Recent clinical successes in gene therapy applications have intensified interest in using adeno-associated viruses (AAVs) as vectors for therapeutic gene delivery. Although prototypical AAV2 shows robust in vitro transduction of human hepatocyte–derived cell lines, it has not translated into an effective vector for liver-directed gene therapy in vivo. This is consistent with observations made in Fah^−/−/Rag2^−/−/Il2rg^−/− (FRG) mice with humanized livers, showing that AAV2 functions poorly in this xenograft model. Here, we derived naturally hepatotropic AAV capsid sequences from primary human liver samples. We demonstrated that capsid mutations, likely acquired as an unintentional consequence of tissue culture propagation, attenuated the intrinsic human hepatic tropism of natural AAV2 and related human liver AAV isolates. These mutations resulted in amino acid changes that increased binding to heparan sulfate proteoglycan (HSPG), which has been regarded as the primary cellular receptor mediating AAV2 infection of human hepatocytes. Propagation of natural AAV variants in vitro showed tissue culture adaptation with resulting loss of tropism for human hepatocytes. In vivo readaptation of the prototypical AAV2 in FRG mice with a humanized liver resulted in restoration of the intrinsic hepatic tropism of AAV2 through decreased binding to HSPG. Our results challenge the notion that high affinity for HSPG is essential for AAV2 entry into human hepatocytes and suggest that natural AAV capsids of human liver origin are likely to be more effective for liver-targeted gene therapy applications than culture-adapted AAV2.

基因治疗应用中的最新临床成功对使用腺相关病毒（AAV）作为治疗性基因传递的载体引起了越来越多的兴趣。尽管原型AAV2在人肝细胞衍生的细胞系中显示出强大的体外转导作用，但尚未转化为用于体内肝定向基因治疗的有效载体。这与Fah ^-/- / Rag2 ^-/- / Il2rg ^-/-中的观察结果一致（FRG）具有人源化肝脏的小鼠，显示AAV2在此异种移植模型中的功能较差。在这里，我们从原代人肝脏样品中获得了天然的肝细胞AAV衣壳序列。我们证明衣壳突变，可能是由于组织培养繁殖的意外结果而引起的，减弱了天然AAV2和相关人类肝脏AAV分离株的固有人类肝向性。这些突变导致氨基酸变化，从而增加了与硫酸乙酰肝素蛋白聚糖（HSPG）的结合，后者被认为是介导人类肝细胞AAV2感染的主要细胞受体。天然AAV变异体在体外的繁殖显示出组织培养适应性，导致人类肝细胞向性丧失。用人源化肝脏在FRG小鼠中对原型AAV2进行体内重配可通过减少与HSPG的结合来恢复AAV2的固有肝向性。我们的结果挑战了对HSPG的高亲和力对于AAV2进入人肝细胞必不可少的观点，并暗示人肝起源的天然AAV衣壳比适应于培养的AAV2对肝脏靶向的基因治疗应用更有效。