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Materials & Methods MQAE (N-(6-methoxyquinolyl) acetoethyl ester) was used as a halide-sensitive fluorescent indicator. Endogenous CFTR expressing CFPAC-1 cells were transfected with QR-010 (100nM) or a non-targeting control oligonucleotide using lipofectamine. Two days after transfection, cultures were loaded with MQAE and subsequently incubated in low chloride buffer containing vehicle control, forskolin (10µM), potentiator (10µM) and/or CFTR inh-172 (25µM). Secretion of chloride increases MQAE fluorescence and the rate of fluorescence increase was used as a measure of CFTR activity. For Ussing chambers, primary human bronchial epithelial cells (hBEs) obtained from the lungs of homozygous ΔF508 patients and grown on air-liquid-interface were used. Cells were treated with QR-010 by culturing the cells in medium containing QR-010 (100nM) . Qualitative assessment of hBE uptake of QR-010 was monitored by fluorescent Cy5-labeled QR-010. CFTR function was measured in the Ussing chamber. The assay was performed to monitor the change in short-circuit current (ΔI sc ) in response to the sequential addition of amiloride (30µM), potentiator (5µM), isoproterenol (100µM) and CFTR inh-172 (30µM). The significance of differences between means was calculated by one-way analysis of variance. Differences were considered significant if p<0.05. Results are expressed as mean ± standard error of the mean (SEM). Jim Swildens, Charlotte van Putten, Patricia Biasutto, Marko Potman, Tita Ritsema ProQR Therapeutics N.V., Darwinweg 24, 2333 CR Leiden, The Netherlands. Introduction • Cystic fibrosis (CF) is caused by mutations in the gene encoding the chloride channel CFTR. • The most common mutation is a three nucleotide deletion resulting in loss of phenylalanine at amino acid position 508 (ΔF508). • To correct CFTR function in patients with this mutation, we are developing QR-010, an RNA oligonucleotide, designed to repair CFTR at the RNA level. • QR-010 is an investigational single-stranded, chemically modified RNA oligonucleotide designed to repair mRNA in CF patients with the ∆F508 mutation and result in translation of wild-type CFTR. Discussion Chloride efflux measurements in CFPAC-1 cells show increased CFTR activity, but the low expression level of CFTR makes this assay cumbersome. The corrective effect of QR-010 as measured in the Ussing chamber appears to be dependent on the amount of QR-010 present in the cell. Delivery of QR-010 to hBEs in vitro was only achieved after prolonged incubation with QR-010 on the basolateral side, indicating that the uptake mechanism of hBE cultures in vitro differs from oligo uptake in vivo. Thank you We would like to thank Prof. Hugo de Jonge, the Cystic Fibrosis Foundation and the Netherlands Enterprise Agency (RVO) for InnovatieKrediet IK12062. Results QR-010 improves CFTR-mediated chloride efflux from CFPAC-1 cells Fluorescence de-quenching as a result of chloride secretion was measured over 15 minutes and the rate of increase over the first 2 minutes was used as a measure of CFTR activity. Comparison of CFTR activity between treatment groups. Chloride efflux was significantly increased in QR-010-treated cells compared to untreated cells or cells transfected with a control oligonucleotide (*p<0.05). Specificity of CFTR activity was assessed using inhibitor inh-172. Chloride efflux was significantly increased in QR-010-treated cultures compared to cultures treated with a control oligonucleotide (****p<0.0001), no significant (ns) difference between the two groups was observed in the presence of CFTR inhibitor. Uptake of QR-010 by hBE cells improves over time and is related to improved short-circuit current measurements Cell borders (F-actin) are shown as green and nuclei are shown as blue. Treatment of hBEs with QR-010 from 2 up to 28 days revealed that oligo uptake (in red) increased over time, with an apparent deterioration of cell viability at 4 weeks as judged from the actin staining. Initial increase in uptake coincided with an increase in I sc . I sc reached a maximum after 2 weeks of QR-010 incubation, with an apparent decline at 4 weeks. QR-010 improves CFTR activity in primary bronchial epithelial cells I sc traces measured in the Ussing chamber on filters of differentiated hBE cultures which were either untreated, or treated with QR-010 or a control oligo. CFTR activity improved significantly after treatment with QR-010 compared to non-treated cultures (p<0.001) and to cultures treated with a negative control oligo ( *p<0.05). Treatment with the negative control oligo did not result in a significantly different I sc compared to non-treated cells. Non-treated Stimulated QR-010 Neg. control 0 5000 10000 15000 ∆ Fluorescence/ ∆ Time (AU/min) * QR-010 QR-010 + CFTR inh-172 Neg.control Neg.control + CFTR inh-172 0 20000 40000 60000 ∆ Fluorescence/ ∆ Time (AU/min) ns **** 0 5 10 15 0 10000 20000 30000 40000 50000 Time (min) Fluorescence (AU) QR-010 Neg. control stimulated Non-treated Non-treated QR-010 Neg. control 0.0 0.5 1.0 1.5 2.0 * ∆I SC inhibited by Inh-172 (fold change over untreated) 0 200 400 600 800 0 1 2 5 10 15 20 25 Time (s) I sc ( μ A/cm 2 ) QR-010 Neg. control ENaC blocker Potentiator Activator Inhibitor Non-treated Objectives The aim of this study was to test the effect of QR‑010 on CFTR functional activity in vitro with two methods: • A chloride efflux assay using a halide‑ sensitive fluorescent indicator (MQAE). • An Ussing chamber short‑circuit current (I sc ) assay. Conclusion QR‑010 restores CFTR mediated chloride efflux in cultures of CFPAC‑1 cells, and CFTR mediated chloride permeation in an Ussing chamber model with hBE cultures derived from ∆F508 homozygote CF patients. Building on passion, heading for success F-actin + nuclei QR-010 Overlay no oligo 2 days 5 days 1 week 2 weeks 3 weeks 4 weeks Non-treated 2 days 5 days 1 week 2 weeks 3 weeks 4 weeks 0.0 0.5 1.0 1.5 2.0 2.5 ∆I SC i nhibited by Inh-172 (fold change over untreated) QR-010, an RNA Therapy, Restores CFTR Function in ΔF508 Cell Cultures See all our QR-010 posters on this conference and get yourself a digital copy QR-010, an RNA Therapy, Restores CFTR Function in ΔF508-CFTR Mice QR-010, an RNA Therapy, is Taken up by Airway Epithelial Cells Showing Systemic Exposure After Oro-Tracheal Dosing
