PRINT, a brand new gene remedy method, employs bird-derived retrotransposons to insert entire genes right into a secure zone of the human genome, providing a complementary strategy to CRISPR-Cas9 by doubtlessly enabling the therapy of illnesses with out the chance of gene disruption or most cancers. Credit: SciTechDaily.com
Retrotransposons can insert new genes right into a “safe harbor” within the genome, complementing CRISPR gene enhancing.
The recent greenlighting of a CRISPR-Cas9 therapy for sickle cell illness underscores the efficacy of gene enhancing applied sciences in deactivating genes to heal inherited sicknesses. However, the potential to combine complete genes into the human genome as replacements for defective or dangerous ones stays unachievable.
A brand new method that employs a retrotransposon from birds to insert genes into the genome holds extra promise for gene remedy, because it inserts genes right into a “safe harbor” within the human genome the place the insertion gained’t disrupt important genes or result in most cancers.
Retrotransposons, or retroelements, are items of DNA that, when transcribed to RNA, code for enzymes that replicate RNA again into DNA within the genome — a self-serving cycle that clutters the genome with retrotransposon DNA. About 40% of the human genome is made up of this “selfish” new DNA, although many of the genes are disabled, so-called junk DNA.
The new method, referred to as Precise RNA-mediated INsertion of Transgenes, or PRINT, leverages the flexibility of some retrotransposons to effectively insert complete genes into the genome with out affecting different genome capabilities. PRINT would complement the acknowledged skill of CRISPR-Cas know-how to disable genes, make level mutations, and insert quick segments of DNA.
An outline of PRINT, which was developed within the laboratory of Kathleen Collins, a professor of molecular and cell biology on the University of California, Berkeley, was not too long ago revealed within the journal Nature Biotechnology.
PRINT includes the insertion of recent DNA right into a cell utilizing supply strategies just like these used to ferry CRISPR-Cas9 into cells for genome enhancing. For PRINT, one piece of delivered RNA encodes a typical retroelement protein referred to as R2 protein, which has a number of lively elements, together with a nickase — an enzyme that binds and nicks double-stranded DNA — and reverse transcriptase, the enzyme that generates the DNA copy of RNA. The different RNA is the template for the transgene DNA to be inserted, plus gene expression management components — a whole autonomous transgene cassette that R2 protein inserts into the genome, Collins mentioned.
Retrotransposons discovered within the genomes of the white-throated sparrow and the zebra finch are proven to soundly shepherd transgenes into the human genome, offering a gene remedy strategy complementary to CRISPR-Cas9 gene enhancing. Credit: Briana Van Treeck, UC Berkeley
A key benefit of utilizing R2 protein is that it inserts the transgene into an space of the genome that incorporates lots of of equivalent copies of the identical gene — every coding for ribosomal RNA, the RNA machine that interprets messenger RNA (mRNA) into protein. With so many redundant copies, when the insertion disrupts one or a couple of ribosomal RNA genes, the lack of the genes gained’t be missed.
Putting the transgene right into a secure harbor avoids a significant drawback encountered when inserting transgenes through a human virus vector, which is the widespread methodology at this time: The gene is commonly inserted randomly into the genome, disabling working genes or messing with the regulation or perform of genes, doubtlessly resulting in most cancers.
“A CRISPR-Cas9-based approach can fix a mutant nucleotide or insert a little patch of DNA — sequence fixing. Or you can just knock out a gene function by site-specific mutagenesis,” mentioned Collins, who holds the Walter and Ruth Schubert Family Chair. “We’re not knocking out a gene function. We’re not fixing an endogenous gene mutation. We’re taking a complementary approach, which is to put into the genome an autonomously expressed gene that makes an active protein —to add back a functional gene as a deficit bypass. It’s transgene supplementation instead of mutation reversal. To fix loss-of-function diseases that arise from a panoply of individual mutations of the same gene, this is great.”
‘The real winners were from birds’
Many hereditary illnesses, similar to cystic fibrosis and hemophilia, are brought on by a lot of totally different mutations in the identical gene, all of which disable the gene’s perform. Any CRISPR-Cas9-based gene enhancing remedy must be tailor-made to a person’s particular mutation. Gene supplementation utilizing PRINT might as a substitute ship the proper gene to each person with the illness, permitting every affected person’s physique to make the traditional protein, it doesn’t matter what the unique mutation.
Many tutorial labs and startups are investigating the usage of transposons and retrotransposons to insert genes for gene remedy. One common retrotransposon underneath research by biotech corporations is LINE-1 (Long INterspersed Element-1), which in people has duplicated itself and a few hitchhiker genes to cowl about 30% of the genome, although fewer than 100 of our genome’s LINE-1 retrotransposon copies are purposeful at this time, a minuscule fraction of the genome.
Collins, together with UC Berkeley postdoctoral colleague Akanksha Thawani and Eva Nogales, UC Berkeley Distinguished Professor within the Department of Molecular and Cell Biology and a Howard Hughes Medical Institute investigator, revealed a cryoelectron microscopy construction of the enzyme protein encoded by the LINE-1 retroelement on Dec. 14 within the journal Nature.
That research made it clear, Collins mentioned, that the LINE-1 retrotransposon protein could be arduous to engineer to soundly and effectively insert a transgene into the human genome. But earlier analysis demonstrating that genes inserted into the repetitive, ribosomal RNA encoding area of the genome (the rDNA) get expressed usually recommended to Collins {that a} totally different retroelement, referred to as R2, may work higher for secure transgene insertion.
Because R2 is just not present in people, Collins and senior researcher Xiaozhu Zhang and postdoctoral fellow Briana Van Treeck, each from UC Berkeley, screened R2 from greater than a rating of animal genomes, from bugs to the horseshoe crab and different multicellular eukaryotes, to discover a model that was extremely focused to rDNA areas within the human genome and environment friendly at inserting lengthy lengths of DNA into the area.
“After chasing dozens of them, the real winners were from birds,” Collins mentioned, together with the zebra finch and the white-throated sparrow.
While mammals should not have R2 of their genomes, they do have the binding websites wanted for R2 to successfully insert as a retroelement — doubtless an indication, she mentioned, that the predecessors to mammals had an R2-like retroelement that in some way acquired kicked out of the mammalian genome.
In experiments, Zhang and Van Treeck synthesized mRNA-encoding R2 protein and a template RNA that may generate a transgene with a fluorescent protein expressed by an RNA polymerase promoter. These had been cotransfected into cultured human cells. About half the cells lit up inexperienced or pink as a result of fluorescent protein expression underneath laser gentle, demonstrating that the R2 system had efficiently inserted a working fluorescent protein into the genome.
Further research confirmed that the transgene did certainly insert into the rDNA areas of the genome and that about 10 copies of the RNA template may very well be inserted with out disrupting the protein-manufacturing exercise of the rDNA genes.
An enormous ribosome biogenesis middle
Inserting transgenes into rDNA areas of the genome is advantageous for causes aside from it provides them a secure harbor. The rDNA areas are discovered on the stubby arms of 5 separate chromosomes. All of those stubby arms huddle collectively to type a construction referred to as the nucleolus, through which DNA is transcribed into ribosomal RNA, which then folds into the ribosomal equipment that makes proteins. Within the nucleolus, rDNA transcription is very regulated, and the genes bear fast repairs, since any rDNA breaks, if left to propagate, might shut down protein manufacturing. As a consequence, any transgene inserted into the rDNA area of the genome could be handled with child gloves contained in the nucleolus.
“The nucleolus is a giant ribosome biogenesis center,” Collins mentioned. “But it’s also a really privileged DNA repair environment with low oncogenic risk from gene insertion. It’s brilliant that these successful retroelements — I’m anthropomorphizing them — have gone into the ribosomal DNA. It’s multicopy, it’s conserved, and it’s a safe harbor in the sense that you can disrupt one of these copies and the cell doesn’t care.”
This makes the area a perfect place to insert a gene for human gene remedy.
Collins admitted that lots remains to be unknown about how R2 works and that questions stay in regards to the biology of rDNA transcription: How many rDNA genes might be disrupted earlier than the cell cares? Because some cells flip off lots of the 400+ rDNA genes within the human genome, are these cells extra vulnerable to unintended effects of PRINT? She and her workforce are investigating these questions, but additionally tweaking the assorted proteins and RNAs concerned in retroelement insertion to make PRINT work higher in cultured cells and first cells from human tissue.
The backside line, although, is that “it works,” she mentioned. “It’s just that we have to understand a little bit more about the biology of our rDNA in order to really take advantage of it.”
Reference: “Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci” by Xiaozhu Zhang, Briana Van Treeck, Connor A. Horton, Jeremy J. R. McIntyre, Sarah M. Palm, Justin L. Shumate and Kathleen Collins, 20 February 2024, Nature Biotechnology.
DOI: 10.1038/s41587-024-02137-y
Other co-authors of the Nature Biotechnology paper are UC Berkeley graduate college students Connor Horton, Jeremy McIntyre, Sarah Palm, and Justin Shumate. The work was supported by the National Institutes of Health (F32 GM139306, DP1 HL156819, T32 GM07232) and the Shurl and Kay Curci Foundation. Collins has filed for patents on PRINT, and co-founded an organization, Addition Therapeutics, to develop PRINT additional as a gene remedy.
