Optimizing Enhanced Hammerhead Ribozymes for Retinal Nucleic Acid Therapeutics

优化用于视网膜核酸治疗的增强型锤头核酶

基本信息

批准号：
10638529
负责人：
JOHN M. SULLIVAN
金额：
$ 56.71万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10638529
关键词：
Antisense Oligonucleotides Binding Biophysics Calorimetry Catalysis Catalytic RNA Cells Cellular Stress Chemistry Clinical Clinical Trials Dependovirus Development Digestion Disease Disease model Elements Engineering Enzymes Evaluation Exonuclease Eye Eye diseases Frequencies Genes Goals Human Incentives Injectable Kinetics Lead Length Macular degeneration Measures Mediating Messenger RNA Mission Modality Modeling Modification Molecular Mus Mutation National Eye Institute Nucleic Acids Nucleotides Opsin Performance Pharmaceutical Preparations Photoreceptors Physiological Population Property Proteins Protons RNA RNA Interference RNA Stability Resistance Resources Retina Retinal Degeneration Retinitis Pigmentosa Rhodopsin Risk Rodent Model Site Specificity Speed Structure Surface Plasmon Resonance Synthesis Chemistry Testing Therapeutic Therapeutic Agents Titrations Toxic effect Transduction Gene Transgenic Mice Translating Work autosome biophysical techniques cellular targeting cofactor combinatorial comparative design drug discovery experimental study gain of function mutation gene therapy hammerhead ribozyme human model humanized mouse improved in vivo inherited retinal degeneration innovation intravitreal injection knock-down molecular dynamics mouse model mutant novel nuclease nucleic acid-based therapeutics peripherin pharmacologic photoreceptor degeneration pre-clinical preservation prevent promoter protein expression reconstitution single molecule small hairpin RNA stem success synthetic nucleotide therapeutic RNA therapeutic candidate therapeutic evaluation therapeutic gene trafficking transcriptome sequencing vector

项目摘要

Many mutations in the human rod opsin (hRHO) and peripherin genes cause autosomal dominant retinitis pigmentosa (adRP) and macular degenerations. The folded mRNAs are targets for mutation-independent hammerhead ribozyme (hhRz) gene therapy. Our long-range goal is to translate effective hhRz therapeutics for hRHO adRP and other genes into human clinical trials. We have discovered a potent form of hhRz, which we call Enhanced-hhRz (EhhRz) therapeutics. All or most known mutations in a given dominant disease gene could be treated with a mutation-independent EhhRz strategy that pairs knockdown (KD) of mutant (and WT) mRNA with reconstitution (RECON) of WT protein with expression of a cleavage-resistant mRNA. The current stage of development of EhhRzs (against hRHO) lead to kinetic turnover rates greater than 2-log orders improved over historical minimal hhRzs (mhhRz); one mhhRz partially rescued mutant RHO retinal degeneration in rodent models. EhhRzs function under substrate (target) excess conditions and physiological levels of cofactor Mg2+, which are optimum for intracellular KD therapeutics. If this exciting kinetic potential can be harnessed for the photoreceptor, retinal, or ocular cells then there is potential for nucleic acid drugs (injectable EhhRzs) as therapeutics without vector-based gene therapy, for diverse ocular diseases and inherited retinal degenerations. Recent clinical success with intravitreal injection of antisense agents incentivizes this development. The objective is to optimize EhhRzs for intracellular performance in cultured human cells and mouse photoreceptor cells that are “humanized” for RHO mRNAs (same targets as in clinical trials). The central hypothesis is that optimization of EhhRz kinetic performance and delivery will maximize target mRNA/protein KD; optimization requires quantitation of biophysical variables that underlie target: EhhRz interaction, rational and/or evolutionary engineering of RNA structure-related function, and identification of the optimum delivery materials and approach. Our rationale is that EhhRzs can be optimized through synthetic chemistry and/or natural modifications, while retaining or enhancing kinetic function, to support robust intracellular KD. Treatment occurs through vector-mediated delivery (AAV) of expression constructs, or direct delivery of synthetic EhhRz therapeutics (injectables). To test the central hypothesis the Specific Aims are: Aim 1. Optimize current EhhRzs at lead hRHO target sites (266 CUC, 725 GUC, 1362 GUC) for rate enhancement, cellular stability, target-colocalization, and knockdown performance. Aim 2. Optimize EhhRz Knockdown strategy for: (i) rescue (efficacy) of retinal degeneration and (ii) lack of toxicity with AAV-EhhRz gene therapy and injectable synthetic EhhRzs in transgenic mouse models humanized for RHO. Innovation occurs through optimization of EhhRz design, and optimization of strategies for therapeutic delivery in a humanized adRP model. Significance- The study optimizes EhhRz function and delivery as an initial step toward a KD/RECON rescue strategy for dominant photoreceptor degenerations.

人棒蛋白（HRHO）和周围基因的许多突变引起常染色体显性视网膜炎色素（ADRP）和黄斑变性。折叠的mRNA是非依赖突变的靶标锤头核酶（HHRZ）基因治疗。我们的远程目标是翻译有效的HHRZ理论对于HRHO ADRP和其他基因进行人类临床试验。我们发现了HHRZ的潜在形式，我们称之为增强的HHRZ（EHHRZ）疗法。给定显性疾病基因中的所有或最著名的突变可以通过独立于突变的EHHRZ策略来处理，该策略将突变体（和WT）配对（KD） mRNA具有WT蛋白的重建（RECER），表达裂解的mRNA。电流 EHRZS的发展阶段（反对HRHO）导致动力学周转率大于2 log Orders 改善了历史最小HHRZ（MHHRZ）；一个MHHRZ部分反应了突变的Rho视网膜啮齿动物模型中的变性。 Ehhrz在底物（目标）下的功能超出条件和生理辅因子MG2+的水平，对于细胞内KD疗法是最佳的。如果这种令人兴奋的动力学潜力可以用于光感受器，视网膜或眼细胞的利用，然后有核酸药物（可注射的EHHRZ）作为无基于媒介基因疗法的治疗，针对大型眼疾病和继承的视网膜变性。玻璃室内注射反义剂的最新临床成功激励这一发展。目的是优化EHRZS在培养中的细胞内性能用于Rho mRNA的“人性化”的人类细胞和小鼠感光细胞（与临床相同的靶标试验）。中心假设是EHHRZ动力学性能和交付的优化将最大化靶mRNA/蛋白质KD；优化需要定量目标的生物物理变量：EHRZ RNA结构相关功能的相互作用，合理和/或进化工程，并鉴定优化送货材料和方法。我们的理由是可以通过合成来优化Ehrzs 化学和/或自然修饰，同时保留或增强动力学功能，以支持鲁棒细胞内KD。通过矢量介导的表达构建体的介导（AAV）或直接进行治疗递送合成EHHRZ理论（注射剂）。为了检验中心假设，具体目的是： AIM 1。在铅HRHO目标位点（266Cuc，725Guc，1362Guc）上优化电流Ehrzs的速率增强，细胞稳定性，靶标性和敲低性能。目标2。优化 EHHRZ敲低策略：（i）残留变性（ii）缺乏毒性（效力） AAV-EHHRZ基因疗法和可注射的合成EHRZS中的转基因小鼠模型人性化的罗。创新是通过优化EHHRZ设计和治疗策略的优化而发生的以人源化的ADRP模型交付。意义 - 研究优化EHRZ功能和传递朝着KD/RENCER救援策略迈出的主要光感受器变性的第一步。