Characterization of microglia polarization states under targeted AAV transduction using bulk and single cell RNA sequencing

The study of microglia biology and the development of microglia-based gene therapies are in urgent need of efficient and safe vehicles for microglia transgene delivery. To address this, we developed adeno-associated virus (AAV) variants that mediate efficient in vitro and in vivo microglia transduction via directed evolution of the AAV capsid protein. To assess the effect of AAV transduction on microglia, we carried out bulk RNAseq in primary microglia and found that microglia transduced by AAV remain close to homeostatic state. Furthermore, single-cell RNA sequencing showed that the AAV-MG variants mediate safe in vivo transgene delivery without inducing microglia immune activation. These AAV variants should facilitate the applications of various genetically-encoded sensors and effectors in studying microglia-related biology and therapeutic interventions. RNAseq: mRNA profiles of cultured microglia treated with blank, LPS, IL4 or/and AAV variants. scRNAseq: mRNA profiles of cold-dissociated microglia from mice treated with Saline, LPS, or Poly(i:c), using 10X Chromium (reference dataset); mRNA profiles of cold-dissociated microglia from mice treated with LPS or stereotaxically injected with AAV, using Smart-seq2 (query dataset).

小胶质细胞生物学研究以及基于小胶质细胞的基因疗法开发，迫切需要高效且安全的小胶质细胞转基因递送载体。为解决这一需求，我们通过对腺相关病毒（adeno-associated virus, AAV）衣壳蛋白的定向进化，开发了可在体外和体内高效介导小胶质细胞转导的AAV变体。为评估AAV转导对小胶质细胞的影响，我们对原代小胶质细胞开展了批量RNA测序（bulk RNAseq），结果发现经AAV转导的小胶质细胞仍接近稳态状态。进一步的单细胞RNA测序（single-cell RNA sequencing）结果显示，AAV-MG变体可在体内安全递送转基因，且不会诱导小胶质细胞免疫激活。这些AAV变体将推动各类遗传编码传感器和效应器在小胶质细胞相关生物学研究及治疗干预中的应用。RNAseq：经空白处理、脂多糖（lipopolysaccharide, LPS）、白细胞介素4（interleukin 4, IL4）或联合AAV变体处理的培养小胶质细胞的mRNA表达谱。scRNAseq：包含两类mRNA表达谱数据，其一为采用10X Chromium平台获取的经生理盐水、LPS或聚肌胞苷酸（polyinosinic-polycytidylic acid, Poly(I:C)）处理的小鼠冷解离小胶质细胞的表达谱（参考数据集）；其二为采用Smart-seq2平台获取的经LPS处理或立体定向注射AAV的小鼠冷解离小胶质细胞的表达谱（查询数据集）。