Development of AAV Variants with Human Hepatocyte Tropism and Neutralizing Antibody Escape Capacity

Xiaolei Pei; Wenwei Shao; Allene Xing; Charles Askew; Xiaojing Chen; Caibin Cui; Yasmina L Abajas; David A Gerber; Elizabeth P Merricks; Timothy C Nichols; Wuping Li; R Jude Samulski; Chengwen Li

doi:10.1016/j.omtm.2020.06.003

Development of AAV Variants with Human Hepatocyte Tropism and Neutralizing Antibody Escape Capacity

Mol Ther Methods Clin Dev. 2020 Jun 3:18:259-268. doi: 10.1016/j.omtm.2020.06.003. eCollection 2020 Sep 11.

Authors

Xiaolei Pei^{1

2}, Wenwei Shao², Allene Xing², Charles Askew², Xiaojing Chen², Caibin Cui³, Yasmina L Abajas⁴, David A Gerber³, Elizabeth P Merricks⁵, Timothy C Nichols⁵, Wuping Li⁶, R Jude Samulski^{2

7}, Chengwen Li^{2

4

8}

Affiliations

¹ Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
² Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
³ Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁴ Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁵ Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁶ Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China.
⁷ Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁸ Carolina Institute for Developmental Disabilities, University of North Carolina at Chapel Hill, NC 27599, USA.

Abstract

Adeno-associated virus (AAV) vectors have been successfully used in patients with bleeding disorders and blindness. For human liver targeting, two major factors restrict effective AAV transduction after systemic administration of AAV vectors: human hepatocyte tropism and neutralizing antibodies (Nabs). In this study, we attempted to isolate AAV variants with the ability to transduce human hepatocytes and escape Nabs using a directed evolution approach in vivo. After four cycles of selection, 14 AAV capsid mutants were identified from a capsid shuffling library selected in the presence of human Intravenous Immunoglobulin (IVIG) and isolated from human hepatocytes xenografted into chimeric mice. AAV neutralization assays using IVIG showed that most of the mutants showed the Nab escape pattern in a manner similar to that of AAV8 or AAV9 and better than that of other AAV serotypes. Different mutants displayed varying capacities to escape Nab activity from individual serum samples collected from healthy subjects or hemophilia patients. The mutant AAV LP2-10 was found in 12 colonies out of 25, which was composed of capsids from AAV serotypes 2, 6, 8, and 9, with VP3 subunits derived from AAV8 swapped with AAV6 from residues 261 to 272. The mutant AAV LP2-10 manifested a higher ability than that of other serotypes to escape Nabs in IVIG and most human serum samples. After injection of AAV vectors encoding a self-complementary GFP cassette into chimeric mice, LP2-10 transduced human hepatocytes with efficiency similar to that of AAV8. In summary, AAV mutants can be isolated in humanized mice with both human hepatocyte tropism and the ability to evade Nab activity.

Keywords: AAV; Nabs; chimeric mice; human hepatocyte; tropism.

Abstract

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