Abstract
Engineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at high resolution. Using this platform, we have both corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Transcriptomic analysis revealed that this neuronal bias is due mainly to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific transitory responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. The presented experimental and computational approaches for parallel characterization of AAV tropism will facilitate the advancement of safe and precise gene delivery vehicles, and showcase the power of understanding responses to gene therapies at the single-cell level.
Highlights
Recombinant AAVs have become the preferred gene delivery vehicles for many clinical and research applications [1, 2] owing to their broad viral tropism, ability to transduce dividing and non-dividing cells, low immunogenicity, and stable persistence as episomal DNA ensuring long-term transgene expression [3,4,5,6,7,8]
To address the current bottleneck in AAV tropism profiling, we devised an experimental and computational workflow (Figure 1A) that exploits the transcriptomic resolution of scRNA-seq to profile the tropism of multiple AAV variants across complex cell-type hierarchies
Single or multiple barcoded Recombinant AAVs (rAAVs) are injected into the retro-orbital sinus of mice followed by tissue dissociation, single-cell library construction using the 10X Genomics Chromium system, and sequencing with multiplexed Illumina next-generation sequencing (NGS) [69]
Summary
Recombinant AAVs (rAAVs) have become the preferred gene delivery vehicles for many clinical and research applications [1, 2] owing to their broad viral tropism, ability to transduce dividing and non-dividing cells, low immunogenicity, and stable persistence as episomal DNA ensuring long-term transgene expression [3,4,5,6,7,8]. Increased target specificity of rAAVs would reduce both the necessary viral dose and off-target effects; there is an urgent need for AAV gene delivery vectors that are optimized for cell-type-specific delivery [13]. Shaping the tropism of existing AAVs to the needs of a specific disease has the potential to reduce activation of the immune system by detargeting cell types, such as dendritic cells, that have an increased ability to activate T-cells [21,22,23,24,25,26]
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