[Post-translational ligation and function of dual-vector transferred split CFTR gene]

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Context:

• Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
• Gene therapy for CF faces limitations due to the cargo capacity of adeno-associated virus (AAV) vectors.
• Delivering the full-length CFTR gene via AAV vectors is challenging.

Purpose:

• To investigate the feasibility of using a dual-vector system with intein-mediated protein trans-splicing to deliver split CFTR genes.
• To assess the post-translational ligation and chloride channel function of CFTR half-proteins.
• To overcome the AAV vector size limitations for CFTR gene delivery.

Summary:

• Human CFTR cDNA was split before Ser712 and fused with Ssp DnaB intein coding sequences, inserted into eukaryotic expression vectors.
• These vectors were co-transfected into baby hamster kidney (BHK) cells, and protein ligation and chloride channel activity were analyzed.
• Western blotting confirmed CFTR protein ligation, while patch-clamp recordings demonstrated functional chloride currents.

Impact:

• The study demonstrates that protein splicing is a viable strategy for delivering split CFTR genes using a dual-vector system.
• This approach shows promise for overcoming AAV vector size constraints in gene therapy for cystic fibrosis.
• Successful functional recovery of the chloride channel supports further research into dual AAV vector systems for CF gene therapy.