Successful Implantation of Multi-Kilobase Gene Therapy Materials Without Genetic Damage Using Prime Assembly Technology
Background
The ability to replace or insert large-scale gene fragments within human cells has long been a challenge in genome editing. Conventional gene scissors technologies are effective for minor corrections of a few base pairs but show significantly reduced efficiency during the insertion of functional genes. The genotoxicity associated with inducing double-strand breaks (DSBs) to implant large genes is a major limiting factor. In particular, gene therapy delivery is nearly impossible in non-dividing differentiated cells. Prime Editing (PE) has emerged as a promising alternative, yet it is still limited to insertions of a few hundred base pairs. Therefore, developing a platform that can precisely insert large gene fragments without genome damage is an urgent task. A research team led by Professor Bae Sangsu at the School of Medicine, Seoul National University, has proposed a novel approach to overcome these limitations by applying the principles of prime editing.
Key Findings
The research team developed a correction technology using prime editors to generate one or two pairs of single-stranded 3'-flap overhangs on both the target genome and donor DNA, which they named Prime Assembly (PA). These 3'-flaps are designed to precisely anneal to each other, enabling precise joining within the cell in a manner similar to the Gibson Assembly technique used for assembling multiple DNA fragments in vitro. Using this technology, the team successfully edited megabase-scale genomic regions and precisely implanted kilobase-scale genes at desired locations. PA is characterized by its broad versatility in delivering plasmid DNA and linear double-stranded DNA as donor genes, ranging in size from 1.0 to 6.5 kilobases (kb). In experiments using human embryonic kidney 293T cells (HEK293T), the team achieved a maximum replacement efficiency of 57.8% using a 2.9-kb donor DNA fragment, with an integration accuracy exceeding 90%. Furthermore, in experiments involving primary human T cells, the team achieved an insertion efficiency of 28.1% for site-specific integration of a chimeric antigen receptor (CAR) gene. When PA carrying a green fluorescent protein (GFP) gene was delivered to mice via hydrodynamic injection, an average integration efficiency of 4.3% was observed in hepatocytes expressing GFP. These results demonstrate that large gene insertions are functional not only in in vitro cell studies but also in in vivo animal tissue, marking a significant advancement. Notably, PA generates minimal harmful mutagenic byproducts, distinguishing it from existing methods.
Significance and Outlook
Conventional gene insertion technologies have faced limitations in clinical applications due to risks such as cellular toxicity and off-target integration. PA, by joining genes without double-strand breaks, is expected to contribute to enhanced safety by eliminating mutations and abnormal recombination. This development provides a valuable tool for the development of therapies for rare genetic disorders requiring genome-wide correction. However, since the in vivo gene insertion efficiency in hepatocytes was only 4.3%, further research to improve efficiency is necessary to maximize therapeutic effects. Challenges remain in optimizing delivery methods such as lipid nanoparticles (LNPs) and adeno-associated viruses (AAVs) to maximize in vivo delivery efficiency.
Replacing large-scale fragments in human cells remains a substantial challenge. Here, we present a programmable gene replacement tool, named prime assembly (PA), which adapts prime editors to produce one or two pairs of 3'-flaps on both the genome and donor DNA. These 3'-flaps anneal to each other precisely, similar to Gibson assembly in DNA oligonucleotides, allowing megabase-scale genomic excision and/or kilobase-scale donor insertion at the gene of interest. PA accepts DNA plasmids and linear double-stranded DNA as donors, ranging from 1.0 to 6.5 kb in size. We demonstrate an efficiency of up to 57.8% in replacing endogenous sequences with a 2.9-kb donor DNA fragment in HEK293T cells, with an accuracy of >90% for integrated PA fragments. Furthermore, PA enables site-specific chimeric antigen receptor integration with up to 28.1% efficiency in primary human T cells. When PA containing a GFP donor is delivered to mice by hydrodynamic injection, an average integration efficiency of 4.3% is measured in GFP-positive hepatocytes.
PA technology has significant potential to simplify ex vivo cell therapy manufacturing processes in clinical settings. A representative application scenario is the production of next-generation immunotherapies such as chimeric antigen receptor T-cell (CAR-T) therapies. Previously, lentiviruses were used to introduce CAR genes into patient T cells, but this approach carried the risk of random genomic integration, potentially causing cancer. In contrast, PA enables precise insertion of CAR genes at specific genomic locations, providing a foundation for the mass production of safe and consistently effective therapies. The 28.1% T-cell correction efficiency observed in clinical research marks an important milestone toward the commercialization of non-viral CAR-T therapies. Additionally, from an industrial perspective, the ability to flexibly utilize large plasmids and linear DNA is expected to reduce production costs and simplify manufacturing processes.