Pharmacological and pre-clinical safety profile of rSIV.F/HN, a hybrid lentiviral vector for cystic fibrosis gene therapy

RATIONALE AND OBJECTIVE: Cystic fibrosis (CF) is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. CFTR modulators offer significant improvements, but approximately 10% of patients remain nonresponsive or are intolerant. This study provides an analysis of rSIV.F/HN, a lentiviral vector optimized for lung delivery, including CFTR protein expression, functional correction of CFTR defects and genomic integration site analysis in preparation for a first-in-human clinical trial.METHODS: Air-liquid interface cultures of primary human bronchial epithelial cells (HBEC) from CF patients (F508del/F508del), as well as a CFTR-deficient immortalized human lung epithelial cell line mimicking Class I (CFTR-null) homozygous mutations, were used to assess transduction efficiency. Quantification methods included a novel proximity ligation assay (PLA) for CFTR protein expression. For assessment of CFTR channel activity, Ussing chamber studies were conducted. The safety profile was assessed using integration site analysis and in vitro insertional mutagenesis studies.RESULTS: rSIV.F/HN expressed CFTR and restored CFTR-mediated chloride currents to physiological levels in primary F508del/F508del HBECs as well as in a Class I cells. In contrast, the latter could not be achieved by small-molecule CFTR modulators, underscoring the potential of gene therapy for this mutation class. Combination of rSIV.F/HN-CFTR with the potentiator ivacaftor showed a greater than additive effect. The genomic integration pattern showed no site predominance (frequency of occurrence ≤10%), and a low risk of insertional mutagenesis was observed in an in vitro immortalization assay.CONCLUSIONS: The results underscore rSIV.F/HN as a promising gene therapy vector for CF, providing a mutation-agnostic treatment option.</p

Structure-based and property-driven optimization of N-aryl imidazoles towards potent and selective oral RORγt inhibitors

RORγt is considered the master transcription factor for the development of Th17 cells that produce pro-inflammatory cytokines such as IL-17A. Overproportionate Th17 cell abundance is associated with the pathogenesis of many inflammatory conditions including psoriasis. In a high-throughput FRET screen, we identified compound 1 as hit with promising lipE. Using structure-based drug design based on a number of X-ray co-crystal structures, we morphed this hit class into potent imidazoles, exemplified by compound 3. To improve the poor ADME properties of neutral imidazoles, we extended our ligands with carboxylic acid substituents towards a polar, water rich area of the protein. This highly lipophilicity efficient modification ultimately led to the discovery of compound 14, a potent and selective inhibitor of RORγt with good ADME properties and excellent in vivo pharmacokinetics. This compound showed good efficacy in an in vivo delayed-type hypersensitivity pharmacology model in rats