Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.

中文翻译：

---

GPR108是高度保守的AAV进入因子。

腺相关病毒（AAV）是一种很有前途的基因转移载体，但人们对AAV进入的主要细胞要求知之甚少。使用全基因组CRISPR筛查进入进化趋异血清型AAVrh32.33，我们将G蛋白偶联受体超家族成员GPR108鉴定为AAV进入因子。在人类细胞系中测试的所有AAV进化枝中，有超过20种不同的AAV发生，在GPR108基因敲除（KO）中只有AAV5转导不受影响。在鼠和原代细胞中进一步显示了GPR108依赖性。这些发现在体内得到了进一步验证，因为Gpr108 KO小鼠证明AAV8和rh32.33的表达降低了10到100倍，但AAV5却没有。从机理上讲，GPR108 N和C末端域都是转导所必需的，在衣壳上，可以转移一个在AAV5上不保守的VP1唯一域，以将GPR108独立性赋予AAV2嵌合体。体外结合和分级分离研究表明，在不存在GPR108的情况下，核输入和胞浆积累减少。因此，我们已经鉴定了在小鼠和人类之间在体内和体外相关的两个AAV进入因子中的第二个，这进一步为AAV基因治疗载体的向性提供了机理上的理解。