CFTR Project

The long-term goal of the CFTR project is to design a high-activity mutant of CFTR for the purpose of gene therapy. Several gene therapies (Luxturna, Kymriah, Zolgensma etc) based on lentiviral and adeno-associated viral vectors have recently been approved by FDA. Lentivral vectors have been used on more than one thousand patients, demonstrating its safety. Cooney et al. (2016) tested a lentiviral vector for CFTR on cystic fibrosis pigs and demonstrated partial restoration of anion channel activity in the lungs although the level of DNA integration was too low to be quantified. In order to have a clinically significant impact, the anion channel activity needs to be restored to more than 30% of the WT level. Using a high activity CFTR mutant in the gene therapy should help us achieve this goal.

Recently, cryo-EM structures of the activated (6MSM) and dephosphorylated (5UAK) human CFTR as well as a structure with ivacaftor bound (6O2P) have been published. The extracellular opening of 6MSM is too small to conduct chloride ions although water molecules may be able to get through. The structure of the true open state is currently unknown, and we are building a molecular model for this state. It will be used to find a way to lock CFTR into the true open state. Experimentally this can be achieved by introduing two engineered cysteines that can be connected by a disulfide bond or a molecular linker.

We are currently working on two projects for CFTR: CFTR Bottleneck Project, GlyH-101 Project and the Open Project.

1. A summary of CFTR mutant progress can be found here: CFTR Project Mutant Summary

2. Background information for CFTR projects. Please help us write the manuscript by doing a literature review.

Box 16 ??

Summary: Experiments testing the significance of R104C and L102C mutants of CFTR are done using electrophysiology experiments, allowing us to test the conductance across a membrane. Comparing the conductance of these two mutants to the WT CFTR, we can determine how effective the R104C and L102C mutants. Priority experiments are Anion Substitution and NaSCN Block. GlyH101 experiments are on pause until Anion Substitution and NaSCN Block experiments are completed.

Secondary Analysis (tutorial below, video and written protocol) needs to be performed on Anion Substitution experiments. Check the last table on the doc below to see which experiments need to be analyzed. Talk to Emma with any questions.