Methods and compositions for treating disease using recombinant self-complementary adeno-associated viruses - Patents.com

Abstract

Methods and compositions for treating disease using recombinant self-complementary adeno-associated viruses are provided. [Solution] Methods and compositions for treating symptoms of diseases such as, but not limited to, osteoarthritis and rheumatoid arthritis. The methods may feature direct intra-articular injection of a recombinant self-complementary adeno-associated virus (sc-rAAV) containing a vector adapted to express a modified IL-1Ra peptide. The methods of the invention may express a therapeutically effective amount of the modified IL-1Ra peptide to ameliorate symptoms associated with the disease being treated.

Description

[Technical field]

[0001] cross reference This application is a continuation of U.S. Provisional Patent Application No. 62 / 377,297, filed August 19, 2016. No. 6,313,623, filed on May 13, 2003, the disclosure of which claims priority from US Pat. No. 6,313,623, the specification of which is incorporated herein by reference in its entirety. Can be enjoyed.

[0002] Sequence Listing Reference The applicant is CALIM_16_02_PCT_Sequence_Listing_S Annex C filed under Rule 13ter.1(a), entitled T25.txt The information recorded in the form of a / ST.25 text file is included in one of the international applications filed. The contents of the sequence listing are hereby incorporated by reference in their entirety. will be incorporated into the subsection.

[0003] The present invention relates to gene therapy and compositions for gene therapy, more particularly recombinant self-complementary and the use of sc-rAAV to treat a disease or condition The present invention relates to a method for treating the symptoms of [Background technology]

[0004] Osteoarthritis (OA) affects more than 27 million Americans and is a common condition affecting older adults. OA is the leading cause of disability in the United States. Patients with OA are also at higher risk of death. The cost of OA to the health care system is estimated to exceed $100 billion annually Statistics like these highlight the fact that OA is incurable and highly resistant to treatment. Reflect.

[0005] The earliest and most dominant symptom of OA is pain. This usually occurs late in the disease process and By that time, the bone has developed symptoms including loss of articular cartilage, sclerosis of the subchondral bone, formation of osteophytes, and synovial inflammation. There is often significant structural change in the affected joint. In the knee, meniscal damage is also present. There are no disease-modifying osteoarthritis drugs (DMOADs) that can halt or reverse disease progression. Currently, there are no effective methods to intervene in the disease process. Many patients progress to the stage where they require total joint replacement because there is no effective treatment. Although this procedure is affordable, it requires significant and expensive surgery with extensive rehabilitation. In many cases, revision surgery is required to replace a malfunctioning artificial joint.

[0006] In the absence of DMOAD, the current standard of care is palliative. The American College of Orthopaedic Surgeons (ACR) and the American Academy of Orthopaedic Surgeons (AAOS) published a joint research paper on knee joints in 2013. As reflected in the latest guidelines for treating osteoarthritis of the joints covered by the IND, Current approaches to treatment fall into three progressive categories: Treatment includes a range of strategies, including patient education and self-help, exercise programs and weight loss. Modified anti-inflammatory drugs include acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), and opiates. and the use of intra-articular injections of glucocorticoids or hyaluronic acid. , which provides partial relief for many patients, but because many individuals with OA are elderly, These are associated with upper gastrointestinal bleeding and renal failure, which are of particular concern in the current situation. Intra-articular injections of acetaminophen often provide rapid relief, but the effects usually last only a few weeks. Repeated glucocorticoid injections are not recommended due to concerns about infection and the need for sustained high doses of glucocorticoids. Impractical and contraindicated due to evidence that corticosteroids damage articular cartilage The benefit of intra-articular injections of hyaluronic acid (viscosupplementation) is controversial; the ACR has The AAOS no longer recommends this. Intra-articular injections of autologous blood products such as MSCs and platelet-rich plasma are becoming increasingly popular, but Not approved by the FDA for the treatment of osteoarthritis of the knee The latest recommendations from the International Research Council and the European League Against Rheumatism are consistent with those of the ACR and AAOS. The recommendations of various agencies are based on the scarcity and lack of reliable treatment options for OA. This highlights the complete lack of highly effective pharmacological interventions. However, it only addresses the signs and symptoms, not the progression of the disease. If symptoms and progression cannot be controlled, surgical intervention may be indicated.

[0007] Arthroscopic irrigation and debridement have been widely used to provide symptomatic relief. However, its use has declined following evidence that its effectiveness is comparable to that of a placebo. Osteotomy is sometimes performed to realign the forces in the joint, resulting in areas of intact cartilage. This procedure is often used to treat joint pain, as it can lead to erosion of the newly weight-bearing joint cartilage and recurrence of symptoms. Generally, osteotomy is the surgical procedure for the replacement of the knee joint. It is seen as a delaying measure to buy time until transplantation. Many patients require total joint replacement. It has progressed to the point where more than 700,000 artificial knee joints were surgically implanted in the United States last year.

[0008] IL-1 was found to be potent in both chondrocyte chondrolysis and inhibition of matrix synthesis by chondrocytes. Together, these two processes are highly destructive to cartilage. IL-1 also inhibits chondrogenesis, but also induces osteophyte formation and sclerosis of the subchondral bone. It has also been shown to promote certain aspects of osteogenic differentiation that may help explain the paradox. In addition, IL-1 also promotes osteoclast activity by stimulating both bone formation and osteolysis. Thus, IL-1 enhances bone turnover, as seen in the subchondral bone during OA. Later, IL-1 is in a favorable position to induce the inflammatory changes seen in OA. The pyrogenic activity is known, and negligible amounts of IL-1 in the knee joints of rabbits are Expression of 1 is sufficient to induce marked synovitis.

[0009] When examining cartilage harvested from human joints with OA, chondrocytes secreted IL-1. Production was found to be highly elevated and sustained in an autocrine manner. IL-1Ra was not produced in OA, which is consistent with the absence of its main physiological inhibitor in OA. These results suggest that IL-1 enhances autocrine and paracrine activation of chondrocytes under conditions of OA. Enhanced responsiveness of chondrocytes to IL-1 in mice was also due to the signaling receptor This was also shown by increased expression of type I IL-1 receptors in OA chondrocytes. Local production and consumption of IL-1 may explain why the concentration of IL-1 in synovial fluid is low even in OA. Genetic analysis may also help explain why IL-1Ra (IL Single nucleotide polymorphisms (SNPs) in the human gene encoding L1RN and the incidence of certain types of OA We identified regulatory elements that correlate with disease severity.

[0010] Targeted drug delivery is a key issue for intra-articular treatment of joint diseases. Molecules and particles are rapidly transported out of the joint via lymphatics, subsynovial capillaries, or both. This makes it difficult to achieve a sustained therapeutic dose of anti-OA drugs in the joint. To address this, small molecules can be delivered systemically but not to the synovial capillaries. Due to size-dependent constraints in crossing the fenestrated endothelium, it may be difficult to deliver proteins in this manner. Moreover, systemic delivery exposes non-targeted sites to high doses of therapeutic agents, which can be undesirable. Rapid release of proteins from the joints, which typically have half-lives of a few hours, As an example, intra-articular delivery of cyclosporine is often used to treat the symptoms of RA. Recombinant IL-1Ra (Kineret, Amgen Biologicals) was administered daily. It is delivered by subcutaneous injection. However, daily delivery can result in increased IL-1R activity between injections. cannot maintain therapeutic serum levels of a (Evans et al., 1996, Human Gene Therapy,7:1261-1290;Evans et al., 2005, PNAS 102(24):8698-8703). Several studies used ex vivo gene transfer to introduce IL-1Ra to treat OA. However, these approaches are time-consuming and labor-intensive and do not provide long-term gene expression. (Frisbie et al., 2002, Gene The Erapy 9(1):12-20. Also, some studies have shown that it is effective in treating osteoarthritis. Dual variable domain targeting IL-1α and IL-1β (e.g., ABT-981) for have described the use of DVD-Ig (Kamath et al. .,2011,Osteoarthritis and Cartilage 19S1 :S64;Wang et al.,2015,Osteoarthritis and Cartilage 23:A398-399;Wang et al.,2014, Osteoarthritis and cartilage 22:S462-S46 3;Lacy et al.,2015,mAbs 7(3):605-619;Wu et al.,2009,mAbs 1(4):339-347;Wang et al. .,2014,Scientific Abstracts SAT0448 pg.7 56;Goss et al.,2014,Scientific Abstracts SAT0447 pg.755-756; U.S. Patent Application Publication No. 2015 / 005023 Issue 8;Wang et al.,2014 ACR / ARHP Annual Meet ing Abstract Number 2237;Wang et al.,201 5 ACR / ARHP Annual Meeting Abstract Number However, due to their relatively short half-lives, the entire repeat structure of these peptides is Intravenous administration (e.g., 4 doses every 2 weeks or 3 doses every 4 weeks, e.g., subcutaneous injection or (Wang et al., 2015, Osteoa rthritis and Cartilage 23:A398-399;Wang et al.,2014,Osteoarthritis and cartilage 22:S462-S463; Evans et al., 2005, PNAS 102 (24):8698-8703).

[0011] The present invention provides a method for targeting a therapeutic gene product (e.g., IL-1Ra) at a location The present invention features methods and compositions for sustained delivery of therapeutic agents to a variety of therapeutic interests, such as to joints. The invention also provides a method for treating diseases such as, but not limited to, osteoarthritis and rheumatoid arthritis. The present invention also features methods and compositions for treating a condition in an individual (e.g., a human). Methods and compositions for providing a therapeutically effective amount of a therapeutic gene product (eg, IL-1Ra) The method and composition are characterized by the use of recombinant self-complementary adeno-associated virus (sc-rAA). V), where the sc-rAAV is a genetically engineered capsid and The vector includes a vector (e.g., a sc-rAAV vector) packaged within the vector. The vector comprises a transgene (e.g., a nucleotide sequence) operably linked to a promoter (e.g., a constitutive promoter). For example, a protein at a targeted site, such as a therapeutic gene product, e.g., IL-1Ra or its The therapeutic gene product may comprise a nucleotide sequence encoding a codon-altered variant. For example, to treat osteoarthritis, c-rAAV was directly injected into the joint to deliver it to cells in the joint (e.g., chondrocytes, synovial cells, etc.). The present invention relates to a method for the delivery of a medicament to any of the aforementioned diseases or targeted sites (e.g., joints). is not limited.

[0012] Goodrich et al.(Molecular Therapy-Nucle ic Acids, 2013, 2:e70) was altered using scAAV-delivered IL-1Ra. It is noted that the present invention generally discloses a method for treating osteoarthritis. oodrich et al. describe specific IL-1Ra sequences, e.g., the IL- In particular, the field of gene therapy has not specifically identified or enabled the 1Ra sequence. It is possible to assume that a particular gene sequence for a protein at a particular site will be efficiently expressed. Furthermore, gene therapy is also a very unpredictable field in which the results are not consistent with animal models compared to humans. It is also impossible to predict the efficacy of a particular method in an animal model. If it is effective, it cannot be assumed that it will be effective in humans. Summary of the Invention

[0013] The present invention features recombinant self-complementary adeno-associated viruses (sc-rAAV). In some embodiments, the sc-rAAV comprises an engineered AAV capsid and a capsid. The vector is packaged in a plasmid, the vector being operable to encode a promoter. operably linked to a modified IL-1Ra gene, the modified IL-1Ra gene being represented by SEQ ID NO: 2. In some embodiments, the promoter is a CMV promoter. In some embodiments, the engineered capsid comprises a serotype AAV2 and at least a portion of serotype AAV6. The engineered capsids are available for the serotypes AAV1, AAV2, AAV3, AAV4, and AAV5. AV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or any of the above. In some embodiments, the vector comprises at least a portion of a combination of the SV4 0 and the bovine growth hormone (bGH) polyadenylation sequence. In this embodiment, the vector contains an SV40 splice donor (SD) and splice acceptor ( In some embodiments, the vector further comprises a sc-rAAV2.5H (SA) site. In some embodiments, the sc-rAAV is part of a composition. do.

[0014] In some embodiments, the sc-rAAV comprises an engineered AAV capsid and The vector packaged in the capsid comprises a vector encoding a promoter. operably linked to a modified IL-1Ra gene, the modified IL-1Ra gene having the sequence It encodes the IL-1Ra protein according to number 6.

[0015] The present invention relates to a method for treating a patient having an IL-1Ra or interleukin-1 receptor agonist (IL-1Ra) peptide comprising administering to said patient a therapeutically effective amount of said peptide. The method comprises administering the therapeutic agent to a human in need thereof (e.g., a human diagnosed with or having osteoarthritis or rheumatoid arthritis). In some embodiments, the method comprises providing a human subject to a pulmonary bypass graft (HPA) treatment, the human subject being at risk for a pulmonary bypass graft (PGA) treatment. The method includes administering to a subject a composition comprising a recombinant self-complementary adeno-associated virus (sc-rAAV) according to the present invention. It involves introducing an entity into a targeted location (e.g., by intra-articular injection). The rAAV transduces cells at the site of the vector targeting the target site, where the modified IL-1R The a gene is expressed to provide a human with a therapeutically effective amount of the IL-1Ra peptide. .

[0016] The present invention also features a method for ameliorating symptoms of osteoarthritis or rheumatoid arthritis in a human. In some embodiments, the method comprises the step of producing a recombinant self-complementary adenovirus according to the invention. A composition containing the associated virus (sc-rAAV) is introduced into the location of the targeted site (e.g., sc-rAAV is a vector that targets the target site. and transducing the modified IL-1Ra gene into cells at the site of osteoarthritis or rheumatoid arthritis. The present invention provides a method for providing an IL-1Ra peptide to a human in an amount effective to ameliorate symptoms associated with arthritis. It is expressed as follows.

[0017] The present invention also relates to a method for treating cartilage damage in a human subject in need of cartilage repair (e.g., a subject suffering from osteoarthritis or rheumatoid arthritis). The present invention features a method for repairing cartilage in a human diagnosed with or at risk for developing glaucoma. In some embodiments, the method comprises administering a recombinant self-complementary adeno-associated virus according to the invention. A composition containing sc-rAAV is introduced into the location of the cartilage (e.g., by direct intra-articular injection). The sc-rAAV transduces vectors into cells at the site of cartilage, The modified IL-1Ra gene expresses IL-1Ra peptides that are effective in repairing cartilage in humans. is expressed to provide

[0018] The present invention also relates to a method for treating inflammation by administering an interleukin-1 receptor agonist (IL-1Ra) peptide to a subject. In some embodiments, the method comprises providing a method of administering to a region of the subject invention Introducing a composition containing a recombinant self-complementary adeno-associated virus (sc-rAAV) into the site of inflammation The sc-rAAV delivers the vector to the site of inflammation. The modified IL-1Ra gene is then transduced into cells, where the modified IL-1Ra gene is an effective therapeutic agent for reducing inflammation. The IL-1Ra peptide is expressed in a manner that provides effective amounts of the IL-1Ra peptide to cells at the site of inflammation.

[0019] In some embodiments, the location of the targeted site is a human joint, synovium, subsynovium, intervertebral column, or endothelial cell. In some embodiments, the cells are from a joint capsule, tendon, ligament, cartilage, or periarticular muscle. The cells may be chondrocytes, synovial cells, or a combination thereof.

[0020] In some embodiments, the method is performed a second time after the first time the method is performed. In some embodiments, the time point is at least 3 months. In some embodiments, the method includes administering a secondary therapy to the location of the targeted site in combination with the composition. (e.g., glucocorticoids, hyaluronan, platelet-rich plasma, recombinant human IL-1Ra, or a combination thereof).

[0021] The invention also features a method for delivering an IL-1Ra peptide to a chondrocyte or synovial cell. In some embodiments, the method comprises culturing chondrocytes or synovial cells in a culture medium according to the present invention. Recombinant self-complementary adeno-associated virus (sc-rAAV), e.g., at least one of serotype 2 A genetically engineered adeno-associated virus (AAV) comprising at least a portion of serotype 6 and contacting the capsid with the vector packaged within the capsid. wherein the vector comprises a modified IL-1Ra gene operably linked to a CMV promoter. The modified IL-1Ra gene is at least 95% identical to SEQ ID NO:2. The rAAV transduces the vector into chondrocytes or synovial cells, and the modified IL-1Ra gene is delivered to the chondrocytes or synovial cells. It is expressed to provide IL-1Ra peptides to bone cells or synovial cells.

[0022] The aforementioned methods and compositions (e.g., a therapeutically effective amount of an interleukin 1 receptor agonist ( Methods for providing IL-1Ra peptides to humans in need thereof, Method for improving symptoms of rheumatoid arthritis or rheumatoid arthritis, and administration of IL-1Ra peptide to chondrocytes or synovium Methods for delivering recombinant self-complementary adeno-associated virus (sc-rAAV) to cells Composition, a recombinant autologous vector comprising a modified IL-1Ra gene operably linked to a CMV promoter. Self-complementary adeno-associated virus (sc-rAAV) vectors for cartilage repair in dogs A method for repairing cartilage in the inflamed area by administering an interleukin-1 receptor agonist (IL- Regarding the method of providing IL-1Ra peptide, etc., the modified IL-1Ra gene is represented by SEQ ID NO:2. and may be at least 95% identical to and encode an IL-1Ra according to SEQ ID NO:6. The present invention provides, for example, the following items. (Item 1) A method for providing a therapeutically effective amount of an interleukin-1 receptor agonist (IL-1Ra) peptide to a human in need thereof, comprising introducing into a targeted site a composition comprising a recombinant self-complementary adeno-associated virus (sc-rAAV), wherein the sc-rAAV comprises: a. an engineered AAV capsid; and b. A vector packaged within said capsid, said vector comprising a modified IL-1Ra gene operably linked to a promoter, said modified IL-1Ra gene being at least 95% identical to SEQ ID NO:2. Including, wherein the sc-rAAV transduces the vector into cells at the location of the targeted site and the modified IL-1Ra gene is expressed to provide the human with the therapeutically effective amount of the IL-1Ra peptide. method. (Item 2) 2. The method of claim 1, wherein the human has been diagnosed with or is at risk of developing osteoarthritis or rheumatoid arthritis. (Item 3) 2. The method according to item 1, wherein the location of the targeted site is a human joint, synovium, subsynovium, joint capsule, tendon, ligament, cartilage, or periarticular muscle. (Item 4) 2. The method of claim 1, wherein the composition is introduced into the targeted site location by direct intra-articular injection. (Item 5) 2. The method of claim 1, wherein the cells are chondrocytes, synovial cells, or a combination thereof. (Item 6) 2. The method of claim 1, wherein the method is performed a second time at a time later than the time the method was performed the first time. (Item 7) 2. The method of claim 1, wherein the time point is at least 3 months. (Item 8) 2. The method of claim 1, wherein the method further comprises simultaneously delivering a secondary therapy to the location of the targeted site in combination with the composition. (Item 9) 9. The method of claim 8, wherein the secondary therapy comprises glucocorticoids, hyaluronan, platelet-rich plasma, recombinant human IL-1Ra, or a combination thereof. (Item 10) 2. The method of item 1, wherein the promoter comprises a CMV promoter. (Item 11) 2. The method of claim 1, wherein the genetically engineered capsid comprises at least a portion of serotype AAV2 and at least a portion of serotype AAV6. (Item 12) 2. The method of claim 1, wherein the engineered capsid comprises at least a portion of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or a combination thereof. (Item 13) 2. The method of claim 1, wherein the vector further comprises SV40 and bovine growth hormone (bGH) polyadenylation sequences. (Item 14) 14. The method of claim 13, wherein the vector further comprises SV40 splice donor (SD) and splice acceptor (SA) sites. (Item 15) The method of item 1, wherein the vector comprises sc-rAAV2.5Hu-IL-1Ra. (Item 16) 1. A method for ameliorating a symptom of osteoarthritis or rheumatoid arthritis in a human, comprising introducing into a location of a targeted site a composition comprising a recombinant self-complementary adeno-associated virus (sc-rAAV), wherein the sc-rAAV: a. an engineered AAV capsid; and b. A vector packaged within said capsid, said vector comprising a modified IL-1Ra gene operably linked to a promoter, said modified IL-1Ra gene being at least 95% identical to SEQ ID NO:2. Including, wherein the sc-rAAV transduces the vector into cells at the location of the targeted site and the modified IL-1Ra gene is expressed to provide a human with an IL-1Ra peptide in an amount effective to ameliorate symptoms associated with osteoarthritis or rheumatoid arthritis. method. (Item 17) 17. The method according to item 16, wherein the targeted site location is a human joint, synovium, subsynovium, joint capsule, tendon, ligament, cartilage, or periarticular muscle. (Item 18) 17. The method of item 16, wherein the composition is introduced into the targeted site location by direct intra-articular injection. (Item 19) 17. The method of claim 16, wherein the cells are chondrocytes, synovial cells, or a combination thereof. (Item 20) 17. The method of claim 16, wherein the method is performed a second time at a time later than the time the method was performed the first time. (Item 21) 17. The method of item 16, wherein the time point is at least 3 months. (Item 22) 17. The method of claim 16, wherein the method further comprises simultaneously delivering a secondary therapy to the location of the targeted site in combination with the composition. (Item 23) 23. The method of claim 22, wherein the secondary therapy comprises glucocorticoids, hyaluronan, platelet-rich plasma, recombinant human IL-1Ra, or a combination thereof. (Item 24) 17. The method of item 16, wherein the promoter comprises a CMV promoter. (Item 25) 17. The method of claim 16, wherein the genetically engineered capsid comprises at least a portion of serotype AAV2 and at least a portion of serotype AAV6. (Item 26) 17. The method of claim 16, wherein the engineered capsid comprises at least a portion of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or a combination thereof. (Item 27) 17. The method of claim 16, wherein the vector further comprises SV40 and bovine growth hormone (bGH) polyadenylation sequences. (Item 28) 28. The method of claim 27, wherein the vector further comprises SV40 splice donor (SD) and splice acceptor (SA) sites. (Item 29) The method of item 16, wherein the vector comprises sc-rAAV2.5Hu-IL-1Ra. (Item 30) 1. A method for delivering an IL-1Ra peptide to a chondrocyte or synovial cell, comprising administering to said chondrocyte or synovial cell: a. an engineered adeno-associated virus (AAV) capsid comprising at least a portion of serotype 2 and at least a portion of serotype 6; and b. a vector packaged within said capsid, said vector comprising a modified IL-1Ra gene operably linked to a CMV promoter, said modified IL-1Ra gene being at least 95% identical to SEQ ID NO:2; with a recombinant self-complementary adeno-associated virus (sc-rAAV) comprising wherein the sc-rAAV transduces the vector into the chondrocytes or synovial cells, and the modified IL-1Ra gene is expressed to provide the IL-1Ra peptide to the chondrocytes or synovial cells. method. (Item 31) The method of item 30, wherein the vector comprises sc-rAAV2.5Hu-IL-1Ra. (Item 32) 31. The method of item 30, wherein the vector further comprises SV40 and bovine growth hormone (bGH) polyadenylation sequences. (Item 33) 31. The method of item 30, wherein the vector further contains SV40 splice donor (SD) and splice acceptor (SA) sites. (Item 34) A composition comprising a recombinant self-complementary adeno-associated virus (sc-rAAV), wherein the sc-rAAV comprises: a. an engineered capsid comprising at least a portion of serotype 2 and at least a portion of serotype 6; and b. A vector packaged within said capsid, said vector comprising a nucleic acid sequence encoding a modified IL-1Ra peptide operably linked to a CMV promoter, said nucleic acid sequence encoding said modified IL-1Ra peptide being at least 90% identical to SEQ ID NO:2. A composition comprising: (Item 35) 35. The composition of item 34, wherein the vector further comprises SV40 and bovine growth hormone (bGH) polyadenylation sequences. (Item 36) 36. The composition of item 35, wherein the vector further comprises SV40 splice donor (SD) and splice acceptor (SA) sites. (Item 37) 35. The composition of item 34, wherein the vector comprises sc-rAAV2.5Hu-IL-1Ra. (Item 38) A recombinant self-complementary adeno-associated virus (sc-rAAV) vector comprising a modified IL-1Ra gene operably linked to a CMV promoter, wherein the modified IL-1Ra gene is at least 95% identical to SEQ ID NO:2. (Item 39) 39. The vector according to item 38, further comprising SV40 and bovine growth hormone (bGH) polyadenylation sequences. (Item 40) 40. The vector of item 39, further comprising an SV40 splice donor (SD) and splice acceptor (SA) site. (Item 41) 39. The vector of item 38, comprising sc-rAAV2.5Hu-IL-1Ra. (Item 42) 1. A method of repairing cartilage in a human in need thereof, comprising introducing a composition comprising a recombinant self-complementary adeno-associated virus (sc-rAAV) to a location of cartilage, wherein the sc-rAAV comprises: a. an engineered AAV capsid; and b. A vector packaged within said capsid, said vector comprising a modified IL-1Ra gene operably linked to a promoter, said modified IL-1Ra gene being at least 95% identical to SEQ ID NO:2. Including, wherein the sc-rAAV transduces the vector into cells at the location of the cartilage and the modified IL-1Ra gene is expressed to provide a human with an IL-1Ra peptide effective to repair cartilage. method. (Item 43) 43. The method of claim 42, wherein the human has been diagnosed with or is at risk of developing osteoarthritis or rheumatoid arthritis. (Item 44) 43. The method according to item 42, wherein the composition is introduced into the location of the cartilage by direct intra-articular injection. (Item 45) 43. The method of claim 42, wherein the cells are chondrocytes, synovial cells, or a combination thereof. (Item 46) 43. The method of claim 42, wherein the method is performed a second time at a time later than the time the method was performed the first time. (Item 47) 43. The method of claim 42, wherein the time point is at least 3 months. (Item 48) 43. The method of claim 42, wherein the method further comprises co-introducing a secondary therapy to the location of the cartilage in combination with the composition. (Item 49) 49. The method of claim 48, wherein the secondary therapy comprises glucocorticoids, hyaluronan, platelet-rich plasma, recombinant human IL-1Ra, or a combination thereof. (Item 50) 43. The method of item 42, wherein the promoter comprises a CMV promoter. (Item 51) 43. The method of claim 42, wherein the genetically engineered capsid comprises at least a portion of serotype AAV2 and at least a portion of serotype AAV6. (Item 52) 43. The method of claim 42, wherein the engineered capsid comprises at least a portion of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or a combination thereof. (Item 53) The method of item 42, wherein the vector comprises sc-rAAV2.5Hu-IL-1Ra. (Item 54) 1. A method for providing an interleukin 1 receptor agonist (IL-1Ra) peptide to an area of ​​inflammation, comprising introducing into the site of inflammation a composition comprising a recombinant self-complementary adeno-associated virus (sc-rAAV), wherein the sc-rAAV comprises: a. an engineered AAV capsid; and b. A vector packaged within said capsid, said vector comprising a modified IL-1Ra gene operably linked to a promoter, said modified IL-1Ra gene being at least 95% identical to SEQ ID NO:2. Including, wherein the sc-rAAV transduces the vector into cells at the site of inflammation and the modified IL-1Ra gene is expressed to provide a therapeutically effective amount of an IL-1Ra peptide to the cells at the site of inflammation effective to reduce inflammation. method. (Item 55) 55. The method according to item 54, wherein the site of inflammation is a human joint, synovium, subsynovium, joint capsule, tendon, ligament, cartilage, or periarticular muscle. (Item 56) 55. The method of item 54, wherein the composition is introduced into the site of inflammation by direct intra-articular injection. (Item 57) 55. The method of item 54, wherein the cells are chondrocytes, synovial cells, or a combination thereof. (Item 58) 55. The method of item 54, wherein the promoter comprises a CMV promoter. (Item 59) 55. The method of claim 54, wherein the genetically engineered capsid comprises at least a portion of serotype AAV2 and at least a portion of serotype AAV6. (Item 60) 55. The method of claim 54, wherein the engineered capsid comprises at least a portion of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or a combination thereof. (Item 61) The method of item 54, wherein the vector comprises sc-rAAV2.5Hu-IL-1Ra. (Item 62) 1. A method for providing a therapeutically effective amount of an interleukin 1 receptor agonist (IL-1Ra) to a human in need thereof, comprising introducing into a targeted site a composition comprising a recombinant self-complementary adeno-associated virus (sc-rAAV), wherein the sc-rAAV comprises: a. an engineered AAV capsid; and b. A vector packaged within said capsid, said vector comprising a modified IL-1Ra gene operably linked to a promoter, said modified IL-1Ra gene encoding an IL-1Ra according to SEQ ID NO:6. Including, wherein the sc-rAAV transduces the vector into cells at the location of the targeted site and IL-1Ra is expressed to provide the human with a therapeutically effective amount of the IL-1Ra. method. (Item 63) 1. A method for ameliorating a symptom of osteoarthritis or rheumatoid arthritis in a human, comprising introducing into a location of a targeted site a composition comprising a recombinant self-complementary adeno-associated virus (sc-rAAV), wherein the sc-rAAV: a. an engineered AAV capsid; and b. A vector packaged within said capsid, said vector comprising a modified IL-1Ra gene operably linked to a promoter, said modified IL-1Ra gene encoding an IL-1Ra according to SEQ ID NO:6. Including, wherein the sc-rAAV transduces the vector into cells at the location of the targeted site and IL-1Ra is expressed to provide the human with an amount of IL-1Ra effective to ameliorate symptoms associated with osteoarthritis or rheumatoid arthritis. method. (Item 64) 1. A method for delivering an IL-1Ra peptide to a chondrocyte or synovial cell, comprising administering to said chondrocyte or synovial cell: a. an engineered adeno-associated virus (AAV) capsid comprising at least a portion of serotype 2 and at least a portion of serotype 6; and b. A vector packaged within said capsid, said vector comprising a modified IL-1Ra gene operably linked to a CMV promoter, said modified IL-1Ra encoding an IL-1Ra according to SEQ ID NO:6. with a recombinant self-complementary adeno-associated virus (sc-rAAV) comprising wherein the sc-rAAV transduces the vector into the chondrocytes or synovial cells, and IL-1Ra is expressed to provide IL-1Ra to the chondrocytes or synovial cells. method. (Item 65) A composition comprising a recombinant self-complementary adeno-associated virus (sc-rAAV), wherein the sc-rAAV comprises: a. an engineered capsid comprising at least a portion of serotype 2 and at least a portion of serotype 6; and b. A vector packaged within said capsid, said vector comprising a nucleic acid sequence encoding a modified IL-1Ra peptide operably linked to a CMV promoter, said nucleic acid sequence encoding an IL-1Ra according to SEQ ID NO:6. A composition comprising: (Item 66) 1. A method of repairing cartilage in a human in need thereof, comprising introducing a composition comprising a recombinant self-complementary adeno-associated virus (sc-rAAV) to a location of cartilage, wherein the sc-rAAV comprises: a. an engineered AAV capsid; and b. A vector packaged within said capsid, said vector comprising a modified IL-1Ra gene operably linked to a promoter, said modified IL-1Ra gene encoding an IL-1Ra according to SEQ ID NO:6. Including, wherein the sc-rAAV transduces the vector into cells at the location of the cartilage, and IL-1Ra is expressed to provide the human with IL-1Ra effective to repair cartilage. method. (Item 67) 1. A method for providing an interleukin 1 receptor agonist (IL-1Ra) peptide to an area of ​​inflammation, comprising introducing into the site of inflammation a composition comprising a recombinant self-complementary adeno-associated virus (sc-rAAV), wherein the sc-rAAV comprises: a. an engineered AAV capsid; and b. A vector packaged within said capsid, said vector comprising a modified IL-1Ra gene operably linked to a promoter, said modified IL-1Ra gene encoding an IL-1Ra according to SEQ ID NO:6. Including, wherein the sc-rAAV transduces the vector into cells at the site of inflammation and IL-1Ra is expressed to provide a therapeutically effective amount of IL-1Ra to the cells at the site of inflammation effective to reduce inflammation. method.

[0023] As will be apparent from the context, the specification, and the knowledge of one of ordinary skill in the art, any of the compounds described herein may be used without departing from the spirit and scope of the present invention. Any feature or combination of features may be used without the need for the features in such combination to be mutually exclusive. Further advantages and aspects of the present invention are described in detail below. This is evident in the description and claims. [Brief description of the drawings]

[0024] [Figure 1] Figure 1 shows the plasmid sc-rAAV2.5Hu-IL-1Ra, which contains a modified cDNA encoding the human IL-1Ra protein under the control of the CMV promoter. The gene insert also contains SV40 and bovine growth hormone (bGH) polyadenylation sequences, as well as SV40 splice donor (SD) and splice acceptor (SA) sites. The region between the inverted terminal repeats (TR) has been confirmed by sequencing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] term Unless otherwise explained, all technical and scientific terms used herein are intended to be understood as meaning the have the same meaning as commonly understood by a person skilled in the art to which the invention pertains. The singular terms "a," "an," and "the" are used interchangeably unless the context clearly dictates otherwise. Similarly, unless the context clearly dictates otherwise, the word "or" is intended to include "and." "Include" means "include." However, Therefore, "including A or B" means "including A" or "including B" or "including A and B." means.

[0026] Suitable methods and materials for practicing and / or testing embodiments of the present disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described can be used. Conventional methods well known in the art to which this invention pertains include, for example, Sambrook et al., Molecular Clo ning:A Laboratory Manual,2d ed.,Cold Spr. ing Harbor Laboratory Press,1989;Sambroo k et al.,Molecular Cloning: A Laboratory Manual,3d ed.,Cold Spring Harbor Press,2 001;Ausubel et al.,Current Protocols in Molecular Biology,Greene Publishing Asso ciates,1992(and Supplements to 2000);Aus ubel et al.,Short Protocols in Molecular Biology:A Compendium of Methods from Cu rrent Protocols in Molecular Biology,4th ed.,Wiley&Sons,1999;Harlow and Lane,Ant ibodies:A Laboratory Manual,Cold Spring Harlow Laboratory Press, 1990; and Harlow an d Lane,Using Antibodies:A Laboratory Man ual,Cold Spring Harbor Laboratory Press, 1999, and other related references.

[0027] All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. The entirety is incorporated by reference.

[0028] To practice or test the disclosed technology, Although equivalent or equivalent methods and materials can be used, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0029] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided: R:

[0030] Adeno-associated virus (AAV), recombinant AAV (rAAV), and recombinant self-complementary AAV AV (sc-rAAV): AAV is a small virus of the Parvoviridae family. AAV is a type virus (20 nm). AAV is not known to cause disease. V has been shown to have low immunogenicity, the ability to efficiently transduce non-dividing cells, and the ability to efficiently transduce a variety of cells and tissues. The gene has recently been identified for a variety of reasons, including the fact that it has been shown to have the ability to transmit hepatitis B virus to other organisms. Recombinant AAV (rAAV) does not contain native viral coding sequences and is used therapeutically. Recombinant AAV DNA is expressed as a single-stranded molecule approximately 4600 nucleotides long. Following infection of a cell by the virus, the virus is packaged into a capsid. A molecular mechanism converts single-stranded DNA into a double-stranded form. Only the double-stranded form is present in the single or double stranded form. is useful for cellular proteins that transcribe multiple genes into RNA. rAAV (sc-rAAV) is capable of forming intramolecular double-stranded DNA templates. Thus, upon infection, the two complementary fragments of sc-rAAV are expressed in the The fragments associate to form a single double-stranded DNA unit that is ready for immediate replication and synthesis. do.

[0031] Expression: Translation of a nucleic acid sequence into a protein. Once expressed, the protein is retained within the cell and is then transformed into a protein. They may be components of the cell surface membrane or may be secreted into the extracellular matrix or medium.

[0032] Operably linked: A first nucleic acid sequence is placed into a functional relationship with a second nucleic acid sequence. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when A promoter acts on a coding sequence if it affects the transcription or expression of the coding sequence. are movably connected.

[0033] Pharmaceutically acceptable vehicle: A pharma- ceutically acceptable carrier (vehicle), such as a solution, The vehicle may be any conventional vehicle, but is not limited to any conventional vehicle. n,Remington's Pharmaceutical Sciences,Ma ck Publishing Co., Easton, PA, 15th Edition (1975) and DBTroy, ed. Remington: The Scienc. e and Practice of Pharmacy, Lippincott Wi lliams&Wilkins,Baltimore MD and Philadel phia,PA,21 stEdition (2006) is a method for identifying one or more therapeutic compounds or describes compositions and formulations suitable for pharmaceutical delivery of molecules. In general, the nature of the carrier will depend on the In addition to biologically neutral carriers, the pharmaceutical composition to be administered may also contain The compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, e.g. For example, they may include sodium acetate or sorbitan monolaurate.

[0034] Preventing, Treating, Managing, or Ameliorating a Disease: "Preventing" a disease means "improving" the disease or "Treating" can mean inhibiting the complete development of a disease or pathological condition. It can mean a therapeutic intervention that ameliorates a sign or symptom after it begins to develop. "Ameliorating" can mean a therapeutic intervention that does not worsen the signs or symptoms of a disease or condition. " may refer to a reduction in the number or severity of signs or symptoms of a disease or condition.

[0035] Sequence identity: The identity (or similarity) between two or more nucleic acid sequences is determined by the identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity. the higher the percentage, the more identical the sequences are. Sequence similarity is measured by the percentage similarity. (which takes into account conservative amino acid substitutions); The higher the stage, the more similar the sequences are. Methods of aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are available. , Smith & Waterman,Adv.Appl.Math.2:482,1981 ;Needleman&Wunsch, J.Mol.Biol.48:443,1970 ;Pearson&Lipman,Proc.Natl.Acad.Sci.USA 8 5:2444,1988;Higgins&Sharp,Gene,73:237-44 ,1988;Higgins&Sharp,CABIOS 5:151-3,1989; Corpet et al.,Nuc.Acids Res.16:10881-90, 1988;Huang et al.Computer Appls.in the B iosciences 8, 155-65, 1992; and Pearson et al. ., Meth. Mol. Bio. 24:307-31, 1994. tschul et al., J. Mol. Biol. 215:403-10, 1990 presents a detailed discussion of sequence alignment methods and homology calculations. Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is a sequence analysis program called bl Related to astp, blastn, blastx, tblastn, and tblastx For use, the National Center for Biotechnology Information (NCBI, National Library of Medicine, Bui lding 38A, Room 8N805, Bethesda, MD 20894) Further information is available from several sources, including the National Institute of Standards and Technology (NIS), and on the Internet. BLASTN is used to compare nucleic acid sequences. BLASTP can be used to compare amino acid sequences. If the sequences share homology, the specified output file will align those regions of homology. If the two compared sequences do not share homology, the sequence is presented as specified. The output file does not present aligned sequences. AT) can also be used to compare nucleic acid sequences (Kent, Genome Res. 1 2:656-664,2002). BLAT is a joint venture with Kent Informatics (S From several sources, including Santos Cruz, CA, and on the Internet ( nome.ucsc.edu). Once aligned, identical nucleotides Or the number of matches is determined by counting the number of positions where an amino acid residue occurs in both sequences. The percent sequence identity is determined based on the length or length of the sequence shown in the identified sequence. The length of the identified sequence (e.g., 100 consecutive nucleotides from the sequence shown in the identified sequence) Divide the number of matches by the number of amino acid residues (or amino acid residues) and then multiply the result by 100. For example, a test sequence having 1554 nucleotides is aligned to the When the nucleic acid sequence has 1166 matches, it is 75.0 percent identical to the test sequence. (1166 ÷ 1554 × 100 = 75.0). The percent sequence identity value is The number is rounded to one decimal place.

[0036] Therapeutically effective amount: Amount sufficient to achieve a desired effect in a subject being treated with a particular drug. The amount of the agent. Such agents may include IL-1Ra. For example, treatment with IL-1Ra An effective amount is an amount sufficient to prevent, treat, or ameliorate the symptoms of osteoarthritis or rheumatoid arthritis. A therapeutically effective amount of an agent useful for preventing, ameliorating, and / or treating a subject. The efficacy and safety of the therapeutic composition will depend on the subject being treated, the type and severity of the disease, and the method of administration of the therapeutic composition. do.

[0037] Transduced: A transduced cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transduction refers to the transfer of a virus or viral vector. Transfection with vectors, transformation with plasmid vectors, and electroporation including naked DNA introduction by troporation, lipofection, and particle gun acceleration. This includes all techniques by which a nucleic acid molecule can be introduced into such cells. These are often called transformed cells.

[0038] Vector: A nucleic acid that is introduced into a host cell, thereby producing a transformed host cell. A vector contains nucleic acid sequences, such as an origin of replication, that enable it to replicate in a host cell. A vector may lack a nucleic acid sequence that enables it to replicate in a host cell. The vector may also contain a gene for the targeted site, one or more selectable marker genes. , other genetic elements known in the art, or any other suitable insertion.

[0039] The present invention relates to the location of a site targeted to a therapeutic gene product (e.g., IL-1Ra), e.g. The present invention also features methods and compositions for sustained delivery to osteoarthritis of joints. For treating symptoms of diseases such as, but not limited to, rheumatoid arthritis or rheumatoid arthritis. The invention also features methods and compositions for administering to an individual (e.g., a human) a therapeutically effective amount of a therapeutic gene. The present invention features methods and compositions for providing gene products (e.g., IL-1Ra). The compositions may feature recombinant self-complementary adeno-associated viruses (sc-rAAV), Here, sc-rAAV is a recombinant AAV that contains a genetically engineered capsid and a gene packaged within the capsid. The vector includes a vector that is a recombinant rAAV vector (sc-rAAV vector). A transgene (e.g., a gene that expresses a protein at a targeted site) operably linked to a constitutive promoter. The nucleic acid sequence may include a nucleic acid sequence encoding a therapeutic gene product, such as IL-1Ra or a modified form thereof. The nucleic acid sequence may include a nucleotide sequence.

[0040] As mentioned above, the present invention provides a recombinant self-complementary adeno-associated virus (sc-rAAV) vector. Non-limiting examples of sc-rAAV vectors are shown in the table below. The sc-rAAV vector of SEQ ID NO:1 contains a modified IL-1R a gene. In some embodiments, the vector contains an SV40 polyadenylation sequence. In some embodiments, the vector comprises a bovine growth hormone (bGH) polyadenylation sequence. In some embodiments, the vector comprises an SV40 splice donor (SD) and The sc-rAAV vector contains a splice acceptor (SA) site. Not determined.

[0041] The sc-rAAV vector contains a nucleic acid encoding a peptide at the targeted site. In some embodiments, the nucleic acid is at least 90% identical to SEQ ID NO:2. In some embodiments, the nucleic acid is at least 92% identical to SEQ ID NO:2. In some embodiments, the nucleic acid is at least 94% identical to SEQ ID NO:2. is at least 95% identical to SEQ ID NO: 2. In some embodiments, the nucleic acid is In some embodiments, the nucleic acid is at least 96% identical to SEQ ID NO:2. In some embodiments, the nucleic acid is at least 97% identical to SEQ ID NO:2. In some embodiments, the nucleic acid is at least 99% identical to SEQ ID NO:2. Non-limiting examples of such nucleic acid sequences can be found in Table 1 below. For example, , SEQ ID NO:3 is the sequence of modified human IL-1Ra which is about 98% identical to SEQ ID NO:2; SEQ ID NO:4 is the sequence of a modified human IL-1Ra that is about 99% identical to SEQ ID NO:2; and and (in Table 1, boldface type indicates nucleotide substitutions compared to SEQ ID NO:2, codons are underlined) (Note that [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0042] In some embodiments, the IL-1Ra peptide encoded by the IL-1Ra insert The peptide includes IL-1Ra (see SEQ ID NO: 6 in Table 2 below). [Table 2]

[0043] The transgene (e.g., a nucleotide sequence encoding a protein at the targeted site) In some embodiments, the promoter is operably linked to a site. The present invention is not limited to the CMV promoter. Any suitable promoter or portion of a variety of promoters may be featured. Examples of promoters include The CMV promoter, hybrid CMV promoter, CAG promoter, and human β-activator promoter are actin promoter, hybrid β-actin promoter, EF1 promoter, U1a promoter Data, U1b promoter, Tet inducible promoter, VP16-LexA promoter, Chicken beta actin (CBA) promoter, human elongation factor 1α promoter, Simian virus SV40 promoter and herpes simplex virus thymidine kinase promoter Includes:

[0044] In some embodiments, the promoter comprises a hybrid promoter. Table 3 shows the IL-1β / IL-6 hybrid promoter (van de Loo See also, et al., 2004, Gene Therapy 11:581-590. The present invention is also not limited to the hybrid promoters shown in Table 3. [Table 3]

[0045] In some embodiments, the sc-rAAV vector is packaged within a capsid. In some embodiments, the capsid is selected from AAV serotype 1 (AAV1), AA V serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), their derivatives, or For example, in some embodiments, the capsid comprises at least a portion of AAV serotype 2 and at least a portion of AAV serotype 6, e.g., AA Includes V2.5.

[0046] The composition, e.g., a composition comprising sc-rAAV, can be delivered to a target site location (e.g., a human For example, in some embodiments, the therapeutic agent may be introduced into cells in the body to treat symptoms of osteoarthritis. In the treatment of osteoarthritis, cells from within the joint (e.g., chondrocytes, synovial cells, e.g. In some embodiments, the composition may be introduced into a human joint (type A, type B, etc.). The present invention is administered to the synovium, subsynovium, joint capsule, tendon, ligament, cartilage, or periarticular muscle. The method may be selected from the above-mentioned diseases (e.g., osteoarthritis), administration methods (e.g., intra-articular injection), and target areas. The present invention is not limited to a particular site (e.g., joint), or cell type (e.g., chondrocytes, synovial cells). For example, in some embodiments, other cell types that may be transduced may include mesenchymal stem cells. .

[0047] The sc-rAAV transduced the vector into cells, and the modified IL-1Ra peptide was expressed. In some embodiments, the IL-1Ra peptide is and a therapeutically effective amount of the modified IL-1R effective to ameliorate symptoms associated with various diseases, such as a peptide is expressed in humans.

[0048] In some embodiments, the composition (e.g., sc-rAAV) is introduced once. In some embodiments, the composition (e.g., sc-rAAV) is introduced twice, e.g., once In some embodiments, the composition is introduced more than twice. The second introduction of the composition is carried out in a similar manner to the first introduction of the composition, for example three, four, five, etc. After the time point, e.g., 3 months, 4 months, 5 months, 6 months, 7 months, 8 months It may be performed months, nine months, ten months, eleven months, a year, more than a year, etc.

[0049] The composition may comprise any suitable pharmaceutical composition. In some embodiments, the composition comprises a buffer. In some embodiments, the buffer comprises phosphate buffered saline (PBS). In some embodiments, the composition further comprises sorbitol, e.g., 5% sorbitol. In some embodiments, the composition further comprises a salt, such as NaCl. The concentration of the salt may be any Any suitable concentration, e.g., 350 mM NaCl, more than 350 mM NaCl, It may be less than M etc.

[0050] In some embodiments, the composition (e.g., sc-rAAV) is administered concomitantly with a second line therapy. In some embodiments, the secondary therapy is a drug to treat OA or RA, or a symptom of the disease. Non-limiting examples of secondary therapies for OA include: glucocorticoids, hyaluronan (viscosupplement), platelet-rich plasma, and recombinant human I L-1Ra (Anakinra; Kineret (registered trademark)). In some embodiments, the sc-rAAV is co-administered with glucocorticoids or platelet-rich plasma. It is given.

[0051] The disclosures of the following United States patents are incorporated herein by reference in their entireties: Publication No. 2008 / 0187576, U.S. Patent Application Publication No. 2009 / 0104155 , Korean Patent Application Publication No. 2012041139, Japanese Patent Application Publication No. 201551881 6, International Publication No. 2013151672, International Publication No. 2008088895, U.S. Patent No. No. 8529885, U.S. Patent No. 7037492, U.S. Patent Application Publication No. 200701 No. 28177, U.S. Patent No. 6,491,907, U.S. Patent No. 8,999,948, U.S. Patent No. Publication No. 20150218586, U.S. Patent No. 7,892,824, U.S. Patent Application Publication No. No. 20130295614, Japanese Patent Application Publication No. 2002538770, Japanese Patent Application Publication No. 2010516252, Korean Patent Application Publication No. 2002027450, Korean Patent Publication No. 2003028080, U.S. Patent No. 6,482,634, U.S. Patent Application Publication No. No. 20090105148, U.S. Patent Application Publication No. 20120232130, U.S. Patent No. Application Publication No. 20140234255, U.S. Patent No. 5756283, U.S. Patent No. 6083 716, International Publication No. 2002038782, International Publication No. 2007039699, International Publication No. 2012047093, International Publication No. 2014170470, International Publication No. 15018860, International Publication No. 2015044292, International Publication No. 20151587 No. 49, U.S. Patent No. 7,452,696, U.S. Patent No. 6,943,153, U.S. Patent No. 642 9001, International Publication No. 2015031392, and International Publication No. 2004092211.

[0052] Example 1 Example 1 describes the administration of a sc-rAAV (encoding IL-1Ra) of the present invention. The invention is not limited to the disclosure of Example 1. Five patients were considered for administration of the sc-rAAV of the present invention. The patient is enrolled in a clinical trial to determine whether osteoarthritis of the right knee is caused by osteoarthritis. (1) a 65-year-old male with osteoarthritis of the left knee; (2) a 59-year-old male with osteoarthritis of the left knee; (4) a 51-year-old man with osteoarthritis of the right knee; and (5 ) A 48-year-old male with osteoarthritis of the right knee. Each patient received 1 × 10 1 2 sc-rAAV is administered by intra-articular injection of the viral gene. IL-1Ra is It is expressed in chondrocytes and synovial cells. Patient 1 developed OA-related symptoms within 2 weeks. Patient 2 described improvement in OA-related symptoms within 1 week. Patient 3 described improvement in OA-related symptoms within 5 weeks. Patient 4 described improvement within 1 week. At 6 weeks, patient 5 reported improvement in OA-related symptoms. It does not mention any improvements.

[0053] Example 2 Example 2 shows a first administration of sc-rAAV (encoding IL-1Ra) of the present invention and The present invention describes a second administration of the same sc-rAAV of the present invention after a certain period of time. The present invention is not limited to the above. A 55-year-old man presents with osteoarthritis in his right knee. Patients were administered a single intra-articular injection of a clear sc-rAAV vector (encoding IL-1Ra). The patient's symptoms resolved within two months. Six months later, the physician administered the same sc-rAAV vector of the present invention to A second (single) intra-articular injection of IL-1Ra-encoding IL-1Ra is administered. is still absent six months after the second injection.

[0054] Example 3 Example 3 shows a first administration of sc-rAAV (encoding IL-1Ra) of the present invention and After a certain period of time, the sc-rAAV of the present invention (containing IL-1Ra) was administered. The present invention is not limited to the disclosure of Example 3. The woman presented with osteoarthritis in her right ankle. Her physician administered the sc-rAAV vector of the present invention to The patient received a single intra-articular injection of IL-1Ra-encoding IL-1Ra. The patient's symptoms improved within 5 months. The condition improved but was not resolved. Six months later, the doctor decided to use a different sc-rAAV vector of the present invention. A second (single) intra-articular injection of IL-1Ra-encoding vector is then administered. Six months after the shot, the patient's symptoms resolve.

[0055] Example 4 Example 4 shows co-administration of a sc-rAAV (encoding IL-1Ra) of the invention with a second-line therapy. The present invention is not limited to the disclosure of Example 4. A 68-year-old male presented with osteoarthritis of the left knee. The doctor of the man described above administered the sc-rAAV vector of the present invention (carrying IL-1Ra) The patient received a single intra-articular injection of both thrombus (a sphincter graft) and platelet-rich plasma. The patient's symptoms resolved within 2 months. It will be erased.

[0056] Various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. It will be apparent to those skilled in the art that such modifications are intended to fall within the scope of the appended claims. Each reference cited in this application is incorporated herein by reference in its entirety. is incorporated into the book.

[0057] Although embodiments of the present invention have been shown and described, modifications may be made without departing from the spirit and scope of the appended claims. It will be readily apparent to those skilled in the art that the reference numerals in the claims are It is illustrative and intended only to facilitate consideration by the Patent Office and is not to be construed as a substitute for any other purpose. In some embodiments, the drawings presented in this patent application are not limiting. The drawings, including angles, dimensional ratios, etc., are drawn to scale. In some embodiments, the drawings may be used interchangeably. The drawings are merely representational and the claims are not limited by the dimensions of the drawings. In embodiments, the description of the invention described herein using the term "comprises" may be understood to include "or "comprising" includes embodiments that may be described as "comprising" and therefore uses the phrase "comprising" This satisfies the written description requirement for claiming one or more embodiments of the invention.

[0058] Any reference numbers recited in the following claims are merely for ease of reference in reviewing this patent application. The drawings are for illustrative purposes only and are not intended to in any way supersede the claims. The disclosure is not intended to be limiting to the specific features having corresponding reference numbers therein. [Sequence List Free Text]

[0059] Sequence Listing 1 <223> The modified human IL1Ra insert and SacII / NotI restriction sites were The sequence of the Hu-IL-1Ra plasmid containing the entire viral sequence Sequence Listing 2 <223> Modified human IL-1Ra insert Sequence Listing 3 <223> Modified human IL-1Ra insert (98% identical to SEQ ID NO:2; Sequence Listing 4 <223> Modified human IL-1Ra insert (99% identical to SEQ ID NO:2; Sequence Listing 5 <223> Modified human IL-1Ra insert (95% identical to SEQ ID NO:2; Sequence Listing 7 <223> IL-1β / IL-6 hybrid promoter

Claims

1. 1. A composition for use in treating a disease in a human subject, comprising a codon-modified IL-1Ra gene comprising a nucleic acid sequence encoding human IL-1Ra, wherein when administered to the human subject, the gene is efficiently expressed to provide a therapeutically effective amount of human interleukin-1 receptor agonist (IL-1Ra), and the gene comprises a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:

2.

2. The composition of claim 1 , wherein the disease is osteoarthritis or rheumatoid arthritis.

3. 3. The composition of claim 1 or 2, wherein administration results in amelioration of one or more symptoms of the disease.

4. The composition of claim 3, wherein administration results in the elimination of one or more symptoms of the disease.

5. The composition of any one of claims 1 to 4, wherein administration comprises intra-articular injection.

6. The composition of any one of claims 1 to 5, wherein the gene is administered to the subject once.

7. The composition according to any one of claims 1 to 5, wherein the gene is administered to the subject at least once and twice.

8. The composition of claim 7, wherein the second administration is more than one year after the first administration.

9. The composition according to any one of claims 1 to 8, wherein the codon-modified IL-1Ra gene encodes a human IL-1Ra comprising the amino acid sequence of SEQ ID NO:

6.

10. The composition of any one of claims 1 to 9, wherein the gene comprises a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:2.

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