Screening for rAAV transduction enhancers

筛选 rAAV 转导增强子

• 批准号: 10556347
• 负责人: Terry L. Bowlin
• 金额: $ 98.78万
• 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 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10556347
• 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; Innate Immune Response; Laboratories; Lead; Libraries; Link; 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; cost; cytotoxicity; delivery vehicle; design; efficacy testing; gene product; genome analysis; high throughput screening; improved; in vivo; inhibitor; lead candidate; lead series; liquid chromatography mass spectrometry; luminescence; member; meter; 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) 是一种用于基因治疗和治疗的成熟载体 疫苗有可能从根本上改变现代医疗保健的面貌。 rAAV 疗法前景广阔，有两个例子已被 FDA 批准用于临床 受到大媒介剂量的必要性以及严重的后勤和安全问题的阻碍 这些剂量带来的挑战，包括先天性疼痛和适应性免疫 该提案的总体目标是优化衣壳和转基因产品的反应。 增强 rAAV 转导并实现治疗应用的小分子药物 该技术的载体剂量低于有害反应的阈值。 第一阶段的成功是 Microbiotix, Inc. 共同努力的结果。 (MBX) 和 TSRI 的 Michael Farzan 博士实验室，对 5 个化学系列的 11 种化合物进行了研究 经过验证测试后，呋喃嘧啶优先作为经过验证的 rAAV 转导增强剂。 支架被选为最高优先级系列，异吲哚啉和 选择吡啶并嘧啶支架作为备用材料。呋喃并嘧啶MBXC-4409。 多个有利的特征，包括：(a) 高度显着的剂量依赖性活性 (EC50 <10 µM) 和独立于转基因的转导增强（≥3倍）（Gaussia或 萤火虫荧光素酶）、检测信号（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 ≥100 µM；SI (CC50/EC50) >20； 类似结构的纯度 (NMR) 和正确质量 (LC/MS) (d) 在其他方面不混杂； 不相关的筛查，对其他病毒（ZIKV）的感染没有显着抑制或增强作用 (e) 添加时间研究表明早期作用；以及 (f) 有利的体外 ADME 特性（溶解度≥100 µM；在鼠血清中稳定；血清蛋白结合率≤93%）。 第二阶段的工作将继续包括 Michael Farzan 博士在 TSRI 的实验室。 呋喃嘧啶抑制剂系列的效力和类药特性将得到优化， 异吲哚啉和吡啶并嘧啶系列作为备份，产生体内验证的临床前 开发作为辅助药物以增强 rAAV 类似物的候选药物将是。 通过分析漏斗进行评估，该漏斗旨在根据效力、选择性和特异性对它们进行优先排序 将制定优先的类似物用于体内研究。 最有希望的先导化合物的最大耐受剂量将在小鼠中确定 耐受性研究和药代动力学分析将用于进一步优先考虑化合物 将测试抑制剂与 rAAV-gLuc 组合的疗效。 采用全身发光和基因组 qRT-PCR 分析的小鼠转导模型 该提案的主要里程碑是选择一种体内验证的 rAAV。 转导增强剂先导系列将在 IIb 期和 进入 IND 毒理学和安全药理学研究。