Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to quantify efficiency of AAV-mediated gene expression across all cell types. scAAVengr allows for definitive, head-to-head comparison of vectors in the same animal. To demonstrate proof-of-concept for the scAAVengr workflow, we quantified - with cell-type resolution - the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. These results validate scAAVengr as a powerful method for development of AAV vectors.

中文翻译：

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scAAVengr：非人灵长类动物视网膜中工程AAV的单细胞转录组定量

腺相关病毒（AAV）介导的基因疗法正在迅速向临床发展，并且AAV工程技术已导致载体具有更高的递送治疗基因的能力。尽管选择载体至关重要，但尤其是在大型动物中，AAV的定量比较仍具有挑战性。在这里，我们开发了一种有效的单细胞AAV工程流水线（scAAVengr），以量化所有细胞类型中AAV介导的基因表达的效率。scAAVengr允许对同一只动物中的载体进行确定的头对头比较。为了证明scAAVengr工作流程的概念验证，我们通过细胞类型的分辨率对玻璃体内注射后天然存在的和新近构建的AAV介导灵长类动物视网膜中基因表达的能力进行了定量。表现最出色的型号K912 被用于递送SaCas9并编辑猕猴视网膜中的视紫红质基因，从而导致编辑效率类似于scAA​​Vengr工作流程检测到的感染率。这些结果验证了scAAVengr是开发AAV载体的有力方法。