ChR2 delivery to the neuroretina to circumvent age-related neurodegeneration

描述（由申请人提供）：最近在基因治疗领域取得的成功突出了基因转移方法治疗许多疾病的可行性和有效性。尽管这些成就令人鼓舞，但在我们能够希望获得更广泛地应用这项技术的全部好处之前，仍存在许多重大障碍。年龄相关性黄斑变性（AMD）是一种无法治愈的疾病，仅在美国就造成了大约175万例老年失明病例，(4)对美国老年人来说是一个巨大的公共卫生负担。AMD的一个重要特征是光感受器的病理性变性，否则它将成为许多AMD治疗的主要目标。（7-8）这种常见的病理也可能提供一个机会，通过单一的治疗方法广泛地解决所有形式的AMD。通过在视网膜的双极细胞中表达蛋白channelrhodopin -2 (ChR2)，每个双极细胞都可以转化为自己的光感受器，避免视杆细胞和视锥细胞的变性，为AMD提供了一种通用的治疗方法。（15-17）虽然这种方法的可行性已在小动物模型中得到证明，但双极细胞对AAV转导的抗性仍有待解决。（18）体外和体内研究表明，通过改变AAV与细胞表面受体的相互作用，AAV可以成功地靶向难以转导的细胞类型。（33-38）重组糖尿病体（scDb），即一种能够结合两种不同表位的抗体衍生分子，设计用于结合AAV和在双极细胞上优先表达的受体，可以用来实现这种重靶向。我们建议生产这样的scDb，并在体外证明其重靶向AAV的能力。该系统的进一步发展可能允许AAV特异性重靶向视网膜双极细胞。[对ChR2（一种藻类衍生的转基因）的视网膜表达的免疫反应将对该技术的进一步发展至关重要。为了研究这一点，我们提出，作为我们的第二个目标，一项研究，以彻底检查在小鼠视网膜下给药的高、中、低剂量AAV2/8TC-ChR2（我们与合作者开发的一种新的AAV血清型，更有效地靶向视网膜双极细胞）的局部毒性、全身免疫反应和转基因表达。本研究旨在表征组成型CAG启动子和ON双极细胞特异性mGluR6启动子驱动的ChR2表达在免疫原性和表达模式上的差异。此外，如果Aim 1中描述的scDb被生产和验证，Aim 2的设计是为了适应将该scDb纳入单独的动物队列中进行体内验证和测试。因此，这项研究不仅将提供关于ChR2免疫反应及其在视网膜中的表达的重要信息，而且将为未来基于ChR2的基因治疗研究提供信息。在实现这些目标的过程中，我们希望解决基于ChR2的眼部基因治疗的两个主要障碍——递送和免疫原性，这样做可以专门推进AMD的基因治疗领域，更广泛地为基于scdb的AAV重靶向治疗其他疾病奠定基础。

DESCRIPTION (provided by applicant): Recent successes in the field of gene therapy have highlighted the feasibility and efficacy of gene transfer approaches in treating numerous diseases.(1-3) While these achievements are encouraging, many significant hurdles remain before we can hope to reap the full benefits of applying this technique more generally. Age-related macular degeneration (AMD), an incurable disease responsible for an estimated 1.75 million cases of blindness in the aging population of the United States alone,(4) represents a huge public health burden for our population of older Americans. An important feature of AMD is the pathologic degeneration of the photoreceptors, which would otherwise serve as the primary target for many AMD therapeutics.(7-8) This common pathology may also provide an opportunity to broadly address all forms of AMD with a single therapeutic approach. By expressing the protein Channelrhodopsin-2 (ChR2) in the bipolar cells of the retina, each bipolar cell could be transformed into its own photoreceptor, circumventing the degeneration of rods and cones, providing a universal cure for AMD.(15-17) While the feasibility of this approach has been shown in small animal models, the resistance of the bipolar cells to AAV transduction remains to be addressed.(18) Successful retargeting of AAV to difficult-to-transduce cell types by modifying its interactions with cell surface receptors has been shown in vitro and in vivo.(33-38) A recombinant diabody (scDb), i.e., an antibody-derived molecule capable of binding two different epitopes, designed to bind both AAV and a receptor preferentially expressed on the bipolar cells, could be employed to achieve such retargeting. We propose to produce such a scDb and demonstrate its ability to retarget AAV in vitro. The further development of this system could allow for specific retargeting of AAV to the retinal bipolar cells. [The immune response to retinal expression of ChR2, an algal-derived transgene, will be critical to the further development of this technique. To investigate this, we propose, as our second aim, a study to thoroughly examine local toxicity, systemic immune response, and transgene expression for high, medium, and low doses of AAV2/8TC-ChR2 (a novel AAV serotype we have developed with our collaborators that more efficiently targets retinal bipolar cells) administered subretinaly in mice. This study is designed to characterize the difference in immunogenicity and expression pattern between ChR2 expression driven by either the constitutive CAG promoter or the ON bipolar cell specific mGluR6 promoter. Additionally, should the scDb described in Aim 1 be produced and validated, Aim 2 is designed to accommodate the inclusion of this scDb in a separate animal cohort for in vivo validation and testing. Thus, this study will not only provide important information about the immune response to ChR2 and its expression in the retina, but will also inform any future ChR2-based gene therapy studies.] In pursuing these aims, we hope to address two major obstacles regarding ChR2 based ocular gene therapy - delivery and immunogenicity - and in doing so to specifically advance the field of gene therapy for AMD, and more generally to lay the foundation for effective scDb-based AAV retargeting for other diseases.