Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, United States.
Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, United States; Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China.
J Control Release. 2017 Sep 28;262:348-356. doi: 10.1016/j.jconrel.2017.08.005. Epub 2017 Aug 5.
Adeno-associated virus (AAV) vectors have been used successfully in clinical trials for patients with hemophilia or blindness, but pre-existing neutralizing antibodies (Nab) are common in the general population and exclude many patients from clinical trials. Exploration of effective strategies to enhance AAV transduction and escape from Nab activity is still imperative. Previous studies have shown the compatibility of capsids from AAV serotypes and homology of recognition sites of AAV Nab located on different capsid subunits from one virion. In this study, we co-transfected AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1 and 1:3) to assemble haploid capsids and study both their transduction efficiency and Nab escape activity. After muscular injection, all of the haploid viruses induced higher transduction than their parental AAV vectors (2- to 9-fold over AAV2), with the highest of these being the haploid vector AAV2/8 3:1. After systemic administration, a 4-fold higher transduction in the liver was observed with haploid AAV2/8 1:3 than that with AAV8 alone. We then packaged the therapeutic factor IX cassette into haploid AAV2/8 1:3 capsids and injected them into FIX knockout mice via the tail vein. Higher FIX expression and improved phenotypic correction were achieved with the haploid AAV2/8 1:3 virus vector when compared to that of AAV8. Additionally, the haploid virus AAV2/8 1:3 was able to escape AAV2 neutralization and did not increase capsid antigen presentation capacity when compared to AAV8. To improve the Nab evasion ability of the haploid virus, we produced the triploid vector AAV2/8/9 by co-transfecting AAV2, AAV8 and AAV9 helper plasmids at a ratio of 1:1:1. After systemic administration, a 2-fold higher transduction in the liver was observed with the triploid vector AAV2/8/9 than that with AAV8. Nab analysis demonstrated that the triploid AAV2/8/9 vector was able to escape Nab activity from mouse sera immunized with parental serotypes. These results indicate that polyploid viruses might potentially acquire advantages from parental serotypes for enhancement of AAV transduction and evasion of Nab recognition without increasing capsid antigen presentation in target cells. Polyploid AAV vectors can be generated from any AAV serotype, whether natural, rational, library derived or a combination thereof, providing a novel strategy that should be explored in future clinical trials in patients with neutralizing antibodies.
腺相关病毒（AAV）载体已成功应用于血友病或失明患者的临床试验，但在普通人群中普遍存在预先存在的中和抗体（Nab），这使许多患者无法参加临床试验。探索增强 AAV 转导和逃避 Nab 活性的有效策略仍然至关重要。先前的研究表明，AAV 血清型的衣壳具有兼容性，并且来自同一病毒粒子的不同衣壳亚基上的 AAV Nab 的识别位点具有同源性。在这项研究中，我们以不同的比例（3:1、1:1 和 1:3）共转染 AAV2 和 AAV8 辅助质粒，组装单倍体衣壳，并研究其转导效率和 Nab 逃逸活性。肌肉注射后，所有单倍体病毒的转导均高于其亲本 AAV 载体（2-9 倍于 AAV2），其中最高的是 AAV2/8 3:1 单倍体病毒。全身给药后，与单独使用 AAV8 相比，AAV2/8 1:3 单倍体在肝脏中的转导提高了 4 倍。然后，我们将治疗因子 IX 盒包装到 AAV2/8 1:3 单倍体衣壳中，并通过尾静脉将其注入 FIX 敲除小鼠体内。与 AAV8 相比，AAV2/8 1:3 单倍体病毒载体可实现更高的 FIX 表达和改善表型校正。此外，与 AAV8 相比，AAV2/8 1:3 单倍体病毒能够逃避 AAV2 中和，并且不会增加衣壳抗原呈递能力。为了提高单倍体病毒的 Nab 逃逸能力，我们通过以 1:1:1 的比例共转染 AAV2、AAV8 和 AAV9 辅助质粒，产生了三倍体载体 AAV2/8/9。全身给药后，与 AAV8 相比，三倍体载体 AAV2/8/9 在肝脏中的转导提高了 2 倍。Nab 分析表明，三倍体 AAV2/8/9 载体能够逃避来自用亲本血清型免疫的小鼠血清中的 Nab 活性。这些结果表明，多倍体病毒可能通过从亲本血清型中获得优势，增强 AAV 转导并逃避 Nab 识别，而不会增加靶细胞中的衣壳抗原呈递。多倍体 AAV 载体可以由任何 AAV 血清型产生，无论是天然的、合理的、文库衍生的还是它们的组合，为具有中和抗体的患者提供了一种新的策略，这应该在未来的临床试验中进行探索。
