| Bio-IT World News Bulltin|
By Joe Stanganelli
February 22, 2017 | CRISPR/Cas9 gene-editing technology has dominated both headlines and genomics laboratories alike over the past year. Furthermore, it is likely to continue to do so throughout 2017 because of a bevy of new announcements related to the science and continued development of CRISPR/Cas9 as well as both the likely-to-be-appealed USPTO ruling on the seminal CRISPR/Cas9 patents and the still undetermined ownership of European patent rights to seminal CRISPR/Cas9 technology—factors that continue to be a concern for healthcare and life-science organizations, which may have CRISPR/Cas9 licenses from only one party or set of parties in interest in these patent disputes.
Nevertheless, impressive and game-changing as it has been, CRISPR/Cas9 is far from the be-all and end-all of current gene-editing innovations. CRISPR proteins other than Cas9 are showing promise in gene-editing therapy, new discoveries in gene-editing demonstrate potentially safer and more accurate genetic-modification methodologies, and other gene-editing technologies that have been around for years continue to be relevant and useful alternatives to CRISPR.
With all the hoopla over CRISPR/Cas9 in the headlines, however, the casual industry observer would hardly know it.
ZFNs and TALENs Taken Down by CRISPR
Before the CRISPR revolution, along with zinc-finger nucleases (ZFNs) and some other then-popular options, gene-editing and gene therapy researchers relied heavily on transcription activator-like effector nucleases ("TALENs"). In the age of CRISPR/Cas9, however, demand for ZFN- and TALEN-based solutions is far less visible (despite, among other advances, a ZFN technique representing a major milestone in HIV gene therapy in 2014).
"Most people will consider using a CRISPR before they consider using a TALEN [or other gene-editing alternative] because of the ease of design and implementation [of CRISPR]," said Ross Whittaker, a life-sciences solutions product manager at Thermo Fisher Scientific, in an interview with Bio-IT World. "It's not just hype; it's the utility of the tool—and it's a little bit more accessible sometimes."
As the person who manages Thermo Fisher Scientific's CRISPR library program (including the company's recently launched Invitrogen LentiArray CRISPR Libraries), Whittaker has been around the genomics block long enough to see and understand how demand in the gene-editing market has fundamentally changed. For Whittaker—and his customers and colleagues—CRISPR is winning out over TALENs and other "legacy" gene-editing techniques because of the sheer power of CRISPR gene editing as not just a process, but also a platform. Because CRISPR has enjoyed such skyrocketing popularity, and because there are more resources available related to CRISPR, younger genomics researchers are largely cutting their teeth on CRISPR instead of on older solutions like TALENs. Meanwhile, TALEN prototyping is known for requiring highly specific expertise in the complex molecular operations of protein engineering—thus having a higher barrier to entry. Additionally, skills and experience aside, TALENs can sometimes be more burdensome to work with than CRISPR/Cas9.
It is of little surprise, therefore, that Whittaker reports seeing a broader and better customer experience for CRISPR tools.
"There are a couple of parts to it. The design and construction of the [CRISPR] tools themselves is much easier—and the popularity means there are more resources… within institutions [and] within companies, where people may not have used TALENs before but they have used CRISPR at this point," said Whittaker. "When people start hunting around for help on how to do a genome-editing project, they are usually finding, more often than not, that [more] people are using CRISPR than TALENs—so that is driving CRISPR adoption."
To be sure, TALENs are more expensive and complex to deal with than CRISPR, and yet reports of the death of the TALEN market have been greatly exaggerated.
Turning Back to TALEN
For starters, as a rising tide lifts all ships, the enormous interest in CRISPR has helped bring researchers to the TALEN table.
"When TALENs were the only solution, it was more of a niche market. The ease of implementation… of CRISPR has actually grown the interest in genome-editing markets substantially [overall]," said Whittaker, speaking of the gene-editing vertical. "They are still interested in TALENs, but a lot of those new customers are coming in… because of the CRISPR hype."
To be certain, interest in using TALENs has continued to grow in the face of CRISPR/Cas9 hype because of how permissive TALEN technology is compared to that CRISPR/Cas9. In particular, whereas Cas-related proteins will not make cuts on invading DNA without the accompaniment of different protospacer adjacent motif (PAM) sequences for different nucleases, TALENs do not require PAM sequences at all. Thus, TALENs make gene edits accessible to genomicists when guide sequences are not readily available—often enough to support the expansion of the TALEN market.
"With the TALENs, we've definitely been expanding our R&D programs there and looking at some of the advances of using TALENs with a design space because they don't require a PAM sequence," said Whittaker. "So your design space is relatively unlimited, which is interesting when it comes to knock-in models when you need to get your double-stranded break—your DNA break—in a very specific place. So [TALENs] definitely have their place in the genome-editing toolbox."
Despite the revolutionary strength and efficiency of CRISPR, the at-times burdensomeness of working with TALENs truly does have yet a bigger payoff; TALEN precision has been found to be the most precise of nuclease technologies; multiple studies have demonstrated that TALENs have lower levels of "off-target activity" than do ZFNs and CRISPR nucleases. Therefore, because TALEN-based gene editing is both much safer (because it is far more precise) and has been around much longer than CRISPR-based gene editing, an eventual bevy of TALEN-based therapeutics may come to market several years before CRISPR-based gene-editing therapeutic solutions ever see the light of day.
RNA Solutions Inspiring—and Complementing—CRISPR and TALEN Permanence
This is not to say that the choice between CRISPR and TALENs (or other "legacy" gene-editing technologies) in a particular given situation is a binary one. Gene knock-ins and knockouts—effective and exciting, yet risky—are but one way of using CRISPR. Thermo Fisher Scientific is but one of numerous life-science companies working on new and alternative ways of using CRISPR for gene-editing and gene-therapy purposes to complement primary Cas9 gene-editing uses. For starters, the company is in the process of introducing new CRISPR-related technologies, CRISPRi ("CRISPR interference") and CRISPRa ("CRISPR activation"), to help would-be gene editors gain the benefits of CRISPR while blunting the permanent edit effect that CRISPR/Cas9-based gene editing tends to have.
"[W]e are trying to inhibit [or activate, respectively] the mRNA to be produced from the gene without trying to introduce a permanent edit… [CRISPRi] is very analogous to using RNAi, except we're using CRISPR to do this," explained Whittaker. "That's a little bit more in the future as we spend time learning more about the design tools that work best for that type of technology."
Currently, RNA-based screening technologies are used in cases where permanent edits are deemed too risky or are otherwise not desirable. RNAi is often the current go-to for such aforementioned gene knockdown uses, "without introducing a permanent edit," according to Whittaker. The "i" in RNAi stands for "interference," but Whittaker likens it to "interrogating" individual genes and their roles—with the eventual aim of performing complete gene or gene-expression knockouts later with CRISPR. This measure-twice-cut-once validation process thereby allows genomicists more confidence in the results of a screen.
During such screens, however, the visibility of a genetic phenotype is much stronger with a complete knockout of an attendant gene expression. Sometimes, the phenotypes cannot be identified at all via these weaker RNA-based technologies, thus the demand for CRISPRi and CRISPRa.
"From the patient perspective, [CRISPRi] potentially opens up the number of opportunities" for applications, said Whittaker, "because we are able to knockout the gene expression—and, therefore, we are able to identify that gene."
Don't expect CRISPR to "knockout" RNAi from genomicists' toolboxes, however.
For one thing, RNAi has a speed advantage over CRISPR technologies. Whittaker reports that RNAi can be applied and the assays can be conducted "within 48 hours"—compared to the "weeks to months" it can take to isolate a gene or aspect thereof via CRISPR.
For another, CRISPR-based edits are far more lasting than RNA-based ones. As such, there are times when genomicists should use the two in tandem.
"RNAi is a complementary technology; there are certain targets, particularly what we call essential genes—these are genes the cells need to survive—we wouldn't want to knock out completely, so we can still understand those genes," cautioned Whittaker. "You could have an idea, you could test it very quickly with RNAi, [and] then [use] CRISPR to knockout [the gene]."
Meanwhile, CRISPRa does "completely the opposite" of CRISPRi—by knocking up gene expressions that are absent, silent, or otherwise underexpressed.
"With CRISPRa, we're… trying to activate the expressions of gene and use that in a screen mode," explained Whittaker, "which is pretty new since mostly screening is knockout or knock-in based[.]"
Knocked Up: mRNA's Pregnant Place in the Therapeutics Market
Hence, CRISPRi is to RNAi as CRISPRa is to mRNA; the use of mRNA itself to "turn on" gene expressions has been explored for more than four years as a potentially safer alternative to lasting gene edits.
"Our thesis is that you're not making a permanent change to anyone's genome. Our mRNA is gone within a period of time. If there was some sort of negative affect to the mRNA or the protein—some sort of safety issue—well, it's going to be gone in some period of time," Lorence Kim, CFO of Moderna Therapeutics, told Bio-IT World in an interview on Moderna's own mRNA research and solutions. "We think that's got utility because you don't have quite as high a bar, and the two technologies can co-exist."
Kim went on to address mRNA's other uses beyond the knockup of genetic expressions to create missing proteins and otherwise right genetic imbalances, such as its role in finding a Zika vaccine (for which, Kim reports, CRISPR/Cas9 has no use case). He hinted, however, at the added bottom-line benefit the temporary "on switch" of mRNA gene-expression knockup offers for biopharmaceutical companies—as they attempt to bring chronic, likely lifelong drug therapies (in lieu of outright genetic cures via CRISPR/Cas9, TALEN, or the like—for which the acquisitions of safety approvals are potentially more difficult and more expensive) to what they anticipate will be a demanding segment of the therapeutics market.
"We do see new opportunity for this new class of [mRNA] instruction sets to become a major part off the pharmaceutical industry as we look long term, 10 to 20 years out," said Kim. "That's what the pharmaceutical industry has done for its entire history: come back to… a patient, and you can treat somebody chronically."
Trends and Perspectives on CRISPR and Company
Indeed, the item of primary importance to remember, despite all of the hullaballoo over CRISPR and even its gene-editing complements/competitors, is that translating gene-editing technology to a therapeutic is an extremely long—and expensive—row to hoe for pharmaceutical companies, clinicians, and patients alike. Even RNA-based gene therapy has its lingering safety hurdles—leading to market setbacks that have caused pharmaceutical researchers to go back to the drawing board on their drug programs.
As it stands now, only a couple of gene therapies have even been approved by regulatory bodies—and both on the European side of the pond. The first, Glybera (developed by UniQure) was approved in 2012 for treating lipoprotein lipase deficiency, a rare kidney disorder. It has been blasted as "a bust"; carrying a $1 million price tag, Glybera has successfully treated a patient but once. The second, Strimvelis (developed by GlaxoSmithKline), was approved just last year for treating ADA severe combined immune deficiency (ADA-SCID, a.k.a. "bubble boy disease")—bearing a price tag well north of half a million dollars. GlaxoSmithKline has admitted that it does not expect Strimvelis, in and of itself, to bring a profit—but the company has indicated that it hopes to achieve long-term gains via a platform-based economy as gene-based therapeutics continue to develop.
Accordingly, given the sheer number of gene edits that researchers are considering, according to Whittaker—especially as researchers are "moving away from just making one or two knockouts to knockouts of whole pathways, or doing a whole lot more knock-in mutation or snip mutation, making different mutants or different proteins"— the only way for gene-editing innovation to work, economically, is with enhanced scalability and streamlining.
"There are three areas that we're really focused on and we're seeing as trends. First, it's scale," said Whittaker of the gene-editing market. "The second is really focused on workflow optimization, and that's something that, as a company, we spend a lot of time doing—especially as we see rapid adoption of the tools … And that speaks directly to the third part, which is efficiency… making it easier to isolate the clone parts you wanted at the end of the day."
The gene therapy market of the late 20th and early 21st century, for want of these aspects, could not realize great success. Now, genetic-modification market demands are made possible by the efficient, optimized, and scalable nature of both CRISPR/Cas9 and nascent CRISPR/Cas9 complements.
"I'm really looking at this as an amazing time to be involved in biology with what we're seeing and what we've seen develop over the last ten years," noted Whittaker. "It's really giving us a whole new toolbox to ask questions in a way that we weren't able to do before—so it's a fantastic time to be a life scientist."