Development of a Targetable Transposase Platform for Precision Gene Integration in Human Cells

PROJECT SUMMARY Current genome editing technologies, such as CRISPR nucleases, are effective for gene knock-outs but face limitations in the precise and efficient insertion of large DNA fragments into specific genomic sites, particularly in non-dividing cells. These limitations arise from the reliance on double-strand breaks and error-prone host repair mechanisms, which often lead to unintended mutations and off-target effects. Additionally, the inefficiency of homology-directed repair further constrains the potential of these methods for gene therapies requiring stable and accurate gene insertion. Consequently, a more efficient, safe, and precise system is needed. This project seeks to address these challenges by developing a platform technology based on the piggyBac (pB) transposase, which facilitates precise DNA insertion with minimal off-target effects. The approach incorporates rational design and directed evolution to optimize the transposase, enabling the delivery of large transgenes to specific genomic sequences. Unlike traditional gene editing methods, this technology actively integrates large DNA fragments without relying on double-strand breaks or homology-directed repair, making it a versatile tool for both therapeutic and research applications. Preliminary data indicate that the pB transposase, when modified with custom DNA-binding domains, achieves targeted DNA integration. Key mutations in the transposase’s native DNA-binding domain have significantly reduced off-target insertions while preserving its ability to efficiently deliver transgenes to specific genomic sites. This proposal aims to further refine the system through phage- assisted continuous evolution (PACE) to enhance both the efficiency and specificity of the transposase, establishing a highly effective gene therapy platform. The project will optimize the pB transposase for site-specific integration by tethering it to custom DNA-binding proteins. Structural modeling will guide these modifications, reducing off-target effects and enhancing targeted insertion into the human genome. PACE will be applied to evolve pB transposase variants with improved efficiency and specificity, rapidly scanning large sequence spaces to identify variants that are suitable for therapeutic applications. The clinical potential of this technology will be demonstrated by using the evolved pB transposase to deliver a chimeric antigen receptor (CAR) gene to human T-cells, highlighting its ability to efficiently and precisely deliver therapeutic genes. This platform technology represents a significant advancement over existing genome editing methods by enabling the precise, efficient, and safe integration of large DNA fragments into non-dividing cells. It offers broad applications in the treatment of genetic diseases and biomedical research, where the ability to integrate large or multiple genes at specific genomic locations is essential. By addressing the limitations of current methods, this approach has the potential to revolutionize gene therapy, providing a robust and versatile tool for a wide range of genetic interventions.