Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany.
Center for Infectious Diseases, Virology, Medical Faculty, Heidelberg University, Im Neuenheimer Feld 344, 69120, Heidelberg, Germany.
Sci Rep. 2022 May 19;12(1):8356. doi: 10.1038/s41598-022-12404-0.
Human brain cells generated by in vitro cell programming provide exciting prospects for disease modeling, drug discovery and cell therapy. These applications frequently require efficient and clinically compliant tools for genetic modification of the cells. Recombinant adeno-associated viruses (AAVs) fulfill these prerequisites for a number of reasons, including the availability of a myriad of AAV capsid variants with distinct cell type specificity (also called tropism). Here, we harnessed a customizable parallel screening approach to assess a panel of natural or synthetic AAV capsid variants for their efficacy in lineage-related human neural cell types. We identified common lead candidates suited for the transduction of directly converted, early-stage induced neural stem cells (iNSCs), induced pluripotent stem cell (iPSC)-derived later-stage, radial glia-like neural progenitors, as well as differentiated astrocytic and mixed neuroglial cultures. We then selected a subset of these candidates for functional validation in iNSCs and iPSC-derived astrocytes, using shRNA-induced downregulation of the citrate transporter SLC25A1 and overexpression of the transcription factor NGN2 for proofs-of-concept. Our study provides a comparative overview of the susceptibility of different human cell programming-derived brain cell types to AAV transduction and a critical discussion of the assets and limitations of this specific AAV capsid screening approach.
体外细胞编程生成的人类脑细胞为疾病建模、药物发现和细胞治疗提供了令人兴奋的前景。这些应用通常需要高效且符合临床要求的工具来对细胞进行基因修饰。重组腺相关病毒 (AAV) 因其多种原因满足这些先决条件，包括具有不同细胞类型特异性（也称为趋向性）的无数种 AAV 衣壳变体。在这里，我们利用可定制的平行筛选方法来评估一系列天然或合成的 AAV 衣壳变体在与其谱系相关的人类神经细胞类型中的有效性。我们确定了常见的先导候选物，适合转导直接转化的早期诱导神经干细胞 (iNSC)、诱导多能干细胞 (iPSC) 衍生的晚期、放射状胶质样神经祖细胞以及分化的星形胶质细胞和混合神经胶质培养物。然后，我们选择了其中一些候选物用于 iNSC 和 iPSC 衍生的星形胶质细胞中的功能验证，使用 shRNA 诱导柠檬酸转运蛋白 SLC25A1 的下调和转录因子 NGN2 的过表达来证明概念。我们的研究提供了不同人类细胞编程衍生脑细胞对 AAV 转导易感性的比较概述，并对这种特定的 AAV 衣壳筛选方法的优势和局限性进行了批判性讨论。
