Division of Nephrology, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
J Am Soc Nephrol. 2018 Sep;29(9):2287-2297. doi: 10.1681/ASN.2018040426. Epub 2018 Jul 5.
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 ; however, protocols for transduction of kidney mesenchymal cells have not been established.
We evaluated the transduction profiles of various pseudotyped AAV vectors expressing either GFP or Cre recombinase reporters in mouse kidney and human kidney organoids.
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 -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.
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.
肾脏间质中的间充质祖细胞在受伤后分化为肌成纤维细胞，这些细胞在肾脏纤维化中起着关键作用。将遗传物质递送到肌成纤维细胞祖细胞的能力可以为治疗肾脏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）能够有效地将遗传物质递送到各种组织靶标，但是尚未建立将遗传物质递送到肾脏间充质细胞的方案。
我们评估了表达 GFP 或 Cre 重组酶报告基因的各种假型 AAV 载体在小鼠肾脏和人类肾脏类器官中的转导情况。
在测试的 6 种 AAV 中，一种称为 Anc80 的合成 AAV 特异性且高效地转导了肾脏基质和肾小球系膜细胞。我们对其细胞特异性、剂量依赖性和表达动力学进行了表征，并通过将 Anc80-Cre 病毒注射到Gli2 纯合或杂合敲除小鼠中来敲除肾脏间充质细胞中的 Gli2，证明了这种方法的有效性。在单侧输尿管梗阻后，Gli2 纯合敲除小鼠的纤维化程度低于 Gli2 杂合子。我们在进行梗阻性损伤前将 Anc80-Cre 病毒注射到β-catenin 敲除小鼠中，观察到了相同的抗纤维化作用，这强烈支持了经典 Wnt 信号通路在肾脏肌成纤维细胞激活中的核心作用。最后，我们表明 Anc80 合成病毒可以转导人类肾脏类器官中的间充质谱系。
这些研究建立了一种在小鼠肾小球系膜细胞、周细胞和血管周围成纤维细胞中诱导敲除 floxed 基因的新方法，为将来治疗肾脏纤维化的基因治疗方法奠定了基础。
