Crosson Sean M, Bennett Antonette, Fajardo Diego, Peterson James J, Zhang Hangning, Li Wei, Leahy Matthew T, Jennings Colin K, Boyd Ryan F, Boye Sanford L, Agbandge-McKenna Mavis, Boye Shannon E
Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida, Gainesville, FL, USA.
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL, USA.
J Virol. 2021 Apr 26;95(10). doi: 10.1128/JVI.02440-20. Epub 2021 Mar 3.
Adeno-associated viruses (AAVs) have recently emerged as the leading vector for retinal gene therapy. However, AAV vectors which are capable of achieving clinically relevant levels of transgene expression and widespread retinal transduction are still an unmet need. Using rationally designed AAV2-based capsid variants, we investigate the role of capsid hydrophilicity and hydrophobicity as it relates to retinal transduction. We show that hydrophilic, single amino acid (aa) mutations (V387R, W502H, E530K, L583R) in AAV2 negatively impact retinal transduction when heparan sulfate proteoglycan (HSPG) binding remains intact. Conversely, addition of hydrophobic point mutations to an HSPG binding deficient capsid (AAV2ΔHS) lead to increased retinal transduction in both mouse and macaque. Our top performing vector, AAV2(4pMut)ΔHS, achieved robust rod and cone photoreceptor (PR) transduction in macaque, especially in the fovea, and demonstrates the ability to spread laterally beyond the borders of the subretinal injection (SRI) bleb. This study both evaluates biophysical properties of AAV capsids that influence retinal transduction, and assesses the transduction and tropism of a novel capsid variant in a clinically relevant animal model.Rationally guided engineering of AAV capsids aims to create new generations of vectors with enhanced potential for human gene therapy. By applying rational design principles to AAV2-based capsids, we evaluated the influence of hydrophilic and hydrophobic amino acid (aa) mutations on retinal transduction as it relates to vector administration route. Through this approach we identified a largely deleterious relationship between hydrophilic aa mutations and canonical HSPG binding by AAV2-based capsids. Conversely, the inclusion of hydrophobic aa substitutions on a HSPG binding deficient capsid (AAV2ΔHS), generated a vector capable of robust rod and cone photoreceptor (PR) transduction. This vector AAV2(4pMut)ΔHS also demonstrates a remarkable ability to spread laterally beyond the initial subretinal injection (SRI) bleb, making it an ideal candidate for the treatment of retinal diseases which require a large area of transduction.
腺相关病毒（AAV）最近已成为视网膜基因治疗的主要载体。然而，能够实现临床相关水平的转基因表达和广泛的视网膜转导的AAV载体仍是未被满足的需求。我们使用合理设计的基于AAV2的衣壳变体，研究衣壳亲水性和疏水性与视网膜转导的关系。我们发现，当硫酸乙酰肝素蛋白聚糖（HSPG）结合保持完整时，AAV2中的亲水性单氨基酸（aa）突变（V387R、W502H、E530K、L583R）对视网膜转导产生负面影响。相反，在缺乏HSPG结合的衣壳（AAV2ΔHS）中添加疏水性点突变会导致小鼠和猕猴的视网膜转导增加。我们表现最佳的载体AAV2(4pMut)ΔHS在猕猴中实现了强大的视杆和视锥光感受器（PR）转导，尤其是在中央凹，并显示出能够横向扩散到视网膜下注射（SRI）泡边界之外的能力。本研究既评估了影响视网膜转导的AAV衣壳的生物物理特性，又评估了一种新型衣壳变体在临床相关动物模型中的转导和嗜性。对AAV衣壳进行合理指导的工程改造旨在创造具有增强人类基因治疗潜力的新一代载体。通过将合理设计原则应用于基于AAV2的衣壳，我们评估了亲水性和疏水性氨基酸（aa）突变与载体给药途径相关的对视网膜转导的影响。通过这种方法，我们确定了基于AAV2的衣壳的亲水性aa突变与经典HSPG结合之间在很大程度上是有害关系。相反，在缺乏HSPG结合的衣壳（AAV2ΔHS）上包含疏水性aa替代物，产生了一种能够进行强大的视杆和视锥光感受器（PR）转导的载体。这种载体AAV2(4pMut)ΔHS还显示出一种非凡的能力，即能够横向扩散到最初的视网膜下注射（SRI）泡之外，使其成为治疗需要大面积转导的视网膜疾病的理想候选者。
