Stromal Gene Therapy Mediates Prolonged Protection Against Corneal Neovascularization Induced by an Aggressive Angiogenic Insult.

IF 3.9 3区 医学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Mark Basche, Scott Robbie, D Frank P Larkin, Alexander J Smith, Rachael A Pearson, Robin R Ali
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Abstract

Corneal neovascularization (CoNV) is both a sight-threatening condition in and of itself and a major risk factor associated with corneal graft failure. Here, we determine the effectiveness of an adeno-associated viral vector (AAV)-based gene therapy targeting both hematic and lymphatic neovascularization in a murine model of severe CoNV. We first assessed the profile of transgene expression mediated by intrastromal injection of AAV2/8[Y733F] via longitudinal visualization of an enhanced Green Fluorescent Protein (eGFP) transgene and found that this serotype mediates a temporary (∼18 day) transduction of the corneal epithelium and sustained (≥148 day) transduction within the stroma. Constitutively expressed sFlt1 or sFlt4 were prophylactically delivered via intrastromal injection of AAV2/8[Y733F] vector at various intervals prior to aggressive induction of CoNV in a murine model. The extent of CoNV induced was quantified by fluorescein angiography and immunohistochemistry 17 days after induction. AAV2/8[Y733F]-CMV-sFlt1 was highly effective in the prevention of hemangiogenesis (HA) induced at 3, 28, and 210 days after intrastromal injection, but ineffective in the prevention of lymphangiogenesis. Two variants of AAV2/8[Y733F]-CMV-sFlt4 were ineffective in the prevention of angiogenesis when delivered alone, but combined delivery of AAV2/8[Y733F]-CMV-sFlt1 and AAV2/8[Y733F]-CMV-sFlt4 suggested a synergistic effect. Our results show that a single intrastromal injection of AAV2/8[Y733F]-CMV-sFlt1 is sufficient to protect against a robust stimulus for corneal HA over the long term. This technique could also be applied ex vivo to reduce the risk of failure in cases of "high-risk" corneal transplantation.

基质基因治疗介导对侵袭性血管生成损伤诱导的角膜新生血管的长期保护。
角膜新生血管(CoNV)是一种威胁视力的疾病,也是角膜移植失败的主要危险因素。在这里,我们确定了一种基于腺相关病毒载体(AAV)的基因治疗在严重CoNV小鼠模型中针对血液和淋巴新生血管的有效性。我们首先通过纵向可视化增强的绿色荧光蛋白(eGFP)转基因评估了细胞内注射AAV2/8[Y733F]介导的转基因表达谱,发现该血清型介导角膜上皮的暂时(~ 18天)转导和持续(≥148天)转导。基质内的转导。在小鼠模型中,在侵袭性诱导CoNV之前,通过不同间隔的AAV2/8[Y733F]载体,预防性地将组成性表达的sFlt1或sFlt4传递给小鼠。诱导后第17天,采用荧光素血管造影和免疫组化方法定量测定小鼠的CoNV诱导程度。AAV2/8[Y733F]-CMV-sFlt1对细胞内注射后3、28和210天诱导的血管生成(HA)非常有效,但对淋巴管生成无效。单独递送AAV2/8[Y733F]-CMV-sFlt4两种变体对血管生成的预防作用无效,但联合递送AAV2/8[Y733F]-CMV-sFlt1和AAV2/8[Y733F]-CMV-sFlt4显示出协同作用。我们的研究结果表明,单次角膜内注射AAV2/8[Y733F]-CMV-sFlt1足以长期保护角膜HA免受强劲刺激。这项技术也可以在体外应用,以降低“高风险”角膜移植的失败风险。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Human gene therapy
Human gene therapy 医学-生物工程与应用微生物
CiteScore
6.50
自引率
4.80%
发文量
131
审稿时长
4-8 weeks
期刊介绍: Human Gene Therapy is the premier, multidisciplinary journal covering all aspects of gene therapy. The Journal publishes in-depth coverage of DNA, RNA, and cell therapies by delivering the latest breakthroughs in research and technologies. Human Gene Therapy provides a central forum for scientific and clinical information, including ethical, legal, regulatory, social, and commercial issues, which enables the advancement and progress of therapeutic procedures leading to improved patient outcomes, and ultimately, to curing diseases.
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