(434) In vitro evaluation of TAVT-135, an artificial pan-genotypic chloride ion transporter

Background: Cystic fibrosis transmembrane conductance regulator (CFTR) modulators have revolutionized the treatment of cystic fibrosis (CF). However, a significant unmet need remains for patients with ineligible genotypes, inadequate responses, or intolerance. Alternative agents, including those capable of mediating chloride (Cl^–) transport, are therefore being investigated. TAVT-135 is a novel cell-penetrating peptide derivative that facilitates passive ion transport of Cl^–, which could benefit all patients with CF, independent of CFTR mutation. A series of in vitro and ex vivo studies were performed to characterize the electrophysiological and mucus-penetrating properties of TAVT-135.

Methods: Intracellular Cl^– levels were determined by measuring fluorescence following exposure of HEK293 cells to TAVT-135 (0, 1, 10, 100 µM). Ion transport in cultured primary airway cells with the W1282X/R1162X mutation was measured in a modified Ussing chamber system. TAVT-135 (0, 1, 10, 100 µM) was delivered to the apical side of the cultures, and the impact of acute or chronic (24-hour) exposure on short-circuit current (Isc) was measured with amiloride-induced suppression of sodium channels. Responses were studied in the presence of Cl^– transport agonists (forskolin, uridine triphosphate [UTP]) and an antagonist (CFTR inhibitor-172). Mucus-penetration times for the diffusion of fluorescently labeled TAVT-135 through 100 µm mucus layers (harvested from human bronchial epithelial cell cultures) were calculated. Mucus was prepared to concentrations to mimic healthy airways, mild and severe CF airway disease, with 2%, 4%, and 8% solids (w/w), respectively. The impact of TAVT-135 on the viability of HEK293 cells was assessed using a commercially available apoptosis/necrosis detection kit.

Results: In HEK293 cells, intracellular Cl^– levels were decreased by TAVT-135 in a dose-dependent manner due to the transport of Cl^– from the cytosol to the extracellular space. In W1282X/R1162X CFTR primary airway cultures, acute exposure to TAVT-135 (10 and 100 µM) induced small, albeit significant, increases in Isc [μA/cm²; mean (SD)] compared with control [10 µM: 0.16 (0.01); 100 µM: 2.20 (0.21); control: –0.012 (0.006); p< 0.001]. The effect on Isc was partially sustained over 10 minutes and was independent of CFTR. UTP-induced activity of Ca²⁺-activated Cl^– channels was increased 1.7- and 2-fold in the presence of TAVT-135 (10 and 100 µM), respectively. After chronic exposure to TAVT-135, the mean potential difference of cultures exposed to TAVT-135 (100 μM) was significantly decreased compared with control, suggesting changes in the permeability of cultures by ion conductance. TAVT-135 readily penetrated mucus layers, with penetration times of 81.7, 146.7, and 120.5 s through 2%, 4%, and 8% solid-containing mucus, respectively. In cell-viability assays, no in vitro toxicity was observed following exposure to TAVT-135 (1, 10, or 100 µM).

Conclusions: Initial investigations show promising signs of a CFTR-independent effect of this candidate on short-circuit current and Cl^– transport, and evidence of it penetrating CF-like mucus. Together, these data suggest TAVT-135 could potentially alleviate symptoms associated with highly viscous mucus by increasing electrolyte levels of the airway surface liquid layer and facilitating water transport out of the epithelial cells. Additional studies into this novel artificial chloride ion transporter are ongoing.

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