Intein technology to deliver CFTR to CF airway using AAV

The aim of our proposal is to deliver intact, functional CFTR using AAV into CF patient cells to relieve the symptoms of CF regardless of the CFTR mutations that they carry. Previous approaches for delivering CFTR using AAV have been conducted using truncated CFTR constructs due to the cargo size limitation of AAV. This approach was not successful, potentially due to dysfunction of the truncated protein. To circumvent this limitation of AAV and deliver a full-length intact CFTR into cells, we have employed intein technology. The CFTR gene is split into two constructs and attached to coding segments for intein, and delivered using two AAVs (a dual AAV approach). This delivered gene is then translated in the cell, and the resulting protein halves are spliced together by inteins to create a functional, full-length CFTR protein. We have tested this dual AAV system using primary airway epithelial cells from six CF patients who do not respond to CFTR modulators and have seen significantly improved CFTR function in vitro. In this proposal, we will optimize delivery of this split CFTR, dual AAV system in airway epithelial cultures from people with CF that do not respond to modulators. This system will also be applied to a CF-relevant mouse model to study efficiency and durability of gene transduction. Since AAV is clinically approved for gene therapy to deliver a therapeutic gene (e.g., Hemophilia gene therapy) and a dual AAV approach was also successfully applied in a recent clinical trial for deaf patients lacking functional otoferlin (OTOF), we expect in the future that our dual AAV with intein may be used to restore CFTR activity in patients with CF, which is especially important for those CF patients who do not respond to CFTR modulators.