A Ribozyme Rescue Strategy for Autosomal Dominant Retinitis Pigmentosa

描述（由申请人提供）：人视杆细胞视蛋白基因（RHO）含有许多导致视网膜色素变性（RP）的突变。折叠的视蛋白mRNA是不依赖于突变的锤头状核酶（hhRz）基因治疗常染色体显性（ad）RP的靶点。由于所有已知的人RHO突变，我们已经实现了用于adRP的单一高效hhRz候选治疗剂。长期目标是将这种用于RHO adRP的有效hhRz治疗转化为人类临床试验。目的是在人突变型和野生型（WT）RHO基因表达的基础上，在视网膜变性小鼠模型中进行该hhRz试剂的临床前材料验证试验，以在不存在小鼠RHO基因表达的情况下模拟人adRP。中心假设是毒性突变体RHO mRNA和蛋白的减少将减少感光细胞应激并降低由于突变体RHO等位基因表达引起的视网膜变性的速率。其基本原理是，通过突变非依赖性hhRz试剂减少突变体（P347 S）RHO mRNA和蛋白质必须与重建WT RHO表达（也被hhRz减少）的方法和策略相结合，以避免也会损害光感受器活力的单倍不足。为了检验中心假设并实现目标，将追求三个具体目标：目标1。通过检测在小鼠WT RHO背景下表达人突变视杆视蛋白转基因的部分人源化adRP鼠模型中视网膜变性的挽救，对敲低hhRz策略进行简单的临床前试验。预期的结果是抑制毒性突变体RHO mRNA/蛋白将拯救光感受器并改善视网膜变性。目标2.在小鼠视杆光感受器中仅表达正常人RHO的完全人源化小鼠模型中测试hhRz PTGS试剂的毒性。预期的结果是知识的最低量的野生型人类视紫红质，需要保持哺乳动物的视杆细胞存活和重要的。目标3.在人源化adRP模型中测试前导hhRz试剂和重建的人WT RHO等位基因用于拯救，其中人突变体和WT RHO mRNA在小鼠视杆光感受器中表达。在adRP的完全人源化小鼠模型中的预期结果是从视网膜变性中拯救，而没有持续的治疗性单倍不足。所提出的计划是创新的，因为它使用本实验室开发的针对靶mRNA进行人源化的小鼠模型来模拟人类临床试验，它使用适体技术来调节治疗性hhRz的活性，以优化体内敲低与毒性，并且它使用最先进的载体递送方法。重要的是，在adRP的人源化小鼠模型中的成功拯救将提供安全性和有效性结果，以支持这种用于人RHO adRP的基因治疗方法的临床转化。我们的技术平台使我们能够快速开发针对任何已知疾病靶mRNA的有效hhRz试剂，其中敲低预期会改善疾病过程。 公共卫生相关性：拟议的实验计划是重要的，并与国家眼科研究所的使命，以开发治疗遗传性视网膜变性。在这里，我们对本实验室开发的高效转录后基因沉默剂（例如核酶）进行了临床前测试，以挽救具有视杆视蛋白突变的人源化小鼠模型中的视网膜变性。在一个独特的完全人源化的常染色体显性视网膜色素变性小鼠模型中成功挽救视网膜变性，可能会支持将成功的治疗药物转化为人类临床试验。

DESCRIPTION (provided by applicant): The human rod opsin gene (RHO) harbors many mutations that cause retinitis pigmentosa (RP). The folded opsin mRNA is a target for mutation-independent hammerhead ribozyme (hhRz) gene therapy for autosomal dominant (ad) RP. We have achieved a single highly potent hhRz candidate therapeutic for adRP due to all known human RHO mutations. The long-range goal is to translate this effective hhRz therapeutic for RHO adRP into human clinical trials. The objective is to conduct a preclinical proof-of-materials test of this hhRz agent in mouse models that have retinal degeneration on the basis of expression of human mutant and wild type (WT) RHO genes to model human adRP in the absence of mouse RHO gene expression. The central hypothesis is that the reduction of toxic mutant RHO mRNA and protein will reduce photoreceptor stresses and reduce the rate of retinal degeneration due to mutant RHO allele expression. The rationale is that reduction of the mutant (P347S) RHO mRNA and protein by the mutation-independent hhRz agent must be combined with approaches and strategies to reconstitute WT RHO expression (also reduced by the hhRz) to avoid haploinsufficiency that would also compromise photoreceptor vitality. To test the central hypothesis and accomplish the objective three Specific Aims will be pursued: Aim 1. Conduct a simple preclinical test of the Knockdown hhRz strategy by testing for rescue of retinal degeneration in a partially humanized murine model of adRP that expresses a human mutant rod opsin transgene on the mouse WT RHO background. Expected results are that suppression of toxic mutant RHO mRNA/protein will rescue photoreceptors and ameliorate retinal degeneration. Aim 2. Test the hhRz PTGS agent for toxicity in a fully humanized mouse model that expresses only normal human RHO in mouse rod photoreceptors. Expected results are knowledge of the minimum amount of WT human rhodopsin that is needed to keep a mammalian rod photoreceptor alive and vital. Aim 3. Test the lead hhRz agent and a reconstituting human WT RHO allele for rescue in a humanized adRP model in which human mutant and WT RHO mRNAs are expressed in mouse rod photoreceptors. Expected results in the fully humanized mouse model of adRP are rescue from retinal degeneration without sustained therapeutic haploinsufficiency. The proposed plan is innovative in that it uses mouse models developed in this lab that are humanized for target mRNA to simulate human clinical trial, it uses aptamer technology to modulate activity of the therapeutic hhRz using to optimize knockdown vs. toxicity in vivo, and it uses state-of-the-art vector delivery approaches. The significance is that successful rescue in humanized mouse models of adRP would provide safety and efficacy outcomes to support clinical translation of this gene therapy approach for human RHO adRP. Our technology platform allows us to rapidly develop potent hhRz agents to any known disease target mRNA in which knockdown is expected to ameliorate the disease process. PUBLIC HEALTH RELEVANCE: The proposed experimental plan is significant and relevant to a mission of the National Eye Institute to develop treatments for hereditary retinal degenerations. Here we conduct a preclinical test of highly potent post-transcriptional gene silencing agents (e.g. ribozymes) developed in this lab to rescue retinal degeneration in humanized mouse models with rod opsin mutations. A success to rescue retinal degeneration in a unique fully humanized mouse model of autosomal dominant retinitis pigmentosa could potentially support translation of successful therapeutic agents into human clinical trials.