Assessment of Lentiviral Vector Mediated CFTR Correction in Mice Using an Improved Rapid in vivo Nasal Potential Difference Measurement Protocol

P. Cmielewski,J. Delhove,D. Parsons,M. Donnelley

doi:10.3389/fphar.2021.714452

Abstract

Cystic Fibrosis (CF) is caused by a defect in the CF transmembrane conductance regulator (CFTR) gene responsible for epithelial ion transport. Nasal potential difference (PD) measurement is a well established diagnostic technique for assessing the efficacy of therapies in CF patients and animal models. The aim was to establish a rapid nasal PD protocol in mice and quantify the efficacy of lentiviral (LV) vector-based CFTR gene therapy. Anaesthetised wild-type (WT) and CF mice were non-surgically intubated and nasal PD measurements were made using a range of buffer flow rates. Addition of the cAMP agonist, isoproterenol, to the buffer sequence was then examined. The optimised rapid PD technique was then used to assess CFTR function produced by second and third generation LV-CFTR vectors. V5 epitope tagged-CFTR in nasal tissue was identified by immunohistochemistry. When intubated, mice tolerated higher flow rates. Isoproterenol could discriminate between WT and CF mice. Improved chloride transport was observed for the second and third generation LV-CFTR vectors, with up to 60% correction of the cAMP-driven chloride response towards WT. V5-CFTR was located in ciliated epithelial cells. The rapid PD technique enables improved functional assessment of the bioelectrical ion transport defect for both current and potential CF therapies.

Highlights

Cystic fibrosis (CF) is one of the most common autosomal recessive genetic disorders in the Caucasian population, with the majority of the mortality and morbidity due to lung disease
For the higher flow rates there was no significant difference in potential difference (PD) measurements (Figure 1) for Basal, low chloride (LC) Krebs-Ringer Buffers (KRB), or the change in nasal PD (ΔPD) in normal mice under LC conditions, when compared to our standard infusion rate of 1 μl/min (n.s., RM analysis of variance (ANOVA), n 4)
The optimal flow rate for rapid and consistently stabilised PD recordings was the 20 μl/min infusion rate, which resulted in a typical mean reduction in PD assessment time for each solution to reach a stable plateau averaging from 16.4 min/solution, to 6.5 min/solution, i.e., 2.5 times faster

Summary

Introduction

Cystic fibrosis (CF) is one of the most common autosomal recessive genetic disorders in the Caucasian population, with the majority of the mortality and morbidity due to lung disease. The CFTR protein is primarily located on the apical surface of epithelial cell membranes, where the chloride ion channel transports salt and fluid across the epithelium to maintain surface hydration (i.e., transepithelial transport) (Riordan et al, 1989; Welsh et al, 1992). CFTR electrolyte transport can be measured electrically as the transepithelial potential difference (PD). This technique involves using a sensitive high-impedance voltmeter to measure the voltage between a fluid-filled sensing electrode in the nose and a reference electrode placed elsewhere into the body (Knowles et al, 1981). The use of a fluid-filled electrode enables solutions that alter ion channel conductances to be introduced into the nasal cavity, and the resulting electrical responses related to specific ion channels can be assessed

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Results

Conclusion