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

• 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
• 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

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.

人类视杆蛋白(HRHO)和外周蛋白基因的许多突变导致常染色体显性视网膜炎 色素变性(ADRP)和黄斑变性。折叠的mRNAs是不依赖突变的靶标。 锤头状核酶(HhRz)基因治疗。我们的长期目标是将有效的hhrz疗法 用于hRHO、adrp和其他基因进入人体临床试验。我们已经发现了一种有效的hhrz形式，它 我们称之为增强型高频辐射(EhhRz)疗法。给定显性疾病基因的全部或大部分已知突变 可以用与突变无关的EhhRz策略来治疗，该策略将突变(和WT)的敲除(KD)配对 重组(Recon)WT蛋白，表达抗切割的mRNA.海流 EhhRzs的发展阶段(相对于hRHO)导致动态周转率大于2对数量级 改善了历史最低hhRz(MhhRz)；一个mhhRz部分挽救了突变的Rho视网膜 啮齿动物模型的退化。EhhRzs在底物(靶)过量条件和生理条件下的作用 辅因子镁离子水平，这是细胞内KD治疗的最佳选择。如果这种激动势能够 被用于光感受器、视网膜或眼细胞那么就有可能开发出核酸药物 (可注射EhhRzs)作为治疗方法，无需基于载体的基因治疗，适用于各种眼病和 遗传性视网膜变性。玻璃体腔内注射反义药物近期临床成功 刺激了这一发展。目的是优化EhhRzs在培养的细胞内的性能 人类细胞和小鼠感光细胞对Rho mRNAs进行人源化(与临床相同的靶点 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KD；优化需要量化作为靶标的生物物理变量：EhhRz RNA结构相关功能的相互作用、理性和/或进化工程，以及鉴定 最佳的输送材料和途径。我们的理论基础是Ehhrzs可以通过合成来优化 化学和/或自然修饰，同时保留或增强动力功能，以支持强健 胞内KD。治疗通过载体介导的表达载体传递(AAV)进行，或直接 提供合成的EhhRz疗法(注射剂)。为了检验中心假设，具体目标是： 目标1.优化领先目标位置的当前EhhRZ(266GUC、725GUC、1362GUC)的比率 增强、细胞稳定性、目标共定位和击倒性能。目标2.优化 EhhRz击倒策略：(I)挽救视网膜变性(有效)和(Ii)无毒性 AAV-EhhRz基因治疗和可注射合成EhhRzs在人源化转基因小鼠模型中的应用 罗。通过优化EhhRz设计和优化治疗策略来实现创新 在人性化的adrp模型中交付。意义-该研究将EhhRz的功能和交付优化为 向KD/Recon抢救显性光感受器退行性变策略迈出的第一步。