Directed Evolution of AAV for Efficient Gene Delivery to Canine and Primate Retina - Raw counts of variants from deep sequencing

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 ...高效的腺相关病毒（AAV）介导的基因递送，仍是实现成熟视网膜基因治疗的重大阻碍。本研究采用定向进化技术，以深度测序为引导，并通过绿色荧光蛋白（Green Fluorescent Protein，GFP）条形码标记的文库对高性能载体开展体内第二轮直接筛选，由此构建具备全新功能的AAV病毒衣壳，可将基因递送至大型动物的视网膜外层组织。所得载体可在犬视网膜中高效靶向光感受器细胞、双极细胞及视网膜色素上皮（Retinal Pigment Epithelium，RPE）细胞，并大幅提升基因向灵长类动物视网膜外层的递送效率。此外，跨物种基因递送的直接对比研究揭示了载体性能所呈现的新兴物种特异性。这些新型病毒载体将支持针对大型临床前动物模型中视网膜外层细胞类型的长效全视网膜基因治疗，并确立以深度测序引导的定向进化技术作为开发适配多元应用场景专用AAV载体的高效研究手段……