In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses

TLDR

AAV serotypes differ widely in transduction efficiency and tissue tropism, offering great potential for gene therapy, yet their clinical use is limited by anti‑AAV immunity and poor performance in specific tissues such as the liver. The study sought to combine desirable traits of multiple natural AAV isolates by DNA family shuffling to generate hybrid capsids. An adapted DNA family shuffling approach produced a library of hybrid capsids from eight wild‑type viruses, and selection on primary or transformed human hepatocytes yielded hybrids from five serotypes (2, 4, 5, 8, 9). Selection with pooled human antisera produced a single type 2/8/9 chimera, AAV‑DJ, which outperformed eight standard serotypes in vitro and in mouse livers, exhibited restricted biodistribution due to a heparin‑binding domain, and, via peptide display, was retargeted to alveolar cells, demonstrating the power of DNA shuffling and viral peptide display for evolving novel AAV vectors.

Abstract

Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications.