Novel adeno-associated virus (AAV) vectors for improved retinal gene therapy

用于改进视网膜基因治疗的新型腺相关病毒（AAV）载体

• 批准号: 399445958
• 负责人: Professor Dr. Stylianos Michalakis
• 金额: --
• 依托单位: Augenklinik (Innenstadt)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/399445958?language=en
• 关键词: Novel; adeno; associated; virus; AAV

Recombinant adeno-associated virus (AAV) vectors have become the “gold standard” gene delivery tool for targeting retinal cells. AAV-based vectors were tested in a number of human clinical trials and have proven safe for the use in the retina. The most advanced gene therapy is AAV-RPE65, a AAV vector for gene supplementation treatment of RPE65-linked Leber congenital amaurosis (LCA2). In clinical studies subretinal delivery of AAV-RPE65 has proven to be safe and showed some efficacy in improving vision. However, the AAV-RPE65 treatment was not able to halt or slow photoreceptor degeneration in LCA2. The reasons for this are not known, but could include disease-specific factors or issues related to the AAV vector like inadequate levels of transduction and expression. For efficient transduction, conventional AAV vectors require direct application to the target cell surface and interaction with receptors for internalization. Most inherited blinding retinal diseases are due to mutations in genes that are specifically expressed in photoreceptors or in retinal pigment epithelium (RPE). Targeting these cells requires surgical detachment of the neuroretina from the RPE and subretinal injection of the AAV vectors into a temporally formed cavity. This procedure can be deleterious to an already compromised retina. In addition, subretinal injections result only in a very localized transduction of retinal cells within the subretinal bleb area. Therefore, there is a critical unmet need to develop novel AAV vectors with improved transduction properties that enable transretinal gene expression through less invasive routes of delivery such as intravitreal (IVT) injection. The major goal of the present grant proposal is to further develop and characterize innovative AAV vectors that show great promise for targeting retinal photoreceptors after IVT administration. The work program includes in vitro characterization of the novel AAVs in 661w cone photoreceptor-like cells and human iPS-derived retinal organoids. In vivo experiments in wild type and retinal degeneration mice with vector versions expressing eGFP driven by cell type-specific promoters will assess their transduction efficiency of mouse rod or cone photoreceptors. These experiments will be followed by analogous experiments in dogs and non-human primates (NHP) to confirm the ability of the vectors to transduce rods and cones from the vitreous in large animal models and to define the optimal dose range in large eyes. The NHP and/or dog studies will also assess any immune response to the novel capsids, effects of re-administration of the vectors to the same eye and their basic biodistribution profile. Finally, the optimal vectors will be tested regarding their efficiency to restore vision and delay degeneration by IVT-delivered gene supplementation therapy in the Cnga3 knockout (KO) mouse model of achromatopsia and in the Cngb1 KO mouse model of retinitis pigmentosa.

重组腺相关病毒（AAV）载体已成为靶向视网膜细胞的“黄金标准”基因递送工具。基于AAV的载体在许多人类临床试验中进行了测试，并已被证明可安全用于视网膜。最先进的基因治疗是AAV-RPE 65，一种用于RPE 65连锁Leber先天性黑蒙（LCA 2）基因补充治疗的AAV载体。在临床研究中，AAV-RPE 65的视网膜下递送已被证明是安全的，并显示出改善视力的一些功效。然而，AAV-RPE 65处理不能停止或减缓LCA 2中的光感受器变性。其原因尚不清楚，但可能包括疾病特异性因素或与AAV载体相关的问题，如转导和表达水平不足。为了有效转导，常规AAV载体需要直接应用于靶细胞表面并与受体相互作用以进行内化。大多数遗传性致盲性视网膜疾病是由于感光细胞或视网膜色素上皮细胞（RPE）中特异性表达的基因突变所致。靶向这些细胞需要将神经视网膜从RPE手术分离，并将AAV载体视网膜下注射到暂时形成的空腔中。这个过程可能对已经受损的视网膜有害。此外，视网膜下注射仅导致视网膜下泡区域内的视网膜细胞的非常局部的转导。因此，存在开发具有改善的转导性质的新型AAV载体的关键的未满足的需求，所述新型AAV载体能够通过侵入性较小的递送途径（例如玻璃体内（IVT）注射）进行经视网膜基因表达。本拨款提案的主要目标是进一步开发和表征创新的AAV载体，这些载体在IVT给药后显示出靶向视网膜光感受器的巨大前景。该工作计划包括在661 w视锥光感受器样细胞和人iPS衍生的视网膜类器官中体外表征新型AAV。在野生型和视网膜变性小鼠中进行的体内实验将评估它们对小鼠视杆或视锥光感受器的转导效率，所述小鼠具有表达由细胞类型特异性启动子驱动的eGFP的载体形式。这些实验之后将在犬和非人灵长类动物（NHP）中进行类似实验，以确认载体在大型动物模型中从玻璃体中清除视杆细胞和视锥细胞的能力，并确定大眼睛中的最佳剂量范围。NHP和/或犬研究还将评估对新衣壳的任何免疫应答、对同一只眼睛重新施用载体的影响及其基本生物分布特征。最后，将在色盲的Cnga 3敲除（KO）小鼠模型和视网膜色素变性的Cngb 1 KO小鼠模型中测试最佳载体通过IVT递送的基因补充疗法恢复视力和延迟变性的效率。