Abstract
Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy.
Highlights
Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation
Its hallmark is that AAV vector genomes packaged into capsid variants of interest or wellknown benchmarks are first barcoded, and qualitatively and quantitatively tracked in transduced animals by next-generation sequencing (NGS) at both, the DNA and RNA level
Each barcode was assigned to a unique AAV capsid from the list of 183 variants in Supplementary Table 1, comprising 12 AAV wild types (AAV1 to AAV9, AAVrh.[10], AAVpo.[1], AAV12), as well as 94 peptide display mutants and 71 chimeric capsids created through DNA family shuffling[2]
Summary
The fact that all of the shown lead capsids that were present in all three libraries were in an identical position relative to each other confirms the robustness, and reproducibility of our experimental and bioinformatic workflows Additional proof of their validity is provided by the data, which confirms the tissue specificity previously reported for the benchmarks AAV-DJ (liver)[7], AAV2 modified with peptide ESGHGFY (lung)[4], and AAV-PHP.B (brain)[3]. Another capsid that we used as benchmark and whose performance was recapitulated in our own data is AAV2-BR1, which consistently ranked among the best candidates in the brain with off-targeting in the lung Brain 0.8 0.6 0.4 0.2 0.0 AAAAVV2-P-EHSPG.BHAGAYVF2-BARA1V9APA5V9P4AAAVA9V9AAA1V6P4AAAVA4V4L1
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