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)包含许多导致视网膜色素变性(RP)的突变。折叠的视蛋白mRNA是常染色体显性遗传(Ad)RP的非突变锤头状核酶(HhRz)基因治疗的靶点。我们获得了一种有效的治疗adrp的hhRz候选药物，可以治疗所有已知的人类Rho突变。长期目标是将治疗Rho adrp的有效hhRz疗法转化为人类临床试验。目的是对这种HHZ制剂和进一步优化的HHRZ制剂在ADRP小鼠模型中进行临床前材料验证测试，这些ADRP模型是RHO的人源化模型。中心假说是，毒性突变体Rho的mRNA和蛋白的减少将减少光感受器应激，减缓或阻止突变Rho引起的视网膜变性(P347S)。其基本原理是，通过非突变的hhRz降低P347S Rho的mRNA和蛋白，必须与野生型(WT)Rho表达的重建(也降低)相结合，以避免光感受器活力的损害。为了验证中心假设并实现目标，具体目标是：目标1.使用理性和进化方法进一步优化我们的725GUC↓人Rho hhRz在当前RNA支架上的催化活性。预期结果：通过严格处理hhRz RNA构效关系(SAR)，我们希望在临床前试验中进一步证明效力优化的hhRzs，并确定用于临床翻译的最佳制剂。目标2.继续一个简单的 通过检测在小鼠WT Rho背景上表达人突变杆视蛋白转基因的部分人源化adrp小鼠模型对视网膜变性的挽救作用，以及在仅表达WT人Rho基因的完全人源化小鼠模型中测试小鼠杆光感受器的毒性，用rAAV包装的基因治疗剂对击倒hhRz策略进行临床前测试。Rho hhRz基因由重组腺相关病毒(RAAV)传递。Adrp组小鼠视网膜外侧变性缓慢(6个月后视网膜变性约50%)。这为治疗性救援提供了一个时间窗口。预期结果：抑制有毒突变体Rho将挽救光感受器和视网膜退化。WT人类Rho模型没有退化，并允许测试与Rho降低相关的毒性。预期结果：ERG的敏感度变化和“WT”光感受器的可能消亡将取决于Rho减少的水平。目的3.在人源化ADRP模型中检测先导hhRz试剂和重组人WT Rho等位基因在人源化ADRP模型中的拯救作用。预期结果：在完全人性化的ADRP中挽救视网膜变性。该计划具有创新性，采用了新的理性和定向进化策略来改进现有的Lead hhRz，并为目标mRNAs人源化了新的小鼠模型以模拟人类临床试验。意义-人源化adrp小鼠模型的成功抢救将提供安全和有效的结果，以支持这种针对人rho adrp的基因治疗方法的临床翻译。

 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 achieved a potent hhRz candidate therapeutic for adRP that could treat all known human RHO mutations. The long-range goal is to translate effective hhRz therapeutics for RHO adRP into human clinical trials. The objective is to conduct a preclinical proof-of-materials test of this hhz agent and further optimized hhRz agents in mouse models of adRP that are "humanized" for RHO. The central hypothesis is that the reduction of toxic mutant RHO mRNA and protein will reduce photoreceptor stresses and reduce the rate or stop retinal degeneration due to mutant RHO (P347S). The rationale is that reduction of the P347S RHO mRNA and protein by the mutation-independent hhRz must be combined with reconstitution of wild type (WT) RHO expression (also reduced) to avoid compromise of photoreceptor vitality. To test the central hypothesis and accomplish the objectives the Specific Aims are: Aim 1. Use rational and evolutionary approaches to further optimize catalytic activity of our 725 GUC↓ human RHO hhRz within the current RNA scaffold. Expected results: By rigorously addressing hhRz RNA structure activity relations (SAR) we expect to manifest further potency-optimized lead hhRzs for preclinical trial and identify the optimum agent for clinical translation. Aim 2. Continue a simple preclinical test of the Knockdown hhRz strategy with rAAV packaged gene therapeutic agents 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, and test toxicity in a fully humanized mouse model expressing only WT human RHO in mouse rod photoreceptors. RHO hhRz genes are delivered by recombinant adeno-associated virus (rAAV). The adRP mouse has a slow outer retinal degeneration (~50% loss in 6 mo.) that offers a temporal window for therapeutic rescue. Expected results: suppression of toxic mutant RHO will rescue photoreceptors and retinal degeneration. The WT human RHO model has no degeneration and allows test of toxicity related to RHO reduction. Expected results: ERG sensitivity shifts and possible demise of "WT" photoreceptors will depend on levels of RHO reduction. 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: rescue of retinal degeneration in the fully humanized adRP. The plan is innovative with novel rational and directed evolutionary strategies to improve the existing lead hhRz, and novel mouse models humanized for target mRNAs to simulate human clinical trial. Significance- successful rescue in humanized adRP mouse models would provide safety and efficacy outcomes to support clinical translation of this gene therapy approach for human RHO adRP.