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。我们获得了一种有效的hhRz候选治疗药物，可以治疗所有已知的人类RHO突变。长期目标是将RHO adRP的有效hhRz疗法转化为人类临床试验。目的是在针对RHO“人源化”的adRP小鼠模型中进行该hhz试剂和进一步优化的hhRz试剂的临床前材料验证测试。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或停止由突变体RHO（P347 S）引起的视网膜变性。基本原理是，通过突变非依赖性hhRz减少P347 S RHO mRNA和蛋白质必须与野生型（WT）RHO表达的重建（也减少）相结合，以避免光感受器活力的损害。为了检验中心假设并实现目标，具体目标是：目标1。使用合理和进化的方法进一步优化我们的725 GUC↓人RHO hhRz在当前RNA支架内的催化活性。预期成果：通过严格解决hhRz RNA结构活性关系（SAR），我们希望进一步证明临床前试验的效力优化的先导hhRz，并确定临床翻译的最佳药物。目标二。继续一个简单 通过在小鼠WT RHO背景上表达人突变视杆视蛋白转基因的adRP的部分人源化鼠模型中测试视网膜变性的拯救，并在小鼠视杆光感受器中仅表达WT人RHO的完全人源化小鼠模型中测试毒性，使用rAAV包装的基因治疗剂进行敲低hhRz策略的临床前测试。RHO hhRz基因通过重组腺相关病毒（rAAV）递送。adRP小鼠具有缓慢的外部视网膜变性（6个月内约50%损失）。为治疗性抢救提供了一个时间窗口预期结果：抑制毒性突变体RHO将挽救光感受器和视网膜变性。WT人RHO模型没有退化，并且允许测试与RHO减少相关的毒性。预期结果：ERG敏感性变化和“WT”光感受器的可能死亡将取决于RHO降低的水平。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。预期结果：在完全人源化的adRP中拯救视网膜变性。该计划是创新的，具有新的理性和定向进化策略，以改善现有的前导hhRz，以及针对靶mRNA人源化的新小鼠模型，以模拟人类临床试验。意义-人源化adRP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。

 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.