A Ribozyme Rescue Strategy for Autosomal Dominant Retinitis Pigmentosa

常染色体显性遗传性色素性视网膜炎的核酶救援策略

• 批准号: 8294751
• 负责人: JOHN M. SULLIVAN
• 金额: $ 30.24万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8294751
• 关键词: Alleles; Animals; Aptamer Technology; Award; Biological Assay; Candidate Disease Gene; Catalytic RNA; Cell Death; Cleaved cell; Clinical; Clinical Trials; Complementary DNA; Disease; Engineering; Evaluation; Gene Expression; Genes; Genetic; Genetic Variation; Goals; Green Fluorescent Proteins; Hereditary Disease; Human; Image; Inherited; Knock-out; Lead; Mammalian Cell; Materials Testing; Measures; Mediating; Messenger RNA; Mission; Modeling; Mus; Mutation; National Eye Institute; Nuclear; Opsin; Outcome; Outcome Measure; Photoreceptors; Physiological; Plasmids; Population; Preclinical Testing; Process; Property; Proteins; RNA Interference; Recombinant adeno-associated virus (rAAV); Recovery; Retina; Retinal; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Safety; Simulate; Site; Stress; Technology; Testing; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Thick; Toxic effect; Transgenes; Transgenic Organisms; Translating; Translations; Variant; Wild Type Mouse; base; design; gain of function; gene therapy; hammerhead ribozyme; high throughput screening; in vivo; innovation; knowledge of results; mRNA Expression; mouse model; mutant; nanoparticle; novel; pre-clinical; preclinical evaluation; protein expression; public health relevance; reconstitution; retinal rods; small hairpin RNA; success; tool; vector

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.

描述（由申请人提供）：人棒OPSIN基因（RHO）具有许多引起色素性视网膜炎（RP）的突变。折叠蛋白mRNA是非依赖突变的锤头核酶（HHRZ）基因治疗常染色体显性率（AD）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度有效的HHRZ候选药物治疗方法。远程目标是将这种有效的HHRZ治疗性转化为RHO ADRP的治疗性，为人类的临床试验。目的是在小鼠模型中对这种HHRZ药物进行临床前材料测试，这些模型基于人类突变体和野生型（WT）Rho基因具有视网膜变性，以在没有小鼠Rho基因表达的情况下对人ADRP进行建模。中心假设是，毒性突变体mRNA和蛋白质的还原将减少感光应力，并降低因突变体Rho等位基因表达而引起的视网膜变性速率。理由是，必须将非依赖性HHRZ药物的突变体（p347s）RHO mRNA和蛋白质降低与重新建立WT RHO表达（也降低HHRZ）的方法和策略相结合，以避免单倍不足易度效率，这也将损害光感受器的活力。为了测试中心假设并实现目标，将追求三个特定目标：目标1。通过测试ADRP的部分人源化的鼠模型中的视网膜变性，对敲低HHRZ策略进行简单的临床前测试，该模型表达了人类突变的ropsin opsin opsin opsin the Backendent the wt wt rho wt rho。预期的结果是，抑制有毒突变体mRNA/蛋白质将挽救感光体并改善视网膜变性。 AIM 2。在完全人性化的小鼠模型中测试HHRZ PTGS毒性的毒性，该模型仅表达在小鼠杆感光体中的正常人Rho。预期的结果是对保持哺乳动物杆光感受器保持活力和至关重要的最低WT人类视紫红质的了解。 AIM 3。在人源化的ADRP模型中测试铅HHRZ剂和重建人WT Rho等位基因进行营救，其中人类突变体和WT RHO mRNA在小鼠杆杆光感受器中表达。完全人性化的ADRP小鼠模型中的预期结果是从视网膜变性中拯救出来的，而无需持续的治疗单倍度不足。该计划具有创新性，因为它使用了该实验室中开发的小鼠模型，该模型被人性化用于目标mRNA来模拟人类的临床试验，它使用APTAMER技术来调节使用治疗性HHRZ的活性，以优化体内的敲低与毒性，并采用了最新的毒性。意义在于，在人源化的ADRP小鼠模型中，成功的营救将提供安全和功效结果，以支持人类Rho ADRP的这种基因治疗方法的临床翻译。我们的技术平台使我们能够迅速开发出有效的HHRZ药物到任何已知的疾病目标mRNA，预计敲低可以改善疾病过程。 公共卫生相关性：拟议的实验计划很重要，并且与国家眼科研究所的任务有关，以开发遗传性视网膜变性治疗。在这里，我们对本实验室中开发的高度有效的转录后基因沉默剂（例如核酶）进行了临床前测试，以挽救具有ROD OPSIN突变的人源性小鼠模型中的视网膜变性。在常染色体显性视网膜炎的独特全人文化小鼠模型中，挽救视网膜变性的成功可能会支持将成功的治疗剂转化为人类临床试验。