Modified rAAV capsid protein for gene therapy
The invention relates to recombinant adeno-associated virus (rAAV) virions for gene therapy, wherein the rAAV virions comprise a novel capsid protein. In particular, the invention relates to the use of such virions in gene therapy for the treatment of an arthritic disease, such as for example rheumatoid arthritis, or symptoms thereof, preferably by intraarticular administration.
1 . A method for delivering a transgene to a fibroblast-like synoviocyte (FLS), the method comprising contacting the FLS with a recombinant adeno-associated virus (rAAV) virion comprising the transgene and a modified VP1 capsid protein, wherein:
the modified VP1 capsid protein comprises
(SEQ ID NO: 27)
EEEIxxxxPVATExxGxxxxNxQy-Z-(x) n LPGMVWQxRDVYLQGPI
WAKIPHTDG,
wherein:
(a) n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;
(b) Z comprises
(SEQ ID NO: 23)
y-G-Q-x-G-(x) 3 -R-(x) 3 -y-A-Q-A-A;
(c) x represents a single amino acid residue; and
(d) y represents 0, 1 or 2 amino acid residues.
2 . The method according to claim 1 , wherein the modified VP1 capsid protein comprises a capsid amino acid sequence selected from the group consisting of:
i) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, wherein amino acids at positions 588-602 of SEQ ID NO: 1 consist of SEQ ID NO: 11,
ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 2, wherein amino acids at positions 585-599 of SEQ ID NO: 2 consist of SEQ ID NO: 10,
iii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3, wherein amino acids at positions 587-601 of SEQ ID NO: 3 consist of SEQ ID NO: 9,
iv) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 4, wherein amino acids at positions 586-600 of SEQ ID NO: 4 consist of SEQ ID NO: 8,
v) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 5, wherein amino acids at positions 588-602 of SEQ ID NO: 5 consist of SEQ ID NO: 9,
vi) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 6, wherein amino acids at positions 588-602 of SEQ ID NO: 6 consist of SEQ ID NO: 8, and
vii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7, wherein amino acids at positions 587-601 of SEQ ID NO: 7 consist of SEQ ID NO: 12;
wherein sequence identity is calculated based on the full length of the capsid amino acid sequence.
3 . The method according to claim 1 , wherein the modified VP1 capsid protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs:1-7.
4 . The method according to claim 1 , wherein the transgene comprises a nucleotide sequence encoding a gene product of interest and wherein the nucleotide sequence encoding the gene product of interest is located between two AAV inverted terminal repeat (ITR) sequences.
5 . The method according to claim 1 , wherein the transgene encodes for a gene product of interest, wherein the gene product of interest is selected from the group consisting of interleukins, immune-modulators, antibodies, shRNA, miRNA, guide RNA, growth factors, proteases, nucleotidases/nucleosidases, peptides, protease inhibitors, inhibitors, enzymes, and combinations thereof.
6 . The method according to claim 2 , wherein the gene product of interest is selected from the group consisting of CD39, CD73, and IFN-β.
7 . The method according to claim 1 , wherein the transgene comprises at least one of:
(i) a polynucleotide comprising a sequence encoding at least one guide RNA; wherein each guide RNA is substantially complementary to a target polynucleotide sequence(s) in a genome; and
(ii) a polynucleotide comprising a sequence encoding a nuclease; wherein the nuclease forms a ribonuclease complex with the guide RNA, and wherein the ribonuclease complex makes site-specific double-stranded DNA breaks (DSDB) in the genome.
8 . The method according to claim 1 , wherein the rAAV virion is administered to the subject in need thereof in a composition comprising the rAAV virion and a pharmaceutically acceptable carrier.
9 . The method according to claim 1 , wherein the rAAV composition comprises empty AAV capsids to mature AAV capsids in a ratio of at least 1:1.
10 . The method according to claim 1 , wherein the rAAV virion is administered systemically and/or locally.
11 . The method according to claim 5 , wherein the gene product of interest is a tumor necrosis factor alpha (TNFα) inhibitor.
12 . The method according to claim 11 , wherein the TNFα inhibitor is selected from the group consisting of etanercept, infliximab, adalimumab, certolizumab pegol and golimumab.
13 . The method according to claim 12 , wherein the TNFα inhibitor is etanercept.