Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants

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.

In vivo high-throughput screening of novel adeno-associated viral capsids targeting adult neural stem cells in the subventricular zone

“…These AAV variants comprise 12 AAV wts, 94 newly generated peptide display mutants based on these wts and 71 chimeric capsids generated through DNA family shuffling. Among the synthetic capsids are 24 previously published benchmarks, the remaining ones were generated as described in the Methods section, Table S4 and in greater detail in 50 . All AAV capsid variants were uniquely barcoded (with a 15 nt long random DNA sequence) and packaged into an AAV vector expressing a CMV promoter-controlled eYFP (enhanced yellow fluorescent protein) that harbors the barcode in its 3’UTR.…”

“…All AAV capsid variants were uniquely barcoded (with a 15 nt long random DNA sequence) and packaged into an AAV vector expressing a CMV promoter-controlled eYFP (enhanced yellow fluorescent protein) that harbors the barcode in its 3’UTR. A library comprising either 91 (library #1 from 50 ) or 157 (library #3 from 50 ) capsid variants was directly injected into the lateral ventricles of the adult mouse brain (10 10 viral genomes in 2μl per mouse (vg), Fig 1a, Fig S2a).…”

“…These synthetic capsids include a set of 12 AAV serotypes, that were previously modified by insertion of over 20 different peptides in exposed capsid loops and that were recently characterized in established or primary cells 74 . In the work of 50 , all barcoded capsids were pooled in different combinations to finally obtain three distinct libraries (#1, #2 (not used in the present work) and #3), with 91, 82 and 157 variants. Further details on library composition are found in the Supplement of 50 .…”

“…In the work of 50 , all barcoded capsids were pooled in different combinations to finally obtain three distinct libraries (#1, #2 (not used in the present work) and #3), with 91, 82 and 157 variants. Further details on library composition are found in the Supplement of 50 . All capsid variants are detailed in Table S4.…”

“…For RNA sequencing, NSCs were seeded in 48-well plates (Greiner Bio-One) and incubated overnight. AAV library #1 or library #3 (same libraries as in 50 , multiplicity of infection (MOI): 10000) were added to the media and remained for the duration of seven days. For IHC, Labtek chambers (ThermoFisher) were coated with PDL (Sigma) / Laminin (Sigma) and NSCs were seeded at a density of 2×10 4 cells per cm 2 overnight.…”