A3 - AAV Vectors - Capsid Engineering
215: Identification of Natural Human-Derived AAV2 Variants with Muscle Tropic Properties in Non-Human Primate Screens
Type: Oral Abstract Session
Presentation Details
Session Title: Developing the Next Generation of Muscle-Targeted Gene Delivery Vehicles via AAV Capsid Engineering
Muscle-targeting AAV gene therapy is hampered by high-dose-associated toxicity, immunogenicity, and nonspecific targeting to other organs. While methods like directed evolution and iterative rounds of selection from a complex library have recently produced several promising muscle-tropic capsids, the screening process has several inherent limitations, such as the potential for cross-packaging, capsid mosaicism, and collateral transduction. Furthermore, promising capsids discovered in this manner may have poor production yields.In this study, we sought to evaluate the ability of AAV capsid variants isolated from human tissues to robustly transduce skeletal muscles of non-human primates (NHPs) following direct muscle injections. The rationale for pursuing this route of delivery was to employ the muscle as a continuous and reversible therapeutic bio-factory, distributing secreted transgene products to diseased cells via the bloodstream or the affected muscle itself. One highlighted application is the use of rAAV to deliver genes encoding broadly neutralizing antibodies against deadly viruses causing pandemic infections. Despite being efficacious in rodent and non-human primate studies, anti-antibody responses in NHPs (and humans) present a significant hurdle, potentially hindering initial protection. To advance this strategy, we have screened our library of natural capsid for their potential to robustly transduce muscle tissues following direct intramuscular (IM) injection.Taking advantage of a relatively low complexity library, we have been employing a single variant to single barcoded non-coding RNA transgene production scheme to identify AAV capsids with favored tropism profiles. In the current study, we injected into cynomolgus macaques (n =3) a pool of ~100 AAV2 variants via intramuscular administration (vastus lateralis). After one month, tissues at the site of injection and livers were collected, and subjected to targeted amplicon-sequencing and analyses. Barcode counts among vector DNA and transgene transcripts revealed three variants that displayed strong transduction in muscle, resulting in over 400x higher levels than conferred by AAV2. We then evaluated their transduction performances in mice. The three variants showed over 12x better transduction than achieved by AAV2, and comparable to those of AAV1. This data suggests that the variants’ remarkable transduction of muscle is exclusive to NHPs.In conclusion, our research has identified three AAV2 variants with enhanced transduction of non-human primate skeletal muscle. Subsequent investigations will focus on a detailed analysis, comparing the amino acid sequences of these high-performance variants, and delving into the mechanisms that permit improved tropism in muscle. This effort aims to advance our understanding of AAV capsid biology and ultimately improve muscle targeting for gene therapy. G.G. and P.T. are corresponding authors.
Fang Zhang1, Mengtian Cui1, Jackson McGowan1, Dan Wang1,2, Jialing Yang Liang1, Guangping Gao1,3, Phillip Tai1,3
1Horae Gene Therapy Center, UMass Chan Medical School, Worcester, MA,2RNA Therapeutics Institute, UMass Chan Medical School, Worcester, MA,3Microbiology and Physiological Systems, UMass Chan Medical School, Worcester, MA"
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