Transgenesis Achieved Without Exogenous DNA: Harnessing Retroelement Proteins

Mar 18, 2024

In recent years, CRISPR-Cas9 gene editing technology has rapidly advanced, serving not only precise gene editing but also gene introduction in transgenic systems. However, the introduction of exogenous DNA, a necessity for these tools, poses risks such as immune responses and negative mutations. With the COVID-19 pandemic highlighting the potential of RNA-based therapies, scientists now seek an RNA tool for efficient transgenesis or gene editing with reduced risks.

On February 20, 2024, a research team from the University of California, Berkeley, reports on their development of a novel transgenic technology called PRINT, utilizing retroelement proteins for gene insertion.

Treasures within “Junk Genes”

The breakthrough isn't the first major publication from this research team in the field of reverse transcriptase proteins. Just three months ago, they reported their study on LINE-1 retrotransposons in Nature. LINE-1, when combined with a cleaving enzyme, can insert genes through its reverse transcription mechanism. However, due to potential disruptions at insertion sites and low editing efficiency, researchers deem LINE-1 unsuitable for gene therapy. Nevertheless, research into the LINE-1 mechanism has sparked new ideas for the team. If they can find a retrotransposon capable of selectively introducing genes into safe regions and enhance its editing efficiency, they could potentially unveil a novel transgenic tool.

Transposons, capable of retrotransposing RNA into DNA within the genome, facilitate jumping and replication, a phenomenon not uncommon in nature. About 40% of the human genome comprises these “selfish” sequences, yet most retrotransposons have lost their physiological function, rendering them “junk genes.” Amidst this genetic debris, researchers, through relentless pursuit, uncovered a retrotransposon named R2. Unlike other “junk gene,” the R2 family can integrate genes into regions encoding ribosomal RNA (rRNA), where hundreds of copies of rRNA genes typically reside due to the crucial role of ribosomes in cells. Inserting genes into this region merely inactivates a few copies, without significantly affecting ribosomal function. In essence, the rRNA region serves as a “safe harbor” for transgenic applications.

Although humans have R2 insertion sites, the R2 element was lost during human evolution. Researchers had to search among animals such as insects and crabs to find the most suitable R2 for transgenic operations. Their perseverance paid off as, after extensive screening, scientists discovered the coveted R2 element in the Zebra Finch and White-throated Sparrow.

Compared to the previously reported Bombyx mori (BoMo) R2, which can achieve target-primed reverse transcription (TPRT), researchers have found that TaGu R2 and ZoAl R2 discovered in birds are more 'specific.' BoMo R2 can almost execute TPRT in any RNA template, while TaGu R2 and ZoAl R2 only undergo TPRT in the presence of specific sequences. Further experiments have shown that the 3' end of the RNA template used by TaGu R2 and ZoAl R2 needs to be complementary to four nucleotides in rRNA (R4) to achieve TPRT, indicating a better specificity of TaGu R2 and ZoAl R2. Introducing adenosine (A22) at the 22nd nucleotide after R4 significantly enhances the efficiency of TPRT. Disrupting the primer region R4 sequence completely abolishes the TPRT function of TaGu R2 and ZoAl R2, highlighting the crucial role of R4 complementary to rRNA for TPRT by TaGu R2 and ZoAl R2. The presence of R4 confers high specificity to TaGu R2 and ZoAl R2.

Avian R2 Achieves Transgenesis

After confirming the in vitro functionality and specificity of TaGu R2 and ZoAl R2, researchers directly constructed an RNA template capable of introducing fluorescent proteins. This template was co-transfected with R2 protein mRNA. Results demonstrated remarkably high GFP expression in human cells, exceeding 10% under certain conditions. Adding A22 to the template's 3' UTR sequence significantly enhanced transgenic efficiency, but extending the complementary sequence by 20 nucleotides did not yield noticeable gains in efficiency.

In clinical applications, modifications of uridine are often required to reduce the immunogenicity of exogenous RNA. Researchers subsequently investigated whether these modifications would affect the system's efficiency. Results showed that using modified uridines still achieved transgenesis, with pseudouridine modifications providing the highest efficiency. Further experiments proved the system's capability to introduce disease-related genes such as TERT into human cells, successfully exhibiting telomerase activity. These findings demonstrate the potential of the dual RNA transgenic system based on TaGu R2 and ZoAl R2 to transfect RNA templates of up to 4.5kb, holding significant promise for gene therapy.

To a Higher Level

Despite the impressive performance of TaGu R2 and ZoAl R2, researchers have observed rapid declines in gene expression transferred by these proteins within cells. Taking GFP as an example, its expression gradually increased to a peak within one day of transfection, followed by a gradual decline. To enhance transgenic stability, researchers developed multiple variants of ZoAl R2, resulting in ZoAl-R1103A with reduced endonuclease activity, named ZoAl-ENT. Although ZoAl-ENT exhibited significantly lower transgenic efficiency compared to the wild-type, it demonstrated remarkable stability, maintaining approximately 6% GFP expression even after 20 days, whereas the wild-type ZoAl, while achieving higher GFP expression in the initial days, dropped below ZoAl-ENT levels after five days, rapidly declining to around 1%. ZoAl-ENT facilitates more stable gene expression within cells, with copy numbers of rRNA sequences ranging from 1 to 7 copies. These findings suggest that modifications to ZoAl R2 can lead to stable transgenesis.

At the culmination of the study, the research team delved into the specificity of TaGu R2 and ZoAl R2. Results revealed that the majority of cleavage sites aligned as expected, allowing for a 'seamless integration' of inserted genes, with only 1% deviating by a single base, occurring at the -1 position. Researchers also identified four potential 'operational modes' for this insertion. Concerning specificity, TaGu R2 exhibited approximately 1% off-target insertions, while ZoAl R2 showed only 0.1%. Regarding human applications, there is still room for further enhancement of the PRINT technology.

Although more research is needed to elucidate the specific mechanisms of R2 proteins, the emergence of PRINT technology has initially validated their significant potential for gene therapy, further enriching humanity's toolkit for transgenesis. It is believed that in the near future, RNA-based transgenesis technology will achieve even greater success.

Reference

[1] Thawani, A., Ariza, A.J.F., Nogales, E. et al. Template and target-site recognition by human LINE-1 in retrotransposition. Nature 626, 186–193 (2024). https://doi.org/10.1038/s41586-023-06933-5

[2] 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