Guiding genes to a “safe harbor” with retrotransposons
The ability to deliver whole genes without causing deleterious effects has been out of reach for regenerative medicine, but recent developments using retrotransposons could change that.
A team of researchers from the University of California, Berkeley (CA, USA) has recently pioneered a retrotransposon-based gene delivery method, using a protein screened from avian genomes, which could be used to successfully deliver whole genes into the genome of cells, a shortfall of current gene therapy techniques like CRISPR-Cas9 [1].
Concerning methods for whole gene integration into a genome, current techniques, including CRISPR-Cas and viral vector systems are either too inefficient or carry too high a risk of mutagenesis.
To find a more suitable whole-gene insertion method, the researchers turned to retrotransposons, DNA sequences that can excise themselves from and reintegrate themselves into a genome [2]. They operate through a copy-and-paste mechanism, whereby they produce an intermediate RNA that is then reverse-transcribed into copy DNA and then integrated into the genome at a different location.
When the researchers were examining how to design a transposon-based gene insertion method, the literature suggested that the well-understood retrotransposon found in humans, LINE-1, would be too difficult to engineer into an efficient and safe gene insertion tool. Instead, prior research pointed to the potential of using a retrotransposon that inserts genes into the repetitive, ribosomal-RNA encoding sections of the genome (rDNA).
The rDNA regions, which form the nucleolus, are considered a “safe harbor” as gene insertions into these highly repetitive and multicopy rDNA regions do not compromise cellular function. It also overcomes the problem of gene insertions into other regions of the genome, which can interrupt and impair normal function of other genes.
As a result, the team turned to the R2 protein – a retrotransposon protein that inserts genetic material into the rDNA region. Despite retaining the target binding site of the protein, mammals have evolutionarily lost R2. The researchers, therefore, screened a multitude of animal genomes to find an R2 protein with the greatest specificity to rDNA regions in humans, which was found in avian genomes (white-throated sparrows and zebra finches).
Next, they synthesized R2 protein-encoding mRNA and co-transfected this with a fluorescent reporter gene into human cells (human hTERT RPE-1 cells) and observed, through flow cytometry, that functional copies of the reporter protein were being produced, indicating that the 2-part RNA system worked. The insertion of the target gene into the rDNA region was also confirmed with polymerase chain reaction methods.
The finalized technique is called PRINT, which stands for Precise RNA-mediated INsertion of Transgenes. It consists of a two-part RNA system, whereby one RNA encodes the R2 protein and the other encodes a transgene along with its regulatory machinery. The R2 protein facilitates this through recognition of the target site, nicking the DNA strand before reverse transcribing the RNA of the transgene cassette for insertion.
While the authors believe that using PRINT in a gene therapy approach could complement existing methods, further research, such as understanding how many rDNA genes can be interrupted before the cell becomes dysfunctional or how the length of the transgene impacts the insertion efficiency, is required before PRINT is utilized as a fully-fledged gene therapy method. In the meantime, however, this research represents a large step towards expanding the gene therapy repertoire.
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