Christina Bennett Freelance Writer GEN
CRISPR Is Not Yet King of the Clinic
A few weeks ago, for the first time, a patient had his genes edited in vivo to treat a genetic disorder. His veins were infused with the investigational genome editing therapy SB-913 to treat his disease, mucopolysaccharidosis type II (MPS II). MPS II, often called Hunter syndrome, is a rare genetic disorder caused by a mutation in the iduronate 2-sulfatase (IDS) gene and occurs almost exclusively among males. The therapy uses the gene-editing tool called zinc finger nucleases to cut out the IDS mutated gene and replace it with the healthy gene.
The patient received the therapy as part of a Phase I/II clinical trial. Eight more adult males are expected to receive the therapy. Sangamo Therapeutics is the manufacturer of the investigational genome-editing therapy and sponsor of the clinical trial. Sangamo tells GEN it hopes to have preliminary data “sometime in 2018, around the first half of the year.”
However, amidst the enthusiasm of this notable event, criticism has emerged regarding the gene-snipping tool used. Critics say that the zinc finger nuclease platform is an outdated technology; a relic of the pre-CRISPR era. GEN talked with Sangamo, as well as other experts, to explore this criticism and the current state of the technology.
Twenty-Year vs. Four-Year Track Record
“What we know is the zinc finger nuclease technology works. There’s no question about that,” Charles Gersbach, Ph.D., tells GEN when questioned about whether the zinc finger nuclease platform is an outdated technology. Dr. Gersbach is Rooney Family Associate Professor of Biomedical Engineering at Duke University and has worked with several genome-editing platforms: zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR).
He disclosed no conflicts of interest with Sangamo but does have financial interests in CRISPR technology and works with other companies performing gene editing. “Fundamentally, all these technologies do the same things. They cut DNA, and once you get past the point of cutting the DNA, all of the rest of the aspects of genome editing are the same.”
Sangamo has been studying zinc finger nucleases for 20 years and is the primary patent holder for this technology. As a result of research and development efforts at Sangamo, zinc finger nucleases have achieved several firsts in the gene-editing space: the first to edit genes in human cells, the first to edit genes ex vivo (i.e., cells removed from the body, modified, and then then infused back in), and now the first to edit genes in vivo (i.e., editing genes in cells still intact in the body). Zinc finger nucleases also have applications in drug discovery, but Sangamo tells GEN it is not currently exploring that application; Sigma-Aldrich holds that license.
“The first key difference that sets zinc finger nucleases aside, apart from all the other platforms, is the length of time that people have been working with them,” says Fyodor Urnov, Ph.D., associate director of Altius Institute for Biomedical Sciences, a nonprofit research organization funded by GlaxoSmithKline. Dr. Urnov was an employee of Sangamo for more than 16 years and helped develop the zinc finger nucleases platform. He is no longer an employee of and no longer owns equity in Sangamo. He disclosed that he has no relevant financial conflicts of interest. “The essential goal of any clinical experimental medicine program is safety first, so it's essential to appreciate that zinc finger nucleases have been used on over 100 subjects in clinical trials, and the safety record has been a good one so far.”
Sangamo has completed several early-phase clinical trials for patients with human immunodeficiency virus (HIV) as well as for other diseases.
CRISPR entered the gene-editing scene in 2013, and while several U.S. gene editing companies are on the cusp of bringing CRISPR-based therapies to clinical trials, clinical development hasn’t started yet. CRISPR Therapeutics announced plans to begin the first U.S. clinical trial evaluating a CRISPR-based therapy in 2018. Back in October 2016, China began the first clinical trial for a CRISPR-based therapy.
The primary criticism of zinc finger nucleases as a gene-editing tool is the time and skill it takes to develop a high-quality gene snipping product compared with CRISPR.
“In order to build your own guide RNA to use CRISPR/Cas9, you need basically a high school education in biology,” says Dr. Urnov. “Zinc finger [nuclease] requires sophisticated engineering.”
While this clear difference still exists, Sangamo has tweaked the development process, thereby shortening the development timeline for zinc finger nucleases and making it less laborious.
Edward Rebar, Ph.D., vice president of technology at Sangamo, tells GEN that the past three years has been “a major transition for us.” He has worked at Sangamo for about 18 years.
“Historically, as the field was originally practiced many years ago, zinc finger proteins often were designed and optimized using methods that were, in their day, labor intensive,” Dr. Rebar says. He recalls that in the past, lead development cycle times could run up to three months and the total time to identify the final therapeutic ZFN would typically take a year. Now, he says, the lead development cycles have been shortened to as few as 10 days, during which constructs are designed, assembled, and tested in cells, and the total time required to produce a final therapeutic ZFN has been shortened to three months. “These days, we make our proteins using a completely different approach.”
Dr. Rebar explains that they assemble the zinc finger nucleases using precharacterized modular parts, which allows then to rapidly develop proteins. He adds that they have also recently developed ways of optimizing specificity that are systematic and predictable. In 2016, Sangamo published a paper in Nature Communications in which they developed a system that could select which zinc finger nucleases cleave genes at the correct location at a high rate and have a low rate of off-target activity.
“People have talked about us being an old technology, a technology that’s not as versatile or as specific as CRISPRs, and with some of the more recent publications and presentations that we've done, we've really tried to dispel those myths and take them head on,” Michael Holmes, Ph.D., vice president of research at Sangamo, tells GEN. Dr. Holmes has worked at Sangamo for about 16 years. For example, earlier this year the company published preclinical data showing the successful knockout of the gene in an attempt to treat sickle-cell disease.
Apples to Oranges, for Now
Genome-editing platforms—in this case, zinc finger nucleases and CRISPR—are often compared head-to-head based on several parameters, but experts cite several reasons to avoid such blanket comparisons.
“The simple answer I can give as far as whether there’s a general difference between them, this is like asking whether there is a difference between the Apple iPhone and the Google Pixel. So, they're both smartphones. They work really well. They run many different kinds of apps, and you can use them to do many wonderful things. The differences that exist are differences of the nuanced and specific context,” says Dr. Urnov.
“Until we have a record for what Cas9 has done clinically—it’s a little bit difficult to compare. It’s not an apples-to-apples comparison because one platform has been in the clinic and the other platform has not,” Dr. Urnov says, referring to CRISPR and zinc finger nucleases, respectively. He says in general, he believes both platforms have comparable specificity and efficiency.
Dr. Gersbach echoes the sentiment: “Everyone wants to play this game of which one’s better, which one’s more specific, which one’s more active, and it all depends on the exact reagents.”
“Sangamo has been doing the zinc finger nuclease stuff for a long time, and they've reported a lot of non-human primate studies and they've already done ex vivo gene-editing studies in humans,” says Dr. Gersbach. “CRISPR…we don't have as much data for.”
He adds, “To my knowledge, as of right now, it's not clear that one technology is advantageous over another.”
Dr. Urnov notes that one disadvantage of CRISPR is the inflexibility to target any gene sequence, unlike zinc finger nucleases. He explains that for CRISPR to work, the target gene sequence must be immediately upstream of a protospacer adjacent motif (PAM). “Zinc fingers have no restriction of that type.”
But within the research setting, CRISPR is likely the preferred platform. Dr. Gersbach says, if you’re new to the field or an academic performing a lot of experiments, “there's no reason to work with zinc finger nucleases because CRISPR is just much easier, cheaper, and faster to work with.”
“Despite the broad excitement about CRISPR, the earlier platforms—ZFNs and TALENs—still have utility,” Dana Carroll, Ph.D., tells GEN. He is a distinguished professor in the department of biochemistry at the University of Utah School of Medicine, and as a one of the original founders of the zinc finger nuclease technology, he receives license royalties from Sangamo.
CRISPR for In Vivo Gene Editing
Dr. Carroll points out that the zinc finger nuclease platform is not the only gene-editing tool that could work for in vivo modification of patients with Hunter’s syndrome, referring to the first patient treated a few weeks ago.
“This could, of course, be done with the CRISPR platform as well,” he says. “Delivery of both the zinc finger nucleases and the donor DNA was accomplished with adeno-associated viral (AAV) vectors, and zinc finger nuclease coding sequences fit comfortably into such vectors. Because AAV has a limited DNA carrying capacity, it is a bit more difficult to squeeze the genes for Cas9 protein and a guide RNA into a single vector, but modifications have been made that make this more feasible. I wouldn’t be surprised to see similar uses with CRISPR in the future.”