Efficient AAV-mediated gene delivery remains a significant obstacle to effective retinal gene therapies. Here, we apply the process of directed evolution – guided by deep sequencing and followed by direct in vivo secondary selection of high-performing vectors with a GFP-barcoded library – to create AAV viral capsids with new capabilities to deliver genes to the outer retina in large animals. The resulting vectors resulted in efficient targeting of photoreceptors, bipolar cells, and RPE cells in dog retina and substantially increased efficiency of gene delivery to primate outer retina. In addition, direct comparison of gene delivery across animals revealed emergent species specificities in vector performance. These new viral vectors will enable long-term and pan-retinal gene therapies targeting outer retina cell types in large preclinical animal models and establish deep sequencing-guided directed evolution as a powerful approach for developing AAV vectors specialized for a multitude of physical barriers and cellular targets.

Highly diverse (~1E+7) libraries of AAV variants were packaged such that each virus contained a genome encoding its own capsid. Libraries were pooled and injected intravitreally in canines or primates. After AAV infection had occurred, retinal tissue and RPE cells were collected, and cap gene variants were PCR amplified recloned, and repackaged for the subsequent round of injection. Five rounds of selection were performed, and error prone PCR was performed after the third round to introduce additional diversity into the library. Following the selections, each pool was subjected to deep sequencing in order to analyze the dynamics of each individual variant and overall convergence of the library. Based on their increase in representation relative to the original library, individual variant capsids were chosen. Files contain raw data counts from deep sequencing of AAV2-7mer libraries (in canine RPE and primate ONL) and LoopSwap~588 library (in primate ONL).