METHODS AND COMPOSITIONS FOR TREATING CONDITIONS USING RECOMBINANT SELF-COMPLEMENTARY ADENO-ASSOCIATED VIRUS

Abstract

Methods and compositions for treating symptoms of conditions such as but not limited to osteoarthritis and rheumatoid arthritis. The methods may feature direct intraarticular injection of a recombinant self-complementary adeno-associated virus (sc-rAAV) with a vector adapted to express a modified IL-1Ra peptide. The methods of the present invention may express a therapeutically effective amount of the modified IL-1Ra peptide so as to ameliorating symptoms associated with the condition being treated.

Claims

1-67. (canceled)

68. A method comprising administering to a human subject into a location of interest a composition comprising a recombinant adeno-associated virus (rAAV) comprising a capsid and a nucleic acid vector comprising a modified IL-1Ra gene comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 2.

69. The method of claim 68, wherein the nucleic acid sequence that is at least 90% identical to SEQ ID NO: 2 is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

70. The method of claim 68, wherein the rAAV is self-complementary rAAV.

71. The method of claim 68, wherein the nucleic acid vector further comprises an SV40 and bovine growth hormone (bGH) polyadenylation sequence; and/or SV40 splice donor (SD) and splice acceptor (SA) sites.

72. The method of claim 68, wherein the nucleic acid sequence that is at least 90% identical to SEQ ID NO: 2 is operably linked to a promoter.

73. The method of claim 68, wherein the capsid comprises at least a portion of AAV1, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or combinations thereof.

74. The method of claim 68, wherein the location of interest is a joint, synovium, subsynovium, joint capsule, tendon, ligament, cartilage, or peri-articular muscle of the human subject.

75. The method of claim 68, wherein the composition is administered via direct intraarticular injection.

76. The method of claim 68, wherein the human subject is diagnosed with or is at risk for developing osteoarthritis or rheumatoid arthritis.

77. (canceled)

78. A method of delivering an IL-1Ra peptide to a chondrocyte or synoviocyte, comprising contacting the chondrocyte or synoviocyte with a composition comprising a recombinant adeno-associated virus (rAAV) comprising a capsid and a nucleic acid vector comprising a modified IL-1Ra gene comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 2.

79. The method of claim 78, wherein the nucleic acid sequence encodes a biologically active interleukin-1 receptor agonist (IL-1Ra) protein.

80. The method of claim 78, wherein the sequence that is at least 90% identical to SEQ ID NO: 2 is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

81. The method of claim 78, wherein the rAAV is self-complementary rAAV.

82-85. (canceled)

86. The method of claim 68, wherein the method is a method of repairing cartilage or reducing inflammation, comprising administering the composition comprising the rAAV into a location of cartilage.

87. The method of claim 86, wherein the human subject has been diagnosed with or is at risk for developing osteoarthritis or rheumatoid arthritis.

88. The method of claim 86, wherein the capsid comprises at least a portion of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or combinations thereof.

89. The method of claim 68, further comprising co-administering a secondary therapy to the location of interest.

90. The method of claim 68, wherein the method is a method of ameliorating symptoms of osteoarthritis or rheumatoid arthritis in a human; wherein the rAAV transduces the vector into cells in the location of interest, and wherein the modified IL-1Ra gene is expressed so as to provide the human with an amount of IL-1 Ra peptide effective for ameliorating symptoms associated with osteoarthritis or rheumatoid arthritis.

91. The method of claim 86, wherein the rAAV is a recombinant self-complementary adeno-associated virus (sc-rAAV), wherein said sc-rAAV comprises: a. an engineered AAV capsid; and b. a vector packaged within the capsid, said vector comprising a modified IL-1 Ra gene operably linked to a promoter, and wherein the modified IL-1Ra gene is at least 95% identical SEQ ID NO 2.

92. A method of providing interleukin-1 receptor agonist (IL-1Ra) peptide to an area of inflammation in a subject, said method comprising: introducing into a location of inflammation a composition comprising a recombinant self-complementary adeno-associated virus (sc-rAAV), wherein said sc-rAAV comprises: a. an engineered AAV capsid; and b. a vector packaged within the capsid, said vector comprising a modified IL-1 Ra gene operably linked to a promoter, the modified IL-1 Ra gene is at least 95% identical to SEQ ID NO: 2 or the IL-1Ra gene encodes an IL-1Ra protein as set forth in SEQ ID NO: 6; wherein the sc-rAAV transduces the vector into cells in the location of inflammation, wherein the modified IL-1 Ra gene is expressed so as to provide the cells in the location of inflammation a therapeutically effective amount of IL-1 Ra peptide effective for reducing inflammation.

93-96. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows Plasmid sc-rAAV2.5Hu-IL-1Ra, which contains a modified cDNA encoding the human IL-1Ra protein under 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 inverted terminal repeats (TR) has been verified by sequencing.

[0025] TERMS

[0026] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. “Comprising” means “including.” Hence “comprising A or B” means “including A” or “including B” or “including A and B.”

[0027] Suitable methods and materials for the practice and/or testing of embodiments of the 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 herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, the disclosures of which are incorporated in their entirety herein by reference.

[0028] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0029] Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0030] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

[0031] Adeno-associated virus (AAV), Recombinant AAV (rAAV), and Recombinant Self-Complementary AAV (sc-rAAV): AAV is a small virus (20 nm) in the family Parvoviridae. AAV is not known to cause disease. AAV has recently been used to gene therapy for a variety of reasons including that it has been shown to have low immunogenicity, the ability to effectively transduce non-dividing cells, and the ability to infect a variety of cell and tissue types. Recombinant AAV (rAAV) does not contain native viral coding sequences. Recombinant AAV DNA is packaged into the viral capsid as a single stranded molecule about 4600 nucleotides in length. Following infection of the cell by the virus, the molecular machinery of the cell converts the single DNA strand into a double-stranded form. Only the double stranded DNA form is useful to the proteins of the cell that transcribe the contained gene or genes into RNA. Self-complementary AAV (sc-rAAV) is an engineered form of rAAV that can form an intra-molecular double stranded DNA template. Thus, upon infection, the two complementary halves of sc-rAAV will associate to form one double stranded DNA unit that is ready for immediate replication and synthesis.

[0032] Expression: The translation of a nucleic acid sequence into a protein. Proteins may be expressed and remain intracellular, become a component of the cell surface membrane, or be secreted into the extracellular matrix or medium.

[0033] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0034] Pharmaceutically acceptable vehicles: Pharmaceutically acceptable carriers (vehicles), e.g., solutions, may be conventional but are not limited to conventional vehicles. For example, E. W. Martin, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 15th Edition (1975) and D. B. Troy, ed. Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore MD and Philadelphia, PA, 21.sup.st Edition (2006) describe compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules. In general, the nature of the carrier will depend on the particular mode of administration being employed. In addition to biologically-neutral carriers, pharmaceutical compositions administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0035] Preventing, treating, managing, or ameliorating a condition: “Preventing” a disease may refer to inhibiting the full development of a condition. “Treating” may refer to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. “Managing” may refer to a therapeutic intervention that does not allow the signs or symptoms of a disease or condition to worsen. “Ameliorating” may refer to the reduction in the number or severity of signs or symptoms of a disease or condition.

[0036] Sequence identity: The identity (or similarity) between two or more nucleic acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 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 Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site. BLASTN may be used to compare nucleic acid sequences, while BLASTP may be used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences. The BLAST-like alignment tool (BLAT) may also be used to compare nucleic acid sequences (Kent, Genome Res. 12:656-664, 2002). BLAT is available from several sources, including Kent Informatics (Santa Cruz, Calif.) and on the Internet (genome.ucsc.edu). Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1554 nucleotides is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth.

[0037] Therapeutically effective amount: A quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. Such agents may include IL-1Ra. For example, a therapeutically effective amount of IL-1Ra may be an amount sufficient to prevent, treat, or ameliorate symptoms of osteoarthritis or rheumatoid arthritis. The therapeutically effective amount of an agent useful for preventing, ameliorating, and/or treating a subject will be dependent on the subject being treated, the type and severity of the affliction, and the manner of administration of the therapeutic composition.

[0038] 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 encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viruses or viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. Such cells are sometimes called transformed cells.

[0039] Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may lack the nucleic acid sequences that permit it to replicate in a host cell. A vector may also include a gene of interest, one or more selectable marker genes, other genetic elements known in the art, or any other appropriate insert.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention features methods and compositions for delivering a therapeutic gene product (e.g., IL-1Ra) in a sustained manner to a location of interest, e.g., a joint. The present invention also features methods and compositions for treating symptoms of conditions such as but not limited to osteoarthritis or rheumatoid arthritis. The present invention also features methods and compositions for providing an individual (e.g., a human) a therapeutically effective amount of a therapeutic gene product (e.g., IL-1Ra). The methods and compositions may feature a recombinant self-complementary adeno-associated virus (sc-rAAV), wherein the sc-rAAV comprises an engineered capsid and a vector (an sc-rAAV vector) packaged within the capsid. The vector may comprise a transgene (e.g., a nucleotide sequence encoding a protein of interest, e.g., a therapeutic gene product, e.g., IL-1Ra or a modified version thereof) operably linked to a promoter (e.g., a constitutive promoter).

[0041] As previously discussed, the present invention features compositions comprising a recombinant self-complementary adeno-associated virus (sc-rAAVs) vector. A non-limiting example of a sc-rAAV vector is shown in SEQ ID NO: 1 of Table 1 below. The sc-rAAV vector of SEQ ID NO: 1 comprises a modified IL-1Ra gene. In some embodiments, the vector comprises SV40 polyadenylation sequences. In some embodiments, the vector comprises bovine growth hormone (bGH) polyadenylation sequences. In some embodiments, the vector comprises SV40 splice donor (SD) and splice acceptor (SA) sites. The sc-rAAV vector is not limited to SEQ ID NO: 1.

[0042] The sc-rAAV vectors comprise a nucleic acid that encodes a peptide of interest. 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. In some embodiments, the nucleic acid is at least 95% identical to SEQ ID NO: 2. 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 98% 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 a sequence for a modified human IL-1Ra that is about 98% identical to SEQ ID NO: 2; SEQ ID NO: 4 is a sequence for a modified human IL-1Ra that is about 99% identical to SEQ ID NO: 2; and (note that the bold letters in Table 1 are nucleotide substitutions as compared to SEQ ID NO: 2, and the codon underlined).

TABLE-US-00001 TABLE 1 SEQ ID NO: DESCRIPTION SEQUENCE 1 Sequence of Hu-IL-1Ra CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGG plasmid containing entire CGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGG viral sequence with CTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCC modified human IL1Ra ACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAG insert (underlined) and CAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT the SacII/NotI restriction AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG sites in bold italics. AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGA TACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGC TCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCA CTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA AGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGA AAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGC GGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGA TCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGG AACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG ATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATC TAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTT GCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCA TCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCT CCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGA AGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGC CGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATG GCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCC CCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTT GTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTG ACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGA CCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACAT AGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGA AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCC ACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTT TCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATA AGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATAT TATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTT GAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCC CGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTA ACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTC GGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCLGGAGACGGTC ACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGC GCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCA TCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCG CACAGATGCGTAAGGAGAAAATACCGCATCAGGAATTCCAACATCCAA TAAATCATACAGGCAAGGCAAAGAATTAGCAAAATTAAGCAATAAAGC CTCAGAGCATAAAGCTAAATCGGTTGTACCAAAAACATTATGACCCTG TAATACTTTTGCGGGAGAAGCCTTTATTTCAACGCAAGGATAAAAATT TTTAGAACCCTCATATATTTTAAATGCAATGCCTGAGTAATGTGTAGG TAAAGATTCAAACGGGTGAGAAAGGCCGGAGACAGTCAAATCACCATC AATATGATATTCAACCGTTCTAGCTGATAAATTCATGCCGGAGAGGGT AGCTATTTTTGAGAGGTCTCTACAAAGGCTATCAGGTCATTGCCTGAG AGTCTGGAGCAAACAAGAGAATCGATGAACGGTAATCGTAAAACTAGC ATGTCAATCATATGTACCCCGGTTGATAATCAGAAAAGCCCCAAAAAC AGGAAGATTGTATAAGCAAATATTTAAATTGTAAACGTTAATATTTTG TTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAA TAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAG ATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAG AACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGAT GGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGG TGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGA GCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAA GCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTG CGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCG TACTATGGTTGCTTTGACGAGCACGTATAACGTGCTTTCCTCGTTAGA ATCAGAGCGGGAGCTAAACAGGAGGCCGATTAAAGGGATTTTAGACAG GAACGGTACGCCAGAATCCTGAGAAGTGTTTTTATAATCAGTGAGGCC ACCGAGTAAAAGAGTCTGTCCATCACGCAAATTAACCGTTGTCGCAAT ACTTCTTTGATTAGTAATAACATCACTTGCCTGAGTAGAAGAACTCAA ACTATCGGCCTTGCTGGTAATATCCAGAACAATATTACCGCCAGCCAT TGCAACAGGAAAAACGCTCATGGAAATACCTACATTTTGACGCTCAAT CGTCTGGAATTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGG CGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGCGCTCGCTC GCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGG TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA CTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTA CTTATCTACGTAGCCATGCTCGATCTGAATTCGGTACCCGTTACATAA CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCA TTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACT TTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTG GCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTC AATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTA TGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATT ACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGG AGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAAC AACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAG GTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA CGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCC AGCCTCCGGACTCTAGAGGATCCGGTACTCGAGGAACTGAAAAACCAG AAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCG GATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGC CTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTG CGGAATTGTAC CCACCATGGAAATCTGCAGAGGCCTGCGGA GCCACCTGATTACCCTGCTGCTGTTCCTGTTCCACAGCGAGACAATCT GCCGGCCCAGCGGCCGGAAGTCCAGCAAGATGCAGGCCTTCCGGATCT GGGACGTGAACCAGAAAACCTTCTACCTGCGGAACAACCAGCTGGTGG CCGGATACCTGCAGGGCCCCAACGTGAACCTGGAAGAGAAGATCGACG TGGTGCCCATCGAGCCCCACGCCCTGTTTCTGGGCATCCACGGCGGCA AGATGTGCCTGAGCTGCGTGAAGTCCGGCGACGAGACAAGACTGCAGC TGGAAGCCGTGAACATCACCGACCTGAGCGAGAACCGGAAGCAGGACA AGAGATTCGCCTTCATCAGAAGCGACAGCGGCCCCACCACCAGCTTTG AGAGCGCCGCCTGCCCCGGCTGGTTCCTGTGTACAGCCATGGAAGCCG ACCAGCCCGTGTCCCTGACAAACATGCCCGACGAGGGCGTGATGGTCA CCAAGTTCTATTTTCAAGAAGATGAGTAATAA CGGGATCC AGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAAT GCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTT ATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTG CATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTA GTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCA GCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGG TGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCA TTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGA CAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGAACCCCACTCCCTC TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCG ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGA GCGAGCGAGCGCGCAGCTGCTG 2 Modified human IL-1Ra ATGGAAATCTGCAGAGGCCTGCGGAGCCACCTGATTACCCTGCTGCTG insert TTCCTGTTCCACAGCGAGACAATCTGCCGGCCCAGCGGCCGGAAGTCC AGCAAGATGCAGGCCTTCCGGATCTGGGACGTGAACCAGAAAACCTTC TACCTGCGGAACAACCAGCTGGTGGCCGGATACCTGCAGGGCCCCAAC GTGAACCTGGAAGAGAAGATCGACGTGGTGCCCATCGAGCCCCACGCC CTGTTTCTGGGCATCCACGGCGGCAAGATGTGCCTGAGCTGCGTGAAG TCCGGCGACGAGACAAGACTGCAGCTGGAAGCCGTGAACATCACCGAC CTGAGCGAGAACCGGAAGCAGGACAAGAGATTCGCCTTCATCAGAAGC GACAGCGGCCCCACCACCAGCTTTGAGAGCGCCGCCTGCCCCGGCTGG TTCCTGTGTACAGCCATGGAAGCCGACCAGCCCGTGTCCCTGACAAAC ATGCCCGACGAGGGCGTGATGGTCACCAAGTTCTATTTTCAAGAAGAT GAGTAATAA 3 Modified human IL-1Ra ATGGAAATCTGCAGAGGACTGCGGAGCCACCTAATTACCCTACTCCTT insert (98% identical to TTCCTGTTCCACAGCGAGACAATCTGCCGGCCCAGCGGCCGGAAGTCC SEQ ID NO: 2; bold AGCAAGATGCAGGCTTTCCGGATCTGGGACGTGAACCAGAAAACCTTC letters are nucleotide TACCTCCGGAACAACCAGCTGGTGGCGGGATACCTCCAGGGCCCCAAC substitutions within a GTGAACCTAGAAGAGAAGATCGACGTGGTGCCCATCGAGCCCCACGCC codon (codon is CTGTTTCTGGGCATCCACGGCGGCAAGATGTGCCTGAGCTGCGTGAAG underlined)) TCCGGCGACGAGACAAGACTGCAGCTGGAAGCCGTGAACATCACCGAC CTGAGCGAGAACCGGAAGCAGGACAAGAGATTCGCCTTCATCAGAAGC GACAGCGGCCCCACCACCAGCTTTGAGAGCGCCGCCTGCCCCGGCTGG TTCCTGTGTACAGCCATGGAAGCCGACCAGCCCGTGTCCCTGACAAAC ATGCCCGACGAGGGCGTGATGGTCACCAAGTTCTATTTTCAAGAAGAT GAGTAATAA 4 Modified human IL-1Ra ATGGAGATCTGCAGAGGCCTGCGGAGCCATCTGATTACCCTACTGCTT insert (99% identical to TTCCTGTTCCATAGCGAGACAATCTGCCGGCCCAGCGGCCGGAAGTCC SEQ ID NO: 2; bold AGCAAAATGCAGGCCTTCCGGATCTGGGACGTGAACCAGAAAACCTTC letters are nucleotide TACCTGCGGAACAACCAGCTGGTGGCCGGATACCTGCAGGGCCCCAAG substitutions within a GTGAACCTGGAAGAGAAGATCGACGTGGTGCCCATCGAGCCCCACGCC codon (codon is CTGTTTCTGGGCATCCACGGCGGCAAGATGTGCCTGAGCTGCGTGAAG underlined)) TCCGGCGACGAGACAAGACTGCAGCTGGAAGCCGTGAACATCACCGAC CTGAGCGAGAACCGGAAGCAGGACAAGAGATTCGCCTTCATCAGAAGC GACAGCGGCCCCACCACCAGCTTTGAGAGCGCCGCCTGCCCCGGCTGG TTCCTGTGTACAGCCATGGAAGCCGACCAGCCCGTGTCCCTGACAAAC ATGCCCGACGAGGGCGTGATGGTCACCAAGTTCTATTTTCAAGAAGAT GAGTAATAA 5 Modified human IL-1Ra ATGGAGATCTGCAGAGGACTGCGGAGCCACCTAATTACCCTACTCCTT insert (95% identical to TTCCTGTTCCATAGCGAGACAATCTGCCGGCCCAGCGGCCGGAAGTCC SEQ ID NO: 2; bold AGCAAAATGCAGGCTTTCCGGATCTGGGATGTGAACCAGAAGACCTTC letters are nucleotide TACCTCCGGAACAACCAGCTGGTGGCGGGATACCTCCAGGGCCCCAAC substitutions within a GTGAACCTAGAAGAGAAGATCGACGTGGTGCCCATCGAGCCCCACGCC codon (codon is CTGTTTCTGGGCATCCATGGCGGCAAGATGTGTCTGAGTTGCGTGAAG underlined)) TCAGGCGACGAGACAAGACTGCAGCTGGAAGCCGTGAACATCACCGAC CTGAGCGAAAACCGGAAGCAGGACAAGAGATTCGCCTTCATCAGAAGC GACAGCGGCCCCACCACTAGCTTTGAGAGCGCAGCCTGCCCCGGCTGG TTCCTGTGTACAGCCATGGAGGCCGACCAGCCCGTGTCCCTGACAAAC ATGCCTGACGAAGGCGTGATGGTCACCAAGTTCTACTTTCAAGAAGAT GAATAATAA

[0043] In some embodiments, the IL-1Ra peptide encoded by the IL-1Ra insert comprises IL-1Ra (see SEQ ID NO: 6 in Table 2 below).

TABLE-US-00002 TABLE 2 SEQ ID NO: DESCRIPTION SEQUENCE 6 IL-1Ra MEICRGLRSH LITLLLFLFH SETICRPSGR KSSKMQAFRI (UNIPROT WDVNQKTFYL RNNQLVAGYL QGPNVNLEEK P18510) IDVVPIEPHA LFLGIHGGKM CLSCVKSGDE TRLQLEAVNI TDLSENRKQD KRFAFIRSDS GPTTSFESAA CPGWFLCTAM EADQPVSLTN MPDEGVMVTK FYFQEDE

[0044] The transgene (e.g., nucleotide sequence encoding protein of interest) is operably linked to a promoter. In some embodiments, the promoter comprises the cytomegalovirus (CMV) promoter. The present invention is not limited to the CMV promoter and may feature any appropriate promoter or portions of various promoters. Examples of promoters include CMV promoter, hybrid CMV promoter, CAG promoter, human beta-actin promoter, hybrid beta-actin promoter, EF1 promoter, U1a promoter, U1b promoter, a Tet-inducible promoter, a VP16-LexA promoter, chicken beta-actin (CBA) promoter, human elongation factor-1alpha promoter, simian virus 40 (SV40) promoter, and herpes simplex virus thymidine kinase promoter.

[0045] In some embodiments, the promoter comprises a hybrid promoter. As an example, Table 3 shows an IL-1 beta/IL-6 hybrid promoter (see also van de Loo et al., 2004, Gene Therapy 11:581-590). The present invention is also not limited to the hybrid promoter shown in Table 3.

TABLE-US-00003 TABLE 3 SEQ ID NO: DESCRIPTION SEQUENCE 7 IL-1 beta/ atccaagag ggagaagaag cccattggag atgatgccat aaaggaagtg IL-6 hybrid gaagcgatat gataaaaatc atagtgccca ttcccaaata atcccagaag promoter cagaagggaa aggagagaaa tatccacaaa gacaggtgtg ggtacacaca acatttttca tactttaaga tcccagagga ctcatggaaa tgatacaaga aaatgactca taagaacaaa tattaggaag ccagtgccaa gaatgagatg ggaaattggg gaaaatgttg ggggcagatt gcttagttct gttctaagca agagggtgaa caaggaagga acagctcact acaaagaaca gacatcactg catgtacaca caataatata agaactaacc catgattatt ttgcttgtct tcttgttcaa aatgattgaa gaccaatgag atgagatcaa ccttgataac tggctggctt cggcatgatt agacacaaga tggtatcagg gcacttgctg ctttgaataa tgtcagtctc ctgtcttgga agaatgacct gacagggtaa agaggaactt gcagctgaga aaggctttag tgactcaaga gctgaataat tccccaaaag ctggagcatc ctggcatttc cagctcccca tctctgcttg ttccacttcc ttggggctac atcaccatct acatcatcat cactcttcca ctccctccct tagtgccaac tatgtttata gcgagatatt ttctgctcat tggggatcgg aaggaagtgc tgtggcctga gcggtctcct tgggaagaca ggatctgata catacgttgc acaacctatt tgacataaga ggtttcactt cctgagatgg atgggatggt agcagatttg ggtccaggtt acagggccag gatgagacat ggcagaactg tggagactgt tacgtcaggg ggcattgccc catggctcca aaatttccct cgagc ctctggccc caccctcacc ctccaacaaa gatttatcaa atgtgggatt ttcccatgag tctcaatatt agagtctcaa cccccaataa atataggact ggagatgtct gaggctcatt ctgccctcga gcccaccggg aacgaaagag aagctctatc tcccctccag gagcccagct atgaactcct tc

[0046] In some embodiments, the sc-rAAV vector is packaged within a capsid. In some embodiments, the capsid comprises at least a portion of AAV serotype 1 (AAV1), AAV serotype 2, (AAV2), AAV serotype 3, (AAV3), AAV serotype 4, (AAV4), AAV serotype 5, (AAV5), AAV serotype 6, (AAV6), derivatives thereof, or combination thereof. 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., AAV2.5.

[0047] The composition, e.g., the composition comprising the sc-rAAV, may be introduced into cells in a location of interest (e.g., in a human). For example, in some embodiments when treating symptoms of osteoarthritis, the composition may be introduced into cells (e.g., chondrocytes, synoviocytes, e.g., type A, type B, etc.) in a joint via direct intraarticular injection. In some embodiments, the composition is administered to a joint, synovium, subsynovium, joint capsule, tendon, ligament, cartilage, or peri-articular muscle of the human. The present invention is not limited to the aforementioned conditions (e.g., osteoarthritis), the means of administration (e.g., intraarticular injection), the location of interest (e.g., joint), or cell type (e.g., chondrocytes, synoviocytes). For example, in some embodiments, other cell types that may be transduced may include mesenchymal stem cells.

[0048] The sc-rAAV transduces the vector into cells and the modified IL-1Ra peptide is expressed. In some embodiments, the IL-1Ra peptide is expressed so as to provide the human with a therapeutically effective amount of said modified IL-1Ra peptide effective for ameliorating symptoms associated with various conditions such as osteoarthritis or rheumatoid arthritis.

[0049] In some embodiments, introduction of the composition (e.g., the sc-rAAV) is performed once. In some embodiments, introduction of the composition (e.g., the sc-rAAV) is performed twice, e.g., a first time and a second time subsequent to the first time. In some embodiments, introduction of the composition is performed more than two times, e.g., three times, four times, five times, etc. The introduction of the composition a second time may be performed at a time point after the time when the method is first performed, e.g., after 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, more than one year, etc.

[0050] The composition may comprise any appropriate pharmaceutical composition. In some embodiments, the composition comprises a buffered solution. In some embodiments, the buffered solution 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, e.g., NaCI. The concentration of salt may be any appropriate concentration, e.g., 350 mM NaCI, more than 350 mM NaCI, less then 350 mM, etc.

[0051] In some embodiments, the composition (e.g., the sc-rAAV) is co-administered with a secondary therapy. In some embodiments, the secondary therapy comprises a therapeutic for OA or RA or any other appropriate therapy for treating the symptoms of the condition. Non-limiting examples of secondary therapies for OA include glucocorticoids, hyaluronan (viscosupplementation), platelet-rich plasma, and recombinant, human IL-1Ra (Anakinra; Kineret®). For example, in some embodiments, the sc-rAAV is co-administered with glucocorticoids or platelet-rich plasma.

[0052] The disclosures of the following U.S. Patents are incorporated in their entirety by reference herein: US2008/0187576, US2009/0104155, KR2012041139, JP2015518816, WO2013151672, WO2008088895, U.S. Pat. Nos. 8,529,885, 7,037,492, US20070128177, U.S. Pat. Nos. 6,491,907, 8,999,948, US20150218586, U.S. Pat. No. 7,892,824, US20130295614, JP2002538770, JP2010516252, KR2002027450, KR2003028080, U.S. Pat. No. 6,482,634, US20090105148, US20120232130, US20140234255, U.S. Pat. Nos. 5,756,283, 6,083,716, WO2002038782, WO2007039699, WO2012047093, WO2014170470, WO2015018860, WO2015044292, WO2015158749, U.S. Pat. Nos. 7,452,696, 6,943,153, 6,429,001, WO2015031392, WO2004092211.

EXAMPLE 1

[0053] Example 1 describes administration of a sc-rAAV of the present invention (encoding IL-1Ra). The present invention is not limited to the disclosure of Example 1. Five patients enroll in a clinical trial investigating administration of a sc-rAAV of the present invention. The patients are as follows: (1) a 65 year old male with osteoarthritis in his right knee; (2) a 59 year old male with osteoarthritis in his left knee; (3) a 58 year old female with osteoarthritis in her left knee; (4) a 51 year old male with osteoarthritis in his right knee; and (5) a 48 year old male with osteoarthritis in his right knee. Each patient is administered the sc-rAAV via intraarticular injection at 1×10.sup.12 viral genes per knee. IL-1Ra is expressed in the chondrocytes and synoviocytes. Patient 1 describes amelioration of OA-related symptoms within 2 weeks. Patient 2 describes amelioration of OA-related symptoms within 1 week. Patient 3 describes amelioration of OA-related symptoms within 5 weeks. Patient 4 describes amelioration of OA-related symptoms within 1 week. As of 6 weeks, Patient 5 describes no amelioration of OA-related symptoms.

EXAMPLE 2

[0054] Example 2 describes a first administration of a sc-rAAV of the present invention (encoding IL-1Ra) and a second administration of the same sc-rAAV of the present invention after a period of time. The present invention is not limited to the disclosure of Example 2. A 55-year-old male presents with osteoarthritis in his right knee. His physician performs a single intra-articular injection of the sc-rAAV vector of the present invention (encoding IL-1Ra). The patient's symptoms are eliminated within 2 months. After 6 months, the physician administers a second (single) intra-articular injection of the same sc-rAAV vector (encoding IL-1Ra) of the present invention. The patient's symptoms are still absent 6 months following the second injection.

EXAMPLE 3

[0055] Example 3 describes a first administration of a sc-rAAV of the present invention (encoding IL-1Ra) and a second administration of a sc-rAAV of the present invention (encoding IL-1Ra) different from the first sc-rAAV after a period of time. The present invention is not limited to the disclosure of Example 3. A 49-year-old female presents with osteoarthritis in her right ankle. Her physician performs a single intra-articular injection of the sc-rAAV vector of the present invention (encoding IL-1Ra). The patient's symptoms have improved within 5 months but are not eliminated. After 6 months, the physician administers a second (single) intra-articular injection of a different sc-rAAV vector (encoding IL-1Ra) of the present invention. Six months following the second injection, the patient's symptoms are eliminated.

EXAMPLE 4

[0056] Example 4 describes co-administration of a sc-rAAV of the present invention (encoding IL-1Ra) and a secondary therapy. The present invention is not limited to the disclosure of Example 4. A 68-year-old male presents with osteoarthritis in his left knee. His physician performs a single intra-articular injection of both a sc-rAAV vector of the present invention (encoding IL-1Ra) and platelet-rich plasma. The patient's symptoms are eliminated within 2 months.

[0057] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.

[0058] Although there has been shown and described embodiments of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Reference numbers recited in the claims are exemplary and for ease of review by the patent office only, and are not limiting in any way. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting of” is met.

[0059] Any reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.