(672) SP-101 activity increases in a dose dependent manner to both vector and doxorubicin dose

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 cultures (HAE) and encodes a promoter/enhancer that drives expression of a human CFTR minigene (hCFTRΔR). Doxorubicin is a small molecule able to increase the expression of hCFTRΔR mRNA when used in combination with SP-101. To understand the dose-response relationship between SP-101 and doxorubicin, as well as 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 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 to cellular vector copy number (VCN), measured via ddPCR, and mRNA expression, measured by RT-qPCR, at day 7 post transduction. Cellular integrity was measured by evaluating transepithelial electrical resistance (TEER) and lactate dehydrogenase (LDH) levels in culture media. Tropism to CF-HAE 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 to non-CF controls, with a Class I mutation donor showing the largest chloride response at the highest multiplicity of infection (MOI; 1e5 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 without significantly affecting VCN. Low concentrations of doxorubicin (as low as 0.5 uM) were able to restore CFTR-mediated 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 30-40% 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 co-administration with SP-101 to achieve hCFTRΔR mRNA expression levels resulting in the correction of CF-HAE, further supporting the co-development of inhaled SP-101 followed by inhaled doxorubicin for the treatment of CF.

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