(621) Administration of SP-101 and Doxorubicin Results in Robust and Durable hCFTRΔR Transgene Expression in the Airways of Ferrets and Corrects Human CF Airway Epithelia In Vitro

Background: SP-101 (AAV2.5T-SP183-hCFTRΔR) is a recombinant adeno-associated virus gene therapy vector composed of a capsid optimized for efficient apical transduction of human airway epithelial (HAE) cultures and regulatory elements that drive expression of a human CFTR minigene (hCFTRΔR). SP-101 is intended as an inhalation treatment for people with cystic fibrosis (CF) followed by inhaled doxorubicin, a small molecule able to enhance hCFTRΔR mRNA expression when used in combination with SP-101. In order to understand the importance of doxorubicin, the dose relationship between SP-101 and doxorubicin for hCFTRΔR mRNA expression in the airways of ferrets and the functional correction of human CF airway epithelia in vitro were investigated.

Methods: SP-101 was applied to the apical surface of primary human CF-HAE grown at the air-liquid interface. Doxorubicin was applied to the basolateral side. Forskolin-induced CFTR chloride conductance was measured via Ussing chamber assay 7 days post SP-101 and doxorubicin addition. SP-101 followed by doxorubicin was delivered as a fine particle fraction aerosol to the airways of adult ferrets using a nebulizer attached to a plenum exposure system. Expression of hCFTRΔR mRNA and endogenous ferret CFTR mRNA were determined by RT-qPCR optimized for removal of viral genome DNA. Ferrets were chosen because SP-101 has demonstrated tropism to the airways of ferrets and a CF ferret model exists which recapitulates many of the pathophysiological features of CF.

Results: Co-administration of SP-101 with doxorubicin restored forskolin-induced CFTR-mediated chloride conductance in CF-HAE cultures with Class I, II and III mutations. Chloride conductance increased with increasing SP-101 and doxorubicin doses, reaching similar levels as non-CF or small molecule modulator controls. When compared to concomitant doxorubicin administration, SP-101 alone was not sufficient to correct CF-HAE. Similar dose-response relationships were observed in the airways of wild-type ferrets. Inhaled administration of increasing doses of SP-101 and doxorubicin resulted in dose-dependent increases of hCFTRΔR mRNA expression. The highest levels were observed in the lungs and bronchi, followed by trachea and nose. Expression levels of hCFTRΔR mRNA were comparable to those of endogenous ferret CFTR at 14 days post-administration. hCFTRΔR mRNA expression started as early as 48h (earliest time point investigated) and persisted up to 3 months (longest time-point investigated). Comparable hCFTRΔR mRNA expression was also evident in the airways of CF ferrets, indicating successful transduction despite pre-existing mucus accumulation.

Conclusions: These data provide a strong rational for the co-development of inhaled doxorubicin to fully realize the potential of SP-101 gene therapy for individuals with CF who do not benefit from treatment with small molecule modulators.

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