Screening for rAAV transduction enhancers

摘要 重组腺相关病毒（rAAV）是用于基因治疗的已建立的载体， 疫苗有可能从根本上改变现代医疗保健的面貌。虽然极 有希望的，有两个例子已经批准用于临床使用的FDA，rAAV治疗 由于需要大剂量的病媒以及严重的后勤和安全问题， 这些剂量造成的挑战，包括有害的先天性和适应性免疫 对衣壳和转基因产物的反应。该提案的总体目标是优化 增强rAAV转导并使得能够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。 作为第一阶段的成功结果，这是Microbiotix，Inc. (MBX)和迈克尔·法尔赞博士在TSRI的实验室，五个化学系列的11种化合物， 优先考虑作为经验证的rAAV转导增强剂。在验证测试后， 支架被选为最高优先级的系列，异吲哚啉和 选择吡啶并嘧啶支架作为备份。呋喃并嘧啶MBXC-4409表现出 多种有利特征，包括：（a）高度显著的剂量依赖性活性（EC 50 <10 µM） 和不依赖于转基因的转导增强（≥3倍）（Gaussia或 萤火虫荧光素酶）、测定信号（发光或qRT-PCR）、细胞系（HT 1080和HeLa）， 或衣壳血清型（血清型1、2、8和9）;（B）CC 50 ≥100 μM; SI（CC 50/EC 50）>20;（c）药物- 纯度≥98%（NMR）和质量正确（LC/MS）的相似结构;（d）在其他方面不混杂 不相关的筛选，并且没有显著抑制或增强其他病毒（ZIKV）的感染 和VSV）;（e）添加时间研究表明早期作用;（f）体外ADME有利 性质（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。的 呋喃并嘧啶抑制剂系列的效力和药物样性质将得到优化， 异吲哚啉和吡啶并嘧啶系列作为备份，以生产体内验证的临床前 作为增强rAAV治疗剂的开发候选物。类似物将是 通过设计用于根据效价、选择性和特异性对其进行优先排序的测定漏斗进行评价 无毒的作用机制。将配制优先类似物用于体内研究。的 将在小鼠中测定最有希望的先导化合物的最大耐受剂量 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， 优化给药策略。将测试抑制剂与rAAV-gLuc组合在以下中的功效： 具有全身发光和基因组qRT-PCR分析的转导的小鼠模型 在组织中复制。该提案的主要里程碑是选择体内验证的rAAV 转导增强剂铅系列。将在IIb期确定临床前候选药物， 推进IND毒理学和安全药理学研究。

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.