US2020121746A1PendingUtilityA1
Adeno-associated virus virions with variant capsids and methods of use thereof
Est. expiryJun 30, 2037(~10.9 yrs left)· nominal 20-yr term from priority
C12N 2750/14143C12N 15/902C12N 15/63A61K 48/0075C12N 2310/20C12N 2750/14122A61K 35/761C07K 14/075C12N 15/86C07K 14/005C12N 15/113C12N 9/22A61P 27/02A61K 9/0048C12N 2750/14145A61P 27/00A61K 9/0019A61K 48/0041C07K 7/06C07K 7/08C12N 15/1082C12N 15/11C12N 2750/14142C12N 2750/14171C12N 2800/80
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Claims
Abstract
The present disclosure provides recombinant adeno-associated virus (AAV) virions with altered capsid protein, where the recombinant AAV (rAAV) virions exhibit greater ability to cross barriers between intravitreal fluid and retinal cells, and thus greater infectivity of a retinal cell compared to wild-type AAV, and where the rAAV virions comprise a heterologous nucleic acid. The present disclosure provides methods of delivering a gene product to a retinal cell in an individual.
Claims
exact text as granted — not AI-modified1 . A recombinant adeno-associated virus (rAAV) virion comprising:
a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises an insertion of a heterologous peptide of any one of Formulas I-VI, and wherein the variant capsid protein confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by a control AAV virion comprising the corresponding parental AAV capsid protein; and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product.
2 . The rAAV virion of claim 1 , wherein the rAAV virion exhibits at least 5-fold increased infectivity of a retinal cell compared to the infectivity of the retinal cell by a control AAV virion comprising the corresponding parental AAV capsid protein.
3 . (canceled)
4 . The rAAV virion of claim 1 , wherein the insertion of the heterologous peptide replaces a contiguous stretch of from 5 amino acids to 20 amino acids of the parental AAV capsid protein.
5 . The rAAV virion of claim 1 , wherein the insertion site is between amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype.
6 . (canceled)
7 . The rAAV virion of any one of claims 1 - 6 , wherein gene product is an interfering RNA, an aptamer, or a polypeptide.
8 . (canceled)
9 . The rAAV virion of claim 7 , wherein the polypeptide is a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances function of a retinal cell.
10 . The rAAV virion of claim 1 , wherein the gene product is:
a) an RNA-guided endonuclease selected from a type II CRISPR/Cas polypeptide, a type V CRISPR/Cas polypeptide, and a type VI CRISPR/Cas polypeptide; b) an enzymatically inactive type II CRISPR/Cas polypeptide; or c) an RNA-guided endonuclease and a guide RNA.
11 .- 12 . (canceled)
13 . The rAAV virion of claim 1 , wherein the heterologous peptide is:
a) a peptide of Formula I: LA(L/N)(I/Q)(Q/E)(D/H)(S/V)(M/K)(R/N)A (SEQ ID NO: 136); b) a peptide of Formula II: TX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 GLX 9 (SEQ ID NO: 137), where:
X 1 is G, V, or S;
X 2 is V, E, P, G, D, M, A, or S;
X 3 is M, V, Y, H, G, S, or D;
X 4 is R, D, S, G, V, Y, T, H, or M;
X 5 is S, L, G, T, Q, P, or A;
X 6 is T, A, S, M, D, Q, or H;
X 7 is N, G, S, L, M, P, G, or A;
X 8 is S, G, D, N, A, I, P, or T; and
X 9 is S or N;
c) a peptide of Formula III: TGX 1 X 2 X 3 X 4 X 5 X 6 X 7 GLS (SEQ ID NO: 138), where:
X 1 is V, E, P, G, D, M, A, or S;
X 2 is M, V, Y, H, G, S, or D;
X 3 is R, D, S, G, V, Y, T, H, or M;
X 4 is S, L, G, T, Q, P, or A;
X 5 is T, A, S, M, D, Q, or H;
X 6 is N, G, S, L, M, P, G, or A; and
X 7 is S, G, D, N, A, I, P, or T:
d) a peptide of Formula IV: X 1 GX 2 X 3 X 4 X 5 X 6 X 7 X 8 GLSPX 9 TX 10 X 10 X 11 (SEQ ID NO: 139), where
X 1 is T or N;
X 2 is L, S, A, or G;
X 3 is D or V;
X 4 is A, G, or P;
X 5 is T or D;
X 6 is R or Y;
X 7 is D, T, or G;
X 8 is H, R, or T;
X 9 is V or A;
X 10 is G or W; and
X 11 is T or A; or
e) a peptide of Formula V: TGX 1 DX 2 TRX 3 X 4 GLSPVTGT (SEQ ID NO: 140), where
X 1 is L, S, A, or G;
X 2 is A, G, or P;
X 3 is D, T, or G; and
X 4 is H, R, or T.
14 . The rAAV virion of claim 13 , wherein the heterologous peptide comprises;
a)
(21)
(SEQ ID NO: 35)
LALIQDSMRA
or
(22)
(SEQ ID NO: 2)
LANQEHVKNA;
b)
(1)
(SEQ ID NO: 6)
TGVMRSTNSGLN;
(2)
(SEQ ID NO: 7)
TGEVDLAGGGLS;
(3)
(SEQ ID NO: 8)
TSPYSGSSDGLS;
(4)
(SEQ ID NO: 9)
TGGHDSSLDGLS;
(5)
(SEQ ID NO: 98)
TGDGGTTMNGLS;
(6)
(SEQ ID NO: 99)
TGGHGSAPDGLS;
(7)
(SEQ ID NO: 100)
TGMHVTMMAGLN;
(8)
(SEQ ID NO: 101)
TGASYLDNSGLS;
(9)
(SEQ ID NO: 20)
TVVSTQAGIGLS;
(10)
(SEQ ID NO: 21)
TGVMHSQASGLS;
(11)
(SEQ ID NO: 22)
TGDGSPAAPGLS;
or
(12)
(SEQ ID NO: 23)
TGSDMAHGTGLS;
c)
(2)
(SEQ ID NO: 7)
TGEVDLAGGGLS;
(4)
(SEQ ID NO: 9)
TGGHDSSLDGLS;
(5)
(SEQ ID NO: 98)
TGDGGTTMNGLS;
(6)
(SEQ ID NO: 99)
TGGHGSAPDGLS;
(8)
(SEQ ID NO: 101)
TGASYLDNSGLS;
(10)
(SEQ ID NO: 21)
TGVMHSQASGLS;
(11)
(SEQ ID NO: 22)
TGDGSPAAPGLS;
or
(12)
(SEQ ID NO: 23)
TGSDMAHGTGLS;
or
d)
(13)
(SEQ ID NO: 24)
TGLDATRDHGLSPVTGT;
(14)
(SEQ ID NO: 25)
TGSDGTRDHGLSPVTWT;
(15)
(SEQ ID NO: 26)
NGAVADYTRGLSPATGT;
OR
(16)
(SEQ ID NO: 27)
TGGDPTRGTGLSPVTGA.
15 .- 21 . (canceled)
22 . The rAAV virion of claim 1 , wherein the heterologous peptide is a peptide of Formula VI: LQX 1 X 2 X 3 RX 4 X 5 X 6 X 7 X 8 X 9 VNX 10 Q (SEQ ID NO: 141), where
X 1 is K or R; X 2 is N, G, or A; X 3 is A, V, N, or D; X 4 is P, I, or Q; X 5 is A, P, or V; X 6 is S, T, or G; X 7 is T or V; X 8 is E, L, A, or V; X 9 is S, E, D, or V; and X 10 is F, G, T, or C.
23 . The rAAV virion of claim 22 , wherein the heterologous peptide comprises: (17) LQKNARPASTESVNFQ (SEQ ID NO: 28); (18) LQRGVRIPSVLEVNGQ (SEQ ID NO: 29); (19) LQRGNRPVTTADVNTQ (SEQ ID NO: 30); or (20) LQKADRQPGVVVVNCQ (SEQ ID NO: 31).
24 . A pharmaceutical composition comprising:
a) a recombinant adeno-associated virus virion of claim 1 ; and b) a pharmaceutically acceptable excipient.
25 . A method of delivering a gene product to a retinal cell in an individual, the method comprising administering to the individual a recombinant adeno-associated virus (rAAV) virion according to claim 1 .
26 . The method of claim 25 , wherein the gene product is a polypeptide, a short interfering RNA, or an aptamer.
27 . (canceled)
28 . The method of claim 26 , wherein the polypeptide is a neuroprotective factor, an anti-angiogenic polypeptide, an anti-apoptotic factor, or a polypeptide that enhances function of a retinal cell.
29 . The method of claim 26 , wherein the polypeptide is glial derived neurotrophic factor, fibroblast growth factor 2, neurturin, ciliary neurotrophic factor, nerve growth factor, brain derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked inhibitor of apoptosis, retinoschisin, RPE65, retinitis pigmentosa GTPase-interacting protein-1, peripherin, peripherin-2, a rhodopsin, RdCVF, retinitis pigmentosa GTPase regulator (RPGR), Sonic hedgehog, or an RNA-guided endonuclease.
30 . (canceled)
31 . A method of treating an ocular disease, the method comprising administering to an individual in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion according to claim 1 .
32 .- 34 . (canceled)
35 . An isolated nucleic acid comprising a nucleotide sequence that encodes a variant adeno-associated virus (AAV) capsid protein, wherein the variant AAV capsid protein comprises an insertion of from about 5 amino acids to about 20 amino acids in the capsid protein GH loop relative to a corresponding parental AAV capsid protein, and wherein the variant capsid protein, when present in an AAV virion, provides for increased infectivity of the AAV virion of a retinal cell, and wherein the amino acid insertion is in the GH loop of a native AAV capsid, wherein the insertion is a peptide of any one of Formulas I-VI.
36 . The isolated nucleic acid of claim 35 , wherein the insertion site is between amino acids 587 and 588 of AAV2, between amino acids 585 and 598 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.
37 . An isolated, genetically modified host cell comprising the nucleic acid of claim 35 .
38 . A variant adeno-associated virus (AAV) capsid protein, wherein the variant AAV capsid protein comprises an insertion of from about 5 amino acids to about 20 amino acids wherein the amino acid insertion is in the GH loop of a native AAV capsid, wherein the insertion is a peptide of any one of Formulas I-VI.
39 .- 63 . (canceled)Join the waitlist — get patent alerts
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