Method for High-Throughput, In Situ Characterization of AAV Variant Pools in Intact Tissue Using Ultrasensitive Sequential FISH

Abstract

Extensive efforts have been made to engineer adeno-associated viruses (AAVs) with desirable characteristics, such as enhanced transduction efficiency and tissue- or cell-type specific tropisms. In-vivo selection, followed by next-generation sequencing (NGS)-based screening, has enabled us to uncover novel viral capsid variants, such as the AAV-PHP series (Deverman et al., Nat Biotech, 2016; Chan et al., Nat Neurosci, 2017; Kumar et al., Nat Methods, 2020). Despite successful library-based selections, the characterization of viral tropisms is slow and labor-intensive and is thus limited to only a handful of variants. To overcome this bottleneck and allow for high-throughput screening, we introduce an imaging-based approach that detects viral transcripts in intact tissue by using ultrasensitive, sequential fluorescence in situ hybridization (FISH). We first developed a new FISH method to enable detection of relatively low abundance viral transcripts compared to endogenous genes in tissue. Compared to two signal amplification methods, rolling-circle amplification (RCA) and hybridization chain reaction (HCR), our method resulted in a 2.7- or 6.7-fold higher signal-to-background ratio, respectively, with the same number of probes. The high sensitivity of our method also allowed us to detect RNA transcripts with 1 probe and distinguish capsid variants packaging an identical viral genome with a short mutated region (7 amino acids, equivalent to 21 base pairs) transduced in HEK293T cells. We also developed an efficient two-step probe stripping method to enable multiple rounds of labeling (up to 8), which increases the number of targets that can be characterized in the same tissue beyond the spectral limit (e.g., 4 colors x 8 rounds = 32 variants). The high sensitivity and ability for sequential labeling allowed us to examine the cell-type tropism of capsid variants and/or gene regulatory elements in intact tissue. For this purpose, we generated AAV pools, comprising a combination of novel AAV-PHP.B-like capsids and cell-type specific promoters, that package the same coding sequence with a unique barcode in the 3'UTR. The pool was injected into one animal at a low dose (~1e10 for each), and after 3-4 weeks of injection, the transcripts of each variant were detected with a custom probe set targeting the unique barcodes. As a proof-of-concept, we were able to characterize the cell-type tropism of 6 variants in one tissue within 4 hours. Further refinement of barcode designs (e.g., temporal barcoding or in situ sequencing) and single-molecule imaging will allow us to either reduce the screening time or increase the number of variants that can be characterized to hundreds. These approaches enable high-throughput characterization of virally delivered transgenes in intact tissue, thus complementing the active field of viral vector engineering with scalable tropism identification or validation. Moreover, visualizing the distribution of many variants while preserving spatial context will offer insights into AAV biology, which can include entry mechanisms as well as cell- and tissue-type associated expression.

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