Genetic Engineering & Biotechnology News

AUG 2018

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 16 of 33 | Genetic Engineering & Biotechnology News | AUGUST 2018 | 15 forehand and afterwards to really understand the underlying basic biology and genomics." Only then, he suggests, can developers use CRISPR to leverage the natural genetic di- versity in agricultural crops and accelerate plant breeding. "Separate and apart from our work in row crops, we are exploring gene editing applica- tions for a wide variety of items you see every day in the produce aisle," states Tom Adams, Ph.D., CEO of Pairwise. Until recently, gene editing has been primarily involved in elimi- nating traits, yet Pairwise is working, Dr. Ad- ams asserts, toward "enabling more subtle changes like modifying a single amino acid to enable new combinations of natural variants." Arbor Biotechnologies In addition to participating in Beam Therapeutics and Pairwise Plants, Dr. Zhang is contributing his expertise to Arbor Bio- technologies, a CRISPR startup that counts him among its co-founders. This New Eng- land–based early-stage life sciences organiza- tion garnered approximately $15.6 million in Series A funding back in June 2017. In a recent issue of the journal Molecular Cell, the company's researchers reported the discovery of a new CRISPR enzyme, Cas13d. This protein, which is smaller than other Cas proteins, may be more easily packed into the viral vectors that are used to deliver CRISPR components. Arbor's discovery platform, which incorporates genome sequencing and artificial intelligence technologies, is credited with discovering the enzyme. Using the plat- form, Arbor's research team hopes to con- tinue its identification and optimization of CRISPR proteins for constructing innovative biotechnology applications. CasZyme CasZyme, a CRISPR startup based in Vil- nius, Lithuania, boasts a relationship with another CRISPR pioneer: Virginius Šikšnys, Ph.D., one of the first researchers to show that CRISPR-Cas9 can be "programmed" to create double-strand breaks in specific DNA sequences. Dr. Šikšnys founded CasZyme and serves as the company's chair. CasZyme, like other CRISPR startups, embraces collaborations with other bio- technology firms. A partnership between CasZyme and New England Biolabs is fo- cused on identifying and commercializing CRISPR-Cas nucleases. In another partner- ship, CasZyme enjoys access to DuPont Pio- neer's CRISPR-Cas library. CasZyme is ap- plying its biochemical assays and expertise to characterize the Cas nucleases. DuPont Pio- neer intends to use the nucleases to advance its plant breeding efforts, which are aimed at the development of seed products that have greater environmental resiliency, productiv- ity, and sustainability. Locus Biosciences Locus Biosciences was founded three years ago to pursue healthcare-related CRISPR applications, primarily precision antimicrobi- als. The company is particularly focused on using the Cas3 helicase/nuclease. Unlike Cas9, which surgically cuts double-stranded DNA, Cas3 "shreds" single strands of DNA that have been separated from their matching strands. Cas3 works with a riboprotein complex known as Cascade. When Cascade recognizes foreign DNA, it binds to a target sequence and causes double-stranded DNA to form a loop, which attracts the attention of Cas3. Joining Cascade and seizing the loop, Cas3 reels in the target DNA, separates its strands, and cuts one of the strands repeatedly along its length while, at the same time, continuing to yank more DNA through the Cascade-Cas3 machinery. Cas3, which is far more prevalent than is Cas9 in bacteria and archaea, may be ex- ploited, Locus asserts, to therapeutic effect. Essentially, Cas3 can induce a pathogen's CRISPR bacterial immune system to attack the pathogen itself. While Cas3 may one day find use as a precision gene editing tool, it has, to date, been of interest mainly for its antimicrobial potential. For example, Cas3 may prove valuable in countering antibiotic- resistant strains of bacteria. Locus' chief technology officer, Dave Ousterout, Ph.D., emphasizes that currently, Cas3 therapeutic relevance depends on the enzyme's ability to damage targeted DNA beyond repair. "Cas3 behaves as a unique single-stranded exonuclease," he says. "It binds DNA and then rapidly degrades a single strand up to 1,000s of base pairs away from where it originally targeted the DNA." "There are no known systems that can ef- ficiently repair DNA lesions caused by Cas3," See CRISPR Startups on page 16 OMICS Kunwoo Lee, Ph.D. Two years ago, I had the opportunity to meet Roelof Botha, a prominent Silicon Valley venture capitalist, and describe the gene editing–delivery technology I'd been working on dur- ing my Ph.D. as well as its potential applications. After hearing me out, he asked: "How different will the life of a human being be decades from now be cause of this technology?" I thought about this for a minute. "It will be a really differ- ent world," I replied. My CRISPR story begins in 2012. While I was a graduate student at UC Berkeley and UCSF, word spread of a new pro- tein with the ability to precisely cut DNA sequences. Soon after, Jennifer Doudna, Emmanuelle Charpentier, Feng Zhang, George Church, and Jin-Soo Kim became CRISPR pioneers, publishing a series of groundbreaking papers about this novel technology. Their research inspired me to believe that gene editing has the potential to change medicine by curing ge- netic disorders. With the support of Doudna and Stanley Qi, my colleagues and I initiated a research project developing a delivery system for the CRISPR-Cas9 ribonucleoprotein. Upon completing my Ph.D., I had two different career paths in front of me. One was academia, which was my dream enter- ing graduate school. The other was the industrial route, working to enhance CRISPR-based gene therapies through the develop- ment of a unique delivery system. A voice deep inside my heart inspired me to take the road where I could make the biggest impact on people's lives. Envisioning a future where CRISPR- based therapeutics could cure disease, I embarked on a journey of biotechnology, with the aim of revolutionizing gene therapy. Launching a Company In 2016, my co-founders Hyo Min Park, Niren Murthy, and I launched GenEdit, with the goal of creating the next genera- tion of gene editing –based therapeutics. We set our vision, built a scientific team, and pitched our idea on how to shape the future of medicine. It is pure excitement to develop our technology and realize the company's potential. At the same time, we also encountered many of the inherent challenges that come with launching a startup company: financing, management, and responsibility. Con- vincing investors to believe in our vision required translat- ing our scientific concepts into the language of business, including financial returns. I have endured many sleepless nights debating the direction of GenEdit. Velocity matters: high velocity means not only high speed but also the right direction. As CEO, I must decide the right course, which can be a lonely position at times. My advice to other young entrepreneurs is to learn from your mentors. Every meeting and interaction I have is a chance to be taught something new. I am fortunate to have many opportunities to meet with entrepre neurs and pharma executives who have kindly shared their experiences, offered valuable input, and helped me find direction. On the technical front, delivery of CRISPR to a specific target tissue is an unsolved challenge. Our focus is polymer nanopar- ticle technology. Polymers are advantageous in that they can deliver a protein form of CRISPR-Cas, unlike viral delivery or lipid nanoparticles. Moreover, various polymer structures have a unique interaction with cellular receptors and serum pro- teins, which are important parts of targeted delivery. In order to screen for the right polymer nanoparticle for each target tissue, we have synthesized a library of polymer nanoparticles with different sizes, charges, and targeting molecules. Our initial results have been encouraging, as polymer nanoparticles showed efficient delivery of the Cas9 protein and guide RNA in muscle and neural tissues with direct injec- tion. With the support of many superb academic collabora- tors, our talented scientists have delivered novel polymer nanoparticle systems that work in a variety of animal models, including Duchenne muscular dystrophy and fragile X syn- drome (Figure). Our recent publications illustrate that nonviral gene edit- ing induces behavioral or functional changes in disease ani- mal models. Our next objective is to screen for systemically injectable nanoparticles. We are pushing the boundaries of in vivo gene editing and aim to move into the clinic in the near future. A look back at the history of RNA interference, reveals that a huge amount of engineering work was conducted to achieve clinical translation. Even though CRISPR has already proven its potential in many preclinical studies, the CRISPR field is still in its infancy. Therefore, another innovation I antici- pate in this field is the engineering of CRISPR systems, such as enhancement of the guide RNA and Cas9 protein. Additionally, donor DNA is a necessary component if a genetic disorder requires precise gene correction by homol- ogy-directed repair. Multiple components need to work in an orchestrated way to guar antee the highest performance of CRISPR-based thera peutics. This is a field that basic and translational science needs to explore further; we are taking a systematic approach with an engineering mindset. My experience has taught me that there are many paths to being an innovator. As Steve Jobs said, "We are here to put a dent in the universe." With my team at GenEdit, I aim to put a dent in next-gener- ation medicine by transforming CRISPR technology to realize the dream and future of gene editing. Kunwoo Lee, Ph.D. (, is CEO and co-founder of GenEdit ( *Reprinted with permission of The CRISPR Journal, published by Mary Ann Liebert, Inc. (, Vol. 1, No. 3, 2018 Realizing the Dream Figure. Polymer nanoparticles can deliver CRISPR protein and guide RNA to specific tissues. Kunwoo Lee, Ph.D.

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