Retrotransposons found in the genomes of the white-throated sparrow and the zebra finch are shown to safely shepherd transgenes into the human genome, providing a gene therapy approach complementary to CRISPR-Cas9 gene editing. Credit: Briana Van Treeck, UC Berkeley
Key points:
- Researchers developed a new technique called PRINT that employs a retrotransposon from birds to insert genes into the genome for more promising gene therapy.
- The technique inserts the transgene into a “safe harbor” to avoid random insertion that can disable working genes, mess with regulation or function, or lead to cancer.
- Using cell culture experiments, the team confirmed PRINT’s feasibility, but more research into the biology of rDNA is needed before translating PRINT to use in humans.
Gene editing tools can knock out genes to cure hereditary diseases, but it is not yet possible to insert whole genes into the human genome to substitute for defective or deleterious genes. Now, a new technique, outlined in Nature Biotechnology, employs a retrotransposon from birds to insert genes into the genome for more promising gene therapy.
The technique, called Precise RNA-mediated Insertion of Transgenes, or PRINT, uses retrotransposons to insert entire genes into the genome. One piece of delivered RNA encodes the retroelement protein R2, while the other acts a template for the transgene DNA. Importantly, R2 inserts the transgene into a “safe harbor”—an area of the genome that contains hundreds of redundant gene copies—to avoid random insertion that can disable working genes, mess with regulation or function, or lead to cancer.
“We’re taking a complementary approach, which is to put into the genome an autonomously expressed gene that makes an active protein,” explained senior author Kathleen Collins, professor at University of California, Berkeley. “It’s transgene supplementation instead of mutation reversal.”
The research team screened R2 from many animal genomes to find a version that was targeted to ribosomal RNA (rDNA) encoding regions in the human genome. The ideal candidate came from birds, including the zebra finch and the white throated sparrow. The team then synthesized mRNA-encoding R2 and a template RNA that would generate a transgene with a fluorescent protein and cotransfected them into cultured human cells. About half the cells lit up under laser light, indicating that the R2 system successfully inserted a working fluorescent protein into the genome. Next, researchers demonstrated that the transgene did insert into the redundant genome regions and failed to disrupt protein-manufacturing activity of the genes.
For hereditary diseases, gene supplementation with PRINT could deliver the correct gene to every person with the disease no matter the mutation, but many questions remain about how to best leverage R2 and rDNA transcription. The team plans to determine how many rDNA genes can be disrupted before interfering with cell function and to identify which cells are susceptible to PRINT side effects. They also hope to develop their technique as a complement to CRISPR gene editing.
“The bottom line is that it works,” said Collins. “It’s just that we have to understand a little bit more about the biology of our rDNA in order to really take advantage of it.”