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 AAV capsid protein, comprising one or more amino acid modifications, wherein the modification confers heparan sulfate binding to the capsid protein.
2 . The modified AAV capsid protein of claim 1 , wherein the AAV is an AAV2.5T.
3 . The modified AAV capsid protein of claim 1 , wherein the AAV is an AAV2.5T/7m8.
4 . The modified AAV capsid protein of claim 3 , wherein the AAV2.5T/7m8 is an AAV2.5T/7m8(+3), an AAV2.5T/7m8(0), an AAV2.5T/7m8(−12), or an AAV2.5T/7m8(−3).
5 . The modified AAV2.5T capsid protein of claim 1 or claim 2 , 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).
6 . The modified AAV2.5T/7m8 capsid protein of any of claim 1, 3 or 4 , 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 576-583, 576-584, 573-583 or 573-584 in AAV2.5T with the following amino acid residues: NLQRGNRQAATAAP (SEQ ID NO:8).
7 . The modified AAV2.5T/7m8 capsid protein of claim 6 , comprising a capsid sequence set forth in any of SEQ ID NOs:1-3.
8 . The modified AAV2.5T or AAV2.5T/7m8 capsid protein of claim 1 , comprising a sequence shown in FIGS. 3 A- 3 C .
9 . A polynucleotide comprising a nucleic acid sequence encoding the modified AAV2.5T or AAV2.5T/7m8 capsid protein of any of claims 1-8 .
10 . An expression vector comprising the polynucleotide of claim 9 , wherein the nucleic acid sequence encoding the modified AAV2.5 or AAV2.5T is operably linked to a promoter sequence.
11 . A cell comprising the expression vector of claim 10 .
12 . The cell of claim 11 , further comprising a polynucleotide that encodes a therapeutic protein.
13 . The cell of claim 10 or claim 12 , further comprising a polynucleotide that encodes a rep protein.
14 . A recombinant virus or viral vector comprising the modified capsid protein of any one of claims 1-8 .
15 . The recombinant virus or viral vector of claim 14 , wherein the recombinant virus or viral vector is an adeno-associated virus (AAV), optionally AAV2.
16 . The recombinant virus or viral vector of claim 14 or claim 15 , wherein the recombinant virus is eluted from a heparan column at a salt concentration of about 0.2 M to about 0.4 M.
17 . The recombinant virus or viral vector of any of claims 14-16 , wherein the recombinant virus or viral vector is capable of binding to and crossing the inner limiting membrane (ILM) when intravitreally injected into a mammal.
18 . The recombinant virus of any one of claims 14-17 , wherein the recombinant virus or viral vector comprises a polynucleotide sequence that encodes a therapeutic protein.
19 . The recombinant virus or viral vector of claim 18 , wherein the therapeutic protein is an anti-vascular endothelial growth factor (anti-VEGF) agent.
20 . The recombinant virus or viral vector of any one of claims 14-18 , wherein the recombinant virus or viral vector has an altered cellular tropism as compared to AAV2.5T or AAV2.5T/7m8.
21 . The recombinant virus or viral vector of claim 20 , wherein the recombinant virus or viral vector is a AAV2.5T comprising a modified capsid protein comprising: (a) a S576R point mutation: or (b) a T579R point mutation, wherein the recombinant virus or viral vector has a greater tropism for RGC than AAV2.5T.
22 . The recombinant virus of claim 20 , wherein the recombinant virus or viral vector is a AAV2.5T/7m8 comprising a modified capsid protein comprising: (a) a S576R point mutation: and (b) a T579R point mutation, wherein the recombinant virus or viral vector has a greater tropism for Müller cells than AAV2.5T/7m8.
23 . The recombinant virus or viral vector of claim 20 , wherein the recombinant virus or viral vector is a AAV2.5T/7m8 comprising a modified capsid protein comprising a substitution of amino acid residues corresponding to 573-584 in AAV2.5T/7m8 with the following amino acid residues: NLQRGNRQAATA (SEQ ID NO:7), wherein the recombinant virus or viral vector has a greater tropism for retinal cells than AAV2.5T/7m8.
24 . A pharmaceutical composition comprising the recombinant virus or viral vector of any one of claims 14-23 .
25 . A method of providing a protein to a retina of a subject, comprising administering to the subject by intravitreal injection the recombinant virus or viral vector of any one of claims 14-23 or the pharmaceutical composition of claim 24 , wherein the recombinant virus or viral vector comprises a polynucleotide sequence that encodes the protein.
26 . A method of providing a therapeutic protein to a retina of a subject in need thereof, comprising administering to the subject by intravitreal injection a pharmaceutical composition comprising the recombinant virus or viral vector of any one of claims 18-23 .
27 . The method of claim 26 , wherein the subject has been diagnosed with or is suspected of having one or more conditions selected from the group consisting of: age-related macular degeneration (AMD), wet-AMD, dry-AMD, retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branched retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, and diabetic retinal edema.
28 . A method of altering the tropism of an AAV2.5T or AAV2.5T/7m8 virus or viral vector, comprising introducing one or more amino acid modifications that confers heparan sulfate binding to a capsid protein of the virus or viral vector.
29 . The method of claim 28 , wherein the amino acid modification introduces one or more amino acids of AAV2 into the capsid protein.
30 . The method of claim 28 or claim 29 , 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 576-581 in AAV2.5T with amino acid residues: RGNRQA (SEQ ID NO:5) or RGNRQAAP (SEQ ID NO:6); or
(d) a substitution corresponding to a substitution of amino acid residues 573-584 in AAV2.5T with amino acid residues: NLQRGNRQAATA (SEQ ID NO:7) or NLQRGNRQAATAAP (SEQ ID NO:8).
31 . The method of claim 29 , wherein the amino acid modification comprises: (a) a S576R point mutation; or (b) a T579R point mutation, wherein the virus or viral vector has a greater tropism for RGC than AAV2.5T.
32 . The method of claim 29 , wherein the amino acid modification comprises: (a) a S576R point mutation; and (b) a T579R point mutation, wherein the virus or viral vector has a greater tropism for Müller cells than AAV2.5T/7m8.
33 . The method of claim 29 , wherein the amino acid modification comprises a substitution of amino acid residues corresponding to 573-584 in AAV2.5T/7m8 with the following amino acid residues: NLQRGNRQAATA (SEQ ID NO:7), wherein the recombinant virus or viral vector has a greater tropism for retinal cells than AAV2.5T/7m8.
34 . A method for selectively delivering a polypeptide to RGC of a subject, comprising administering to the subject an AAV 2 . 5 T virus or viral vector comprising a polynucleotide that encodes the polypeptide, wherein the virus or viral vector comprises a capsid protein comprising a S576R point mutation or a T579R point mutation.
35 . A method for selectively delivering a polypeptide to Müller cells of a subject, comprising administering to the subject an AAV2.5T/7m8 virus or viral vector comprising a polynucleotide that encodes the polypeptide, where the virus comprises a capsid protein comprising: (a) a S 576 R point mutation; and (b) a T579R point mutation, optionally AAV2.5T/7m8(+3)Δ2.
36 . A method for selectively delivering a polypeptide to retinal cells of a subject, comprising administering to the subject an AAV2.5T/7m8 virus or viral vector comprising a polynucleotide that encodes the polypeptide, where the virus comprises a capsid protein comprising a substitution of amino acid residues corresponding to 573-583 in AAV2.5T/7m8 with amino acid residues: NLQRGNRQAATA (SEQ ID NO:7) or NLQRGNRQAATAAP (SEQ ID NO:8).