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）载体已成功用于血友病或失明患者的临床试验中，但是预先存在的中和抗体（Nab）在普通人群中很常见，并将许多患者排除在临床试验之外。探索增强AAV转导和逃避Nab活性的有效策略仍然势在必行。先前的研究表明，来自AAV血清型的衣壳具有相容性，并且位于一个病毒粒子的不同衣壳亚基上的AAV Nab识别位点具有同源性。在这项研究中，我们以不同比例（3：1、1：1和1：3）共转染AAV2和AAV8辅助质粒，以组装单倍体衣壳，并研究它们的转导效率和Nab逃逸活性。肌肉注射后 所有单倍体病毒都比其亲本AAV载体诱导更高的转导（是AAV2的2到9倍），其中最高的是单倍体载体AAV2 / 8 3：1。全身给药后，单倍体AAV2 / 8 1：3在肝脏中的转导率比单独AAV8高4倍。然后，我们将治疗因子IX盒包装到单倍体AAV2 / 8 1：3衣壳中，并将其注射入FIX基因敲除小鼠中通过尾静脉。与AAV8相比，单倍体AAV2 / 8 1：3病毒载体可实现更高的FIX表达和更好的表型校正。此外，与AAV8相比，单倍体病毒AAV2 / 8 1：3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逃避能力，我们通过按1：1：1的比例共转染AAV2，AAV8和AAV9辅助质粒来生产三倍体载体AAV2 / 8/9。全身给药后，三倍体载体AAV2 / 8/9的肝脏中的转导率比AAV8高2倍。Nab分析表明，三倍体AAV2 / 8/9载体能够从用亲本血清型免疫的小鼠血清中逃脱Nab活性。这些结果表明，多倍体病毒可能会从亲本血清型中获得优势，以增强AAV转导和逃避Nab识别，而不会增加靶细胞中的衣壳抗原呈递。可以从任何AAV血清型中生成多倍体AAV载体，无论是自然的，有理的，文库衍生的还是它们的组合，都提供了一种新的策略，应在以后的中和抗体患者临床试验中进行探索。