Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity

Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity

Blog Article

Limitations to successful gene therapy with adeno-associated virus Microwave Belt (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene.Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy.In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants.The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures.AAV3B-DE5 contains 24 capsid amino acid substitutions compared Shampoos with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII.

In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model.Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v.Ig), as well as individual serum samples from 100 healthy human donors.Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies.