The quest for effective gene therapy has taken a significant leap forward with the development of a new technique that leverages avian retrotransposons to safely insert genes into the human genome. This innovative approach, known as Precise RNA-mediated INsertion of Transgenes (PRINT), was developed at the University of California, Berkeley, under the guidance of Professor Kathleen Collins. Detailed in the journal Nature Biotechnology, PRINT represents a complementary strategy to the CRISPR-Cas9 system, promising a new horizon in treating hereditary diseases.
The Mechanics of PRINT
At the heart of PRINT is the use of a retrotransposon-derived protein, specifically the R2 protein from birds, which exhibits a remarkable capacity to insert entire genes into a genome without disrupting its essential functions. This protein comprises multiple active components, including a nickase and reverse transcriptase, facilitating the integration of a transgene cassette into the genome. This cassette not only contains the gene of interest but also the necessary elements for its expression.
A notable feature of employing the R2 protein is its targeted insertion into the genome's ribosomal RNA (rRNA) encoding regions. These areas house hundreds of copies of rRNA genes, making them an ideal "safe harbor" for gene insertion. The redundancy of these genes ensures that any disruption has minimal impact on the cell's overall function, addressing a significant concern in current gene therapy methods that utilize viral vectors for gene insertion, which can lead to unintended gene disruptions or oncogenesis.
Insights from the Lab
The genesis of PRINT can be traced back to the exploration of various retrotransposons and their potential for gene therapy. While the LINE-1 retrotransposon has been a focus for many, its engineering challenges led Collins and her team to seek alternatives. The discovery that avian genomes harbored a highly efficient version of the R2 retrotransposon, particularly from species like the zebra finch and the white-throated sparrow, marked a turning point.
In laboratory settings, the team demonstrated the efficacy of this system by co-transfecting cultured human cells with mRNA encoding the R2 protein and a template RNA for the desired transgene. The successful expression of fluorescent proteins in a significant portion of the cells confirmed the potential of this approach. Further analysis revealed that the transgenes were indeed integrating into the desired rDNA regions, with minimal impact on the cell's ribosomal functions.
The Nucleolus: A Prime Target for Gene Therapy
The strategic choice of the rDNA region for gene insertion is not merely due to its safety profile. This region's unique role in the cell, particularly in protein synthesis and DNA repair, offers additional advantages. The rDNA genes are located in the nucleolus, a cellular structure that is not only a hub for ribosome production but also a site of meticulous DNA repair mechanisms. This ensures that any inserted genes are maintained with high fidelity, further reducing the risk of oncogenic events.
Moving Forward
Despite the promising results, Collins acknowledges that there are still many unknowns regarding the R2 mechanism and the dynamics of rDNA transcription. The tolerance level of cells to rDNA gene disruption and the potential side effects in cells with inactivated rDNA genes are among the questions that need addressing. Ongoing research aims to refine the components of the PRINT system to enhance its efficiency and applicability to various cell types, including primary human cells.
In conclusion, the development of PRINT, leveraging the unique capabilities of avian retrotransposons, opens new avenues for gene therapy. By offering a safe and efficient method for gene insertion, this technique stands to complement existing gene-editing tools, broadening the scope of treatments for a range of hereditary diseases. As Collins aptly puts it, "it works," laying the foundation for further exploration and potential clinical applications in the field of gene therapy.
Original publication:
Zhang, X., Van Treeck, B., Horton, C.A. et al. Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02137-y