Protein trans-splicing: optimization of intein-mediated GFP assembly as a model for the development of gene therapy

• DOI: 10.3389/fbioe.2024.1488912
• Publication Date: 2024-11-20T04:48:31Z
• Authors: Andrew Brovin; Ekaterina Minskaia; Matvei Sabantsev; Sergey Chuvpilo; Alexander Karabelsky
• Keywords: Intein; Trans-splicing

Adeno-associated virus (AAV)-based gene therapy has become one of the key directions modern translational medicine geared towards treatment hereditary disorders by means replacement. At moment, about 5,000 different syndromes are associated with mutations in large genes, which presents a great problem due to AAV packaging capacity 5 kilobases. The main strategies for overcoming this obstacle were creation truncated versions, overloading viral vector, and separate delivery partial genetic material restore whole at level DNA, RNA, or protein. present, genome editing via prime editors, most effectively delivered AAV, relies on intein pair used protein complex. amazing integration speed intein-based trans splicing technology makes it versatile tool variety applications, albeit not always successful first attempt. This study discusses points working Ssp, Npu, Ava inteins DnaE group, known as effective assembly proteins. Using green fluorescent (GFP) model, we demonstrate that requires only cysteine position C+1 but also certain aminoacid residues either side its immediate environment. Furthermore, conformation extein-intein composition, difficult predict computer modeling, an additional effect, demonstrated experimental tests three split sites optimal amino acid composition. NpuDnaE variant highest kinetics interaction between N C parts group inteins. Optimization conditions using led GFP 80% transfected HEK293 cells 55% AAV5-transduced cells, flow cytometry. efficiency post-plasmid DNA transfection transduction cell line was 15% higher than ARPE19 line. We hope obtained data will facilitate development therapies caused genes.