Isolated modified VP1 capsid protein of AAV5
This application relates to the fields of gene therapy and molecular biology. More specifically, the present invention relates to an isolated altered VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid comprising one or more amino acid substitutions as compared to the VP1 protein of wild-type AAV5 capsid, which increase transduction efficiency, as well as to a capsid and a vector based thereon.
1 . An isolated altered VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid for transduction of target cells, comprising the amino acid sequence of the VP1 protein of a wild-type AAV5 capsid, encoded by the Cap gene, comprising one or more substitutions selected from the group comprising:
a S2A and T711S substitution, or
a S2A, S651A and T711S substitution,
wherein the amino acid sequence of the VP1 protein of a wild-type AAV5 capsid has the amino acid sequence of SEQ ID NO: 1.
2 . The isolated altered VP1 protein of the AAV5 capsid of claim 1 , which comprises the S2A and T711S substitutions.
3 . The isolated altered VP1 protein of the AAV5 capsid of claim 2 , which has the amino acid sequence of SEQ ID NO: 3.
4 . The isolated altered VP1 protein of the AAV5 capsid of claim 1 , which comprises the substitutions S2A, S651A and T711S.
5 . The isolated altered AAV5 VP1 capsid protein of claim 4 , which has the amino acid sequence of SEQ ID NO: 4.
6 . An isolated nucleic acid encoding the altered VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid of claim 1 , which is used for transduction of target cells.
7 . The isolated nucleic acid of claim 6 encoding an altered VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid comprising:
(i) the nucleic sequence of SEQ ID NO: 6; or
(ii) the nucleic sequence of SEQ ID NO: 7.
8 . An isolated capsid for transduction of target cells comprising the altered VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid of claim 1 .
9 . The isolated capsid of claim 8 comprising the altered VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid, a VP2 protein of AAV5 capsid or an altered variant thereof, and a VP3 protein of AAV5 capsid or an altered variant thereof.
10 . The isolated capsid of claim 9 comprising:
(i) the VP2 protein of a wild-type AAV5 capsid; or
(ii) the altered VP2 protein of adeno-associated virus serotype 5 (AAV5) capsid.
11 . The isolated capsid of claim 10 , comprising the VP2 protein of a wild-type AAV5 capsid protein having the amino acid sequence of SEQ ID NO: 8.
12 . The isolated capsid of claim 10 , comprising the altered VP2 protein of AAV5 capsid, comprising the amino acid sequence of the VP2 protein of a wild-type AAV5 capsid, encoded by the Cap gene, comprising one or more substitutions selected from the group comprising:
a T575S substitution, or
a S515A, and a T575S substitution,
wherein the amino acid sequence of the VP2 protein of a wild-type AAV5 capsid has the amino acid sequence of SEQ ID NO: 8 .
13 . The isolated capsid of claim 12 , comprising the altered VP2 protein of AAV5 capsid comprising the T575S substitution and having the amino acid sequence of SEQ ID NO: 9 or the S515A and T575S substitutions and having the amino acid sequence of SEQ ID NO: 10.
14 . The isolated capsid of claim 9 comprising:
(i) the VP3 protein of a wild-type AAV5 capsid; or
(ii) the altered VP3 protein of adeno-associated virus serotype 5 (AAV5) capsid.
15 . The isolated capsid of claim 14 , comprising the VP3 protein of a wild-type AAV5 capsid having the amino acid sequence of SEQ ID NO: 11.
16 . The isolated capsid of claim 14 , comprising the altered VP3 protein of AAV5 capsid, comprising the amino acid sequence of the VP3 protein of a wild-type AAV5 capsid, encoded by the Cap gene, comprising one or more substitutions selected from the group comprising:
a T519S substitution, or
a S459A, and a T519S substitution,
wherein the amino acid sequence of the VP3 protein of a wild-type AAV5 capsid has the amino acid sequence of SEQ ID NO: 11.
17 . The isolated capsid of claim 16 comprising the altered VP3 protein of AAV5 capsid comprising the T519S substitution and having the amino acid sequence of SEQ ID NO: 12; or comprising the S459A and T519S substitutions and having the amino acid sequence of SEQ ID NO: 13.
18 . An isolated nucleic acid encoding the capsid of claim 8 , for transduction of target cells.
19 . A vector based on recombinant adeno-associated virus serotype 5 (rAAV5) for delivery to a subject of a heterologous nucleic acid sequence, which comprises:
1) the capsid of claim 8 , and
2) a heterologous nucleic acid sequence comprising regulatory sequences that promote the expression of a product encoded by the heterologous nucleic acid sequence, in target cells.
20 . The vector based on rAAV5 of claim 19 , wherein the expression product of the heterologous nucleic acid sequence is a therapeutic polypeptide or a reporter polypeptide.
21 . The vector based on rAAV5 of claim 20 , wherein the therapeutic polypeptide is a coagulation factor selected from the group consisting of Factor VIII, Factor IX, or a functional variant thereof.
22 . The vector based on rAAV5 of claim 21 , wherein the therapeutic peptide is Factor VIII or a functional variant thereof; or wherein the therapeutic peptide is Factor IX or a functional variant thereof.
23 . A pharmaceutical composition for the delivery of a gene product to a subject in need thereof, comprising:
a) the vector based on rAAV5 of claim 19 ; and
b) a pharmaceutically acceptable excipient.
24 . The pharmaceutical composition of claim 23 , wherein the subject is a human subject.
25 . A method for the delivery of a gene product to a subject in need thereof, comprising administering to the subject the vector based on rAAV5 of claim 19 or a pharmaceutical composition for the delivery of a gene product to a subject in need thereof, comprising the vector based on rAAV5 and a pharmaceutically acceptable excipient.
26 . The method for the delivery of a gene product of claim 25 , wherein the subject is a human subject.
27 . A method of obtaining of the vector based on rAAV5 of claim 19 comprising the transfection of producer cells with a nucleic acid encoding the capsid, which is used for transduction of target cells.