Deep Tropism Profiling of Barcoded AAV Capsid and Cargo Pools in Intact Tissue Using High-Throughput Ultrasensitive Sequential FISH

Abstract

Genetic access to specific cell types through minimally invasive routes is of particular interest in basic research and clinical applications. Extensive efforts have been made in engineering gene delivery vectors, such as recombinant adeno-associated viruses (rAAVs), and gene regulatory elements to achieve this goal. Despite many interesting candidates, revealed for example from directed evolution via M-CREATE (Sripriya Ravindra Kumar et al., Nature Methods, 2020), histology-based characterization presents a bottleneck due to the limited number of variants and/or cell types that can be investigated at once. To address this, we have developed ultrasensitive sequential FISH (useqFISH) for multiplexed detection of both endogenous and barcoded transgene transcripts in intact tissue with single-molecule resolution. By combining two amplification strategies (rolling circle amplification, RCA, and hybridization chain reaction, HCR), we achieved a 2.7- or 6.7-fold increased signal-to-background ratio of useqFISH in comparison to one with RCA or HCR only amplification, respectively. UseqFISH allowed us to detect endogenous genes with a single probe pair (20-nucleotide (nt) for each) and, in transfected cell cultures, to distinguish capsid variants with genomes differing by only 7-mer peptide modification. We further improved useqFISH by establishing an automated single-molecule imaging and microfluidic solution exchange system and an analytical pipeline for 3D imaging data. To demonstrate the applicability of useqFISH for in vivo AAV profiling, we employed this method to further characterize a pool of 6 AAV capsid variants that we found to be highly efficient for brainwide and/or cell-type biased transduction in the mouse brain following systemic delivery. We designed unique nucleic acid barcodes (160-nt) in the 3'UTR of each viral genome and retro-orbitally injected the pooled AAVs into 2 C57BL6/J mice at a dose of 5e10 viral genomes (vg) per variant (total 3e11 vg/mouse). For transcript detection, 11 canonical cell-type markers (e.g., Slc17a7, Gad1, Pvalb, SST, VIP, etc) were used together with probes against the viral genome barcodes, to characterize the cell-type tropisms of each variant. Next, we designed a pool of 103 barcoded AAV genomes carrying 4 tandem repeats of a unique miRNA target site. We packaged these genomes into AAV-PHP. eB and delivered to 3 C57BL6/J mice at a dose of 1e10 vg/variant (total ~1e12 vg/mouse). Using useqFISH, we were able to assess the ability of each miRNA target site to dampen transgene expression in different cell types, thereby revealing useful intersectional strategies to refine celltype- specific transgene expression with capsid/cargo combinations. These results demonstrate that useqFISH allows for high-throughput characterization of pooled genetic variants of viral capsids and gene regulatory elements in intact tissue and thus enables comprehensive profiling of genetic toolkits for precise access to targets of interest.

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