Xi Zhouhuan, Öztürk Bilge E, Johnson Molly E, Turunç Serhan, Stauffer William R, Byrne Leah C
Department of Ophthalmology, University of Pittsburgh, PA, USA.
Eye Center of Xiangya Hospital, Hunan Key Laboratory of Ophthalmology, Central South University, Changsha, Hunan, China.
Mol Ther Methods Clin Dev. 2022 Apr 30;25:476-489. doi: 10.1016/j.omtm.2022.04.014. eCollection 2022 Jun 9.
Gene therapy is a rapidly developing field, and adeno-associated viruses (AAVs) are a leading viral-vector candidate for therapeutic gene delivery. Newly engineered AAVs with improved abilities are now entering the clinic. It has proven challenging, however, to predict the translational potential of gene therapies developed in animal models due to cross-species differences. Human retinal explants are the only available model of fully developed human retinal tissue and are thus important for the validation of candidate AAV vectors. In this study, we evaluated 18 wild-type and engineered AAV capsids in human retinal explants using a recently developed single-cell RNA sequencing (RNA-seq) AAV engineering pipeline (scAAVengr). Human retinal explants retained the same major cell types as fresh retina, with similar expression of cell-specific markers except for a photoreceptor population with altered expression of photoreceptor-specific genes. The efficiency and tropism of AAVs in human explants were quantified with single-cell resolution. The top-performing serotypes, K91, K912, and 7m8, were further validated in non-human primate and human retinal explants. Together, this study provides detailed information about the transcriptome profiles of retinal explants and quantifies the infectivity of leading AAV serotypes in human retina, accelerating the translation of retinal gene therapies to the clinic.
基因治疗是一个快速发展的领域，腺相关病毒（AAV）是治疗性基因递送的主要病毒载体候选物。具有改进能力的新型工程化AAV目前正在进入临床。然而，由于跨物种差异，预测在动物模型中开发的基因疗法的转化潜力已被证明具有挑战性。人视网膜外植体是完全发育的人视网膜组织的唯一可用模型，因此对于候选AAV载体的验证很重要。在本研究中，我们使用最近开发的单细胞RNA测序（RNA-seq）AAV工程流程（scAAVengr）在人视网膜外植体中评估了18种野生型和工程化AAV衣壳。人视网膜外植体保留了与新鲜视网膜相同的主要细胞类型，除了光感受器特异性基因表达改变的光感受器群体外，细胞特异性标志物表达相似。以单细胞分辨率对人外植体中AAV的效率和趋向性进行了量化。表现最佳的血清型K91、K912和7m8在非人灵长类动物和人视网膜外植体中得到了进一步验证。总之，本研究提供了有关视网膜外植体转录组图谱的详细信息，并量化了主要AAV血清型在人视网膜中的感染性，加速了视网膜基因疗法向临床的转化。
