Modified AAV capsids and uses thereof
The present disclosure provides adeno-associated virus (AAV) virions with altered capsid protein that binds heparan sulfate proteoglycans, where the AAV virions exhibit greater infectivity of retinal cells, altered tropism and/or the ability to bind and cross the inner limiting membrane following intravitreal injection. The present disclosure further provides methods of delivering a gene product to a retinal cell in an individual, and methods of treating ocular disease.
1. A non-naturally-occurring modified adeno-associated virus (AAV) capsid protein, comprising one or more amino acid modification that confers heparan sulfate binding to the capsid protein, wherein the AAV is an AAV2.5T/7m8.
2. The modified AAV capsid protein of claim 1 , wherein the AAV2.5T/7m8 is an AAV2.5T/7m8(+3), an AAV2.5T/7m8(0), an AAV2.5T/7m8(−12), an AAV2.5T/7m8(−9), an AAV2.5T/7m8(−6), or an AAV2.5T/7m8(−3), wherein the AAV2.5T/7m8(+3) comprises an insertion of the amino acids LALGETTRPA between residues 579-580 of AAV2.5T, the AAV2.5T/7m8(0) comprises an insertion of the amino acids LALGETTRPA between residues 578-579 of AAV2.5T, the AAV2.5T/7m8(−12) comprises an insertion of the amino acids LALGETTRPA between residues 574-575 of AAV2.5T, the AAV2.5T/7m8(−9) comprises an insertion of the amino acids LALGETTRPA between residues 575-576 of AAV2.5T, wherein the AAV2.5T/7m8(−6) comprises an insertion of the amino acids LALGETTRPA between residues 576-577 of AAV2.5T, and the AAV2.5T/7m8(−3) comprises an insertion of the amino acids LALGETTRPA between residues 577-578 of AAV2.5T.
3. A non-naturally-occurring modified adeno-associated virus (AAV) capsid protein, comprising one or more amino acid modification that confers heparan sulfate binding to the capsid protein, wherein the AAV is AAV2.5T, and wherein at least one of the one or more amino acid modifications comprises:
(a) a S576R point mutation;
(b) a T579R point mutation;
(c) a substitution of amino acid residues 576-579 or 576-581 with the following amino acid residues: RGNRQA (SEQ ID NO:5);
(d) a substitution of amino acid residues 576-581 with the following amino acid residues: RGNRQAAP (SEQ ID NO:6);
(e) a substitution of amino acid residues 573-579, 573-581 or 573-584 with the following amino acid residues: NLQRGNRQAATA (SEQ ID NO:7); or
(f) a substitution of amino acid residues 573-581 or 573-584 with the following amino acid residues: NLQRGNRQAATAAP (SEQ ID NO:8).
4. The modified AAV2.5T/7m8 capsid protein claim 1 , wherein at least one of the one or more amino acid modifications comprises:
(a) a point mutation corresponding to a S576R point mutation in AAV2.5T;
(b) a point mutation corresponding to a T579R point mutation in AAV2.5T;
(c) a substitution corresponding to a substitution of amino acid residues 576-579 or 576-581 in AAV2.5T with the following amino acid residues: RGNRQA (SEQ ID NO:5);
(d) a substitution corresponding to a substitution of amino acid residues 576-581 in AAV2.5T with the following amino acid residues: RGNRQAAP (SEQ ID NO:6);
(e) a substitution corresponding to a substitution of amino acid residues 573-579, 573-581, 573-583, 573-584, 576-579, 576-581, 576-583 or 576-584 in AAV2.5T with the following amino acid residues: NLQRGNRQAATA (SEQ ID NO:7); or
(f) a substitution corresponding to a substitution of amino acid residues 576-581, 576-583, 576-584, 573-583 or 573-584 in AAV2.5T with the following amino acid residues: NLQRGNRQAATAAP (SEQ ID NO:8).
5. The modified AAV2.5T/7m8 capsid protein of claim 4 , comprising a capsid sequence set forth in any of SEQ ID NOs:1-3.
6. The modified AAV2.5T/7m8 capsid protein of claim 4 , comprising a capsid sequence having at least 90% sequence identity to SEQ ID NO: 1, and further comprising the sequence set forth in SEQ ID NO: 61.
7. The modified AAV2.5T/7m8 capsid protein of claim 4 , comprising a capsid sequence having at least 90% sequence identity to SEQ ID NO: 2, and further comprising the sequence set forth in SEQ ID NO: 68.
8. The modified AAV2.5T/7m8 capsid protein of claim 4 , comprising a capsid sequence having at least 90% sequence identity to SEQ ID NO: 3, and further comprising the sequence set forth in SEQ ID NO: 20.
9. The modified AAV2.5T/7m8 capsid protein of claim 4 , wherein the capsid protein is an AAV2.5T/7m8(+3), further comprising the following amino acid modifications:
(a) a point mutation corresponding to a S576R point mutation in AAV2.5T; and
(b) a point mutation corresponding to a T579R point mutation in AAV2.5T.
10. The modified AAV2.5T/7m8 capsid protein of claim 4 , wherein the capsid protein is an AAV2.5T/7m8(−12), further comprising a substitution corresponding to a substitution of amino acid residues 576-581, 576-583, 576-584, 573-583 or 573-584 in AAV2.5T with the following amino acid residues: NLQRGNRQAATAAP (SEQ ID NO:8).
11. The modified AAV2.5T/7m8 capsid protein of claim 10 , wherein the capsid protein is an AAV2.5T/7m8(−12), further comprising a substitution corresponding to a substitution of amino acid residues 576-581 in AAV2.5T with the following amino acid residues: NLQRGNRQAATAAP (SEQ ID NO:8).
12. The modified AAV2.5T/7m8 capsid protein of claim 4 , wherein the capsid protein is an AAV2.5T/7m8(−3), further comprising a substitution corresponding to a substitution of amino acid residues 573-579, 573-581, 573-583, 573-584, 576-579, 576-581, 576-583 or 576-584 in AAV2.5T with the following amino acid residues: NLQRGNRQAATA (SEQ ID NO:7).
13. The modified AAV2.5T/7m8 capsid protein of claim 12 , wherein the capsid protein is an AAV2.5T/7m8(−3), further comprising a substitution corresponding to a substitution of amino acid residues 573-584 in AAV2.5T with the following amino acid residues: NLQRGNRQAATA (SEQ ID NO:7).