PRINT, a new gene treatment strategy, utilizes bird-derived retrotransposons to insert entire genes into a safe zone of the human genome, providing a complementary technique to CRISPR-Cas9 by possibly enabling the treatment of diseases without the danger of gene disruption or cancer. Credit: SciTechDaily.comRetrotransposons can place new genes into a “safe harbor” in the genome, complementing CRISPR gene editing.The recent greenlighting of a CRISPR-Cas9 treatment for sickle cell disease highlights the efficacy of gene editing technologies in deactivating genes to recover inherited illnesses. The ability to incorporate whole genes into the human genome as replacements for malfunctioning or harmful ones remains unachievable.A new method that uses a retrotransposon from birds to place genes into the genome holds more promise for gene treatment, given that it inserts genes into a “safe harbor” in the human genome where the insertion wont interfere with necessary genes or lead to cancer.Retrotransposons, or retroelements, are pieces of DNA that, when transcribed to RNA, code for enzymes that copy RNA back into DNA in the genome– a self-serving cycle that jumbles the genome with retrotransposon DNA. About 40% of the human genome is made up of this “self-centered” brand-new DNA, though the majority of the genes are disabled, so-called scrap DNA.The new method, called Precise RNA-mediated INsertion of Transgenes, or PRINT, leverages the ability of some retrotransposons to efficiently place entire genes into the genome without affecting other genome functions. PRINT would match the acknowledged capability of CRISPR-Cas technology to disable genes, make point mutations, and insert brief sections of DNA.A description of PRINT, which was developed in the lab of Kathleen Collins, a teacher of molecular and cell biology at the University of California, Berkeley, was just recently released in the journal Nature Biotechnology.PRINT involves the insertion of new DNA into a cell utilizing shipment techniques similar to those utilized to transport CRISPR-Cas9 into cells for genome modifying. For PRINT, one piece of provided RNA encodes a typical retroelement protein called R2 protein, which has several active parts, including a nickase– an enzyme that binds and nicks double-stranded DNA– and reverse transcriptase, the enzyme that creates the DNA copy of RNA. The other RNA is the template for the transgene DNA to be placed, plus gene expression control aspects– a whole autonomous transgene cassette that R2 protein inserts into the genome, Collins said.Retrotransposons found in the genomes of the white-throated sparrow and the zebra finch are revealed to safely shepherd transgenes into the human genome, supplying a gene treatment method complementary to CRISPR-Cas9 gene modifying. Credit: Briana Van Treeck, UC BerkeleyA essential advantage of using R2 protein is that it inserts the transgene into a location of the genome that includes numerous similar copies of the exact same gene– each coding for ribosomal RNA, the RNA device that translates messenger RNA (mRNA) into protein. With many redundant copies, when the insertion disrupts one or a few ribosomal RNA genes, the loss of the genes wont be missed.Putting the transgene into a safe harbor avoids a major problem experienced when placing transgenes via a human infection vector, which is the common approach today: The gene is often inserted arbitrarily into the genome, disabling messing or working genes with the policy or function of genes, potentially leading to cancer.”A CRISPR-Cas9-based technique can fix a mutant nucleotide or place a little patch of DNA– sequence fixing. Or you can simply knock out a gene function by site-specific mutagenesis,” stated Collins, who holds the Walter and Ruth Schubert Family Chair. “Were not knocking out a gene function. Were not repairing an endogenous gene mutation. Were taking a complementary approach, which is to put into the genome an autonomously expressed gene that makes an active protein– to include back a practical gene as a deficit bypass. Its transgene supplementation instead of anomaly reversal. To repair loss-of-function diseases that occur from a panoply of individual mutations of the exact same gene, this is fantastic.”The genuine winners were from birdsMany genetic diseases, such as cystic fibrosis and hemophilia, are brought on by a number of different anomalies in the same gene, all of which disable the genes function. Any CRISPR-Cas9-based gene editing treatment would need to be tailored to a persons specific anomaly. Gene supplements utilizing PRINT could rather deliver the right gene to every individual with the disease, permitting each patients body to make the normal protein, no matter what the initial mutation.Many academic labs and startups are investigating using transposons and retrotransposons to place genes for gene therapy. One popular retrotransposon under study by biotech business is LINE-1 (Long INterspersed Element-1), which in people has actually duplicated itself and some hitchhiker genes to cover about 30% of the genome, though fewer than 100 of our genomes LINE-1 retrotransposon copies are functional today, a minuscule fraction of the genome.Collins, in addition to UC Berkeley postdoctoral colleague Akanksha Thawani and Eva Nogales, UC Berkeley Distinguished Professor in the Department of Molecular and Cell Biology and a Howard Hughes Medical Institute investigator, published a cryoelectron microscopy structure of the enzyme protein encoded by the LINE-1 retroelement on Dec. 14 in the journal Nature.That research study made it clear, Collins said, that the LINE-1 retrotransposon protein would be tough to engineer to safely and effectively insert a transgene into the human genome. However previous research study demonstrating that genes placed into the recurring, ribosomal RNA encoding region of the genome (the rDNA) get revealed normally suggested to Collins that a different retroelement, called R2, might work much better for safe transgene insertion.Because R2 is not found in people, Collins and senior scientist Xiaozhu Zhang and postdoctoral fellow Briana Van Treeck, both from UC Berkeley, screened R2 from more than a rating of animal genomes, from insects to the horseshoe crab and other multicellular eukaryotes, to discover a variation that was highly targeted to rDNA areas in the human genome and efficient at placing long lengths of DNA into the area.”After going after lots of them, the real winners were from birds,” Collins said, including the zebra finch and the white-throated sparrow.While mammals do not have R2 in their genomes, they do have the binding sites needed for R2 to efficiently place as a retroelement– most likely an indication, she stated, that the predecessors to mammals had an R2-like retroelement that in some way got tossed out of the mammalian genome.In experiments, Zhang and Van Treeck manufactured mRNA-encoding R2 protein and a design template RNA that would create a transgene with a fluorescent protein revealed by an RNA polymerase promoter. These were cotransfected into cultured human cells. About half the cells lit up red or green due to fluorescent protein expression under laser light, demonstrating that the R2 system had effectively inserted a working fluorescent protein into the genome.Further studies showed that the transgene did undoubtedly place into the rDNA areas of the genome and that about 10 copies of the RNA template could be placed without disrupting the protein-manufacturing activity of the rDNA genes.A giant ribosome biogenesis centerInserting transgenes into rDNA areas of the genome is helpful for factors besides it provides a safe harbor. The rDNA areas are found on the stubby arms of five different chromosomes. All of these stubby arms huddle together to form a structure called the nucleolus, in which DNA is transcribed into ribosomal RNA, which then folds into the ribosomal equipment that makes proteins. Within the nucleolus, rDNA transcription is highly regulated, and the genes undergo quick repair work, since any rDNA breaks, if left to propagate, might close down protein production. As an outcome, any transgene inserted into the rDNA area of the genome would be treated with kid gloves inside the nucleolus.”The nucleolus is a huge ribosome biogenesis center,” Collins stated. “But its likewise a truly privileged DNA repair environment with low oncogenic threat from gene insertion. Its dazzling that these successful retroelements– Im anthropomorphizing them– have gone into the ribosomal DNA. Its multicopy, its conserved, and its a safe harbor in the sense that you can interrupt among these copies and the cell does not care.”This makes the area an ideal location to insert a gene for human gene therapy.Collins confessed that a lot is still unidentified about how R2 works which questions remain about the biology of rDNA transcription: How numerous rDNA genes can be interfered with before the cell cares? Due to the fact that some cells shut off many of the 400+ rDNA genes in the human genome, are these cells more prone to side effects of PRINT? She and her team are investigating these questions, however likewise tweaking the various proteins and RNAs included in retroelement insertion to make PRINT work much better in cultured cells and main cells from human tissue.The bottom line, however, is that “it works,” she said. “Its just that we have to comprehend a 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-yOther co-authors of the Nature Biotechnology paper are UC Berkeley 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 actually declared patents on PRINT, and co-founded a business, Addition Therapeutics, to develop PRINT further as a gene treatment.
PRINT, a new gene treatment method, employs bird-derived retrotransposons to insert entire genes into a safe zone of the human genome, providing a complementary approach to CRISPR-Cas9 by possibly making it possible for the treatment of diseases without the danger of gene disturbance or cancer. Credit: SciTechDaily.comRetrotransposons can place new genes into a “safe harbor” in the genome, matching CRISPR gene editing.The recent greenlighting of a CRISPR-Cas9 treatment for sickle cell disease highlights the efficacy of gene editing technologies in shutting down genes to heal acquired health problems. The ability to integrate entire genes into the human genome as replacements for hazardous or defective ones stays unachievable.A new method that uses a retrotransposon from birds to place genes into the genome holds more pledge for gene treatment, considering that it inserts genes into a “safe harbor” in the human genome where the insertion wont lead or interfere with important genes to cancer.Retrotransposons, or retroelements, are pieces of DNA that, when transcribed to RNA, code for enzymes that copy RNA back into DNA in the genome– a self-serving cycle that clutters the genome with retrotransposon DNA. With so numerous redundant copies, when the insertion disrupts one or a few ribosomal RNA genes, the loss of the genes will not be missed.Putting the transgene into a safe harbor avoids a significant problem experienced when inserting transgenes via a human virus vector, which is the common method today: The gene is frequently inserted randomly into the genome, disabling messing or working genes with the policy or function of genes, possibly leading to cancer. Gene supplementation utilizing PRINT could instead provide the correct gene to every individual with the illness, enabling each clients body to make the regular protein, no matter what the original mutation.Many academic labs and start-ups are examining the use of transposons and retrotransposons to insert genes for gene therapy.