Peptide-Modified Hybrid Recombinant Adeno-Associated Virus Serotype Between AAV9 and AAVrh74 with Reduced Liver Tropism and Increased Muscle Transduction

Abstract

The invention relates to a recombinant adeno-associated virus (AAV) capsid protein, which is a peptide-modified hybrid between AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins comprising at least one copy of a peptide comprising the RGD motif, wherein said recombinant peptide-modified hybrid AAV capsid protein has a further reduced liver tropism and an increased muscle transduction compared to the recombinant hybrid AAV capsid protein not having said peptide. The invention relates also to the derived peptide-modified hybrid AAV serotype vector particles packaging a gene of interest and their use in gene therapy, in particular for treating neuromuscular genetic diseases, in particular muscular genetic diseases.

1 . A recombinant adeno-associated virus (AAV) capsid protein, which is a peptide-modified hybrid between AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins comprising at least one copy of a peptide comprising the RGD motif, wherein said recombinant peptide-modified hybrid AAV capsid protein has a further reduced liver tropism and an increased muscle transduction compared to the recombinant hybrid AAV capsid protein not having said peptide. 
     
     
         2 . The recombinant hybrid AAV capsid protein according to  claim 1 , which results from the replacement of a variable region in the AAV9 or AAVrh74 capsid sequence with the corresponding variable region of the other AAV serotype capsid sequence,
 wherein the variable region of AAV9 capsid corresponds to the sequence situated from any one of positions 331 to 493 to any one of positions 556 to 736 in AAV9 capsid of SEQ ID NO: 1 or a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 consecutive amino acids of the sequence situated from positions 493 to 556 in AAV9 capsid of SEQ ID NO: 1, and   the variable region of AAVrh74 capsid corresponds to the sequence situated from any one of positions 332 to 495 to any one of positions 558 to 738 in AAVrh74 capsid of SEQ ID NO: 2 or a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 consecutive amino acids of the sequence situated from positions 495 to 558 in AAVrh74 capsid of SEQ ID NO: 2.   
     
     
         3 . The recombinant hybrid AAV capsid protein according to  claim 2 , which results from the replacement of the variable region corresponding to the sequence situated from positions 449 to 609 in AAV9 capsid of SEQ ID NO: 1 or from positions 450 to 611 in AAVrh74 capsid of SEQ ID NO: 2 with the corresponding variable region of the other AAV serotype capsid sequence. 
     
     
         4 . The recombinant hybrid AAV capsid protein according to  claim 1 , which is derived from a hybrid AAV capsid protein comprising a sequence selected from the group consisting of the sequences SEQ ID NO: 3 and SEQ ID NO: 4 and the sequences having at least 85%, 90%, 95%, 97%, 98% or 99% identity with said sequences. 
     
     
         5 - 6 . (canceled) 
     
     
         7 . The recombinant hybrid AAV capsid protein according to  claim 1 , wherein the peptide comprising the RGD motif is of up to 30 amino acids, and comprises or consists of any one of: RGDLGLS (SEQ ID NO: 8), LRGDGLS (SEQ ID NO: 14), LGRGDLS (SEQ ID NO: 15), LGLRGDS (SEQ ID NO: 16), LGLSRGD (SEQ ID NO: 17) and RGDMSRE (SEQ ID NO: 18). 
     
     
         8 - 10 . (canceled) 
     
     
         11 . The recombinant hybrid AAV capsid protein according to  claim 7 , wherein the sequences SEQ ID NO: 8 and 14 to 18 are flanked by GQSG (SEQ ID NO: 9) and AQAA (SEQ ID NO: 10), respectively at the N- and C-terminal end of the peptide. 
     
     
         12 . The recombinant hybrid AAV capsid protein according to  claim 7 , wherein the peptide comprises or consists of SEQ ID NO: 13. 
     
     
         13 . (canceled) 
     
     
         14 . The recombinant hybrid AAV capsid protein according to  claim 1 , wherein the at least one copy of the peptide comprising the RGD motif is inserted into a site exposed on the AAV capsid surface chosen from a site around any of positions 261, 383, 449, 575 or 590 according to the numbering in SEQ ID NO: 3. 
     
     
         15 . (canceled) 
     
     
         16 . The recombinant hybrid AAV capsid protein according to  claim 14 , wherein the at least one copy of the peptide comprising the RGD motif is inserted around position 449 or 590 according to the numbering in SEQ ID NO: 3. 
     
     
         17 . (canceled) 
     
     
         18 . The recombinant hybrid AAV capsid protein according to  claim 16 , wherein the insertion site of the at least one copy of the peptide comprising the RGD motif is from positions 587 to 592 according to the numbering in SEQ ID NO: 3, and wherein the peptide comprising the RGD motif replaces all the residues from positions 587 to 592 of the AAV capsid protein according to the numbering in SEQ ID NO: 3. 
     
     
         19 . (canceled) 
     
     
         20 . The recombinant hybrid AAV capsid protein according to  claim 1 , which comprises a sequence selected from the group consisting of SEQ ID NO: 5 and the sequences having at least 85%, 90%, 95%, 97%, 98% or 99% identity with said sequence. 
     
     
         21 . The recombinant hybrid AAV capsid protein according to  claim 1 , which is a hybrid VP1, VP2 or VP3 protein. 
     
     
         22 . A recombinant chimeric AAV capsid protein, which is selected from the group consisting of:
 a chimeric VP1 protein comprising: (i) a VP1-specific N-terminal region having a sequence from natural or artificial AAV serotype other than AAV9 and AAVrh74, (ii) a VP2-specific N-terminal region having a sequence from AAV9, AAVrh74 or natural or artificial AAV serotype other than AAV9 and AAVrh74, and (iii) a VP3 C-terminal region having the sequence of a hybrid VP3 protein according to  claim 21 , and   a chimeric VP2 protein comprising: (i) a VP2-specific N-terminal region having a sequence from natural or artificial AAV serotype other than AAV9 and AAVrh74, and (ii) a VP3 C-terminal region having the sequence of a hybrid VP3 protein according to  claim 21 .   
     
     
         23 . A polynucleotide encoding the recombinant hybrid AAV capsid protein according to  claim 1 , in expressible form, and eventually further encoding AAV Replicase protein in expressible form. 
     
     
         24 . (canceled) 
     
     
         25 . An AAV vector particle packaging a gene of interest, which comprises the hybrid recombinant AAV capsid protein according to  claim 1 , and eventually also at least one AAV capsid protein from natural or artificial AAV serotype other than AAV9 and AAVrh74. 
     
     
         26 . The AAV vector particle according to  claim 25 , wherein the gene of interest is selected from the group consisting of:
 (i) therapeutic genes;   (ii) genes encoding therapeutic proteins or peptides such as therapeutic antibodies or antibody fragments and genome editing enzymes; and   (iii) genes encoding therapeutic RNAs such as interfering RNAs, guide RNAs for genome editing and antisense RNAs capable of exon skipping.   
     
     
         27 . A pharmaceutical composition comprising a therapeutically effective amount of AAV vector particles according to  claim 26 . 
     
     
         28 . A method of treating a disease by gene therapy, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the recombinant hybrid AAV capsid protein according to  claim 1 . 
     
     
         29 . The method according to  claim 28 , wherein the gene therapy targets a gene responsible for a neuromuscular genetic disorders selected from the group consisting of Duchenne muscular dystrophy, Limb-girdle muscular dystrophies, Spinal muscular atrophy, Myotubular myopathy, Pompe disease and Glycogen storage disease III. 
     
     
         30 . The method according to  claim 29 , wherein the target gene is selected from the group consisting of DMD, CAPN3, DYSF, FKRP, ANO5, SMN1, MTM1, GAA and AGL genes.