Deep Parallel Characterization of AAV Tropism and AAV-Mediated Transcriptional Changes via Single-Cell RNA Sequencing

Frontiers in Immunology

Tatyana Dobreva¹  David Brown²  Viviana Gradinaru²  Sisi Chen²  Michael Altermatt²  Matt Thomson²  Alexander Wang³ 

[1] Andrew and Peggy Cherng Department of Medical Engineering, California Institute of Technology, Pasadena, CA, United States;Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, United States;Division of Engineering and Applied Science, California Institute of Technology, Pasadena, CA, United States;

关键词: gene therapy;    next generation sequencing;    computational biology;    molecular biology;    neuroscience;   

DOI  :  10.3389/fimmu.2021.730825

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown