Prevention of nocturnal elevation of intraocular pressure by gene transfer of dominant-negative RhoA in rats.
Prevention of nocturnal elevation of intraocular pressure by gene transfer of dominant-negative RhoA in rats.
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DOI：
10.1001/jamaophthalmol.2014.4747
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发表时间：
2015-02
影响因子：
8.1
通讯作者：
Carabana J
中科院分区：
文献类型：
--
作者：
Borrás T;Buie LK;Spiga MG;Carabana J
我们开发了一种基因转移工具来控制夜间眼压升高（IOP）。通过在长表达的重组病毒中传递一种突变的、显性阴性的RhoA基因（dnRhoA）来抑制小梁网RhoA通路，可以降低活体动物夜间升高的IOP。我们通过将带有翻译增强子-启动子的突变RhoA互补DNA插入到含有突变病毒末端重复序列的特殊设计质粒中，生成了优化的重组病毒分子。然后，我们产生了携带突变基因（scAAV2.dnRhoA）的病毒颗粒，自互补腺相关病毒血清型2，并通过感染原代人小梁网细胞在体外评估其功能，并通过在活体大鼠体内注射治疗和对照病毒来评估其功能。使用了三种不同的12小时明暗循环模型。当动物出现昼夜暗眼压升高时注射病毒。眼压测量在夜间和白天周期开始后2至4小时用眼压计进行。注射前的数值作为基线。注射后4 ~ 8周实施安乐死。鼠眼内注射重组病毒载体scAAV2.dnRhoA。夜间IOP升高被转移的RhoA基因长时间阻断。通过目测，人小梁网细胞感染了scAAV2。dnRhoA显示应力纤维形成减少。活体大鼠的IOP周期可以通过调节光照条件来重置，从而促进夜间IOP研究。单剂量注射scAAV2。dnRhoA在夜间周期内阻止大鼠眼内眼压升高至少4周（平均[SE]， 9.2 [0.2] mm Hg浅IOP和9.6 [0.4]mm Hg暗IOP），而对照眼的眼压明显高于基线（9.5 [0.4]mm Hg浅IOP和13.5 [0.3]mm Hg暗IOP）。据我们所知，这是基因转移策略的第一个例子，该策略可以长期防止活体动物夜间眼压升高。用一种安全的基因药物抑制Rho激酶上游的RhoA通路可能为夜间IOP升高的长期治疗提供一种新的强化治疗方法。
We developed a gene transfer tool for the control of nocturnal elevated intraocular pressure (IOP). To demonstrate that inhibiting the trabecular meshwork RhoA pathway by delivering a mutated, dominant-negative RhoA gene (dnRhoA) carried inside a long-expressing recombinant virus would reduce nocturnal elevated IOP in a living animal. We generated an optimized recombinant viral molecule by inserting a mutated RhoA complementary DNA with a translation enhancer-promoter into a specially designed plasmid containing mutated viral terminal repeats. We then generated the virus particle, self-complementary adeno-associated virus serotype 2 carrying the mutated gene (scAAV2.dnRhoA) and assessed its function in vitro by infecting primary human trabecular meshwork cells and in vivo by injecting living rats intracamerally with therapeutic and control viruses. Three different models of 12-hour light and dark cycles were used. Viruses were injected when animals showed the circadian dark IOP elevation. The IOP measurements were conducted with a tonometer at 2 to 4 hours after onset of the nocturnal and diurnal cycles. Values at preinjection time were used as baselines. Animals were euthanized at 4 to 8 weeks after injection. Intraocular injection of rodent eyes with the recombinant viral vector scAAV2.dnRhoA. Nocturnal elevation of IOP blocked for prolonged periods by transferred RhoA gene. By visual inspection, human trabecular meshwork cells infected with scAAV2.dnRhoA showed diminished stress fiber formation. Living rats exhibited a circadian IOP cycle that could be reset by adjusting light conditions to facilitate light and dark nocturnal IOP studies. A single-dose injection of scAAV2.dnRhoA into the rat eyes prevented elevation of IOP during the nocturnal cycle for at least 4 weeks (mean [SE], 9.2 [0.2] mm Hg light IOP and 9.6 [0.4] mm Hg dark IOP), while control eyes showed a significantly higher IOP over baseline (9.5 [0.4] mm Hg light IOP and 13.5 [0.3] mm Hg dark IOP). To our knowledge, this is the first example of a gene transfer strategy that prevents nocturnal IOP elevation in living animals for prolonged periods. Inhibiting the RhoA pathway upstream of Rho kinase with a safe gene drug could provide a new enhanced treatment for long-term management of elevated nocturnal IOP.