673 Elexacaftor/tezacaftor/ivacaftor corrects function of H1085R-, N1303K-, and R334W-cystic fibrosis transmembrane conductance regulator and improves clinical status of patients

Background: SP-101 (AAV2.5T-SP183-hCFTRΔR) is a novel recombinant adeno-associated virus (AAV) gene therapy vector being investigated as an inhalation treatment, followed by inhaled doxorubicin, for people with cystic fibrosis (CF) in a mutation-agnostic manner.SP-101 is composed of a novel capsid optimized for efficient apical transduction of human airway epithelial (HAE) cultures and encodes a promoter/enhancer that drives expression of a human CF transmembrane conductance regulator (CFTR) minigene (hCFTRΔR).Doxorubicin is a small molecule able to increase expression of hCFTRΔR messenger ribonucleic acid (mRNA) when used in combination with SP-101.To understand the dose-response relationship between SP-101 and doxorubicin and the amount of mRNA expression required for functional correction, we investigated vector copy number (VCN), hCFTRΔR mRNA expression and vector tropism in relation to functional correction of human CF-HAE epithelia from donors with class I, II, or III mutations.Methods: SP-101 was applied to the apical surfaces of CF-HAE cells cultured at the air-liquid interface with or without doxorubicin added to the basal media.Forskolin-induced CFTR chloride conductance, measured via Ussing chamber assay, was compared with cellular VCN, measured via droplet digital polymerase chain reaction, and mRNA expression, measured by quantitative polymerase chain reaction, 7 days after transduction.Cellular integrity was measured by evaluating transepithelial electrical resistance (TEER) and lactate dehydrogenase (LDH) levels in culture media.Tropism to CF-HAE cells was determined by five-color wholemount immunostaining and confocal microscopy for mCherry-positive cells and various cell-type markers after transduction with an AAV2.5T-mCherry reporter vector.Results: In the presence of doxorubicin, SP-101 restored forskolin-induced CFTR-mediated chloride conductance in all mutation classes tested to levels comparable with those of non-CF controls, with a class I mutation donor showing the largest chloride response at the highest multiplicity of infection (MOI; 1e5 vector genomes [vg]/cell).Functional chloride correction increased with increasing MOI, correlating with increasing VCN and hCFTRΔR mRNA expression.Similarly, increasing doxorubicin concentrations increased chloride conductance and hCFTRΔR mRNA expression in CF-HAE cells without significantly affecting VCN.Low doxorubicin concentrations (as low as 0.5 μM) were able to restore CFTRmediated chloride conductance with as little MOI as 5e3 vg/cell, whereas SP-101 alone at the highest MOI (1e5 vg/cell) was not sufficient to restore chloride conductance.TEER and LDH levels were not significantly different from control epithelia, indicating no obvious toxicity as a result of treatment.Using an AAV2.5T-mCherry reporter vector, we were able to show that approximately 30% to 40% of CF-HAE cells, including ciliated, secretory, and basal-like cells, expressed the reporter gene under the same experimental conditions, providing insight into which and how many cells contribute to the correction of CF observed in vitro.Conclusions: These data demonstrate the importance of doxorubicin coadministration with SP-101 to achieve hCFTRΔR mRNA expression levels resulting in correction of CF-HAE, supporting co-development of inhaled SP-101 followed by inhaled doxorubicin for the treatment of CF.