AAV TRANSFER CASSETTE
Described herein are AAV transfer cassettes and plasmids used in the production of recombinant adeno-associated viral (rAAV) vectors. The disclosed cassettes and plasmids comprise one or more transgenes having therapeutic efficacy in the amelioration, treatment and/or prevention of one or more diseases or disorders, such as Niemann-Pick Disease, type C1 (NPC1).
1 . An Adeno-Associated Virus (AAV) transfer cassette comprising, from 5′ to 3′:
a 5′ inverted terminal repeat (ITR);
a chicken beta-actin promoter;
a transgene;
a polyadenylation signal; and
a 3′ ITR;
wherein the transfer cassette comprises an intronic sequence; and
wherein the transgene encodes the NPC1 protein.
2 . The AAV transfer cassette of claim 1 , wherein the 5′ ITR comprises the sequence of SEQ ID NO: 3.
3 . The AAV transfer cassette of claim 1 , wherein the 3′ ITR comprises the sequence of SEQ ID NO: 4.
4 . The AAV transfer cassette of claim 1 , wherein the promoter comprises the sequence of SEQ ID NO: 5.
5 . The AAV transfer cassette of claim 1 , wherein the intronic sequence is an SV40 intron.
6 . The AAV transfer cassette of claim 1 , wherein the intronic sequence comprises the sequence of SEQ ID NO: 10.
7 . The AAV transfer cassette of claim 1 , wherein the NPC1 protein is the human NPC1 protein.
8 . The AAV transfer cassette of claim 1 , wherein the NPC1 protein comprises the sequence of SEQ ID NO: 1.
9 . The AAV transfer cassette of claim 1 , wherein the transgene comprises the sequence of SEQ ID NO: 2.
10 . The AAV transfer cassette of claim 1 , wherein the polyadenylation signal is the SV40 polyadenylation signal.
11 . The AAV transfer cassette of claim 1 , wherein the polyadenylation signal comprises the sequence of SEQ ID NO: 12.
12 . The AAV transfer cassette of claim 1 , wherein the cassette comprises an enhancer.
13 . The AAV transfer cassette of claim 1 , wherein the AAV transfer cassette comprises the sequence of SEQ ID NO: 14.
14 . The AAV transfer cassette of claim 1 , wherein the AAV transfer cassette comprises the sequence of any one of SEQ ID NO: 15-19.
15 . A plasmid comprising the AAV transfer cassette of any one of claims 1 - 14 .
16 . A nucleic acid comprising, from 5′ to 3′, a 5′ inverted terminal repeat (ITR); a promoter; a transgene; a polyadenylation signal; and a 3′ ITR; wherein the nucleic acid comprises an intronic sequence; wherein the transgene encodes the amino acid sequence of SEQ ID NO: 1.
17 . A cell comprising the AAV transfer cassette of any one of claims 1 - 14 , the plasmid of claim 15 , or the nucleic acid of claim 16 .
18 . A recombinant AAV vector comprising a protein capsid and a nucleic acid encapsidated by the protein capsid:
wherein the nucleic acid comprises the AAV transfer cassette of any one of claims 1 - 14 .
19 . The recombinant AAV vector of claim 18 , wherein the protein capsid comprises a capsid protein subunit that is from an AAV of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, Avian AAV or Bovine AAV.
20 . The recombinant AAV vector of claim 18 , wherein the protein capsid comprises a capsid protein subunit that has one or more amino acid mutations relative to a capsid protein subunit of any one of the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, Avian AAV or Bovine AAV.
21 . A method of producing a recombinant AAV vector, the method comprising contacting an AAV producer cell with the AAV transfer cassette of any one of claims 1 - 14 , the plasmid of claim 15 , or the nucleic acid of claim 16 .
22 . A recombinant AAV vector produced by the method of claim 21 .
23 . A composition comprising the AAV transfer cassette of any one of claims 1 - 14 , the plasmid of claim 15 , the nucleic acid of claim 16 , the cell of claim 17 , or the recombinant AAV vector of any one of claims 18 - 20 or 22 .
24 . A method for treating a subject in need thereof comprising administering to the subject an effective amount of the AAV transfer cassette of any one of claims 1 - 14 , the plasmid of claim 15 , the nucleic acid of claim 16 , the cell of claim 17 , or the recombinant AAV vector of any one of claims 18 - 20 or 22 .
25 . The method of claim 24 , wherein the subject has NPC1.
26 . The method of claim 24 , wherein the subject is a human subject.