Xing Ruxiao, Xu Mengyao, Reil Darcy, Destefano April, Cui Mengtian, Liu Nan, Liang Jialing, Xu Guangchao, Luo Li, Xu Meiyu, Zhang Fang, Tai Phillip W L, Wei Yuquan, Gruntman Alisha M, Flotte Terence R, Gao Guangping, Wang Dan
Department of Genetic and Cellular Medicine, Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
bioRxiv. 2025 Jun 14:2025.03.04.641478. doi: 10.1101/2025.03.04.641478.
AAV capsid serotypes isolated from nature have been widely used in gene delivery and gene therapy. Recently, more than 1,000 distinct AAV capsids were identified from human clinical samples by high-throughput, long-read DNA sequencing. In this study, we tap into this broad natural biodiversity of AAV capsids to develop liver-tropic AAV capsids. We initially screened a subset of variants derived from AAV8 (n=159) for packaging efficiency. The high-yielding variants were subjected to a barcoded vector library screen in mice and ferrets for their ability to mediate liver gene transfer. Although no variant surpassed AAV8 for liver targeting, several exhibited a liver detargeting phenotype. Among these, we focused on the N271D variant (AAV8 VP1 numbering), located in the variable region I (VR-1), which has been previously implicated in influencing liver tropism. The liver detargeting phenotype of AAV8.N271D was confirmed by single vector administration in mice. Additionally, we grafted the N271D variant onto AAV9 and MyoAAV capsids (N270D by AAV9 VP1 numbering). The AAV9.N270D and MyoAAV.N270D vectors showed a similar liver-detargeting phenotype, although muscle targeting was moderately reduced. Although we did not identify any capsid variants that outperform AAV8 in liver transduction, this study reinforces the important role of VR-1 in modulating liver tropism and highlights the potential of engineering VR-1 residues to reduce liver gene transfer and associated toxicity observed in several gene therapy studies.
从自然界分离出的腺相关病毒（AAV）衣壳血清型已广泛应用于基因递送和基因治疗。最近，通过高通量、长读长DNA测序从人类临床样本中鉴定出1000多种不同的AAV衣壳。在本研究中，我们利用AAV衣壳这种广泛的自然生物多样性来开发肝脏靶向性AAV衣壳。我们最初筛选了源自AAV8的一组变体（n = 159）的包装效率。将高产变体在小鼠和雪貂中进行条形码载体文库筛选，以评估它们介导肝脏基因转移的能力。尽管没有变体在肝脏靶向方面超过AAV8，但有几种表现出肝脏脱靶表型。其中，我们重点研究了位于可变区I（VR-1）的N271D变体（按AAV8 VP1编号），该区域先前被认为与影响肝脏嗜性有关。通过在小鼠中单次给予载体证实了AAV8.N271D的肝脏脱靶表型。此外，我们将N271D变体嫁接到AAV9和肌AAV衣壳上（按AAV9 VP1编号为N270D）。AAV9.N270D和肌AAV.N270D载体表现出类似的肝脏脱靶表型，尽管肌肉靶向略有降低。尽管我们没有鉴定出在肝脏转导方面优于AAV8的任何衣壳变体，但本研究强化了VR-1在调节肝脏嗜性中的重要作用，并突出了改造VR-1残基以减少在一些基因治疗研究中观察到的肝脏基因转移和相关毒性的潜力。
