteoblasts, synoviocytes and monocytes, and exerts its effect by binding to the membrane receptor IL-1 receptor (IL-1R). In addition, IL‐1β reduces key extracellular matrix proteins (type II collagen and proteoglycan ) and stimulate the production of matrix-degrading proteases (MMP-3 and ADAMTS-4). IL-1β can also induce apoptosis of chondrocytes by up-regulating Bcl-2 protein family members in chondrocytes, mitochondrial depolarization and production of reactive oxygen species. Therefore, blocking the signaling pathway of IL-1 has become a popular target for the research and treatment of OA.

Currently, many viral vectors have been used in gene therapy, including adenovirus, retrovirus, lentivirus, and adeno-associated virus (AAV). However, retroviruses, lentiviruses, and adenoviruses are not suitable for large-scale production applications due to safety and technical considerations, and AAV with low immunogenicity has been widely recognized. AAV has good safety and high infection efficiency, as well as the characteristics of mediating long-term gene expression. At present, AAV-mediated gene therapy drugs have been approved, and AAV vectors are ideal gene therapy tools.

Although AAV has low immunogenicity, it is often reported clinically that systemic administration of AAV will trigger a severe immune response, which is mostly caused by the use of higher doses. For OA, local injection of viral vectors into the joint cavity can effectively avoid adverse reactions caused by systemic administration. By increasing the level of transcribed protein expression, the amount of viral vector can be reduced and high levels of foreign gene expression can be maintained.

Therefore, in order to more effectively block the signaling pathway of IL1, so as to achieve a better therapeutic effect on malignant tumors and/or inflammatory diseases (such as joint inflammation, especially osteoarthritis), it is expected to obtain IL-1 with a higher expression level. 1Ra coding sequence and expression cassette.

Contents of the invention

In order to solve the above technical problems, in the first aspect, the present disclosure provides a nucleic acid molecule encoding an interleukin 1 receptor antagonist protein, the nucleotide sequence of which is the same as that shown in SEQ ID NO: 7 or SEQ ID NO: 8 The sequences are at least 50% identical, preferably at least 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical.

In one embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8. In a preferred embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 7 or SEQ ID NO: 8.

The nucleic acid molecule encoding the human interleukin 1 receptor antagonist protein of the present disclosure comprises a codon-optimized human interleukin 1 receptor antagonist protein coding nucleic acid sequence (SEQ ID NO: 7 or SEQ ID NO: 8), and an uncoded Compared with the original human interleukin 1 receptor antagonist protein encoding nucleic acid sequence (SEQ ID NO: 6) optimized by codon, the human interleukin 1 receptor antagonist protein encoding nucleic acid sequence (SEQ ID NO: 7 or SEQ ID NO: 7) through codon optimization NO: 8) has a higher white Interlein 1 receptor antagonist protein expression level.

In the second aspect, the present disclosure provides a transgene expression cassette, which includes: a promoter, the nucleic acid molecule according to the first aspect, and polyA.

In a preferred embodiment, polyA is bGH polyA (SEQ ID NO: 2).

In one embodiment, the promoter is selected from: CB promoter, CAG promoter, SV40 promoter, collagen collagen promoter, proteoglycan aggrecan promoter, synoviolin gene promoter, adipose tissue specific promoter PPAR white gene promoter, transcription factor SOX9 promoter and osteocalcin promoter. In a preferred embodiment, the promoter is a CB promoter (SEQ ID NO: 1).

In one embodiment, the transgene expression cassette further includes: an intron located behind the promoter (abbreviated as an intron after the promoter), and/or two ITRs located at both ends, the two ITRs are each independently Normal ITR or shortened ITR. In a preferred embodiment, the intron behind the promoter is selected from the group consisting of: SV40 intron (SEQ ID NO: 3), VH4 intron (SEQ ID NO: 4), Chi intron (SEQ ID NO : 5), U12 intron, RHD intron and other class 1 introns. In a more preferred embodiment, the intron following the promoter is SV40 or VH4.

In a preferred embodiment, the promoter is a CB promoter, and the intron behind the promoter is VH4. In a preferred embodiment, the promoter is a CB promoter, and the intron behind the promoter is SV40.

In one embodiment, the transgene expression cassette further comprises: at least one (eg, 1 or 2) introns inserted into the nucleic acid molecule according to the first aspect. In a preferred embodiment, the inserted intron is selected from the group consisting of: SV40 intron, VH4 intron and Chi intron. The inventors found that inserting such an intron can better enhance the expression and secretion of the target protein (interleukin-1 receptor antagonist protein).

In one embodiment, the nucleotide sequence of the transgenic expression cassette is such as SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16 , SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, preferably SEQ ID NO: 12 and SEQ ID NO: 19, more preferably SEQ ID NO: 19.

In a third aspect, the present disclosure provides a gene delivery system, which includes: the transgene expression cassette according to the second aspect and AAV capsid protein.

In one embodiment, the above-mentioned AAV capsid protein is a natural AAV capsid protein or an artificially modified AAV capsid protein. In a preferred embodiment, the AAV is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ and AAV843. In a more preferred embodiment, the AAV capsid protein is AAV843, and its amino acid sequence is shown in SEQ ID NO: 24.

The transgene expression cassette and gene delivery system of the present disclosure can express higher levels of interleukin-1 receptor antagonist protein (IL1RA), thereby blocking the signaling pathway of IL-1, and achieving better therapeutic effects on malignant tumors and inflammatory diseases (such as joint inflammation, especially osteoarthritis). In addition, the gene delivery system of the present disclosure can achieve long-term stable delivery of therapeutic proteins.

In a fourth aspect, the present disclosure provides the use of the nucleic acid molecule according to the first aspect, the transgene expression cassette according to the second aspect, or the gene delivery system according to the third aspect in the preparation of a drug for treating a disease , the disease is a disease that can be improved by blocking IL-1 signaling pathway, such as acute inflammatory disease, chronic inflammatory disease and malignant tumor.

In a preferred embodiment, the disease is joint inflammation, including osteoarthritis (OA), rheumatoid arthritis (RA), synovitis, hemophilic arthritis and other inflammatory joint inflammations.

In a more preferred embodiment, the disease is osteoarthritis (OA).

In the fifth aspect, the present disclosure provides a medicine, which comprises: the nucleic acid molecule according to the first aspect, the transgene expression cassette according to the second aspect, or the gene delivery system according to the third aspect; and excipient agent.

In one embodiment, excipients include salts, organics and/or surfactants.

In one embodiment, the medicament is used to treat a disease that can be improved by blocking the IL-1 signaling pathway. In a preferred embodiment, the disease is an acute inflammatory disease, a chronic inflammatory disease and/or a malignant tumor. In a more preferred embodiment, the disease is joint inflammation, including joint inflammation caused by osteoarthritis (OA), rheumatoid arthritis (RA), synovitis, hemophilic arthritis and other inflammatory conditions.

In a sixth aspect, the present disclosure provides a method for treating a disease, comprising administering a therapeutically effective amount of the drug according to the fifth aspect to a subject in need, the disease being selected from acute inflammatory diseases, chronic inflammatory diseases and Malignant tumor.

In a preferred embodiment, the disease is joint inflammation, including osteoarthritis (OA), rheumatoid arthritis (RA), synovitis, hemophilic arthritis and other inflammatory joint inflammations.

In a more preferred embodiment, the disease is osteoarthritis (OA).

In one embodiment, the drug is administered systemically or locally, such as intravenous administration, topical contact, and intralesional administration, preferably locally to the joint cavity, such as intra-articular injection.

In a seventh aspect, the present disclosure provides an engineered AAV capsid protein (AAV843), wherein the amino acid sequence of the AAV capsid protein is shown in SEQ ID NO: 24.

In an eighth aspect, the present disclosure provides a nucleic acid molecule encoding the AAV843 capsid protein according to the seventh aspect. In one embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 23.

In one embodiment, an AAV vector packaged with an AAV capsid protein of the present disclosure comprises an exogenous polynucleotide comprising a nucleotide sequence encoding a therapeutic protein. In one embodiment, the therapeutic protein is a protein that blocks the IL1 signaling pathway. In one embodiment, the therapeutic protein is interleukin 1 receptor antagonist protein. In one embodiment, the exogenous polynucleotide comprises a nucleotide sequence encoding an interleukin-1 receptor antagonist protein.

Description of drawings

Figure 1 is a schematic diagram of the B93, B94, B95, B96, B97, B98, B126, B127, B128, B129, B130, B131, B132 and B133 expression cassettes.

Fig. 2A shows the expression level of IL1RA in the cell secreted supernatant after transfection of B94, B95, B96, B97 and B98 expression cassettes to Huh7 cells for 72 hours, and the results of ELISA detection. n=3, *p<0.05, t-test.

Figure 2B shows the expression level of IL1RA in the cell secreted supernatant after B94, B95, B96, B97 and B98 expression cassettes were packaged with AAV843 capsid and infected Huh7 cells for 72 hours, MOI=7E+5vg/cell, ELISA detection results. n=3, **p<0.01, t-test.

Figure 2C shows the expression level of IL1RA in mouse serum after 3 weeks after B93, B94, B95, B96, B97, and B98 expression cassettes were packaged with AAV843 capsids, and the mice were injected with the virus through the tail vein. The dose was 1E+13vg/kg , ELISA test results. n=3, *p<0.05, t-test.

Figure 2D shows the expression level of IL1RA in the cell secreted supernatant after the B126, B127, B128, B129, B130, B131, B132 and B133 expression cassettes were transfected to Huh7 cells for 72 hours, and the ELISA detection results. n=3, *p<0.05, t-test.

Figure 2E shows the expression level of IL1RA in the cell secreted supernatant after the B126, B127, B128, B129, B130, B131, B132 and B133 expression cassettes were packaged with AAV843 capsids and infected with Huh7 cells for 72 hours, MOI=7E+5vg/cell, ELISA test results. n=3, *p<0.05, t-test.

Figure 2F shows the expression level of IL1RA in the mouse serum after 3 weeks after B126, B127, B128, B129, B130, B131, B132 and B133 expression cassettes were packaged with AAV843 capsid, and the virus was injected into the tail vein of mice, with a dose of 1E +13vg/kg, ELISA test result. n=3, *p<0.05, t-test.

Figure 2G shows the expression level of IL1RA in mouse serum after 4 weeks after B126, B127, B128, B129, B130, B131, B132, and B133 expression cassettes were packaged with AAV843 capsids, and the virus was injected into the tail vein of mice at a dose of 1E +13vg/kg, ELISA test result. n=3, *p<0.05, t-test.

Fig. 2H shows the comparison results of IL1RA expression levels of B94, B96, B127, B130 expression cassettes. After the expression cassette was packaged with AAV843 capsid and infected Huh7 cells for 72 hours, the expression level of IL1RA in the cell secretion supernatant was measured, MOI=7E+5vg/cell, and the results were detected by ELISA. n=3, **p<0.01, t-test.

Figure 3A shows the effects of B93, B94, B95, B96, B97 and B98 expression cassettes on IL1β-induced inflammatory factors and soft Inhibition of elevated levels of extraosseous matrix-degrading proteases. The expression cassette was packaged with AAV843 capsid and pre-infected C28/I2 cells (MOI=7E+5vg/cell). After IL1β induction for 48h, the mRNA expression levels of IL1, IL6, TNFα and MMP13 were detected by qPCR. n=3, *p<0.05, **p<0.01, t-test.

Figure 3B shows the inhibitory effects of B126, B127, B128, B129, B130, B131, B132 and B133 expression cassettes on IL1β-induced increase in the levels of inflammatory factors and extrachondral matrix degrading proteases. The expression cassette was packaged with AAV843 capsid and pre-infected C28/I2 cells (MOI=7E+5vg/cell). After IL1β induction for 48 hours, the mRNA expression levels of IL1, IL6, TNFα and MMP13 were detected by qPCR. n=3, *p<0.05, t-test.

Figure 4 shows a rat model of osteoarthritis induced by shearing of the cruciate ligament and removal of part of the meniscus. Two weeks after modeling, SD rats were injected with AAV particles at a dose of 1×10 ¹¹ vg/joint and 1×10 ¹² vg/joint respectively. The particles had AAV843 capsid and packaged B130 expression cassette. At 0, 2, 4, and 8 weeks after virus injection, the joint swelling of the rats was measured. Blood was collected at 2, 4, and 8 weeks after virus injection to measure the inflammation level of rat serum. Eight weeks after virus injection, the lesioned joints of the rats were scanned and photographed by micro-CT. Finally, the rats were euthanized, and the joints were taken for paraffin sections for immunohistochemistry, and mRNA was extracted for qPCR determination.

Figure 5A shows the measurement results of rat joint swelling at 0, 2, 4, and 8 weeks after intra-articular injection of AAV virus particles (AAV843-B130) with AAV843 capsid and packaged B130 expression cassette into the OA rat model, n=4.

Fig. 5B shows the micro-CT scanning results at week 8 after intra-articular injection of AAV843-B130 virus particles into the OA rat model, n=3.

Figure 5C shows the statistical graph of bone parameter analysis of micro-CT scans. Mean±s.d., n=3, *P<0.05.

Figure 6 shows the results of ELISA detection of inflammation levels in serum of rats at 2, 4, and 8 weeks after intra-articular injection of AAV843-B130 virus particles into the OA rat model, n=4.

Figure 7 shows the results of pathological sections of the joints of the rats after intra-articular injection of AAV843-B130 virus particles into the OA rat model. Safranin-O and HE staining, n = 3, scale bar: 200 μm.

Figure 8 shows the results of qPCR detection of the expression levels of target proteins, inflammatory factors, degrading enzymes and cartilage matrix proteins by extracting mRNA from the joints at 8 weeks after intra-articular injection of AAV843-B130 virus particles into the OA rat model. n=3, *p<0.05, **p<0.01, t-test. LD: low dose; HD: high dose.

Figure 9A and Figure 9B show the safety evaluation of the OA rat model injected with AAV843-B130 virus particles intra-articularly. n=3, *p<0.05, t-test.

Figure 10 shows codon-optimized human interleukin-1 receptor antagonist protein encoding nucleic acid optimized sequence one (SEQ ID NO: 7).

Figure 11 shows codon-optimized human interleukin 1 receptor antagonist protein encoding nucleic acid optimized sequence two (SEQ ID NO: 8).

Figure 12 shows the nucleotide sequence (SEQ ID NO: 10) of the B94 expression cassette.

Figure 13 shows the nucleotide sequence (SEQ ID NO: 11) of the B95 expression cassette.

Figure 14 shows the nucleotide sequence (SEQ ID NO: 12) of B96 expression cassette.

Figure 15 shows the nucleotide sequence (SEQ ID NO: 13) of the B97 expression cassette.

Figure 16 shows the nucleotide sequence (SEQ ID NO: 14) of the B98 expression cassette.

Figure 17 shows the nucleotide sequence (SEQ ID NO: 16) of B127 expression cassette.

Figure 18 shows the nucleotide sequence (SEQ ID NO: 17) of the B128 expression cassette.

Figure 19 shows the nucleotide sequence (SEQ ID NO: 18) of the B129 expression cassette.

Figure 20 shows the nucleotide sequence (SEQ ID NO: 19) of the B130 expression cassette.

Figure 21 shows the nucleotide sequence (SEQ ID NO: 20) of the B131 expression cassette.

Figure 22 shows the nucleotide sequence (SEQ ID NO: 21) of the B132 expression cassette.

Figure 23 shows the nucleotide sequence (SEQ ID NO: 22) of the B133 expression cassette.

Figure 24 shows the nucleotide sequence (SEQ ID NO: 23) encoding AAV843 capsid protein.

Figure 25 shows the amino acid sequence of the AAV843 capsid protein (SEQ ID NO: 24).

Detailed ways

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

Unless otherwise indicated, nucleic acid or polynucleotide sequences listed herein are in single-stranded form, oriented 5' to 3', left to right. Nucleotides and amino acids are presented herein using the format recommended by the IUPACIUB Biochemical Nomenclature Commission, using either the single-letter code or the three-letter code for the amino acids.

Unless otherwise stated, "polynucleotide" is synonymous with "nucleic acid" and refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, mixed sequences thereof, or the like. A polynucleotide may include modified nucleotides, such as methylated or restricted nucleotides and nucleotide analogs.

As used herein, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (ie, meaning "including but not limited to").

As used herein, the terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to an organism suffering from or susceptible to a condition that can be prevented or treated by administering the medicaments of the present disclosure, and Humans and non-human animals (eg, rodents or other mammals) are included.

In one embodiment, the subject is a non-human animal (e.g., chimpanzees and other ape and monkey species; farm animals, Such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; chickens and other chickens, ducks, geese, etc.). In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

As used herein, the term "treating" includes: (1) inhibiting the condition, disease or disorder, i.e., arresting, reducing or delaying the development of the disease or its recurrence or the development of at least one clinical or subclinical symptom thereof; or (2) Ameliorating a disease, ie, causing regression of at least one of a condition, disease or disorder, or clinical or subclinical symptoms thereof.

As used herein, the term "therapeutically effective amount" refers to a dose that produces the therapeutic effect for which it is administered. For example, a therapeutically effective amount of a drug suitable for treating an inflammatory disease of the joint may be an amount capable of preventing or ameliorating one or more symptoms associated with the inflammatory disease of the joint.

As used herein, the term "amelioration" refers to an amelioration of a symptom associated with a disease, and may refer to an amelioration of at least one parameter that measures or quantifies the symptom.

As used herein, the term "preventing" a condition, disease or disorder includes preventing, delaying or reducing the incidence and/or likelihood of the occurrence of at least one clinical or subclinical symptom of a developing condition, disease or disorder in a subject, The subject may suffer from or be susceptible to the condition, disease or disorder but has not experienced or exhibited clinical or subclinical symptoms of the condition, disease or disorder.

As used herein, the term "topical administration" or "local route" refers to administration with a local effect.

As used herein, the terms "transduction," "transfection," and "transformation" refer to the process of delivering exogenous nucleic acid into a host cell, followed by transcription and translation of the polynucleotide product, which involves the transfer of exogenous A source polynucleotide is introduced into a host cell.

Herein, the term "gene delivery" refers to the introduction of exogenous polynucleotides into cells for gene delivery, including targeting, binding, uptake, transport, replicon integration and expression.

As used herein, the term "gene expression" or "expression" refers to the process of gene transcription, translation and post-translational modification to produce the gene's RNA or protein product.

As used herein, the term "infection" refers to the process by which a virus or virus particle comprising a polynucleotide component delivers a polynucleotide into a cell and produces its RNA and protein products, and may also refer to the process of virus replication in a host cell .

As used herein, the term "vector" refers to one or a series of macromolecules that encapsulate a polynucleotide, which facilitates the delivery of the polynucleotide to target cells in vitro or in vivo. Types of vectors include, but are not limited to, plasmids, viral vectors, liposomes, and other gene delivery vehicles. The polynucleotides to be delivered are sometimes referred to as "expression cassettes" or "transgenic expression cassettes," which may include, but are not limited to, certain proteins or synthetic polypeptides (which can enhance, inhibit, weaken, protect, trigger, or prevent certain biological biological and physiological functions), coding sequences of interest in vaccine development (e.g. expressing immune response proteins, polynucleotides of polypeptides or peptides), coding sequences of RNAi materials (eg, shRNA, siRNA, antisense oligonucleotides), or alternative biomarkers.

Herein, the terms "expression cassette", "transgene cassette" and "transgene expression cassette" are used interchangeably to refer to a polynucleotide fragment encoding a specific protein, polypeptide or RNAi element, which can be cloned into a plasmid vector.

In some embodiments, a "cassette" can also be packaged into an AAV particle and used as the viral genome to deliver the transgene product into target cells. The "cassette" may also include other regulatory elements, such as specific promoters/enhancers, polyA, introns, etc., to enhance or attenuate the expression of the transgene product.

In one embodiment, the transgene cassette contains a number of regulatory elements to enable packaging of the transgene into the virus, such as a normal ITR of 145 bp, a shortened ITR of approximately 100 bp in length, in addition to the sequence encoding the protein product. In some embodiments, the transgenic cassette further comprises polynucleotide elements for controlling expression of the protein product, such as origins of replication, polyadenylation signals, promoters, and enhancers (e.g., with vertebrate β-actin, β - CMV promoters of globulin or β-globin regulatory elements or other hybrid CMV promoters (called CB and CAG promoters, SV40 promoter). Promoters and enhancers can be activated by chemicals or hormones (such as doxycycline or tamoxifen) to ensure gene expression at specific time points. Furthermore, promoters and enhancers may be natural or artificial or chimeric sequences, ie prokaryotic or eukaryotic sequences.

In some preferred embodiments, the inducible regulatory element for gene expression may be a tissue or organ-specific promoter or enhancer, including but not limited to: promoters specific to various types of joint tissue cells, For example, articular chondrocyte lineage-specific promoters (e.g., collagen promoter, aggrecan promoter), synoviolin gene promoters, adipose tissue-specific promoters (e.g., PPAR white gene promoter), osteoprogenitor cell-specific promoters promoters (such as the transcription factor SOX9 promoter), and specific promoters of the osteoblast lineage (such as the osteocalcin promoter).

As used herein, the term "inverted terminal repeat (ITR)" includes any AAV viral terminal repeat or synthetic sequence that forms a hairpin structure and acts as a cis element to mediate viral replication, packaging and integration. ITRs herein include, but are not limited to, terminal repeats from AAV types 1-11 (avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV). Furthermore, the AAV terminal repeats need not have native terminal repeats, so long as the terminal repeats are available for viral replication, packaging and integration.

As used herein, the term "cis-element" refers to a transgene cassette packaged in AAV particles and expressed in target cells to produce a therapeutic protein product.

As used herein, the term "codon-optimized" refers to a polynucleotide sequence modified from its native form. Such modifications result in differences of one or more base pairs, with or without changes in the corresponding amino acid sequences, which may enhance or inhibit the Expression of the gene and/or cellular response to the modified polynucleotide sequence.

Those skilled in the art know that the AAV capsid protein contains VP1, VP2 and VP3 proteins, and the VP2 and VP3 proteins undergo transcription and translation at the start codon inside the VP1 protein, that is, the VP1 sequence contains the VP2 and VP3 sequences.

In one embodiment, the AAV capsid protein can be any AAV serotype capsid protein, including native AAV capsid proteins (e.g., native AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV). AAV capsid protein) and other engineered AAV capsid proteins (eg, engineered capsid proteins of types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV). The genome sequences, ITR sequences, Rep and Cap proteins of different AAV serotypes are known in the art. These sequences can be found in the literature or in public databases, such as the GenBank database.

In one embodiment, the present disclosure provides a therapeutic tool that can antagonize the IL1 signaling pathway, which can be used to treat various diseases with related pathological mechanisms, including but not limited to: malignant tumors and inflammatory diseases (including acute inflammatory diseases and chronic Inflammatory diseases, such as joint inflammatory diseases, such as osteoarthritis, rheumatoid arthritis, synovitis, hemophilic arthritis or other inflammation-induced joint inflammatory diseases).

In one embodiment, the protein product of the therapeutic tool (such as a transgene expression cassette) includes a protein that antagonizes the IL1 signaling pathway, such as but not limited to, IL-1Ra, recombinant IL-1R soluble receptor, anti-IL-1 antibody.

The inventors found that AAV vectors with the AAV843 capsid (SEQ ID NO: 24) exhibit high transduction efficiency in the joint cavity. Accordingly, in some preferred embodiments, an AAV843 vector is used to deliver a gene expressing IL-IRa.

In one embodiment, with CB as the promoter, the intron behind the promoter is VH4, and the transgenic expression cassette B93 of the nucleic acid sequence encoding the original human interleukin-1 receptor antagonist protein without codon optimization is constructed, and its nucleotide The sequence is shown in SEQ ID NO:9.

In one embodiment, with CB as the promoter, the intron behind the promoter is VH4, and the codon-optimized human interleukin 1 receptor antagonistic protein coding nucleic acid sequence (optimized sequence one (IL1Ra(1)), SEQ ID NO: 7) the transgenic expression cassette B94, its nucleotide sequence is as shown in SEQ ID NO: 10.

In one embodiment, with CB as the promoter, the intron behind the promoter is VH4, and the Chi intron is inserted into the optimized sequence one (SEQ ID NO: 7) to construct the transgenic expression cassette B95, its nucleotide sequence As shown in SEQ ID NO: 11.

In one embodiment, CB is used as the promoter, the intron behind the promoter is VH4, two introns Chi and SV40 are sequentially inserted into the optimized sequence one (SEQ ID NO: 7), and the transgene expression cassette B96 is constructed, Its nucleotide sequence is shown in SEQ ID NO:12.

In one embodiment, with CB as the promoter, the intron behind the promoter is VH4, and the SV40 intron is inserted into the optimized sequence one (SEQ ID NO: 7) to construct the transgenic expression cassette B97, its nucleotide sequence As shown in SEQ ID NO:13.

In one embodiment, CB is used as the promoter, and the intron behind the promoter is VH4, and the two introns SV40 and Chi are sequentially inserted into the optimized sequence one (SEQ ID NO: 7) to construct the transgenic expression cassette B98, Its nucleotide sequence is shown in SEQ ID NO: 14.

In one embodiment, with CB as the promoter, the intron behind the promoter is SV40, and the transgenic expression cassette B126 of the original human interleukin-1 receptor antagonist protein coding nucleic acid sequence without codon optimization is constructed, and its nucleotide The sequence is shown in SEQ ID NO:15.

In one embodiment, with CB as the promoter, the intron behind the promoter is SV40, and the codon-optimized human interleukin 1 receptor antagonistic protein coding nucleic acid sequence (optimized sequence two (IL1Ra(2)), SEQ ID NO:8) the transgenic expression cassette B127, its nucleotide sequence is as shown in SEQ ID NO:16.

In one embodiment, with CB as the promoter, the intron after the promoter is SV40, and the Chi intron is inserted in the optimized sequence two (SEQ ID NO: 8) to construct the transgenic expression cassette B128, its nucleotide sequence As shown in SEQ ID NO:17.

In one embodiment, with CB as the promoter, the intron after the promoter is SV40, the Chi intron is inserted in the optimized sequence two (SEQ ID NO: 8) and the VH4 intron is connected at the end of the optimized sequence two , to construct a transgenic expression cassette B129, the nucleotide sequence of which is shown in SEQ ID NO: 18.

In one embodiment, with CB as the promoter, the intron behind the promoter is SV40, two introns Chi and VH4 are sequentially inserted into the optimized sequence 2 (SEQ ID NO: 8) to construct the transgenic expression cassette B130, Its nucleotide sequence is shown in SEQ ID NO: 19.

In one embodiment, with CB as the promoter, the intron behind the promoter is SV40, and the VH4 intron is inserted into the optimized sequence two (SEQ ID NO: 8) to construct the transgenic expression cassette B131, its nucleotide sequence As shown in SEQ ID NO:20.

In one embodiment, with CB as the promoter, the intron after the promoter is SV40, the VH4 intron is inserted in the optimized sequence two (SEQ ID NO: 8) and the Chi intron is connected at the end of the optimized sequence two , to construct a transgenic expression cassette B132, the nucleotide sequence of which is shown in SEQ ID NO: 21.

In one embodiment, CB is used as the promoter, the intron behind the promoter is SV40, and the two introns VH4 and Chi are sequentially inserted into the optimized sequence 2 (SEQ ID NO: 8) to construct the transgenic expression cassette B133, Its nucleotide sequence is shown in SEQ ID NO:22.

In a preferred embodiment, the transgenic expression cassette expressing interleukin 1 receptor antagonist protein comprises: CB promoter sequence (SEQ ID NO: 1), SV40 intron (SEQ ID NO: 3), bGH polyadenylation ( polyA) sequence (SEQ ID NO: 2) and codon-optimized human IL-1Ra coding sequence (SEQ ID NO: 8), the codon-optimized human IL-1Ra coding sequence is inserted with a Chi intron ( SEQ ID NO: 5) and VH4 intron (SEQ ID NO: 4) to enhance the expression of the protein, thus forming the B130 expression cassette (SEQ ID NO: 19). The B130 expression cassette is flanked by a normal ITR and a shortened ITR to enable packaging of the B130 expression cassette into AAV particles as a self-complementary AAV vector.

In some embodiments, AAV particles of IL-IRa are produced by three-plasmid (plasmid 1: cis-element plasmid; plasmid 2: AAV Rep/Cap plasmid; plasmid 3: helper plasmid) transfection of HEK293 cells.

In one embodiment, to generate therapeutically functional AAV particles, three-plasmid transfection of HEK293 cells is performed as follows: Plasmid 1: cis-element plasmid with ITR (e.g., B130 expression cassette); Plasmid 2: with capsid AAV Rep/Cap plasmid of protein (for example, AAV843 capsid protein) coding sequence; Plasmid 3: a helper plasmid with adenoviral components, which can facilitate the replication, assembly and packaging of AAV virions. In one embodiment, AAV particles produced by HEK293 cells are purified by affinity chromatography and iodixanol density gradient ultracentrifugation (Xiao X et al., J Virol (1998) 72(3):2224-32).

Those skilled in the art can use known standard methods to produce recombinant and synthetic polypeptides or proteins thereof, design nucleic acid sequences, produce transformed cells, construct recombinant AAV mutants, transform capsid proteins, package and express AAV Rep and/or Cap sequences vector, and transiently or stably transfect packaging cells. These techniques are known to those skilled in the art. See, eg, MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, (Cold Spring Harbor, NY, 1989).

In some embodiments, the gene delivery systems of the present disclosure are used to aid in cell transplantation therapy. Specifically, AAV particles with transgenes can be used to transduce various types of cells in vitro to generate stable cell lines expressing protein products, which can then be introduced in vivo for therapeutic purposes. Types of cells include, but are not limited to, chondrocytes, synoviocytes, mesenchymal stem cells.

In one embodiment, the cells used for transplantation are autologous cells of the subject, which allow for in vitro culture. The principles and techniques of introducing or transplanting cells into a subject are known to those skilled in the art.

In one embodiment, AAV particles are harvested from the culture medium and lysates of HEK293 cells. Purification methods such as affinity chromatography, ion exchange chromatography, cesium chloride and iodixanol gradient ultracentrifugation. Chemicals or reagents related to AAV production and purification include, but are not limited to: Chemicals or reagents used in cell culture (e.g., components of cell culture media including bovine, equine, goat, chicken or other vertebrate serum, glutamine Amide, Glucose, Sucrose, Pyruvate sodium, phenol red; antibiotics such as penicillin, kanamycin, streptomycin, tetracycline); chemicals or reagents for cell lysis, polynucleotide precipitation or ultracentrifugation (such as Triton X-100, NP-40, Sodium deoxycholate, Sodium Lauryl Sulfate, Domiphene Bromide, Sodium Lauryl Salicylate, Sodium Chloride, Magnesium Chloride, Calcium Chloride, Barium Chloride, Nitrate, Potassium Chloride, Chloride Ammonium, Ammonium Persulfate, Ammonium Sulfate, PEG-20, PEG-40, PEG-400, PEG-2000, PEG-6000, PEG-8000, PEG-20000, Tris-HCl, Tris-Acetate, Manganese Chloride , phosphate, bicarbonate, cesium chloride, methanol, ethanol, glycerol, iodixanol, isopropanol, butanol, benzoin enzyme, DNase I, RNase); affinity column materials (such as AAVX affinity resin, Heparan sulfate proteoglycans and mucin resins, other materials associated with AAV-specific antibodies); acids, bases, and organics contained in ion-exchange chromatography materials and wash buffers (e.g., hydrochloric, sulfuric, acetic, formic, nitric, urea , acetone, chloroform, acetonitrile, trifluoroacetic acid, sodium hydroxide, potassium hydroxide, barium hydroxide, ammonium hydroxide, Tris base or other organic amines, Poloxamer 188, Tween 20, Tween 40, Tween 80, guanidine hydrochloride).

In one embodiment, the AAV vectors of the present disclosure can be loaded with exogenous polynucleotides for gene delivery into target cells. Thus, the AAV vectors of the present disclosure can be used to deliver nucleic acids to cells in vitro or in vivo.

In one embodiment, the exogenous polynucleotide delivered to the target cell by the AAV vector encodes a native protein for therapeutic use, either codon-optimized or non-codon-optimized.

In one embodiment, the exogenous polynucleotide delivered by the AAV vector to the target cell encodes a synthetic polypeptide.

In one embodiment, the AAV vector or transgene expression cassette or gene delivery system of the present disclosure is made into a pharmaceutical preparation (eg, injection) and administered to humans or other mammals. In addition, pharmaceutical formulations may be delivered in single or multiple doses by systemic or local (eg, intravenous, intra-articular) administration.

In one embodiment, the medicament of the present disclosure is used to transduce cells in vitro or mammals (such as rodents, primates and humans) in vivo to treat diseases that can be improved by blocking the IL-1 signaling pathway , such as acute inflammatory diseases, chronic inflammatory diseases and malignant tumors, preferably joint inflammation.

In one embodiment, the joint inflammation is selected from the group consisting of osteoarthritis, rheumatoid arthritis, synovitis, hemophilic arthritis and other inflammatory joint inflammations. In one embodiment, joint inflammation involves degeneration of joint function.

In one embodiment, treating or improving joint inflammatory disease refers to improving joint pain, cartilage wear and joint function in the treated patient.

The present disclosure will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following examples are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods without specific conditions indicated in the examples were operated according to conventional conditions known in the art, or according to the conditions suggested by the manufacturer.

Example

Example 1: Codon optimization and plasmid construction of IL1Ra gene

First, based on the cDNA gene sequence of human IL1Ra in the NCBI gene bank, the original sequence of IL1Ra (SEQ ID NO: 6) was codon-optimized to obtain two codon-optimized sequences: IL1Ra optimized sequence one (IL1Ra(1 )) (SEQ ID NO: 7) and IL1Ra optimized sequence two (IL1Ra(2)) (SEQ ID NO: 8).

Next, construct expression cassettes based on IL1Ra original sequence, IL1Ra optimized sequence 1 and IL1Ra optimized sequence 2:

With CB as promoter and VH4 as intron, plasmid B93 (SEQ ID NO: 9) was constructed with IL1Ra original sequence; with CB as promoter and VH4 as intron, plasmid B94 (SEQ ID NO: 9) was constructed with IL1Ra optimized sequence one NO: 10); with CB as the promoter, VH4 as the intron, and based on IL1Ra optimized sequence 1, Chi and/or SV40 introns were inserted at the 5' end and 3' end of the transcription unit to construct plasmids B95 to B98 (SEQ ID NO: 11 to SEQ ID NO: 14);

With CB as promoter and SV40 as intron, plasmid B126 (SEQ ID NO: 15) was constructed with the original sequence of IL1Ra; with CB as promoter and SV40 as intron, plasmid B127 (SEQ ID NO: 15) was constructed with the optimized sequence of IL1Ra NO: 16); with CB as the promoter and SV40 as the intron, based on IL1Ra optimized sequence 2, insert Chi and/or VH4 intron at the 5' end and 3' end of the transcription unit to construct plasmids B128 to B133 (SEQ ID NO: 17 to SEQ ID NO: 22).

Example 2: Expression of codon-optimized human IL1Ra-encoding nucleic acid sequence

Spread Huh7 cells in a 12-well plate at a seeding density of 2E+5 cells/well, and replace serum-free DMEM when the cells increase to 80-90%. A transfection system was prepared, the amount of transfection plasmid was 2.5 μg (plasmid: PEI=1:2), added to each well for 12 hours after transfection, and then replaced with DMEM with 10% fetal bovine serum to continue culturing for 48 hours. Huh7 cells were transfected with five plasmids (B94-B98) with IL1Ra optimized sequence one, and the supernatant was collected 72 hours later to measure the concentration of IL1Ra protein.

ELISA results showed that the expression of the four plasmids (B95-B98) obtained by further optimization by inserting Chi and/or SV40 introns was higher than that of the B94 plasmid (Fig. 2A), which indicated that gene expression could be improved by inserting Chi or SV40 introns. expression.

The plasmid with IL1Ra optimized sequence 1 was packaged into AAV843 virus to infect Huh7 cells at an infection MOI of 7.5E+5vg/cell, and the supernatant was collected after 72 hours to measure the IL1Ra protein concentration.

ELISA results showed ( FIG. 2B ) that the results of virus infection were basically consistent with those of plasmid transfection, and the expression of B96 virus was most significantly increased.

The B93 virus packaging the original sequence of IL1Ra and the B94 to B98 virus packaging IL1Ra optimized sequence 1 were injected into C57 mice through the tail vein at a dose of 1E+13vg/kg. Serum was extracted 3 weeks after injection, and the expression of IL1Ra was determined.

The results showed that codon-optimized IL1Ra(1) had increased IL1Ra protein expression, and the protein expression of genes further optimized by inserting Chi and/or SV40 introns was further improved in animals (Fig. 2C).

Seven plasmids (B127-B133) with IL1Ra optimized sequence 2 and an unoptimized control plasmid (B126) were transfected into Huh7 cells, and the supernatant was collected after 48 hours to measure the concentration of IL1Ra protein.

ELISA results showed that the IL1Ra protein expression of codon-optimized B127 (10ng/ml) was significantly higher than that of the unoptimized control plasmid (1.5ng/ml), indicating that codon-optimized IL1Ra(2) increased its protein expression . Moreover, based on IL1Ra(2), the expressions of the 6 plasmids (B128-B133) obtained by further optimizing by inserting Chi and/or VH4 introns were further improved, and the expression of the B130 plasmid increased most significantly (19.6 ng/ml), which was 13.9 times higher than that of the unoptimized original gene (B126), and about 1.9 times higher than that of the B127 plasmid without an intron inserted (Fig. 2D).

The plasmid with IL1Ra optimized sequence 2 was packaged into AAV843 virus to infect Huh7 cells at an infection MOI of 7.5E+5vg/cell, and the supernatant was collected after 48 hours to measure the IL1Ra protein concentration.

The results of ELISA ( FIG. 2E ) showed that the results of virus infection were basically consistent with those of plasmid transfection, indicating that insertion of Chi and/or VH4 introns could further increase gene expression.

The B126 virus packaging the original sequence of IL1Ra and the B127 to B133 virus packaging the optimized sequence II of IL1Ra were injected into the tail vein of C57 mice, and the serum was extracted 3 and 4 weeks after the injection, and the expression of IL1Ra was determined.

The results showed that codon-optimized IL1Ra(2) had increased IL1Ra protein expression, and the protein expression of genes further optimized by insertion of Chi and/or VH4 introns was further improved in animals (Fig. 2F, Figure 2G).

Finally, in order to compare the IL1Ra protein expression efficiency of IL1Ra optimized sequence 1 and IL1Ra optimized sequence 2, B94 (optimized sequence 1), B96 (optimized sequence 1 + intron insertion), B127 (optimized sequence 2) and B130 (optimized sequence Two + intron insertion) viruses for comparison. After transfecting Huh7 cells for 72 hours, the supernatant was collected and the concentration of IL1Ra protein was determined.

ELISA results showed that the expression of IL1Ra protein of B127 virus was significantly higher than that of B94 virus (Fig. 2H, **p<0.01), and the expression of IL1Ra protein of B130 virus was also better than that of B96 virus, indicating that IL1Ra optimized sequence 2 has higher expression than IL1Ra optimized sequence 1 Higher IL1Ra protein expression.

Example 3: In vitro biological function of the codon-optimized IL1Ra gene

Under the stimulation of IL1, C28/I2 chondrocytes will undergo an inflammatory reaction, which will promote the production of inflammatory factors such as IL1, IL6, and TNFa, which in turn will cause the increase of cartilage matrix degrading enzyme MMP13, leading to the degradation of cartilage matrix. In this embodiment, in order to verify the biological activity of the protein expressed by the transcription and translation of the codon-optimized IL1Ra gene, C28/I2 chondrocytes were selected for verification.

C28/I2 cells were plated in 12-well plates at a seeding density of 2e5 cells/well. When the confluence reaches 80-90%, replace the serum-free medium, and the virus infection MOI is 7.5E+5vg/cell. After 24 hours of infection, the DMEM medium containing 10% fetal bovine serum was replaced. In addition to the blank control wells, a certain amount of IL1 (10ng/ml) was added to each well for stimulation and induction. After 48h, the cells were collected to extract mRNA, and the expression levels of IL1, IL6, TNFa and MMP13 were measured.

The results of the virus in vitro functional experiment for optimized sequence 1 showed that under the action of IL1, the levels of IL1, IL6, TNFa and MMP13 in the model group (6.4, 23.8, 2.1, 9.4, respectively) were significantly higher than those in the blank control group (1, 1 , 1, 1) (FIG. 3A). Pre-infection with IL1Ra virus attenuated the stimulating effect of IL1, and the levels of IL1, IL6 and MMP13 were significantly reduced. Under the same virus titer conditions, the B96 group had the best therapeutic effect, and the levels of IL1, IL6, TNFa and MMP13 were reduced to 1.9, 3.5, 1.9, and 2.3, respectively, which was consistent with the result of the highest expression (Figure 2B).

The results of the virus in vitro functional experiment for optimized sequence 2 showed that the levels of IL1, IL6, TNFa, and MMP13 in the B127 group were reduced to 2.5, 8.5, 1.3, and 4.6, respectively, which was better than that of the B126 virus group (5.0, 19.1, 1.7, 10.5). Under the same virus titer condition, the treatment effect of the B130 group was better, and the levels of IL1, IL6, TNFa and MMP13 were reduced to 2.1, 4.6, 1.0, and 3.3, respectively (Fig. 3B).

The results of in vitro functional experiments show that the optimized packaged virus can effectively express the target protein IL1RA, antagonize the inflammatory response caused by IL1 signaling pathway, thereby reducing the degradation of cartilage matrix and protecting cartilage.

Example 4: The therapeutic effect of AAV-mediated expression of IL1Ra on arthritis

As shown in Figure 4, cut the anterior cruciate ligament of one side of the knee joint and remove part of the meniscus to construct the OA model. After 2 weeks of modeling, the joints were injected with AAV843-B130 virus (low dose 1x10 ¹¹ vg/joint, high dose 1x10 ¹² vg/joint), and the treatment lasted 2 months. The joint swelling was measured weekly after administration. As shown in Figure 5A, the joint swelling of the rats in the model group was significantly more severe than that in the blank control group, and both the low-dose virus group and the high-dose group effectively reduced joint swelling.

After the treatment, the Micro-CT machine was used to scan the joints of the rats to observe the therapeutic effect of the virus. The results showed that compared with the model group, the joint surfaces of the rats treated in the low-dose group and the high-dose group were significantly smoother and more complete ( FIG. 5B ), and the effect of the high-dose group was better. Analysis of trabecular bone parameters also showed that both the low-dose and high-dose groups of IL1Ra virus could effectively reduce the wear and tear of the joints of model rats (Fig. 5C), and had a certain protective effect on articular cartilage.

At the 2nd, 4th, and 8th weeks after the intra-articular injection of the drug, serum samples of the rats were collected to measure the levels of inflammatory factors. As shown in Figure 6, the levels of inflammatory factors IL1, IL6, and TNFa in the serum of rats in the model group were significantly increased, and the level of inflammation was reduced after administration, and the reduction effect of high dose was better than that of low dose.

After 8 weeks of treatment, joint tissues were taken for pathological analysis. The results of Safranin O Fast Green staining (Figure 7) showed that the articular cartilage in the normal control group was evenly stained and the surface was smooth and complete; while in the model group, the chondrocytes were reduced, the arrangement was disordered, the cartilage surface defect was serious, and some safranin was lost. After drug treatment, the pathological development of articular cartilage in OA rats was significantly improved. process, cartilage wear is reduced. At the same time, the HE staining results showed ( FIG. 7 ), compared with the model group, the inflammatory infiltration of the joint synovium of the OA rats in the administration group was significantly reduced. In addition, the results of joint qPCR showed that after the injection of AAV843-B130 virus into the joint cavity, the expression of IL1RA was significantly increased in a dose-dependent manner (Figure 8).

The results showed that virus treatment could effectively reduce the expression of inflammatory factors such as IL1, IL6, and TNFα in the model joints, and reduce the production of cartilage matrix degrading enzymes, thereby inhibiting the reduction of cartilage matrix proteoglycan and type II collagen.

The above results show that AAV843-B130 virus can express IL1Ra protein after intra-articular injection, effectively antagonize IL1 inflammatory factor, exert anti-inflammatory effect, effectively protect articular cartilage, and finally delay the pathogenesis of OA.

Finally, the safety evaluation of the virus is carried out. One week after virus joint injection, the blood of rats in each group was collected, and the whole blood was used for blood routine testing, and the serum was used for testing the levels of liver indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

The results of ELISA showed that 1 week after the virus injection, the levels of ALT and AST in the liver function of the rats had no significant changes compared with those in the non-injected virus group ( FIG. 9A ). The results of blood routine testing showed that the number of white blood cells (WBC), percentage of lymphocytes (Lymph), number of platelets (PLT), percentage of neutrophils (Gran), number of red blood cells (RBC) and hemoglobin ( HGB) and the non-injected virus group had no significant difference ( FIG. 9B ), which indicated that AAV virus had no obvious toxic and side effects on rats after joint injection.

Although the present disclosure has been illustrated and described with reference to certain preferred embodiments of the present disclosure, those skilled in the art should understand that the above content is a further detailed description of the present disclosure in conjunction with specific embodiments, and cannot be deemed The specific implementation of the present disclosure is limited only by these descriptions. Those skilled in the art may make various changes in form and details, including several simple deduction or substitutions, without departing from the spirit and scope of the present disclosure.

Claims (15)

1. A nucleic acid molecule encoding an interleukin-1 receptor antagonist protein whose nucleotide sequence has at least 50% identity, preferably at least 60%, 70% identity to the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 %, 80%, 85%, 90%, 95%, 99% or 100% identity.
2. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8, preferably, the nucleotide sequence of the nucleic acid molecule is as SEQ ID NO: ID NO: 7 or SEQ ID NO: 8.
3. A transgene expression cassette, comprising: a promoter, the nucleic acid molecule according to claim 1 or 2, and polyA; preferably, the polyA is bGH polyA.
4. The transgenic expression cassette according to claim 3, wherein the promoter is selected from the group consisting of: CB promoter, CAG promoter, SV40 promoter, collagen collagen promoter, proteoglycan aggrecan promoter, synoviolin gene promoter promoter, adipose tissue-specific promoter PPAR white gene promoter, transcription factor SOX9 promoter and osteocalcin promoter; preferably, the promoter is a CB promoter.
5. The transgene expression cassette according to claim 3 or 4, wherein the transgene expression cassette further comprises: an intron behind the promoter, and/or two ITRs at both ends; preferably, the intron Selected from class 1 introns such as SV40 intron, VH4 intron, Chi intron, U12 intron, RHD intron, and the two ITRs are each independently a normal ITR or a shortened ITR.
6. The transgene expression cassette according to any one of claims 3 to 5, wherein the transgene expression cassette further comprises: at least one intron inserted into the nucleic acid molecule according to claim 1 or 2, preferably, Said intron is selected from: SV40 intron, VH4 intron and Chi intron.
7. The transgene expression cassette according to any one of claims 3 to 6, wherein the nucleotide sequence of the transgene expression cassette is such as SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, preferably, the nucleotide of the transgene expression cassette The sequence is shown in SEQ ID NO: 12 or SEQ ID NO: 19, more preferably in SEQ ID NO: 19.
8. A gene delivery system, comprising: the transgene expression cassette and AAV capsid protein according to any one of claims 3 to 7.
9. The gene delivery system according to claim 8, wherein the AAV capsid protein is a natural AAV capsid protein or an artificially modified AAV capsid protein; preferably, the AAV is selected from: AAV1, AAV2, AAV3, AAV4 , AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, and AAV843.
10. The gene delivery system according to claim 8 or 9, wherein the AAV capsid protein is AAV843, and its amino acid sequence is as shown in SEQ ID NO: 24.
11. The nucleic acid molecule according to claim 1 or 2, the transgene expression cassette according to any one of claims 3 to 7 or the gene delivery system according to any one of claims 8 to 10 are used in the preparation of medicaments for treating diseases In the application in , the disease is a disease that can be improved by blocking the IL-1 signaling pathway, such as acute inflammatory disease, chronic inflammatory disease and malignant tumor; preferably, the disease is joint inflammation, including osteoarthritis, Rheumatoid arthritis, synovitis, hemophilic arthritis and other inflammatory joint inflammations; more preferably, the disease is osteoarthritis.
12. A medicine comprising: the nucleic acid molecule according to claim 1 or 2, the transgene expression cassette according to any one of claims 3 to 7 or the gene delivery system according to any one of claims 8 to 10, and Forming agent.
13. A method for treating a disease, comprising administering a therapeutically effective amount of the drug of claim 12 to a subject in need, the disease being a disease that can be improved by blocking IL-1 signaling pathway.
14. The method according to claim 13, wherein the disease is acute inflammatory disease, chronic inflammatory disease and/or malignant tumor; preferably, the disease is joint inflammation, including osteoarthritis, rheumatoid arthritis, slippery Meningitis, hemophilic arthritis and other inflammatory joint inflammations; more preferably, the disease is osteoarthritis.
15. The method according to claim 13 or 14, wherein the drug is administered systemically or locally, such as intravenous administration, local contact and intralesional administration, preferably locally in the joint cavity, such as through intra-articular injection.