Screening for rAAV transduction enhancers

摘要 重组腺相关病毒(RAAV)是一种成熟的基因治疗载体。 有可能从根本上改变现代医疗保健面貌的疫苗。虽然非常 RAAV疗法很有前途，有两个例子已经被FDA批准用于临床 由于大量媒介剂量的必要性以及严重的后勤和安全问题 这些剂量带来的挑战，包括有害的先天免疫和获得性免疫 对衣壳和转基因产物的反应。这项建议的总体目标是优化 增强rAAV转导并使其在治疗中应用的小分子药物 这项技术在媒介剂量低于有害反应的门槛. 作为第一阶段成功的结果，这是MicroBiotix，Inc.的合作努力。 (MBX)和Michael Farzan博士在TSRI的实验室，五个化学系列中的11个化合物 优先作为有效的rAAV转导增强剂。经过验证测试，呋喃并嘧啶 支架被选为最优先的系列，异吲哚和异吲哚的成员 选用吡啶并嘧啶类支架作为载体。呋喃并嘧啶MBXC-4409展示 多种有利特征，包括：(A)高度显著的剂量依赖性活性(EC50；10微米) 和转导增强(≥3倍)，不依赖于转基因(高斯白或 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1、2、8和9)；(B)CC50≥100uM；SI(CC50/EC50)&GT；20；(C)药物- ≥98%纯度(核磁共振)和正确质量(LC/MS)的相似结构；(D)在其他 与筛查无关，对其他病毒(ZIKV)的感染没有明显的抑制或增强作用 和VSV)；(E)添加时间研究表明早期作用；和(F)有利的体外ADME 性质(溶解度≥为100uM；在小鼠血清中稳定；血清蛋白结合≤为93%)。 这项第二阶段的工作将继续包括Michael Farzan博士在TSRI的实验室。这个 将优化呋喃并嘧啶类抑制剂系列的效力和类药物性能， 异吲哚和吡啶系列作为备份，以产生体内验证的临床前 作为增强rAAV疗法的辅助剂的候选药物。类比将是 通过测试漏斗进行评估，该测试漏斗旨在根据效力、选择性和特异性对它们进行优先排序 无毒的作用机制。将为活体研究制定优先顺序的类似物。这个 最有希望的先导化合物的最大耐受量将在小鼠身上确定 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。抑制剂将与rAAV-gLuc联合进行疗效测试 小鼠全身发光转导模型及基因组的qRT-PCR分析 在组织中复制。这一建议的主要里程碑是选择一种经体内验证的rAAV 转导增强剂导联系列。将在IIb阶段确定临床前候选对象，并 高级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.