CRISPR and beyond: The ins and outs of gene editing and its potential for cures

The big 4 gene-editing platforms and how they could usher in new therapies for HIV, cancer — and other diseases
An image of a megaTAL
MegaTALs are one of four new gene-editing tools that hold promise for treating a variety of inherited genetic disorders. Image courtesy of Dr. Barry Stoddard

It’s unusual for a laboratory method to make global headlines. But CRISPR – the small-but-mighty gene-editing technique — has had more than its moment in the sun.

Science magazine declared the technology its breakthrough of the year in 2015, noting CRISPR’s ease of use that has offered any biologist who wants it a crack at gene editing in their own laboratory. More recently, the tiny technique made big news again as scientists in China gear up to test, for the first time in humans, new therapies based on cells that have been genetically modified with CRISPR.

Amid all of the CRISPR clamor, some may overlook the fact that this flavor of gene editing is not completely unique. CRISPR is by far the easiest to use, said Fred Hutchinson Cancer Research Center protein-engineering expert Dr. Barry Stoddard, which is one reason that it has taken off in small and large research groups alike.

“CRISPR has democratized the entire [gene-editing] field,” Stoddard said.

Dr. Barry Stoddard
Dr. Barry Stoddard is a protein-engineering expert at Fred Hutch. Fred Hutch file

But there are other gene-editing tools based on similar biological principles. CRISPR and its three cousins — known as zinc-finger nucleases, TALENs and megaTALs — all use a targeted nuclease, a specialized protein that cuts DNA at a specific sequence. That targeted cutting is the core requirement of any true gene edit.

All four of the techniques involve some kind of laboratory engineering of the gene-editing molecules to make them home in on the part of DNA that researchers want to change: the mutations that cause inherited genetic disorders such as sickle cell disease or thalassemia, for example, or the gene coding for the molecular doorway HIV uses to enter cells.

Gene editing to cure HIV will be a major focus of the third annual Conference on Cell and Gene Therapy for HIV Cure that starts today at Fred Hutch. The conference is hosted by defeatHIV, a research consortium working on several avenues to HIV cure.

Some engineering of gene-editing platforms is easier than others, Stoddard said. CRISPR’s ease of use comes from the fact that its engineering requires modifying an RNA molecule to match the DNA strand you want to snip — a feat many orders of magnitude easier than protein engineering. But that doesn’t mean the other platforms don’t have merit. The other three gene-editing technologies each have their own particular strengths, he said. Therapeutic approaches using some of them are far enough down the translational path to clinical use — in some contexts, much further along than CRISPR — that he and his colleagues don’t see any of the other three being abandoned any time soon.

“Just because something is hard to make doesn’t make it any less useful once you’ve made it,” Stoddard said.

Dr. Chris Peterson
Dr. Chris Peterson is a Fred Hutch staff scientist and self-proclaimed "zinc-finger aficionado."

First to first-in-human trials

Zinc-finger nucleases were among the first true gene-editing nucleases made and are arguably the closest to clinical use, said Fred Hutch staff scientist and zinc-finger nuclease aficionado Dr. Chris Peterson. Like the other non-CRISPR gene-editing platforms, zinc-finger nucleases are a sort of Franken-protein, stitched together from pieces of naturally existing proteins to make a new kind of gene-targeting machine.

Peterson is working on the laboratory team of stem cell transplant researcher Dr. Hans-Peter Kiem on research to cure HIV infection by disabling a protein known as CCR5, the cell-surface “door” HIV most commonly uses to enter and infect immune cells. Using an animal model of HIV infection, Peterson uses a pair of zinc-finger nucleases to disrupt the gene coding for CCR5 in blood stem cells, which are then transplanted back into the animals to repopulate their immune systems with the modified version of CCR5.

His project is part of the Fred Hutch-headquartered defeatHIV consortium. Gene editing’s potential to cure HIV is ahead of other clinical applications for these technologies, Peterson said, because the CCR5-based approach is focused on mimicking a naturally occurring — and safe — mutation. Some people of Northern European and Middle Eastern descent have the CCR5 mutation that researchers are hoping to copy through gene editing. Those born with this mutation have minimally discernable health effects other than a natural resistance to HIV.

“Since we know how important [CCR5] is to HIV, it was sort of a slam dunk to start there for the gene-editing field,” Peterson said.

Kiem and others on the defeatHIV team are partnering with Sangamo BioSciences, a biotech company that develops and tests many of the zinc-finger nucleases in research use today. And these proteins have one big leg up on CRISPR so far. Researchers from Sangamo, the University of Pennsylvania and the Albert Einstein College of Medicine in New York have published results from a clinical trial using one such set of nucleases to disrupt CCR5 in the immune cells of HIV-positive patients. The technique appears safe and might even be slowing HIV’s progression, although none of the patients in that trial are truly cured of their infections; the gene edit didn’t happen in enough of their cells to protect the rest.

While this early-stage clinical trial is just the first step toward curing a disease with gene-editing tools, this early success in humans is both a boost for the gene-editing field overall and can act as a building block for future trials using other nucleases.

“There’s a lot of excitement that they’re doing these first-in-human trials [with CRISPR], but zinc fingers are already there,” Peterson said. “So there’s a lot we can learn about gene editing in human trials based on what’s already out there.”

Other nuclease technologies have been tested in humans as well.  Last year, the French biotech company Cellectis made worldwide headlines when it was announced that a baby girl in the U.K., Layla Richards, had received the world’s first T cells engineered with a TALEN gene-editing protein to treat her leukemia.

A zinc-finger nuclease
An image of a zinc-finger nuclease Image courtesy of Dr. Barry Stoddard

Dark horses and reducing side effects

Stoddard, Seattle Children’s researcher Dr. Andrew Scharenberg and Dr. Jordan Jarjour, at the time a University of Washington graduate student working in Scherenberg’s lab, engineered megaTALs, yet another gene-editing protein on the scene.

“MegaTALs are sort of the dark horse in the race right now. They don’t get as much press as the other platforms,” Peterson said.

Crafted from a fusion of a TALEN DNA-binding platform and a fungal DNA-cutting enzyme, megaTALs’ potential for clinical impact comes from their small size and particularly high specificity for their gene target. The proteins recognize a piece of DNA that’s much longer than those targeted by other gene-editing platforms, Stoddard said. That’s important because there’s less of a chance that long target sequence would randomly exist elsewhere in the genome than the spot gene researchers want to snip — what’s known as “off-target effects,” where the nucleases cut extra DNA.

MegaTALs are now being developed for clinical use by the Cambridge, Massachusetts, biotech company bluebird bio in collaboration with Stoddard and Scharenberg. Stoddard thinks their technologies should be ready for clinical trials in the next few years. 

That balance between specificity (cutting only where you want to cut and not elsewhere) and efficiency (cutting as close to all of the target DNA as possible) is what will ultimately make any of these gene-editing platforms clinically useful. Right now, researchers have looked for spots elsewhere in the genome they think the nucleases might cut — and many approaches, such as the zinc-finger nucleases with which Peterson works, show no detectable snipping outside the desired gene target.

And, of course, there are the early clinical trials showing that some of these proteins don’t seem to have any noticeable side effects. But Peterson thinks that sequencing the entire genome will be the next step to ensure these techniques are truly safe.

“There could be cutting in places we never imagine for reasons we never imagine,” he said. “Having as extensive an off-target profile as possible is going to be a very important part of permitting any nuclease therapy to go into people.”

As much as researchers may have their gene-editing tactic of choice, what really matters is the end result, Peterson said.

“Being able to show they’re working to treat diseases and make a meaningful impact in patients with those diseases — that’s more important than what the platform is,” he said.

Rachel Tompa is a former staff writer at Fred Hutchinson Cancer Center. She has a Ph.D. in molecular biology from the University of California, San Francisco and a certificate in science writing from the University of California, Santa Cruz. Follow her on Twitter @Rachel_Tompa.

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