腺相关病毒(AAV)的衣壳工程可以克服目前基因治疗的局限性,例如广泛的组织嗜性,低转导效率,或预先存在的限制患者资格的中和抗体(NAb)。我们先前通过整合合理的设计和定向进化来产生AAV3B组合衣壳文库,目的是改善肝性。潜在的隔离,在肝细胞球体培养中,AAV3B-DE5在五轮迭代选择中获得了选择性增殖优势。在这项研究中,我们重新分析了来自AAV3B组合文库的原始数据集和来自早期(一到三轮)选择的分离变体,假设具有更快的复制动力学的变体不一定是最有效的换能器。我们确定了一个潜在的候选人,AAV3B-V04,在小鼠传代的原代人肝细胞以及人源化肝嵌合小鼠中表现出显着增强的转导,与亲本AAV3B或先前描述的分离株相比,AAV3B-DE5.有趣的是,AAV3B-V04衣壳变体对汇集的或个体的人血清样品表现出显著降低的血清反应性。具有AAV3B的预先存在的NAb的血清样品的44%具有比AAV3B-V04低5至20倍的倒数NAb滴度。AAV3B-V04只有9个氨基酸取代,与AAV3B相比,在可变区IV中聚类,表明三重突起顶部的环在确定转导效率和免疫原性方面的重要性。这项研究强调了通过分子进化方法增强AAV转导的合理设计与靶向选择相结合的有效性。我们的发现支持限制选择轮的概念,以分离出最佳的转导AAV3B变体,而不会产生更快的复制候选物。我们得出的结论是,AAV3B-V04提供了诸如改善的人肝细胞嗜性和免疫逃避等优势,并提出了其作为肝脏基因治疗的优越候选者的实用性。
Capsid engineering of adeno-associated virus (AAV) can surmount current limitations to gene therapy such as broad tissue tropism, low transduction efficiency, or pre-existing neutralizing antibodies (NAb) that restrict patient eligibility. We previously generated an
AAV3B combinatorial capsid library by integrating rational design and directed evolution with the aim of improving hepatotropism. A potential isolate,
AAV3B-DE5, gained a selective proliferative advantage over five rounds of iterative selection in hepatocyte spheroid cultures. In this study, we reanalyzed our original dataset derived from the AAV3B combinatorial library and isolated variants from earlier (one to three) rounds of selection, with the assumption that variants with faster replication kinetics are not necessarily the most efficient transducers. We identified a potential candidate, AAV3B-V04, which demonstrated significantly enhanced transduction in mouse-passaged primary human hepatocytes as well as in humanized liver chimeric mice, compared to the parental AAV3B or the previously described isolate,
AAV3B-DE5. Interestingly, the AAV3B-V04 capsid variant exhibited significantly reduced seroreactivity to pooled or individual human serum samples. Forty-four percent of serum samples with pre-existing NAbs to AAV3B had 5- to 20-fold lower reciprocal NAb titers to
AAV3B-V04.
AAV3B-V04 has only nine amino acid substitutions, clustered in variable region IV compared to
AAV3B, indicating the importance of the loops at the top of the three-fold protrusions in determining both transduction efficiency and immunogenicity. This study highlights the effectiveness of rational design combined with targeted selection for enhanced AAV transduction via molecular evolution approaches. Our findings support the concept of limiting selection rounds to isolate the best transducing AAV3B variant without outgrowth of faster replicating candidates. We conclude that AAV3B-V04 provides advantages such as improved human hepatocyte tropism and immune evasion and propose its utility as a superior candidate for liver gene therapy.