Genetic Engineering & Biotechnology News

DEC 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Page 43 of 69

10 | DECEMBER 2017 | and specificity," says Edward Rebar, vice president of technology at San- gamo. The focus will have to be on reducing off-target effects, improving delivery, creating robust reagents with dependable effects, and being able to detect all of any treatment's effects. The Rise of CRISPR While ZFPs, TALENs, and oth- ers might stay in the race, most of the attention now is in the CRISPR arena. Developed approximately five years ago, CRISPR caps almost 30 years of research on gene editing. It contains a dual RNA structure, one part of which binds naturally to a complementary DNA sequence and an endonuclease enzyme that snips and degrades nucleotides. The system is natural to the bacterial immune system, helping them defend against the DNA of attacking viruses. Key details of this process were published in a landmark Science paper in 2012, and it soon became the basis for a widely used DNA-editing technique. The system works with elegant simplicity. The CRISPR-associated (Cas) proteins recognize the sequence, hold the endonucleases in place, and then unwind the target DNA so the sequence becomes visible to the matching CRISPR RNA (also called the "guide" RNA). The most popular version of the system uses the CRISPR/Cas9 complex, although CRISPR/Cpf1 is now also coming into use. When the targeted sequence binds to its target, the Cas protein cuts both DNA strands at the chosen spot. While understanding all this was helpful, the really big break- through was the realization that it is possible to program the protein, using a single RNA, to cleave almost any DNA sequence. That sealed the technology's status as a break- through, and is why CRISPR/Cas9 is often referred to as "programmable molecular scissors." CRISPR/Cas9 can recognize as few as 20 base pairs of comple- mentary sequence, as long as that sequence is followed by a proto- spacer associated motif (PAM). That PAM serves as the binding signal for the complex. By mixing in a short DNA sequence, the system can be used to correct single-base mutations. It can either shut down or ramp up gene expression; it casn alter genes for noncoding RNA; and it can even edit RNA directly (if the Cas9 is suit- ably modified). and to edit messenger RNA or non-coding sequences. This technology has already transformed plant and microbial research, and is having a similar impact on the devel- opment of animal models. CRISPR Contenders Preclinical studies with CRISPR have been enticing. It has, for exam- ple, shrunk human prostate cancer tumors in mouse xenografts and the group, Basel-Duby works with at UT used it to successfully treat mice with Duchenne-like muscular dystrophy. And then there are those studies of embryos, which have shown that it is indeed possible to repair genes even at that early stage. These reports have prompted more labs and compa- nies to look at clinical applications of the technology. But while many large pharmaceutical companies and biotechs are using CRISPR or looking at incorporating it in their pipelines, largely because of patent issues, there are few companies using the platform as the backbone of their business. As noted earlier, one of the most attractive targets is the CAR-T therapeutics that have gotten so much attention. Poseida is one of the com- panies with an eye on that market. CEO Eric Ostertag, M.D., Ph.D., said their NextGEN CRISPR technology solves many of the problems inher- ent in other gene-editing techniques. "First-generation CRISPR is great, but it is an inherently sloppy system," he said. "It will cut your 20–base pair target but it may also make unwanted cuts at similar sites that could differ from the target by as many as five nucleotides." TALENs and ZFPs, he says "Are quite clean in terms of specificity, but they require expertise in reagent design and construction." Poseida's technology uses a Cas9 protein that is mutated so that is has no nuclease activity. It's also bound to a nuclease called Clo051, which Dr. Ostertag describes as an "obligate heterodimer." In this system, the Cas9 guide RNA only works as a binding protein to determine site specificity. And the nuclease only cuts when the two fused proteins come together at the same target site, at the same time. "That increases specificity because it is more similar to the earlier dimeric systems," Dr. Ostertag noted. Poseida is using this technology with the CAR-T therapeutics in their pipeline, which include treatments for multiple myeloma, prostate cancer, and other malignancies. Crispr Therapeutics, Editas Medi- cine, and Intellia Therapeutics are all "pure play" CRISPR-based therapeu- tic developers with cofounders who helped pioneer the technology. All of these are also leaning heavily on deals with pharmaceutical companies. Crispr Tx was cofounded by Emmanuelle Charpentier, Ph.D., now director of the Max Planck Institute for Infection Biology in Berlin. Dr. Charpentier was one of the coauthors, along with Jennifer Doudna, on the pivotal 2012 Science paper describing the CRISPR/Cas9 gene-editing process. Intellia uses that specific approach, and has deals with Vertex Pharma- ceuticals and Bayer AG. Its pipeline contains projects for cystic fibrosis, Duchenne muscular dystrophy, hemo- philia, sickle-cell disease, and beta- thalassemia. All of those programs are preclinical, although an IND has been filed for beta-thalassemia. Editas Medicine was founded by several leading lights in genome edit- A Dose of CRISPR Continued from page 8 B E S T O F C R I S P R 2 017

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