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

重组腺相关病毒（AAV）载体已成为靶向视网膜细胞的“金标准”基因传递工具。基于aav的载体在许多人体临床试验中进行了测试，并已证明在视网膜中使用是安全的。目前最先进的基因疗法是AAV- rpe65，这是一种AAV载体，用于RPE65-linked Leber congenital amaurosis （LCA2）的基因补充治疗。在临床研究中，AAV-RPE65视网膜下递送已被证明是安全的，并显示出一定的改善视力的功效。然而，AAV-RPE65治疗不能阻止或减缓LCA2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，传统的AAV载体需要直接应用于靶细胞表面并与受体相互作用以内化。大多数遗传性致盲性视网膜疾病是由于光感受器或视网膜色素上皮（RPE）中特异性表达的基因突变引起的。针对这些细胞，需要手术将神经视网膜从RPE上剥离，并将AAV载体注射到视网膜下暂时形成的腔中。这个过程可能对已经受损的视网膜有害。此外，视网膜下注射只导致视网膜下水泡区域内视网膜细胞的非常局部的转导。因此，迫切需要开发具有改进转导特性的新型AAV载体，使其能够通过侵入性较小的传递途径（如玻璃体内（IVT）注射）经视网膜基因表达。目前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给药后靶向视网膜光感受器方面表现出很大的希望。工作计划包括在661w锥状光感受器样细胞和人类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的体内实验中，用由细胞类型特异性启动子驱动的表达eGFP的载体版本将评估它们对小鼠杆状或锥状光感受器的转导效率。这些实验之后将在狗和非人灵长类动物（NHP）中进行类似实验，以确认病媒在大型动物模型中从玻璃体中转导视杆细胞和视锥细胞的能力，并确定大眼睛的最佳剂量范围。NHP和/或狗的研究还将评估对新型衣壳的任何免疫反应，对同一只眼睛重新施用载体的影响及其基本生物分布概况。最后，在Cnga3基因敲除（KO）小鼠色盲模型和Cngb1色素性视网膜炎小鼠模型中，通过ivt介导的基因补充治疗，测试最佳载体恢复视力和延缓变性的效率。

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.