Boye Sanford L, Bennett Antonette, Scalabrino Miranda L, McCullough K Tyler, Van Vliet Kim, Choudhury Shreyasi, Ruan Qing, Peterson James, Agbandje-McKenna Mavis, Boye Shannon E
Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, Florida, USA.
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida, USA.
J Virol. 2016 Mar 28;90(8):4215-4231. doi: 10.1128/JVI.00200-16. Print 2016 Apr.
Adeno-associated viruses (AAVs) currently are being developed to efficiently transduce the retina following noninvasive, intravitreal (Ivt) injection. However, a major barrier encountered by intravitreally delivered AAVs is the inner limiting membrane (ILM), a basement membrane rich in heparan sulfate (HS) proteoglycan. The goal of this study was to determine the impact of HS binding on retinal transduction by Ivt-delivered AAVs. The heparin affinities of AAV2-based tyrosine-to-phenylalanine (Y-F) and threonine-to-valine (T-V) capsid mutants, designed to avoid proteasomal degradation during cellular trafficking, were established. In addition, the impact of grafting HS binding residues onto AAV1, AAV5, and AAV8(Y733F) as well as ablation of HS binding by AAV2-based vectors on retinal transduction was investigated. Finally, the potential relationship between thermal stability of AAV2-based capsids and Ivt-mediated transduction was explored. The results show that the Y-F and T-V AAV2 capsid mutants bind heparin but with slightly reduced affinity relative to that of AAV2. The grafting of HS binding increased Ivt transduction by AAV1 but not by AAV5 or AAV8(Y733F). The substitution of any canonical HS binding residues ablated Ivt-mediated transduction by AAV2-based vectors. However, these same HS variant vectors displayed efficient retinal transduction when delivered subretinally. Notably, a variant devoid of canonical HS binding residues, AAV2(4pMut)ΔHS, was remarkably efficient at transducing photoreceptors. The disparate AAV phenotypes indicate that HS binding, while critical for AAV2-based vectors, is not the sole determinant for transduction via the Ivt route. Finally, Y-F and T-V mutations alter capsid stability, with a potential relationship existing between stability and improvements in retinal transduction by Ivt injection.
AAV has emerged as the vector of choice for gene delivery to the retina, with attention focused on developing vectors that can mediate transduction following noninvasive, intravitreal injection. HS binding has been postulated to play a role in intravitreally mediated transduction of retina. Our evaluation of the HS binding of AAV2-based variants and other AAV serotype vectors and the correlation of this property with transduction points to HS affinity as a factor controlling retinal transduction following Ivt delivery. However, HS binding is not the only requirement for improved Ivt-mediated transduction. We show that AAV2-based vectors lacking heparin binding transduce retina by subretinal injection and display a remarkable ability to transduce photoreceptors, indicating that other receptors are involved in this phenotype.
目前正在研发腺相关病毒（AAV），以便在进行非侵入性玻璃体内（Ivt）注射后有效地转导视网膜。然而，玻璃体内递送的AAV遇到的一个主要障碍是内界膜（ILM），这是一种富含硫酸乙酰肝素（HS）蛋白聚糖的基底膜。本研究的目的是确定HS结合对Ivt递送的AAV视网膜转导的影响。建立了基于AAV2的酪氨酸到苯丙氨酸（Y-F）和苏氨酸到缬氨酸（T-V）衣壳突变体的肝素亲和力，这些突变体旨在避免细胞运输过程中的蛋白酶体降解。此外，还研究了将HS结合残基嫁接到AAV1、AAV5和AAV8（Y733F）上以及基于AAV2的载体消除HS结合对视网膜转导的影响。最后，探讨了基于AAV2的衣壳热稳定性与Ivt介导的转导之间的潜在关系。结果表明，Y-F和T-V AAV2衣壳突变体与肝素结合，但亲和力相对于AAV2略有降低。HS结合的嫁接增加了AAV1的Ivt转导，但没有增加AAV5或AAV8（Y733F）的Ivt转导。任何典型HS结合残基的取代都消除了基于AAV2的载体的Ivt介导的转导。然而，当通过视网膜下递送时，这些相同的HS变体载体显示出有效的视网膜转导。值得注意的是，一种缺乏典型HS结合残基的变体AAV2（4pMut）ΔHS在转导光感受器方面非常有效。不同的AAV表型表明，HS结合虽然对基于AAV2的载体至关重要，但不是通过Ivt途径进行转导的唯一决定因素。最后，Y-F和T-V突变改变了衣壳稳定性，稳定性与Ivt注射后视网膜转导的改善之间存在潜在关系。
AAV已成为向视网膜进行基因递送的首选载体，注意力集中在开发能够在非侵入性玻璃体内注射后介导转导的载体。据推测，HS结合在玻璃体内介导的视网膜转导中起作用。我们对基于AAV2的变体和其他AAV血清型载体的HS结合及其与转导的相关性的评估表明，HS亲和力是控制Ivt递送后视网膜转导的一个因素。然而，HS结合并不是改善Ivt介导的转导的唯一要求。我们表明，缺乏肝素结合的基于AAV2的载体通过视网膜下注射转导视网膜，并显示出转导光感受器的显著能力，表明其他受体参与了这种表型。
