Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors.
Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors.
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DOI：
10.1038/mtm.2014.64
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发表时间：
2015
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--
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携带多个基因盒的基于慢病毒的基因递送载体是基因转移研究和基因治疗中的有力工具，其允许多个治疗因子和荧光报告物（如果需要）的共表达。目前从单个载体表达转基因和微小RNA（miRNA）簇的策略具有影响转基因表达水平和/或载体滴度的某些限制。在这项研究中，我们描述了一种新的载体设计，有利于治疗性RNA和蛋白质为基础的抗血管生成因子的联合表达，以及从背靠背RNApolII驱动的表达盒的荧光报告。这种构型允许有效产生内含子嵌入的miRNA，其在靶细胞转导时释放。利用此类多基因慢病毒载体，我们证明了miRNA介导的血管内皮生长因子（VEGF）表达的稳健下调，导致血管生成减少，并通过共同递送编码色素上皮衍生因子（PEDF）的基因平行损害血管生成途径。值得注意的是，视网膜下注射慢病毒载体揭示了由VMD 2启动子驱动的有效的视网膜色素上皮特异性基因表达，验证了多基因慢病毒载体可以以足以用于体内应用的高滴度产生。总之，我们的研究结果表明，组合的miRNA和蛋白质编码慢病毒载体在抗血管生成基因治疗中的潜在适用性，包括改善年龄相关性黄斑变性的新的组合疗法。
Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF) expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF). Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration.
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