Gene-sized DNA insertion at genomic safe harbors in human cells using a site-directed transposase

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Gene therapy faces challenges with precise integration of large DNA sequences into the human genome.
• Current methods often rely on double-strand DNA breaks, increasing risks of unintended genomic alterations.
• The diversity of mutations in monogenic diseases necessitates universal full-gene replacement strategies.

Purpose of the Study:

• To develop a novel system for precise and efficient integration of gene-sized DNA into the human genome.
• To engineer a site-specific DNA insertion tool that avoids double-strand DNA breaks.
• To create a versatile platform for full-gene replacement strategies in gene therapy.

Main Methods:

• Development of INsertion by Targeted Anchoring and Conditional Transposition (INTACT), a mammalian transposase system.
• Engineering mutations in the transposase DNA-binding domain to reduce off-target activity.
• Restoring site specificity by linking programmable DNA-binding proteins to the modified transposase.
• Systematic optimization of INTACT components and linkage strategies.

Main Results:

• Achieved targeted DNA insertion at specific genomic safe harbor loci using INTACT.
• Optimized INTACT demonstrated an average of 1.2 targeted insertions per cell across multiple loci.
• Mutations in the transposase DNA-binding domain significantly reduced off-target integration events.
• Demonstrated precise insertion of >4 kb DNA without inducing double-strand DNA breaks.

Conclusions:

• INTACT enables precise and efficient genomic insertion of large DNA payloads without double-strand DNA breaks.
• The engineered site-directed transposase offers a powerful new tool for genome engineering and gene therapy.
• This technology addresses limitations of current gene-editing approaches, paving the way for safer and more effective genetic interventions.