Deep Parallel Characterization of AAV Tropism and AAV-Mediated Transcriptional Changes via Single-Cell RNA Sequencing.
Deep Parallel Characterization of AAV Tropism and AAV-Mediated Transcriptional Changes via Single-Cell RNA Sequencing.
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DOI：
10.3389/fimmu.2021.730825
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发表时间：
2021
影响因子：
7.3
通讯作者：
Gradinaru V
中科院分区：
文献类型：
--
作者：
Brown D;Altermatt M;Dobreva T;Chen S;Wang A;Thomson M;Gradinaru V
重组腺相关病毒（rAAV）的工程变体正在迅速开发，以满足对具有特定细胞类型和组织嗜性的基因治疗递送载体的需求。虽然高通量AAV工程化和选择方法已经产生了许多变体，但随后的向性和响应表征仍然是低通量的，并且在许多相关细胞和组织类型中缺乏分辨率。为了充分利用跨多个靶标的这些大筛选范例的输出，我们开发了一种实验和计算单细胞RNA测序（scRNA-seq）管道，用于以高分辨率对条形码化的rAAV池进行体内表征。使用这个平台，我们既证实了先前报道的病毒向性，又发现了未鉴定的AAV衣壳靶向偏倚。正如预期的，我们观察到，当与AAV-PHP. eB相比时，AAV.CAP-B10在小鼠中的向性特征朝向神经元而远离星形胶质细胞。转录组学分析表明，这种神经元的偏见主要是由于增加的靶向效率为amatergic神经元，我们证实了RNA荧光原位杂交。我们进一步揭示了AAV变体在血管和神经胶质细胞中的细胞亚型向性，例如周细胞和Myoc+星形胶质细胞的低转导。此外，我们已经观察到对全身性AAV-PHP.eB施用的细胞类型特异性瞬时应答，例如注射后三天内皮细胞中参与p53信号传导的基因的上调，其在第25天恢复到对照水平。所提出的用于AAV向性的平行表征的实验和计算方法将促进安全和精确的基因递送载体的进步，并展示在单细胞水平上理解对基因治疗的反应的能力。
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.
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影响因子：
16.6
作者：
Adachi, Kei;Enoki, Tatsuji;Nakai, Hiroyuki
通讯作者：
Nakai, Hiroyuki
影响因子：
8.8
作者：
Capellera-Garcia S;Pulecio J;Dhulipala K;Siva K;Rayon-Estrada V;Singbrant S;Sommarin MN;Walkley CR;Soneji S;Karlsson G;Raya Á;Sankaran VG;Flygare J
通讯作者：
Flygare J
影响因子：
9.3
作者：
Chui R;Dorovini-Zis K
通讯作者：
Dorovini-Zis K
影响因子：
12.4
作者：
Arruda, VR;Fields, PA;High, KA
通讯作者：
High, KA
影响因子：
25
作者：
Chan KY;Jang MJ;Yoo BB;Greenbaum A;Ravi N;Wu WL;Sánchez-Guardado L;Lois C;Mazmanian SK;Deverman BE;Gradinaru V
通讯作者：
Gradinaru V