Adeno-associated virus virions with variant capsid and methods of use thereof
The present disclosure provides adeno-associated virus (AAV) virions with altered capsid protein, where the AAV virions exhibit greater infectivity of retinal cells, when administered via intravitreal injection, compared to wild-type AAV. 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 method of identifying a novel variant recombinant adeno-associated virus (rAAV) virion having greater infectivity of a cell in vivo as compared to the corresponding parental AAV virion, the method comprising:
a) administering a composition comprising a library of variant rAAV virions in an eye of an animal by intravitreal injection, wherein each variant rAAV virion comprises: i) a variant AAV virion capsid protein comprising a peptide of from five to eleven amino acids in length inserted into the GH loop of the corresponding parental AAV capsid protein; and ii) DNA encoding the variant AAV virion capsid protein;
b) isolating a target photoreceptor cell from non-target ocular cells, wherein the target photoreceptor cell has been successfully infected by a variant rAAV virion;
c) polymerase chain reaction (PCR) amplifying DNA encoding an AAV virion capsid protein from the isolated target photoreceptor cells;
d) sequencing the amplified DNA to identify one or more variant AAV virion capsid sequences that have permissive mutations for high infectivity of the target photoreceptor cell as compared to non-target ocular cells;
e) cloning the DNA encoding the variant AAV virion capsid protein; and
f) carrying out error-prone PCR amplification of the cloned DNA, thereby generating a plurality of additional variant rAAV.
2. The method according to claim 1 , wherein the peptide is located between two adjacent amino acids within amino acids corresponding to amino acids 570-611 of the AAV2 capsid protein or the corresponding position in the capsid protein of any of the following AAV serotypes: AAV1, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.
3. The method according to claim 1 , wherein the variant AAV virion capsid protein comprises 1 to 25 amino acid substitutions.
4. The method according to claim 1 , wherein the variant AAV virion capsid protein is a chimeric capsid protein.
5. The method of claim 1 , further comprising
packaging variant AAV virions from the cloned DNA encoding the variant AAV virion capsid protein.
6. The method according to claim 1 , wherein the increased infectivity is a greater infectivity of said photoreceptor cells following administration of the variant rAAV in vivo as compared to the infectivity of an AAV virion comprising the corresponding parental AAV capsid protein when administered in vivo.