A Ribozyme Rescue Strategy for Autosomal Dominant Retinitis Pigmentosa

描述（由申请人提供）：人类视杆蛋白基因（RHO）包含许多导致色素性视网膜炎（RP）的突变。折叠的视蛋白mRNA是突变非依赖性锤头核酶（hhRz）基因治疗常染色体显性RP的靶标。由于所有已知的人类RHO突变，我们已经获得了一种高效的hhRz候选adRP治疗方法。长期目标是将这种有效的hhRz治疗RHO adRP转化为人体临床试验。目的是在人类突变型和野生型（WT） RHO基因表达的基础上，在具有视网膜变性的小鼠模型中对这种hhRz药物进行临床前材料证明测试，以模拟缺乏小鼠RHO基因表达的人类adRP。核心假设是，毒性突变RHO mRNA和蛋白的减少将减少光感受器应激，降低RHO突变等位基因表达引起的视网膜变性率。其基本原理是，通过突变无关的hhRz药物减少突变体（P347S） 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，以避免单倍性不足，这也会损害光感受器的活力。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。对Knockdown hhRz策略进行简单的临床前测试，通过在小鼠WT RHO背景下表达人类突变杆视蛋白转基因的部分人源化adRP小鼠模型中测试其对视网膜变性的拯救作用。预期的结果是，抑制毒性突变RHO mRNA/蛋白将拯救光感受器并改善视网膜变性。目标2。在小鼠杆状光感受器中仅表达正常人类RHO的完全人源化小鼠模型中测试hhRz PTGS剂的毒性。预期的结果是了解WT人类视紫红质的最小量，这是保持哺乳动物视杆光感受器存活和重要所需要的。目标3。在小鼠杆状光感受器中表达人类突变体和WT RHO mrna的人源化adRP模型中，测试hhRz先导剂和重组人类WT RHO等位基因的救援作用。在完全人源化的adRP小鼠模型中，预期结果是从视网膜变性中恢复，没有持续的治疗单倍功能不全。该计划的创新之处在于，它使用本实验室开发的小鼠模型，将目标mRNA人源化以模拟人体临床试验，它使用适体技术来调节治疗性hhRz的活性，以优化体内的敲除与毒性，并使用最先进的载体递送方法。重要的是，成功拯救人源化adRP小鼠模型将提供安全性和有效性结果，支持该基因治疗方法用于人类RHO adRP的临床翻译。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的强效hhRz药物，其中敲除有望改善疾病过程。

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.