Screening for rAAV transduction enhancers

筛选 rAAV 转导增强子

• 批准号: 10157565
• 负责人: Terry L. Bowlin
• 金额: $ 100万
• 依托单位: MICROBIOTIX, INC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-27 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10157565
• 关键词: Address; Animal Model; Binding Proteins; Biological Assay; CMV promoter; Capsid; Cell Culture Techniques; Cell Line; Cells; Chemicals; Clinical; Development; Dose; Drug Kinetics; Engineering; Enhancers; Exhibits; Face; Firefly Luciferases; Funding; Gene Delivery; Gene Transduction Agent; Goals; Healthcare; Hela Cells; Human; Immune response; In Vitro; Infection; Laboratories; Lead; Libraries; Link; Logistics; Luciferases; Maximum Tolerated Dose; Modernization; Mus; Pharmaceutical Preparations; Pharmacology Study; Phase; Predictive Value; Property; Public Health; Quantitative Reverse Transcriptase PCR; Recombinant adeno-associated virus (rAAV); Reporter; Safety; Series; Serotyping; Serum; Serum Proteins; Signal Transduction; Small Business Innovation Research Grant; Solubility; Structure; Structure-Activity Relationship; Technology; Testing; Therapeutic; Time; Tissues; Toxic effect; Toxicology; Transgenes; United States; Vaccines; Validation; Vesicular stomatitis Indiana virus; Viral; Viral Genome; Virus; Work; Zika Virus; acute toxicity; adaptive immune response; adeno-associated viral vector; analog; base; cost; cytotoxicity; design; efficacy testing; genome analysis; high throughput screening; improved; in vivo; inhibitor/antagonist; lead candidate; lead series; liquid chromatography mass spectrometry; luminescence; member; mouse model; pre-clinical; response; scaffold; screening; small molecule; small molecule libraries; transduction efficiency; vector

ABSTRACT Recombinant adeno-associated virus (rAAV) is an established vector for gene therapy and vaccines with the potential to radically change the face of modern healthcare. Although extremely promising, with two examples already approved for clinical use by the FDA, rAAV therapeutics are hampered by the necessity of large vector doses and the serious logistical and safety challenges resulting from these doses, including detrimental innate and adaptive immune responses to capsid and transgene products. The overall goal of this proposal is to optimizie small molecule drugs that enhance rAAV transduction and enable the therapeutic application of this technology at vector doses below the threshold of deleterious responses. As the result of a successful Phase I, which was a collaborative effort by Microbiotix, Inc. (MBX) and Dr. Michael Farzan’s laboratory at TSRI, 11 compounds in five chemical series were prioritized as validated rAAV transduction enhancers. After validation testing, the furopyrimidine scaffold was selected as the highest priority series, and members of isoindoline and pyridopyrimidine scaffolds were selected as backups. The furopyrimidine MBXC-4409 exhibited multiple favorable features, inlcuding: (a) highly significant dose-dependent activity (EC50 <10 µM) and transduction enhancements (≥3-fold) that are independent of the transgene (Gaussia or Firefly luciferase), the assay signal (luminescence or qRT-PCR), the cell line (HT1080 and HeLa), or the capsid serotype (serotypes 1, 2, 8, and 9); (b) CC50 ≥100 µM; SI (CC50/EC50) >20; (c) drug- like structures of ≥98% purity (NMR) and of correct mass (LC/MS); (d) not promiscuous in other unrelated screens and no significant inhibition or enhancement of infection by other viruses (ZIKV and VSV); (e) time-of-addition studies indicate early action; and (f) favorable in vitro ADME properties (solubility ≥100 µM; stable in murine serum; serum protein binding ≤93%). This Phase II effort will continue to include Dr. Michael Farzan’s laboratory at TSRI. The potency and drug-like properties of the furopyrimidine inhibitor series will be optimized, with the isoindoline and pyridopyrimidine series as backups, to produce in vivo-validated preclinical candidates for development as adjunctive agents to enhance rAAV therapeutics. Analogs will be evaluated through an assay funnel designed to prioritize them by potency, selectivity, and specific non-toxic mechanisms of action. Prioritized analogs will be formulated for in vivo studies. The maximum tolerated dose of the most promising lead compounds will be determined in mouse tolerability studies, and pharmacokinetic analyses will be used to further prioritize compounds and optimize dosing strategies. Inhibitors will be tested for efficacy in combination with rAAV-gLuc in a murine model of transduction with whole body luminescence and qRT-PCR analysis of genome copies in tissues. The major milestone of this proposal is to select an in vivo-validated rAAV transduction enhancer lead series. A preclinical candidate will be identified in Phase IIb and advanced to IND-enabling toxicology and safety pharmacology studies.

抽象的 重组腺相关病毒（RAAV）是基因治疗和 疫苗有可能从根本上改变现代医疗保健的面貌。虽然非常 有希望的，有两个示例已经批准了FDA的临床用途 受到大型矢量剂量的必要性和严重的后勤和安全性的阻碍 这些剂量带来的挑战，包括有害的先天和适应性免疫 对衣壳和转换产品的响应。该提案的总体目标是优化 小分子药物，增强RAAV翻译并实现治疗应用 矢量的这项技术剂量低于有害响应的阈值。 由于成功的第一阶段，这是Microbobiotix，Inc。的合作努力。 （MBX）和TSRI的Michael Farzan博士实验室，五个化学系列中的11种化合物是 优先为经过验证的RAAV转导增强剂。验证测试后，呋喃吡汀 脚手架被选为最高优先级系列，以及Isoindoline的成员和 选择吡啶吡啶支架作为备用。暴露于 多个有利的特征，插入：（a）高度显着的剂量依赖性活性（EC50 <10 µm） 与转基因无关的翻译增强（≥3倍）（高斯或 萤火虫荧光素酶），测定信号（发光或QRT-PCR），细胞系（HT1080和HELA）， 或衣壳血清型（血清型1、2、8和9）； （b）CC50≥100µm; Si（CC50/EC50）> 20; （c）药物 - 纯度≥98％（NMR）和正确质量（LC/MS）的结构； （d）在其他 无关的筛选，没有其他病毒（ZIKV）对感染的显着抑制或增强 和VSV）; （e）陷入困境的研究表明早期作用； （f）在体外Adme中有利 性质（溶解度≥100µm；在鼠血清中稳定；血清蛋白结合≤93％）。 这次第二阶段的努力将继续包括TSRI的Michael Farzan博士的实验室。这 将优化呋喃吡啶抑制剂系列的效力和类似药物样性能，并在 等丁氨酸和吡啶吡啶系列作为备用，以在体内验证临床前产生 作为辅助药物增强RAAV治疗的候选者。类似物会 通过旨在通过效力，选择性和特定的测定漏斗进行评估 无毒作用机制。将对体内研究的优先类似物进行制定。这 最大耐受剂量的最有前途的铅化合物将在小鼠中确定 耐受性研究和药代动力学分析将用于进一步确定化合物和 优化给药策略。将测试抑制剂的效率，以与RAAV-GLUC结合使用 带有全身发光和QRT-PCR分析基因组的鼠模型 在组织中的副本。该提案的主要里程碑是选择一个体内验证的RAAV 转导增强剂铅系列。临床前候选人将在IIB期中确定，并 提高到毒理学和安全药理学研究。