Optimizing The Targeting Of The Piggybac And T2022 Transposon System

Abstract

Gene therapy, which involves correcting pre-existing genetic interruptions, could provide a long-term solution for those suffering from genetic diseases such as cystic fibrosis (CF), a monogenic, autosomal recessive disease. Typical methods, however, for introducing DNA into cells have limitations such as random insertion, limited cargo capacity, and immune response. Thus, this thesis aims to optimize DNA binding domain (DBD)-directed transposition to develop a novel approach that would result in a reliable and effective non-viral method of transporting a gene of interest to a particular location in the genome in non-dividing cells. We hypothesize that the piggyBac (pB) and t2022 transposon systems can be optimized to target and integrate at a specific TTAA site through (a) introducing mutations in the pB and the t2022 transposases to reduce integration at off-target TTAA sites and (b) pairing the pB and the t2022 transposon systems to programmable targetable nucleases that contain a DNA binding domain capable of binding to a DNA sequence near a TTAA site of interest. In Chapter 2, we generate mutations in the dimerization interface and the DNA binding domain of the pB and the t2022 transposases to reduce their natural abilities to bind and integrate at various TTAA sites in the genome. In Chapter 3, we fuse the modified pB and the t2022 transposases to a CRISPR-associated protein 9 (Cas9) and synthesize user-defined sgRNAs. that bind to our region of interest. We also fuse the modified pB and t2022 transposases to custom-built transcription activator-like effectors (TALEs) that can direct integration to specific TTAA sites in the genome. The sgRNAs and TALEs are designed to target a specific TTAA site in the cystic fibrosis transmembrane conductance regulator (CFTR) gene as well as a specific TTAA site in the genomic safe harbors: CCR5, hROSA26, and AAVS1. Overall, the optimization of the pB and t2022 transposon systems will lead to a safer and more efficient approach to delivering therapeutic genes into the genome that have the potential to reverse various genetic diseases.