Gene Therapy for Retinitis Pigmentosa

描述(申请人提供)：这项研究的长期目标是开发一种治疗视网膜色素变性(RP)的方法，这是一种导致光感受器退化和失明的疾病。目前还没有治疗这种疾病的方法。RP是由仅在杆状感光细胞中表达的基因突变引起的。隐性RP的最常见原因是编码cGMP磷酸二酯酶(PDE)亚单位的基因突变。尽管有多种PDE突变的动物模型可用，例如Rd1、Rd10、RCD等，但目前还没有证据表明在这些模型中长期挽救杆状光感受器，这是临床试验的先决条件。这与基因治疗方法在其他眼睛组织，如视网膜色素上皮(RPE)，特别是在Leber先天性黑色素症动物模型中取得的成功形成了鲜明的对比。杆状感光细胞疾病的抢救进展滞后是由于缺乏有效的光感受器基因转移载体。到目前为止，眼部基因治疗中最常用的载体是腺相关病毒(AAV)，其大多数血清型的克隆能力有限，为4.8kb。这一能力不足以包括实现杆状特异性和受调控的转基因表达所需的大基因调控元件。此外，AAV最近被证明通过插入突变在动物中以令人震惊的高达56%的比率引起肝细胞癌。因此，需要能够持续存在的基因治疗载体。AAV载体的这些显著缺陷和本提案中讨论的其他缺陷可以通过使用辅助依赖型腺病毒载体(HD-Ad)来克服，这些载体具有36K的克隆能力，并在非人类灵长类动物中持续存在多年。然而，HD-Ad载体对光感受器没有趋向性，相反，它们只针对RPE。最近，我们已经发现，缺失了五元碱基RGD结构域的腺病毒(Ad)载体或衣壳上显示转铁蛋白的Ad载体可以非常有效地转导感光细胞。这些修改应该可以很容易地转移到HD-ADS。因此，这项建议有三个具体目标。具体目的1：确定在第一代Ad载体的背景下，含有五肽RGD缺失的Ad衣壳结合衣壳上的转铁蛋白是否比单独修饰更有效地转导光感受器。具体目标2：开发一种容量为36Kb的HD-Ad，它已被修改为在其五元碱基上含有RGD-缺失，在其衣壳上含有转铁蛋白。使用非常大的(&gt；10kb)5‘上游和下游基因调控元件，包括10kb的PDE启动子，检测杆状特异的转基因表达。具体目的3：检测光受体靶向的HD-Ad系统能否利用天然的(10kb)PDE 5‘和3’基因调控元件在RD1和RD10小鼠视网膜中特异地表达PDE，从而加强对光感受器变性的长期挽救。与公共健康相关：根据民意调查，在美国人中，失明是仅次于癌症的最可怕的疾病。遗传性失明最常见的原因是视网膜色素变性，目前还没有FDA批准的治疗方法。这项建议旨在开发一种基因疗法，用于治疗视网膜色素变性和导致失明的相关疾病。这项研究中开发的分子工具还将用于开发糖尿病视网膜病变、老年性黄斑变性、青光眼等的治疗方法，这些都是美国最常见的失明原因。

DESCRIPTION (provided by applicant): The long-term objective of this study is to develop a therapy for Retinitis Pigmentosa (RP), a disease that causes photoreceptor degeneration and blindness. There is currently no therapy available for this disease. RP is caused by mutations in genes expressed exclusively in the rod photoreceptors. The most common causes of recessive RP are mutations in the gene encoding the ¿ subunit of cGMP Phosphodiesterase (¿ PDE). Despite the availability of a variety of animal models with mutations in ¿ PDE, e.g. rd1, rd10, rcd etc., no evidence yet exists for long-term rescue of rod photoreceptors in these models, a prerequisite to clinical trials. This is in stark contrast to the success gene therapy approaches have had in other ocular tissues such as the retinal pigment epithelium (RPE) and specifically in animal models of Leber's congenital amaurosis. The lag in progress for rescue of rod photoreceptor diseases is due to the absence of efficacious gene transfer vectors for photoreceptors. The most commonly used vector in ocular gene therapy to date is adeno-associated virus (AAV), most serotypes of which have a limited cloning capacity of 4.8 Kb. This capacity is insufficient for the inclusion of large gene regulatory elements needed to achieve rod-specific and regulated transgene expression. Furthermore, AAV has been recently shown to cause hepatocellular carcinoma at the alarmingly high rate of 56% in animals by insertional mutagenesis. Hence, there is a need for gene therapy vectors that persist episomally. These significant deficiencies with AAV vectors and other deficiencies discussed in the proposal can be overcome through the use of helper-dependent adenovirus vectors (Hd-Ad) that have a 36K cloning capacity and persist episomally for years in non human primates. However, Hd-Ad vectors do not have a tropism for photoreceptors but instead, they target only the RPE. Recently, we have shown that adenovirus (Ad) vectors with deletions in the RGD domain of penton base or Ad vectors that display transferrin on their capsid through can very efficiently transduce photoreceptors. These modifications should readily be transferable to Hd-Ads. Hence, this proposal has 3 specific aims. Specific Aim 1: Determine whether in the context of 1st generation Ad vectors, do Ad capsids containing an RGD deletion in penton base combined with transferrin on their capsid, transduce photoreceptors more efficiently than either modification alone. Specific Aim 2: To develop a 36Kb-capacity Hd-Ad that has been modified to contain an RGD-deletion in it's penton base and transferrin on it's capsid. Examine rod specific transgene expression using very large (>10Kb) 5' upstream and downstream gene regulatory elements, including a 10Kb ¿ PDE promoter. Specific Aim 3: To examine whether the photoreceptor targeted Hd-Ad system can express ¿ PDE in the rd1 and rd10 murine retina specifically in rod photoreceptor cells using native (10Kb) ¿ PDE 5' and 3' gene regulatory elements and potentiate long term rescue of photoreceptor degeneration. PUBLIC HEALTH RELEVANCE: According to public opinion polls, blindness is second only to cancer as the most feared disease amongst Americans. The most common cause of genetic blindness is Retinitis Pigmentosa, for which there is currently no FDA-approved therapy available. This proposal aims to develop a genetic therapy for Retinitis Pigmentosa and allied disorders that cause blindness. The molecular tools developed in this study will also have application in developing therapies for diabetic retinopathy, age related macular degeneration, glaucoma etc., that are collectively the most common causes of blindness in the United States.