The recent approval of a CRISPR-Cas9 therapy to treat sickle cell anemia shows that this gene editing tool can do an excellent job of deleting genes with the aim of curing rare or inherited diseases.
However, it is not yet possible to insert entire genes into the human genome to replace defective or harmful genes.
A new technique that uses an avian retrotransposon to insert genes into the genome holds promise for gene therapy because it inserts genes into a “safe haven” in the human genome where the insertion does not alter significant genes or cause cancer.
Retrotransposons, or retroelements, are DNA fragments that, when transcribed into RNA, encode enzymes that copy the RNA in the genome back into the DNA, a cycle that crowds the genome with retrotransposon DNA. Around 40% of the human genome is made up of this new “selfish” DNA, although most of the genes are unused: the so-called junk DNA.
The new method, called PRINT, exploits the ability of some retrotransposons to efficiently insert entire genes into the genome without affecting other functions of the genome.
The new method, called PRINT (Precise RNA-mediated INsertion of Transgenes), exploits the ability of some retrotransposons to efficiently insert entire genes into the genome without affecting other functions of the genome.
The technique would complement CRISPR-Cas technology’s known ability to inactivate genes, make point mutations and insert short segments of DNA.
The description of PRINT, developed in the laboratory of Kathleen Collins, professor of molecular and cellular biology at the University of California, Berkeley, was published this week in the journal Nature Biotechnology.
PRINT involves introducing new DNA into a cell using similar methods to introducing CRISPR Cas9 into cells to edit the genome. In this new method, an RNA fragment encodes a common retroelement protein called the R2 protein, which has several active parts, including a nicase (an enzyme that binds to and cleaves double-stranded DNA) and a reverse transcriptase, the enzyme this creates the DNA copy of the RNA. The other RNA is the template for the transgenic DNA to be inserted, as well as elements to control gene expression: a complete autonomous transgenic assembly that inserts the R2 protein into the genome, Collins explains.
A key advantage of using the R2 protein is that it inserts the transgene into a region of the genome that contains hundreds of identical copies of the same gene, each of which encodes ribosomal RNA, the RNA machine, messenger RNA (mRNA). translated. . ) in proteins. With so many redundant copies, the loss of genes will not go unnoticed if the insertion destroys one or more ribosomal RNA genes.
Placing the transgene in a safe harbor avoids a major problem that arises when inserting transgenes via a human viral vector, which is the common method today: the gene is often randomly inserted into the genome, thereby inactivating functioning genes or reducing their regulation or function is changed. which can cause cancer.
Placing the transgene in a safe harbor avoids a major problem that occurs when introducing transgenes via a human viral vector, which is the common method today.
“A CRISPR-Cas9-based method can fix a mutated nucleotide or insert a small DNA fragment (sequence fixation). You can also switch off the function of a gene through targeted mutagenesis,” explains Collins. “We don’t eliminate the function of a gene. “We are not correcting an endogenous genetic mutation.”
Complementary approach
In this work, “we are pursuing a complementary approach, which consists of introducing an autonomously expressed gene into the genome that produces an active protein, i.e. adding a functional gene again to circumvent the deficiency.” It is more of a transgenic one addition than a reversal of the mutation. “This is great for solving loss-of-function diseases that arise from a large number of individual mutations in the same gene,” he emphasizes.
Many inherited diseases, such as cystic fibrosis and hemophilia, are caused by several different mutations in the same gene, all of which affect its function. Any CRISPR Cas9-based gene editing therapy would need to be tailored to each person’s specific mutation.
Instead, gene supplementation using PRINT could deliver the correct gene to each person with the disease, allowing each patient’s body to produce the normal protein, regardless of the original mutation, the authors say.
Gene supplementation with PRINT could deliver the correct gene to each person, allowing each patient’s body to produce the normal protein regardless of the original mutation.
Many academic labs and startups are exploring the use of transposons and retrotransposons to insert genes for gene therapy purposes. One of the most studied by biotech companies is LINE-1 (Long INterspersed Element-1), which in humans has duplicated itself and some hitchhiking genes to cover about 30% of the genome, although less than 100 of them. Copies of the LINE -1 retrotransposon in our genome are now functional, a tiny portion of the genome.
Collins, along with UC Berkeley postdoctoral colleagues Akanksha Thawani and Eva Nogales, also at the Howard Hughes Medical Institute, published a cryo-electron microscopy structure of the enzyme protein encoded by the LINE-1 retroelement in Nature in December.
According to Collins, this study made it clear that the LINE-1 retrotransposon protein would be difficult to manipulate to safely and efficiently insert a transgene into the human genome. However, previous research showing that genes inserted into the repetitive region of the genome that encodes ribosomal RNA (rDNA) are expressed normally suggested to Collins that another retroelement called R2 may be better suited for safe transgene insertion is.
The junk DNA chosen came from birds
Because R2 is not found in humans, Collins and lead researcher Xiaozhu Zhang and postdoctoral researcher Briana Van Treeck, both at UC Berkeley, examined R2 from more than two dozen animal genomes, from insects to the horseshoe crab and other multicellular eukaryotes, to find a version , which was highly targeted to rDNA regions in the human genome and could efficiently insert large lengths of DNA into the region.
They examined genomes of insects, horseshoe crabs and other multicellular eukaryotes until they found a version that strongly targeted rDNA regions in the human genome and was efficient at inserting long lengths of DNA.
“After researching dozens of them, the birds were the real winners,” Collins said, including the zebra finch and white-throated sparrow.
Although mammals do not have R2 in their genomes, they do have the binding sites required for R2 to be inserted efficiently as a retroelement, which is likely a sign that mammalian predecessors had an R2-like retroelement that somehow was removed from the mammalian genome, the researchers say.
In the experiments, Zhang and Van Treeck synthesized mRNA encoding the R2 protein and a template RNA that would create a transgene with a fluorescent protein expressed from an RNA polymerase promoter. They were cotransfected into cultured human cells. About half of the cells glowed green or red under laser light due to the expression of the fluorescent protein, showing that the R2 system had successfully inserted a functional fluorescent protein into the genome.
Subsequent studies showed that the transgene was indeed inserted into the rDNA regions of the genome and that about 10 copies of the RNA template could be inserted without altering the protein-making activity of the rDNA genes.
A giant of ribosome biogenesis
The researchers point out that inserting transgenes into the rDNA regions of the genome has other benefits besides providing a safe harbor.
The rDNA regions are located on the thick arms of five different chromosomes. All of these arms roll up and form a structure called the nucleolus, where DNA is transcribed into ribosomal RNA, which is then folded into the ribosomal machinery that produces proteins.
In the nucleolus, rDNA transcription is highly regulated and genes are repaired quickly because any rDNA break, if propagated, could cripple protein production. Consequently, any transgene inserted into the rDNA region of the genome would be treated with “kid gloves” in the nucleolus.
“The nucleolus is a gigantic center of ribosome biogenesis,” explains Collins. “But it is also a really privileged environment for DNA repair, with a low oncogenic risk due to gene insertion. It’s great that these successful retroelements – I anthropomorphize them – have been introduced into ribosomal DNA. It’s a multiple copy, it’s conserved and it’s a safe place in the sense that one of those copies can be changed and the cell doesn’t care.”
This makes the region an ideal place to introduce a gene for gene therapy into humans, the researchers said.
Questions
Collins acknowledges that there is still much unknown about how R2 works and that questions remain about the biology of rDNA transcription: How many rDNA genes can be destroyed before the cell is destroyed? Given that some cells turn off many of the more than 400 rDNA genes in the human genome, are these cells more susceptible to the side effects of PRINT?
She and her team are exploring these questions, but are also modifying the various proteins and RNAs involved in retroelement insertion so that PRINT works better in cultured cells and primary human tissue cells.
The end result, however, is that “it works,” he says. “We just need to understand the biology of our rDNA a little better to really use it.”
Reference:
Xiaozhu Zhang et al. “Junk DNA in birds could hold the key to safe and efficient gene therapy.” Nature Biotechnology (2024)