The recent approval of a CRISPR-Cas9 remedy for sickle cell illness demonstrates that gene modifying instruments can do an outstanding job knocking out genes to remedy hereditary illness. But it is nonetheless not attainable to insert complete genes into the human genome to substitute for faulty or deleterious genes.
A brand new approach 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 “protected harbor” within the human genome the place the insertion will not disrupt important genes or result in most cancers.
Retrotransposons, or retroelements, are items of DNA that, when transcribed to RNA, code for enzymes that duplicate 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 “egocentric” new DNA, although many of the genes are disabled, so-called junk DNA.
The new approach, known as Precise RNA-mediated INsertion of Transgenes, or PRINT, leverages the power of some retrotransposons to effectively insert complete genes into the genome with out affecting different genome features. PRINT would complement the acknowledged skill of CRISPR-Cas expertise 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, might be revealed Feb. 20 within the journal Nature Biotechnology.
PRINT entails the insertion of recent DNA right into a cell utilizing supply strategies just like these used to ferry CRISPR-Cas9 into cells for genome modifying. For PRINT, one piece of delivered RNA encodes a standard retroelement protein known as R2 protein, which has a number of energetic 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 complete autonomous transgene cassette that R2 protein inserts into the genome, Collins stated.
A key benefit of utilizing R2 protein is that it inserts the transgene into an space of the genome that incorporates tons of of an identical 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 will not be missed.
Putting the transgene right into a protected harbor avoids a serious drawback encountered when inserting transgenes through a human virus vector, which is the widespread methodology as we speak: The gene is commonly inserted randomly into the genome, disabling working genes or messing with the regulation or perform of genes, probably resulting in most cancers.
“A CRISPR-Cas9-based method can repair a mutant nucleotide or insert a bit of patch of DNA — sequence fixing. Or you may simply knock out a gene perform by site-specific mutagenesis,” stated Collins, who holds the Walter and Ruth Schubert Family Chair. “We’re not knocking out a gene perform. We’re not fixing an endogenous gene mutation. We’re taking a complementary method, which is to place into the genome an autonomously expressed gene that makes an energetic protein —so as to add again a practical gene as a deficit bypass. It’s transgene supplementation as a substitute of mutation reversal. To repair loss-of-function ailments that come up from a panoply of individual mutations of the identical gene, that is nice.”
‘The actual winners had been from birds’
Many hereditary ailments, comparable to cystic fibrosis and a few types of hemophilia, are attributable to a variety of completely different mutations in the identical gene, all of which disable the gene’s perform. Any CRISPR-Cas9-based gene modifying 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.
A schematic exhibiting how the R2 retroelement protein (teal blobs) copies its DNA into the genome. The protein opens up double-stranded DNA (black and gray spirals) the place the gene might be inserted. It carries with it an RNA template (orange) that in its native context codes for the R2 protein. It then inserts this sequence by transcribing that RNA to DNA instantly into the genome. UC Berkeley researchers changed the R2-encoding RNA within the template with RNA coding for a transgene, turning R2 right into a supply automobile for transgenes that’s extra environment friendly and safer for gene remedy functions — a method they name PRINT.
Many tutorial labs and startups are investigating the usage of transposons and retrotransposons to insert genes for gene remedy. One fashionable retrotransposon below 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 practical as we speak, a miniscule 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, published a cryoelectron microscopy structure of the enzyme protein encoded by the LINE-1 retroelement on Dec. 14 within the journal Nature.
That research made it clear, Collins stated, that the LINE-1 retrotransposon protein could be exhausting to engineer to securely 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 instructed to Collins {that a} completely different retroelement, known as R2, would possibly work higher for protected transgene insertion.
Because R2 isn’t 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 actual winners had been from birds,” Collins stated, together with the zebra finch and the white-throated sparrow.
While mammals don’t have R2 of their genomes, they do have the binding websites wanted for R2 to successfully insert as a retroelement — seemingly an indication, she stated, that the predecessors to mammals had an R2-like retroelement that someway received kicked out of the mammalian genome.
In experiments, Zhang and Van Treeck synthesized mRNA-encoding R2 protein and a template RNA that will 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 because of fluorescent protein expression below laser gentle, demonstrating that the R2 system had efficiently inserted into the genome a gene expressing a fluorescent protein .
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 might insert with out disrupting the protein-manufacturing exercise of the rDNA genes.
An enormous ribosome biogenesis heart
Inserting transgenes into rDNA areas of the genome is advantageous for causes aside from it offers them a protected 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 known as the nucleolus, by 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 huge ribosome biogenesis heart,” Collins stated. “But it is also a extremely privileged DNA restore surroundings with low oncogenic threat from gene insertion. It’s sensible that these profitable retroelements — I’m anthropomorphizing them — have gone into the ribosomal DNA. It’s multicopy, it is conserved, and it is a protected harbor within the sense that you would be able to disrupt certainly one of these copies and the cell would not care.”
This makes the area a perfect place to insert a gene for human gene remedy.
Collins admitted that loads continues to be unknown about how R2 works and that questions stay in regards to the biology of rDNA transcription: How many rDNA genes may 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 crew are investigating these questions, but in addition tweaking the varied 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 really works,” she stated. “It’s simply that we now have to grasp a bit of bit extra in regards to the biology of our rDNA in an effort to actually benefit from it.”
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.
