Junk DNA in birds may hold key to safe, efficient gene therapy

The recent approval of CRISPR-Cas9 therapy for sickle cell disease shows that gene-editing tools can do a great job of knocking out genes to treat an inherited disease. But it is still not possible to insert whole genes into the human genome to replace defective or damaged genes.

A new technique that uses retrotransposons from birds to insert genes into the genome holds more promise. Gene therapyas it inserts the gene into a “safe harbor” in the human genome where the insertion will not disrupt essential genes or cause cancer.

Retrotransposons, or retroelements, are fragments of DNA that, when transcribed into RNA, code for enzymes that copy RNA into DNA in the genome—a self-serving cycle that clutters the genome with retrotransposon DNA. puts About 40% of the human genome is made up of this “selfish” new DNA, although most genes are inactive, so-called junk DNA.

The new technique, called Precise RNA-mediated INsertion of Transgenes, or PRINT, takes advantage of the ability of some retrotransposons to efficiently insert entire genes into the genome without affecting other functions of the genome. PRINT will complement CRISPR-Cas technology’s ability to inactivate genes, create point mutations and insert small segments of DNA.

A description of PRINT, which was developed in the laboratory of Kathleen Collins, professor of molecular and cell biology at the University of California, Berkeley, is published in the journal Feb. 20. Nature Biotechnology.

PRINT involves introducing new DNA into the cell using delivery methods such as those used to carry CRISPR-Cas9 into the cell for genome editing. For imprinting, a piece of delivered RNA encodes a common retroelement protein called the R2 protein, which contains multiple functional domains, including a nickase—an enzyme that binds and unwinds double-stranded DNA. — and reverse transcriptase, the enzyme that replicates DNA. The second RNA is the template for insertion of transgene DNA, as well as gene expression control elements — a completely autonomous transgene cassette that the R2 protein inserts into the genome, Collins said.

An important advantage of using the R2 protein is that it inserts the transgene into a part of the genome that contains hundreds of identical copies of the same gene—for each coding ribosomal RNA, the RNA machine that makes messenger RNA (mRNA ) into proteins. With many redundant copies, when the insertion disrupts one or more ribosomal RNA genes, the gene loss will not be missed.

Placing the transgene in a safe harbor avoids a major problem encountered when introducing a transgene via a human virus vector, which is the common method today: the gene is often randomly inserted into the genome, the functional gene inactivates or disrupts gene regulation or function. , possibly causing cancer.

“A CRISPR-Cas9-based approach can fix a mutant nucleotide or insert a small patch of DNA-sequence fixing,” said Collins, who holds the Walter and Ruth Schubert family chair.

“We’re not knocking out a gene’s function. We’re not fixing an endogenous gene mutation. We’re taking a complementary approach, which is inserting an autonomously expressed gene into the genome that’s functional. Makes protein. Functional gene as a bypass for a deficiency. This is transgene supplementation rather than mutation reversal. For correcting loss-of-function diseases caused by a panoply of individual mutations of the same gene, this is great. Is.”

‘The original conquerors were from the birds’

Many inherited diseases, such as cystic fibrosis and hemophilia, are caused by multiple different mutations in the same gene, all of which disable the gene’s function. Any CRISPR-Cas9-based gene-editing therapy must be tailored to a person’s specific mutation. Gene complementation using PRINT can deliver the correct gene to anyone with the disease, allowing each patient’s body to make the normal protein, regardless of the original mutation.

Several academic labs and startups are investigating the use of transposons and retrotransposons to insert genes for gene therapy. A popular retrotransposon studied by biotech companies is LINE-1 (long interspersed element-1), which has duplicated itself and some hitchhiker genes in humans to cover about 30 percent of the genome, although ours There are fewer than 100 line-1 retrotransposons. Copies are active today, a small part of the genome.

Collins, along with UC Berkeley postdoctoral fellows Akanksha Thavani and Eva Nogales, distinguished professor in UC Berkeley’s Department of Molecular and Cell Biology and investigators at the Howard Hughes Medical Institute, published A cryo-electron microscopy structure of the enzyme protein by the LINE-1 retroelement on December 14 in the journal The nature.

The study made it clear that the line-1 retrotransposon protein would be difficult to engineer to safely and efficiently insert a transgene into the human genome, Collins said. But previous research has shown that genes inserted into the repetitive, ribosomal RNA-encoding region of the genome (rDNA) are normally expressed by the colons to insert a different retroelement-protected transgene called R2. Could do better.

Because R2 is not found in humans, Collins and senior researcher Xiaozhu Zhang and postdoctoral fellow Briana Van Treeck, both from UC Berkeley, compared R2 to scores of animal genomes, from insects to horseshoe crabs and other multicellular organisms. to cellular eukaryotes, to explore. A version that was highly targeted to rDNA regions in the human genome was effective in inserting long lengths of DNA into that region.

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

Although mammalian genomes do not contain R2, they do have binding sites for R2 to efficiently insert as a retroelement, which is a possible sign, he said. The predecessors had a retroelement like the R2 which was somehow kicked out. The mammalian genome

In experiments, Zhang and Van Treeck synthesized mRNA-encoding R2 protein and a template RNA that would produce a transgene with a fluorescent protein expressed by an RNA polymerase promoter. These were transfected into cultured human cells. About half of the cells turn green or red because of the fluorescent protein down. Laser beamdemonstrating that the R2 system successfully inserted a functional fluorescent protein into the genome.

Further studies showed that the transgene had indeed inserted into the rDNA regions of the genome and that about 10 copies of the RNA template could be inserted without disrupting the protein manufacturing activity of the rDNA gene.

A major ribosome biogenesis center

Insertion of transgenes into rDNA regions of the genome is advantageous for reasons other than that it provides them with a safe harbor. The rDNA regions are found on the opposite arms of five separate chromosomes. All these antisense arms come together to form a structure called the nucleolus, where DNA is transcribed into ribosomal RNA, which is then folded into proteins by the ribosomal machinery.

Within the nucleolus, rDNA transcription is highly regulated, and genes are quickly repaired, because any rDNA breaks, if left to proliferate, can stop protein production. As a result, any transgene inserted into the rDNA region of the genome will be treated with kid gloves inside the nucleolus.

“The nucleus is a major ribosome biogenesis center,” Collins said, “but it’s also a really privileged DNA repair environment that has less oncogenic risk than gene insertion.” It’s amazing that these successful retroelements—I’m anthropomorphizing them—have moved into ribosomal DNA. It’s multi-copy, it’s secure, and it’s a safe harbor. The feeling that you can disrupt one of those copies and the cell doesn’t care.”

This region makes an ideal site for gene insertion for human gene therapy.

Collins admits that much is still unknown about how R2 works and questions remain about the biology of rDNA transcription: How many rDNA genes can be disrupted before a cell cares? Because some cells turn off 400+ rDNA genes. The human genomeAre these cells more sensitive to PRINT’s side effects?

He and his team are investigating these questions, but also modifying the various proteins and RNAs involved in retroelement insertion so that PRINT works better in cultured cells and primary cells from human tissues. could

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