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

Christin M Hong,Tyler B Krause,Constance L Cepko,Sean K Wang,Sylvain W Lapan

doi:10.1016/j.omtm.2020.10.003

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 genomes in situ, allowing for studies of AAV vector biology and the tracking of transduced cells following vector administration.

Highlights

Recombinant adeno-associated viral (AAV) vectors derived from adeno-associated viruses have become the vector of choice for gene therapy due to their long-term expression of transgenes and relative lack of pathogenicity.[1,2,3,4] Each AAV virion consists of a protein capsid surrounding a single-stranded DNA genome, which can be up to 4.7 kb.[3,5] capable of integration into host genomes,[6] AAV vectors in infected cells are thought to predominantly exist as extrachromosomal episomes in the nucleus.[7]
Using Signal amplification by exchange reaction (SABER)-fluorescence in situ hybridization (FISH), we show that retinal microglia, a cell type thought to be resistant to infection with AAV vectors, are efficiently transduced but appear to degrade AAV genomes prior to their translocation into the nucleus
Based on a limiting dilution assay, in which a series of vector dilutions were administered in vivo, we determined that each fluorescent punctum detected by SABER-FISH most likely corresponded to an individual AAV genome

Summary

Introduction

Recombinant adeno-associated viral (AAV) vectors derived from adeno-associated viruses have become the vector of choice for gene therapy due to their long-term expression of transgenes and relative lack of pathogenicity.[1,2,3,4] Each AAV virion consists of a protein capsid surrounding a single-stranded DNA genome, which can be up to 4.7 kb.[3,5] capable of integration into host genomes,[6] AAV vectors in infected cells are thought to predominantly exist as extrachromosomal episomes in the nucleus.[7] These episomes are diluted in actively dividing cells, but they can be effectively maintained in post-mitotic cell types, such as those of the brain, retina, muscle, and heart These advances, there remain significant gaps in our understanding of AAV vectors, due in part to the lack of readily deployed methods to track vector capsids and genomes. The sensitivity of conventional FISH for DNA sequences below 5–10 kb is limited, a drawback that has precluded broader use of this technique in AAV and gene therapy research

Methods

Results

Conclusion