Efficient Gene Transfer to Kidney Mesenchymal Cells Using a Synthetic Adeno-Associated Viral Vector
Efficient Gene Transfer to Kidney Mesenchymal Cells Using a Synthetic Adeno-Associated Viral Vector
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DOI：
10.1681/asn.2018040426
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发表时间：
2018-09-01
影响因子：
13.6
通讯作者：
Humphreys, Benjamin D.
中科院分区：
文献类型：
--
作者：
Ikeda, Yoichiro;Sun, Zhao;Humphreys, Benjamin D.
背景 损伤后，肾间质中的间充质祖细胞分化为肌成纤维细胞，这些细胞在肾脏纤维形成中起关键作用。将遗传物质传递给肌成纤维细胞祖细胞的能力可能会为治疗肾纤维化提供新的治疗方法。临床前和临床研究表明，腺相关病毒（AAV）可有效、安全地在体内转导各种组织靶标；然而，肾间充质细胞的转导方案尚未建立。 方法 我们评估了在小鼠肾脏和人肾类器官中表达 GFP 或 Cre 重组酶报告基因的各种假型 AAV 载体的转导概况。 结果 在测试的 6 个 AAV 中，一种名为 Anc80 的合成 AAV 显示出对肾基质和系膜细胞的特异性和高效转导。我们表征了细胞特异性、剂量依赖性和表达动力学，并通过将 Anc80-Cre 病毒注射到纯合或杂合 Gli2-floxed 小鼠中，从肾间充质细胞中敲除 Gli2，展示了该方法的功效。单侧输尿管梗阻后，纯合子 Gli2 floxed 小鼠的纤维化程度低于 Gli2 杂合子小鼠。我们在梗阻性损伤前注射 Anc80-Cre 病毒的 β-连环蛋白 flox 小鼠中观察到相同的抗纤维化作用，强烈支持经典 Wnt 信号在肾脏肌成纤维细胞激活中的核心作用。最后，我们证明 Anc80 合成病毒可以转导人肾类器官中的间充质谱系。结论这些研究建立了一种诱导敲除小鼠系膜、周细胞和血管周围成纤维细胞中 floxed 基因的新方法，并为未来治疗肾纤维化的基因治疗方法奠定了基础。
Background After injury, mesenchymal progenitors in the kidney interstitium differentiate into myofibroblasts, cells that have a critical role in kidney fibrogenesis. The ability to deliver genetic material to myofibroblast progenitors could allow new therapeutic approaches to treat kidney fibrosis. Preclinical and clinical studies show that adeno-associated viruses (AAVs) efficiently and safely transduce various tissue targets in vivo; however, protocols for transduction of kidney mesenchymal cells have not been established.Methods We evaluated the transduction profiles of various pseudotyped AAV vectors expressing either GFP or Cre recombinase reporters in mouse kidney and human kidney organoids.Results Of the six AAVs tested, a synthetic AAV called Anc80 showed specific and high-efficiency transduction of kidney stroma and mesangial cells. We characterized the cell specificity, dose dependence, and expression kinetics and showed the efficacy of this approach by knocking out Gli2 from kidney mesenchymal cells by injection of Anc80-Cre virus into either homozygous or heterozygous Gli2-floxed mice. After unilateral ureteral obstruction, the homozygous Gli2-floxed mice had less fibrosis than the Gli2 heterozygotes had. We observed the same antifibrotic effect in beta-catenin-floxed mice injected with Anc80-Cre virus before obstructive injury, strongly supporting a central role for canonical Wnt signaling in kidney myofibroblast activation. Finally, we showed that the Anc80 synthetic virus can transduce the mesenchymal lineage in human kidney organoids.Conclusions These studies establish a novel method for inducible knockout of floxed genes in mouse mesangium, pericytes, and perivascular fibroblasts and are the foundation for future gene therapy approaches to treat kidney fibrosis.