ChR2 delivery to the neuroretina to circumvent age-related neurodegeneration

ChR2 递送至神经视网膜以避免与年龄相关的神经变性

• 批准号: 8595030
• 负责人: Theodore George Drivas
• 金额: $ 2.88万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8595030
• 关键词: Achievement; Address; Adenoviruses; Affect; Age related macular degeneration; Aging; Alzheimer' s Disease; American; Amino Acid Sequence; Animal Model; Animals; Antibodies; B-Lymphocytes; Binding; Biological Assay; Blindness; Bypass; Cations; Cell Line; Cell Surface Receptors; Cells; Characteristics; Degenerative Disorder; Dependovirus; Disease; Dose; Elderly; Engineering; Enzyme-Linked Immunosorbent Assay; Epitopes; Foundations; Future; Gene Transfer; Genes; Genetic Transcription; Histologic; Human; Immune; Immune response; In Vitro; Individual; Infiltration; Inflammation; Injection of therapeutic agent; Life; Mediating; Membrane; Monitor; Monoclonal Antibodies; Mus; Mutation; Nerve Degeneration; Older Population; Parkinson Disease; Pathologic; Pathology; Patients; Pattern; Peptide Sequence Determination; Photoreceptors; Population; Proteins; Public Health; Recombinants; Resistance; Retina; Retinal; Retinal Cone; Retinal Degeneration; Sensory; Series; Serotyping; Solutions; System; Systems Development; T-Lymphocyte; Techniques; Testing; Therapeutic; Toxic effect; Transgenes; United States; Validation; Viral Vector; Virion; Work; age related neurodegeneration; aged; aging population; base; body system; cell type; cellular transduction; cohort; cone-rod degeneration; design; gene therapy; immunogenic; immunogenicity; in vivo; light gated; mouse model; novel; novel therapeutic intervention; particle; patient population; photoreceptor degeneration; promoter; public health relevance; receptor; response; retinal rods; success; technique development; therapeutic gene; transgene expression; treatment strategy; vector

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.

描述（由申请人提供）：基因治疗领域最近的成功突出了基因转移方法在治疗许多疾病中的可行性和有效性。(1-3)虽然这些成就令人鼓舞，但在我们希望更广泛地应用这项技术获得充分利益之前，仍然存在许多重大障碍。视网膜相关性黄斑变性（AMD）是一种无法治愈的疾病，仅在美国老龄化人口中估计就有175万例失明病例，（4）对我们的老年美国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治疗方法的主要目标。(7-8)这种常见的病理学也可能提供一个机会，以广泛地解决所有形式的AMD与一个单一的治疗方法。通过在视网膜的双极细胞中表达蛋白质视紫红质-2（ChR 2），每个双极细胞可以转化为自己的光感受器，从而避免杆和锥的变性，为AMD提供了通用的治疗方法。（15-17）虽然这种方法的可行性已经在小动物模型中显示，但双极细胞对AAV转导的抗性仍有待解决。(18)已经在体外和体内显示了通过修饰其与细胞表面受体的相互作用将AAV成功地重靶向至难以转染的细胞类型。（33-38）重组双抗体（scDb），即，能够结合两个不同表位的抗体衍生分子，其被设计为结合AAV和在双极细胞上优先表达的受体，可以用于实现这种重靶向。我们建议产生这样的scDb并证明其在体外重新靶向AAV的能力。该系统的进一步发展可以使腺相关病毒特异性重新靶向视网膜双极细胞。[The对ChR 2（藻类衍生的转基因）的视网膜表达的免疫应答对于该技术的进一步发展将是关键的。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，以彻底检查局部毒性，全身免疫反应，和转基因表达的高，中，低剂量的AAV 2/8 TC-ChR 2（一种新的AAV血清型，我们已经开发了与我们的合作者，更有效地靶向视网膜双极细胞）视网膜下给予小鼠。本研究旨在表征由组成型CAG启动子或ON双极细胞特异性mGluR 6启动子驱动的ChR 2表达之间的免疫原性和表达模式的差异。此外，如果目标1中描述的scDb被生产和验证，则目标2被设计为适应将该scDb纳入单独的动物队列中以进行体内验证和测试。因此，这项研究不仅将提供有关对ChR 2的免疫反应及其在视网膜中的表达的重要信息，而且还将为未来任何基于ChR 2的基因治疗研究提供信息。在追求这些目标的过程中，我们希望解决关于基于ChR 2的眼部基因治疗的两个主要障碍-递送和免疫原性-并且在这样做的过程中专门推进AMD的基因治疗领域，并且更普遍地为有效的基于scDb的AAV重靶向用于其他疾病奠定基础。