Junk DNA in birds could be key to safe and effective gene therapy

The recent approval of a CRISPR-Cas9 therapy for sickle cell disease demonstrates that gene editing tools can do an excellent job of eliminating genes to cure inherited diseases. But it is still not possible to insert entire genes into the human genome to replace them with defective or deleterious genes.

A new technique that uses a bird retrotransposon to insert genes into the genome holds more promise for gene therapy because it inserts the genes into a “safe haven” in the human genome where the insertion will not disrupt essential genes or will not lead to cancer.

Retrotransposons, or retroelements, are pieces of DNA that, once transcribed into RNA, encode enzymes that copy the RNA into the genome’s DNA – a selfish cycle that clutters the DNA genome with retrotransposons. About 40% of the human genome is made up of this new “selfish” DNA, although most of the genes are turned off, called junk DNA.

The new technique, called Precise RNA-Mediated Transgene Insertion, or PRINT, exploits the ability of certain retrotransposons to efficiently insert entire genes into the genome without affecting other genome functions. PRINT would complement CRISPR-Cas technology’s proven ability to disable genes, make point mutations and insert short segments of DNA.

A description of PRINT, developed in the laboratory of Kathleen Collins, professor of molecular and cellular biology at the University of California, Berkeley, is published February 20 in the journal Natural biotechnology.

PRINT involves the insertion of new DNA into a cell using delivery methods similar to those used to transport CRISPR-Cas9 into cells for genome editing. For PRINT, a delivered piece of RNA encodes a common retroelement protein called the R2 protein, which has several active parts, including a nickase (an enzyme that binds and cuts double-stranded DNA) and reverse transcriptase, the enzyme that generates DNA’s copy of RNA. The other RNA is the template for the transgenic DNA to be inserted, as well as the gene expression control elements — an entire stand-alone transgenic cassette that the R2 protein inserts into the genome, Collins said.

One of the main advantages of using the R2 protein is that it inserts the transgene into an area of ​​the genome that contains hundreds of identical copies of the same gene, each coding for ribosomal RNA, the RNA machine that translates messenger RNA (mRNA) into protein. With so many redundant copies, when the insertion disrupts one or a few ribosomal RNA genes, loss of the genes will not be missed.

Placing the transgene in a safe zone avoids a major problem encountered when inserting transgenes via a human viral vector, which is the common method today: the gene is often randomly inserted into the genome, disabling the genes functional or disrupting the regulation or function of genes. , which can lead to cancer.

“A CRISPR-Cas9-based approach can repair a mutant nucleotide or insert a small fixing piece of DNA sequence. Or you can simply knock out the function of a gene through site-specific mutagenesis,” said Collins, PhD. from the study Walter and Ruth Schubert. Family chair.

“We do not remove the function of a gene. We do not repair an endogenous genetic mutation. We adopt a complementary approach, which consists of introducing into the genome an autonomously expressed gene which makes an active protein, to add a active protein.” “A functional gene as a bypass of the deficiency. This is transgenic supplementation instead of mutation reversal. To correct loss-of-function diseases that result from a panoply of individual mutations of the same gene, it is great. “

“The real winners were the birds”

Many inherited diseases, such as cystic fibrosis and hemophilia, are caused by a number of different mutations in the same gene, all of which turn off the gene’s function. Any CRISPR-Cas9-based gene editing therapy would need to be tailored to a person’s specific mutation. Genetic supplementation using PRINT could instead deliver the right gene to each person with the disease, allowing each patient’s body to make the normal protein, regardless of the original mutation.

Many academic labs and startups are investigating the use of transposons and retrotransposons to insert genes in gene therapy. A popular retrotransposon studied by biotechnology companies is LINE-1 (Long INterspersed Element-1), which in humans has duplicated itself and some hitchhiker genes to cover about 30% of the genome, although less than 100 LINE-1 retrotransposons in our genome. the copies are functional today, a tiny fraction of the genome.

Collins, along with UC Berkeley postdoctoral colleague Akanksha Thawani and Eva Nogales, professor emeritus in the Department of Molecular and Cellular Biology at UC Berkeley and investigator at the Howard Hughes Medical Institute, published a cryoelectron microscopy structure of the enzymatic protein encoded by the retroelement LINE-1. December 14 in the newspaper Nature.

This study clearly showed, Collins said, that the LINE-1 retrotransposon protein would be difficult to engineer to safely and efficiently insert a transgene into the human genome. But previous research demonstrating that genes inserted into the repetitive ribosomal RNA-coding region of the genome (rDNA) are expressed normally suggested to Collins that a different retroelement, called R2, might work better for safe insertion. of the transgene.

Because R2 is not found in humans, Collins and principal investigator Xiaozhu Zhang and postdoctoral fellow Briana Van Treeck, both of UC Berkeley, examined R2 from more than two dozen animal genomes , from insects to horseshoe crabs and other multicellular eukaryotes, to find a version that is highly targeted to the rDNA regions of the human genome and efficient at inserting long lengths of DNA into the region.

“After hunting dozens of them, the real winners were the birds,” Collins said, including the zebra finch and the white-throated sparrow.

Although mammals do not have R2 in their genome, they do have the necessary binding sites for R2 to insert efficiently as a retroelement, which is likely a sign, she said, that predecessors mammals possessed an R2-like retroelement that somehow got pushed out of the mammalian genome.

In experiments, Zhang and Van Treeck synthesized the mRNA-encoding R2 protein and an RNA template that would generate a transgene with a fluorescent protein expressed by an RNA polymerase promoter. These were cotransfected into cultured human cells. About half of the cells glowed green or red due to the expression of a fluorescent protein under the laser light, demonstrating that the R2 system had successfully inserted a functional fluorescent protein into the genome.

Further studies showed that the transgene actually inserted into the rDNA regions of the genome and that about 10 copies of the RNA template could insert without disrupting the protein-making activity of the rDNA genes. rDNA.

A center for giant ribosome biogenesis

Insertion of transgenes into rDNA regions of the genome is advantageous for reasons other than providing a safe haven. The rDNA regions are found on the truncated arms of five distinct chromosomes. All these stubby arms come together to form a structure called a nucleolus, in which DNA is transcribed into ribosomal RNA, which then folds into the ribosomal machinery that makes proteins.

Within the nucleolus, rDNA transcription is highly regulated and genes undergo rapid repair, because any break in rDNA, if allowed to propagate, could stop protein production. As a result, any transgene inserted into the rDNA region of the genome would be treated like kid gloves inside the nucleolus.

“The nucleolus is a giant center of ribosome biogenesis,” Collins said. “But it’s also a really privileged DNA repair environment with low oncogenic risk from gene insertion. It’s great that these successful retroelements – I anthropomorphize them – got into ribosomal DNA. It’s multi-copy, it’s preserved, and it’s a safe haven in the feeling that you can disrupt one of those copies and the cell doesn’t care.”

This makes the region an ideal place to insert a gene for human gene therapy.

Collins admitted that much is still unknown about how R2 works and that questions remain about the biology of rDNA transcription: how many rDNA genes can be disrupted before the cell care? Since some cells turn off many of the more than 400 rDNA genes in the human genome, are these cells more sensitive to the side effects of PRINT?

She and her team are investigating these questions, but also fine-tuning the various proteins and RNAs involved in retroelement insertion to make PRINT work better in cultured cells and primary cells in human tissues.

The bottom line, though, is that “it works,” she said. “It’s just that we need to understand a little more about the biology of our rDNA in order to really take advantage of it.”