JP-2022529775-A5 -

Dates

Publication Date

20230511

Application Date

20200424

Description

All references cited herein, including this patent application and publications, are incorporated by reference in their entirety. The present invention provides, for example, the following items: (Item 1) (a) A variant AAV capsid protein comprising a modified sequence having one or more amino acid substitutions within amino acid residues 570-579 compared to the parent adeno-associated virus (AAV) capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1) and the amino acid residue numbering corresponds to that of the AAV5 VP1 capsid protein; and (b) Polynucleotide sequences encoding therapeutic gene products Recombinant AAV billions, including (Item 2) The recombinant AAV virion according to item 1, wherein the parent AAV capsid protein is an AAV5 capsid protein or an AAV5 and AAV2 hybrid capsid protein. (Item 3) The recombinant AAV virion according to item 1 or 2, wherein the parent AAV capsid protein is the AAV2.5T capsid protein. (Item 4) The recombinant AAV virion according to any one of items 1 to 3, wherein the parent AAV capsid protein is the AAV2.5T VP1 capsid protein. (Item 5) The recombinant AAV virion described in any one of items 1 to 4, wherein the modified sequence includes LAHKFKSGDA (sequence number 3). (Item 6) The recombinant AAV virion according to any one of items 1 to 5, wherein the variant AAV capsid protein comprises a capsid sequence having at least 85% homology to the amino acid sequence described in SEQ ID NO: 4 or SEQ ID NO: 5. (Item 7) The recombinant AAV virion according to any one of items 1 to 6, wherein the variant AAV capsid protein comprises the capsid sequence described in SEQ ID NO: 6 or SEQ ID NO: 7. (Item 8) A recombinant AAV virion according to any one of items 1 to 7, wherein the rAAV virion is a variant AAV5, or a variant AAV2 and AAV5 hybrid virion. (Item 9) A recombinant AAV virion according to any one of items 1 to 8, wherein the rAAV virion is a variant AAV2.5T virion. (Item 10) Recombinant AAV virions as described in any one of items 1 to 9, which can be transduced into retinal cells when injected intravitreously into mammals. (Item 11) Recombinant AAV virion as described in item 10, which can be transduced into one or more of the following when injected intravitreously into a mammal: photoreceptor cells, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, and retinal pigment epithelial cells. (Item 12) Recombinant AAV virions as described in item 10 or 11, which can be transduced into retinal pigment epithelial cells when injected intravitreously into mammals. (Item 13) A recombinant AAV virion according to any one of items 1 to 12, wherein the therapeutic gene product is siRNA, miRNA, or protein. (Item 14) The recombinant AAV virion according to any one of items 1 to 13, wherein the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product. (Item 15) The recombinant AAV virion according to any one of items 1 to 13, wherein the therapeutic gene product is opsin. (Item 16) The recombinant AAV virion according to any one of items 1 to 15, wherein the polynucleotide encoding the therapeutic gene product is adjacent to the ITR of one or more AAVs. (Item 17) The recombinant AAV virion described in item 16, wherein the ITR of one or more AAVs is the ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, bird AAV, cattle AAV, canine AAV, horse AAV, primate AAV, non-primate AAV, or sheep AAV, or a variant thereof. (Item 18) The recombinant AAV virion according to item 16 or 17, wherein the ITR of one or more AAVs is the ITR of AAV2 or the ITR of AAV5. (Item 19) Recombinant AAV virions according to any one of items 1 to 18, having altered cell-targeting properties compared to AAV2.5T. (Item 20) A pharmaceutical composition comprising recombinant AAV virion as described in any one of items 1 to 19. (Item 21) A method for producing rAAV virions, (a) A step of culturing host cells under conditions in which rAAV virions are produced, wherein the host cells (i) A polynucleotide encoding a variant AAV capsid protein comprising a modified sequence containing one or more amino acid substitutions within amino acid residues 570-579 compared to the parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1); (ii) Polynucleotides that encode rep proteins; (iii) A polynucleotide cassette containing a sequence encoding a therapeutic gene product adjacent to the ITR of at least one AAV; and (iv) AAV Helper function Steps including; and (b) Step of recovering rAAV virions produced by the host cells. Methods that include... (Item 22) A method for providing a therapeutic gene product to the retina of a subject, comprising the step of administering to the subject by intravitreal injection a recombinant AAV virion according to any one of items 1 to 19, or a pharmaceutical composition according to item 20. (Item 23) The aforementioned subjects have conditio