Recombinant adeno-associated virus (rAAV) vectors selected from capsid libraries present enormous advantages in high selectivity of tissue tropism and their potential use in human gene therapy applications. For example, rAAV-LK03, was used in a gene therapy trial for hemophilia A (ClinicalTrials.gov: NCT03003533). However, high doses in patients resulted in severe adverse events and subsequent loss of factor VIII (FVIII) expression. Thus, additional strategies are needed to enhance the transduction efficiency of capsid library-derived rAAV vectors such that improved clinical efficacy can be achieved at low vector doses. In this study, we characterized two commonly used library-derived rAAV vectors, rAAV-DJ and rAAV-LK03. It was concluded that rAAV-DJ shared similar transport pathways (e.g., cell surface binding, endocytosis-dependent internalization, and cytoplasmic trafficking) with rAAV serotype 2, while rAAV-LK03 and rAAV serotype 3 shared similar transport pathways. We then performed site-directed mutagenesis of surface-exposed tyrosine (Y), serine (S), aspartic acid (D), and tryptophan (W) residues on rAAV-DJ and rAAV-LK03 capsids. Our results demonstrated that rAAV-DJ-S269T and rAAV-LK03-Y705+731F variants had significantly enhanced transduction efficiency compared to wild-type counterparts. Our studies suggest that the strategy of site-directed mutagenesis should be applicable to other non-natural AAV variants for their optimal use in human gene therapy.

选自衣壳文库的重组腺相关病毒（rAAV）载体在组织定向性的高选择性及其在人类基因治疗应用中的潜在应用方面显示出巨大优势。例如，rAAV-LK03被用于血友病A的基因治疗试验中（ClinicalTrials.gov：NCT03003533）。但是，高剂量患者会导致严重的不良事件，并继而丧失VIII因子（FVIII）的表达。因此，需要额外的策略来增强衣壳文库衍生的rAAV载体的转导效率，从而可以在低载体剂量下实现改善的临床功效。在这项研究中，我们表征了两个常用的库衍生rAAV载体，rAAV-DJ和rAAV-LK03。结论是，rAAV-DJ具有相似的转运途径（例如，细胞表面结合，内吞作用依赖性内化和细胞质运输）与rAAV血清型2，而rAAV-LK03和rAAV血清型3共享相似的转运途径。然后，我们在rAAV-DJ和rAAV-LK03衣壳上对表面暴露的酪氨酸（Y），丝氨酸（S），天冬氨酸（D）和色氨酸（W）残基进行了定点诱变。我们的结果表明，与野生型对应物相比，rAAV-DJ-S269T和rAAV-LK03-Y705 + 731F变体具有显着增强的转导效率。我们的研究表明，定点诱变策略应适用于其他非天然AAV变异体，以便在人类基因治疗中得到最佳利用。然后，我们在rAAV-DJ和rAAV-LK03衣壳上对表面暴露的酪氨酸（Y），丝氨酸（S），天冬氨酸（D）和色氨酸（W）残基进行了定点诱变。我们的结果表明，与野生型对应物相比，rAAV-DJ-S269T和rAAV-LK03-Y705 + 731F变体具有显着增强的转导效率。我们的研究表明，定点诱变策略应适用于其他非天然AAV变异体，以便在人类基因治疗中得到最佳利用。然后，我们在rAAV-DJ和rAAV-LK03衣壳上对表面暴露的酪氨酸（Y），丝氨酸（S），天冬氨酸（D）和色氨酸（W）残基进行了定点诱变。我们的结果表明，与野生型对应物相比，rAAV-DJ-S269T和rAAV-LK03-Y705 + 731F变体具有显着增强的转导效率。我们的研究表明，定点诱变策略应适用于其他非天然AAV变异体，以便在人类基因治疗中得到最佳利用。