Roman Alejandro J, Boye Sanford L, Aleman Tomas S, Pang Ji-jing, McDowell J Hugh, Boye Shannon E, Cideciyan Artur V, Jacobson Samuel G, Hauswirth William W
Department of Ophthalmology, Scheie Eye Institute, Philadelphia, PA, USA.
Mol Vis. 2007 Sep 18;13:1701-10.
Dramatic restoration of retinal function has followed subretinal viral-mediated gene therapy in RPE65-deficient animal models of human Leber congenital amaurosis (LCA) caused by RPE65 mutations. Progress in early-phase clinical trials of RPE65-LCA prompted us to begin development of an in vivo bioassay of clinical grade vector stability for later-phase trials.
Naturally-occurring Rpe65-mutant rd12 mice (2-4 mo of age) were studied with full-field electroretinograms (ERGs). Flash stimuli (range, -4.1 to 3.6 log scot-cd x s x m(-2)) were used to evoke ERGs in anesthetized, dark-adapted mice. B-wave amplitudes were measured conventionally and luminance-response functions were fit. Leading edges of photoresponses were analyzed with a model of rod phototransduction activation. A unilateral subretinal injection of AAV2-CB(SB)-hRPE65 vector was delivered and therapeutic efficacy of 4 doses spanning a 2 log unit range was studied with ERGs performed about 6 weeks after injection. Uninjected rd12 eyes and wild-type (wt) mice served as controls.
Rd12 mice showed substantially smaller amplitudes and lower sensitivities than wt mice for all measured ERG b-wave and photoresponse parameters. For the dose-response study, there was no difference between 0.01X-dosed mice and untreated mutants. Improved receptoral and post-receptoral function was evident for 0.1X, 0.3X, 1X doses: b-wave semi-saturation constants decreased, b-wave amplitudes increased with dose; photoresponses showed faster kinetics and higher maximum amplitudes. ERG b-wave amplitude to a selected stimulus light intensity could provide evidence of biologic activity of the vector; interocular differences in b-wave amplitude comparing treated versus untreated eyes in the same animal also revealed vector efficacy.
We have taken the first steps toward developing an ERG assay of biologic activity of human grade vector for future clinical trials of RPE65-LCA. Faithful murine models of treatable human disease tested with specific ERG protocols may emerge as valuable in vivo bioassays for future human clinical trials of therapy in many retinal degenerative diseases.
在由RPE65突变引起的人类莱伯先天性黑蒙（LCA）的RPE65缺陷动物模型中，视网膜下病毒介导的基因治疗后视网膜功能得到了显著恢复。RPE65-LCA早期临床试验的进展促使我们开始开发一种临床级载体稳定性的体内生物测定法，用于后期试验。
使用全视野视网膜电图（ERG）对自然发生Rpe65突变的rd12小鼠（2至4月龄）进行研究。在麻醉、暗适应的小鼠中，使用闪光刺激（范围为-4.1至3.6 log scot-cd x s x m(-2)）诱发ERG。按常规测量B波振幅并拟合亮度响应函数。用光杆光转导激活模型分析光反应的前沿。进行单侧视网膜下注射AAV2-CB(SB)-hRPE65载体，并在注射后约6周通过ERG研究了跨越2个对数单位范围的4种剂量的治疗效果。未注射的rd12眼和野生型（wt）小鼠作为对照。
对于所有测量的ERG b波和光反应参数，rd12小鼠的振幅明显小于wt小鼠，敏感性也更低。在剂量反应研究中，0.01X剂量的小鼠与未治疗的突变体之间没有差异。对于0.1X、0.3X、1X剂量，受体和受体后功能得到改善：b波半饱和常数降低，b波振幅随剂量增加；光反应显示出更快的动力学和更高的最大振幅。针对选定刺激光强度的ERG b波振幅可提供载体生物活性的证据；在同一动物中比较治疗眼与未治疗眼的b波振幅的眼间差异也揭示了载体的疗效。
我们已朝着开发用于未来RPE65-LCA临床试验的人源级载体生物活性的ERG测定迈出了第一步。用特定ERG方案测试的可治疗人类疾病的忠实小鼠模型可能会成为未来许多视网膜退行性疾病人类临床试验中有价值的体内生物测定法。
