Adeno-associated virus virions with variant capsid and methods of use thereof

A method of screening a library of variant recombinant adeno-associated virus (rAAV) virions to identify novel variant rAAV virions having greater infectivity of target photoreceptor cells as compared to non-target ocular cells, the method comprising:

• administering a composition comprising a library of at least 10^6 variant rAAV virions in an eye of an animal by intravitreal injection, wherein each variant rAAV virion in the library comprises a variant AAV capsid protein comprising at least one amino acid modification and DNA encoding the variant AAV virion capsid protein;
• isolating target photoreceptor cells from non-target ocular cells, wherein the target photoreceptor cells have been infected by variant rAAV virions;
• cloning and packaging DNA from the isolated target photoreceptor cells to generate a smaller library of variant rAAV virions;
• repeating one or more times the administering, isolating, cloning and packaging steps to generate an enriched population of virions comprising variant rAAV virions with high infectivity of target photoreceptor cells as compared to non-target ocular cells;
• polymerase chain reaction (PCR) amplifying DNA encoding a variant AAV virion capsid from the enriched population of variant rAAV virions with additional mutations, wherein the PCR amplification comprises error-prone PCR;
• and wherein the enriched population of variant rAAV virions with additional mutations undergoes one or more rounds of in vivo screening to generate another enriched population of variant rAAV virions;
• and
• identifying a DNA encoding a variant AAV capsid from a final round of isolated target photoreceptor cells obtained from in vivo screening of a final enriched population of variant rAAV virions with high infectivity of target photoreceptor cells as compared to non-target ocular cells.