In Situ Detection of Adeno-associated Viral Vector Genomes with SABER-FISH

Molecular Therapy. Methods & Clinical Development
Sean K WangConstance L Cepko

Abstract

Gene therapy with recombinant adeno-associated viral (AAV) vectors is a promising modality for the treatment of a variety of human diseases. Nonetheless, there remain significant gaps in our understanding of AAV vector biology, due in part to the lack of robust methods to track AAV capsids and genomes. In this study, we describe a novel application of signal amplification by exchange reaction fluorescence in situ hybridization (SABER-FISH) that enabled the visualization and quantification of individual AAV genomes after vector administration in mice. These genomes could be seen in retinal cells within 3 h of subretinal AAV delivery, were roughly full length, and correlated with vector expression in both photoreceptors and the retinal pigment epithelium. SABER-FISH readily detected AAV genomes in the liver and muscle following retro-orbital and intramuscular AAV injections, respectively, demonstrating its utility in different tissues. Using SABER-FISH, we also found that retinal microglia, a cell type deemed refractory to AAV transduction, are in fact efficiently infected by multiple AAV serotypes, but appear to degrade AAV genomes prior to nuclear localization. Our findings show that SABER-FISH can be used to visualize AAV geno...Continue Reading

Citations

May 19, 2021·Translational Vision Science & Technology·Ying Kai ChanUNKNOWN Ocular Gene Therapy Inflammation Working Group

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Methods Mentioned

BETA
PCR

Software Mentioned

SABER
ImageJ
GraphPad Prism
MATLAB
ACME
ApE
FISH

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