Screening for rAAV transduction enhancers

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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.