F E AT U R E

Nature Biotechnology’s academic spinouts of 2014 Aaron Bouchie & Laura DeFrancesco

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Ventures focusing on drug testing or therapies against rare disease, cancer, gastrointestinal disease, fibrosis and pain are among those selected by the editors in 2014’s crop of startups.

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Unum Therapeutics: a universal cellular immunotherapy

Aaron Bouchie is a freelance writer based in Ithaca, New York. Laura DeFrancesco is Senior Editor, Nature Biotechnology.

Engineering T cells to capture approved or experimental therapeutic monoclonal antibodies. The past few years have seen unprecedented investor and big pharma interest in a particular type of cell therapy: engineered adoptive T-cell immunotherapies, whether chimeric antigen receptor (CAR)- or T-cell receptor (TCR)-engineered T cells. Unum Therapeutics (Cambridge, MA, USA) is developing a new wrinkle on this approach that complements and synergizes with antibody therapies. Unveiled last October with a $12-million series A round, the startup has already begun its first clinical trial: a phase 1 trial in Singapore of its engineered T cells plus Rituxan (an anti-CD20 antibody) to treat patients with B-cell chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma (NHL). Whereas in traditional adoptive T-cell therapies, the targeting moiety is a CAR or TCR expressed on the surface of the engineered cell, Unum’s approach is instead to modify T cells so that they recognize the Fc portion of a free antibody added separately that targets tumors. Being able to titer antibody dosage enables the company to dial up or dial down T-cell activity, which may have advantages in terms of controlling T-cell toxicity. Instead of engineering T cells with a CAR or a TCR, Unum modify T cells to express an Fc receptor (CD16) coupled to T-cell-activating domains (4-1BB and CD3z) through a CD8 transmembrane hinge region. Unum terms the construct an antibodycoupled T-cell receptor (ACTR). Compared with an approved antibody in isolation, Unum thinks this combination of free antibody with an ACTR T cell may have the ability to potentiate treatment efficacy. The company’s technology is based on the work of scientific founder Dario Campana,

n 2014, the biotech sector posted the largest ever influx of private funding since Nature Biotechnology was founded—a total of $9 billion, up 50% from the previous year. At the same time, the past two years have seen more exits through initial public offerings (IPOs) and trade sales than any other two-year period. Eyepopping valuations totaling over $100 million have been obtained for several firms with preclinical stage technology. What were the cutting-edge biotech ventures that raised venture funding during this bumper year? Nature Biotechnology editors have chosen eight areas in which the best-funded startups are pursuing groundbreaking science. Firms were identified first as originating from academic institutions. They were then prioritized on the basis of the amount of series A funding they raised (a measure of commercial excitement) and second on our editors’ assessment of their research. Some firms that received more funding than those in this article were not considered as interesting as those featured; others, whose science was innovative, were ultimately not included because the companies were in ‘stealth mode’ and could not disclose sufficient details about their R&D programs to our reporters. We did make one exception to the academic spinout criterion, however; Lysogene (Paris) was founded by a mother with a mission to develop a drug that could help her daughter. In the following pages, we present several short overviews of the science and technology behind the selected ventures. These 11 companies represent some of the best that academic research had to offer the startup world in 2014.

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who developed the ACTR while at the National University Singapore, where he is currently the director of the Division of Immunopathology and Cell Therapy. In the early 2000s, while at St. Jude Children’s Research Hospital (Memphis, TN, USA), Campana pioneered next-generation CARs, ACTR’s predecessors. Current CARs comprise four elements: inside the cell, a signaling domain CD3z, which is adjacent to a co-stimulatory domain (typically 4-1BB or CD28); a transmembrane domain, typically CD8; and an extracellular targeting domain, typically a single-chain fragment variable region (scFv). Because Campana’s entire research career has focused on childhood cancers, he designed his first CAR to have a scFv that targets CD19, which is a B-cell antigen expressed in various leukemias and lymphomas. That CAR was further developed as an adoptive T-cell immunotherapy and taken into the clinic by the University of Pennsylvania (Philadelphia), which partnered the candidate with Novartis (Basel) in 2012. That product called CTL019 is currently in phase 2 clinical trials for B-cell acute lymphocytic leukemia and NHL. St. Jude’s rights to the resulting CAR-expressing T cells are currently under legal dispute. Unum CEO Charles Wilson was responsible for that deal for Novartis, where he led partnering for the pharma’s research at the time. In the meantime, Campana shifted his research focus to natural killer cells, which are important mediators of antibody-dependent, cell-mediated cytoxicity. He hypothesized that Charles Wilson, putting CD16—a president and CEO of receptor normally Unum Therapeutics 247

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f e atu r e found on natural killer cells, which binds to the Fc portion of antibodies—on the surface of a T cell and then delivering it with a separate therapeutic monoclonal antibody (mAb) could synergize the anticancer potential of T cells and antibody therapies. By replacing the scFv portion of his CAR construct with a CD16, while retaining the basic CD3z /4-1BB/CD8, he created ACTR T cells. Campana and his colleagues subsequently showed that T cells expressing the ACTR plus Rituxan (rituximab) led to a 100% survival rate after 120 days in a mouse model of lymphoma, compared with 0% for all of the other groups (ACTR alone, mAb alone or no treatment; P < 0.01 for all). Furthermore, the researchers showed that ACTR co-administered with therapeutic mAbs lead to statistically significant greater cytotoxicity than the mAbs alone in vitro. Specifically, the group co-administered ACTR with Rituxan in CD20+ lymphoma cells, with trastuzumab in HER2+ breast and gastric cancer cells, and with hu14.18K322A in GD2+ neuroblastoma and osteosarcoma cells. In all three cell lines, the ACTR-mAb combination killed a greater percentage of cancer cells than mAb alone (P < 0.01 for all). The group also showed that ACTR-rituximab had cytotoxicity superior to an anti-CD19 CAR in vitro (Cancer Res. 74, 93–103, 2014). According to Wilson, ACTR-expressing T cells have an advantage over CAR-T cells because, at least in principle, a single ACTR can be combined with any mAb, making it easy to expand the technology to other indications. By contrast, developing a CAR against a new target requires the development of a completely new construct for modifying the T cell. Marcela Maus, at the University of Pennsylvania (Philadelphia), who has run several of the CAR-T clinical trials at Penn, agrees that the flexibility of Unum’s technology would make it more straightforward to standardize and test ACTR-expressing T cells. However, she also notes that in cases where the antibody is not already approved, the combination of cell therapy and antibody may lead to more headaches in terms of technology access and regulatory complexity, in addition to issues concerning dosing, safety and interpretation of clinical data. Wilson counters that developing such ‘novel-novel’ combinations is becoming more standard in oncology; the US Food and Drug Administration (FDA) released guidance on how such trials should be conducted and their regulatory expectations in June 2013. Wilson says another advantage is that all three CARs in the clinic are going after the same target, CD19—largely because side effects caused by loss of normal CD19-bearing cells are manageable—and are thus directly com248

peting with each other. Additionally, the first CAR was created about 25 years ago, making it difficult for companies in that space to get a solid intellectual property (IP) estate; in contrast, Unum’s ACTR technology has clearer IP protection, Wilson claims. Unum also will be able to more easily dial in the correct dosage by simply changing the dose of the mAb, whereas it is impossible to adjust the dose of a CAR once the modified T cells have been infused into a patient; CAR T-cells expand as they contact tumor cells bearing target antigen to drive a robust anti-tumor response, but can also cause cytokine release syndrome or other adverse safety problems, Wilson adds. Based on all of these positive attributes of the ACTR technology, Wilson and Campana formed Unum in March 2014 and closed the $12-million series A round in September with funds from Fidelity Biosciences (Cambridge, MA, USA), Atlas Ventures (Cambridge, MA, USA) and Sanofi-Genzyme BioVentures (Cambridge, MA, USA). The company is looking at developing three different potential forms of the therapy. The safest is transfecting a patient’s own T cells using mRNA encoding ACTR, which results in transient activity of about 5–6 days, according to Wilson. This is the approach Unum is taking in its current single-dose escalation phase 1 trial. If there are no acute toxicities, then the company will begin another trial in late 2015 or early 2016 using a retrovirus to modify patients’ T cells, which can have a half-life of >16 years—comparable to natural T-cell immunity to viruses (Sci. Transl. Med. 4, 132ra53, 2012). Much farther down the road would be an off-the-shelf, genomeengineered allogeneic product prepared from healthy human donors, although Wilson says it is yet not clear whether such a product is technologically possible. Unum is not currently looking to make its own mAbs to combine with ACTR, but instead is in discussions with multiple undisclosed mAb developers as potential partners. Wilson says there are ~30 mAbs targeting tumors in clinical trials, any of which could be combined with ACTR-modified T cells. Given the enthusiasm seen in recent years for CAR product candidates, one or more partnerships could very well be in the cards. In addition to Novartis’ commitment to the Penn CAR candidate, which includes $20 million to build a cell therapy center at the university, investors in the field have ponied up sizeable amounts of cash in the past two years. Juno Therapeutics (Seattle) and Kite Pharma (Los Angeles), each of which has a CAR against CD19 in the clinic, raised a respective $310 million and $85 million as private companies in 2013 and 2014 (Nat. Biotechnol. 32, 229–238, 2014).

Those two biotechs rode this investor enthusiasm into 2014, each going public on the NASDAQ, with market caps now north of $3.8 billion and $2.5 billion, respectively. AB Voyager Therapeutics and Lysogene: gene therapy takes aim at the nervous system Two new gene therapy companies focused on central nervous system (CNS) diseases have hopes of single-dose cures. The approval in 2012 of the Western world’s first gene therapy, uniQure’s (Amsterdam) Glybera (alipogene tiparvovec) for lipoprotein lipase deficiency, has reignited commercial interest in gene therapy. Together with an increasing list of diseases where the underlying genetics are being elucidated, Glybera’s registration established both a delivery vector (adeno-associated virus (AAV)) and a manufacturing system (the baculovirus/sf9 system) deemed safe by a major regulatory agency, the European Medicines Agency (see Nat. Biotechnol. 32, 237–238, 2014). Two promising companies rode these trends to successful series A rounds in 2014: in February, Voyager Therapeutics (Cambridge, MA, USA) unveiled itself with $45 million from Third Rock Ventures (Boston); and Lysogene (Paris) raised €16.5 ($22.6) million in May in a venture round led by Sofinnova Partners (Paris) and joined by InnoBio Fund (Paris) and Novo Seeds (Hellerup, Denmark). Voyager is a typical Third Rock startup; Steven Paul, CEO the venture capital Voyager firm spent more than two years evaluating technologies for delivering gene therapies with AAV vectors to the brain and spinal cord. Third Rock venture partner and Voyager CEO Steven Paul led this process, tapping into leading academic researchers and expert advisors to perform diligence on various technologies, followed by lining up four essential scientific founders: Krystof Bankiewicz, a translational neurosurgeon at the University of California, San Francisco (UCSF), who has brought numerous new AAV-delivered gene therapies into the clinic; Guanping Gao, a virologist at the University of Massachusetts Medical School (UMMS; Worcester, MA, USA), who has helped discover and characterize many new AAV serotypes; Mark Kay, a gene therapy and AAV biologist at Stanford University Medical School (Stanford, CA,

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f e atu r e USA); and Phillip Zamore, co-director of the RNA Therapeutics Institute at UMMS. The company is focusing primarily on CNS diseases largely because of Paul’s long and distinguished career in neuroscience, including as scientific director of the National Institute of Mental Health and heading up neuroscience discovery and R&D at Eli Lilly (Indianapolis) for 17 years. Another factor that influenced the CNS focus is the strong decade-plus safety and efficacy track record of AAV serotypes, which have been injected directly into the brain or intrathecal space to the cerebrospinal fluid of about 1,000 patients. Stéphane Palfi, professor of neurosurgery and neuroscience at the University of Paris, noted that because AAV vectors do not integrate into genomic DNA, they do not have the safety problems of early gene therapies that caused cancers due to activation of oncogenes. Palfi and Paul both feel that CNS diseases are a good fit for gene therapy; because neurons do not replicate, using AAV to deliver genes to neurons could result in life-long expression with a single dose. Indeed, Bankiewicz has shown that Voyager’s lead gene therapy has remained active in non-human primate brains for more than 10 years and in human brains for four years and counting (Hum. Gene Ther. 23, 377–81, 2012). Paul adds that the compartmentalization of the brain and the spinal canal, which keeps the delivered products from entering systemic circulation, is another advantage of targeting CNS diseases. Palfi, an advisor for Oxford BioMedica (Oxford, UK) and principal investigator for the company’s clinical trial of ProSavin, a lentivirus-delivered gene therapy to treat Parkinson’s disease, notes that some clinical trial subjects have exhibited an immune response to AAV vectors, which could pose a safety problem for gene therapies using the vector. For this reason, Paul says Voyager is screening clinical trial subjects to eliminate patients with preexisting antibodies against their capsids. Additionally, the likelihood of antibodies causing an immunologic reaction to the vector in the brain is slim because the brain is an immunologically privileged site. He adds that subjects also are unlikely to develop antibodies against the encoded therapeutics because they are “self ” proteins. Voyager’s lead product, VY-AADC01, encodes the gene for aromatic l-amino acid decarboxylase (AADC), which catalyzes the final step in the dopamine biosynthetic pathway, delivered with the AAV2 serotype vector to treat Parkinson’s disease. Parkinson’s disease patients experience a progressive loss of dopamine-producing neurons in the brain, resulting in symptoms such as trem-

ors, rigidity and slowness of movement. The main therapy for Parkinson’s disease patients is levodopa, the chemical precursor to dopamine. Unfortunately, as the disease progresses, patients must take larger doses of levodopa, which causes side effects like dyskinesia and motor fluctuations. Voyager hopes that by providing the enzyme, patients will be able to reduce the amount of levodopa they take and thus have better symptom control. VY-AADC01 was initially developed by Bankowitz and is now in a phase 1b study at UCSF. Paul believes that the permanent nature of the therapy may preclude the need to conduct traditional phase 1, 2 and 3 clinical trials. He notes that FDA is “giving signals” that one large adaptive clinical trial could be possible, and he hopes to talk with the agency about that possibility early this year. He says that large trials might not be needed for rare diseases, such as Friedrich’s ataxia and monogenic amyotrophic lateral sclerosis (ALS), for which Voyager has products in preclinical testing. Voyager in-licensed the AAV2 vector from REGENXBIO (Washington, DC, USA), but the company is not relying on that vector for its entire pipeline. They also licensed AAV vector serotypes developed and characterized by Gao and are developing their own synthesized vectors using two approaches. One approach is to discover new capsids or new viral vectors that are better at crossing the blood-brain barrier, can express genes throughout the brain and spinal cord after injection into the intrathecal space or have a certain proclivity for different cell types, like glia or neurons. The company can screen natural capsids and vectors in a multiplexed way, and then perform phenotypic screening in animals. Another way Voyager is expanding its vector option is through its ability to make synthetic capsids (as yet unpublished). Paul thinks the company can come up with vectors and capsids that cross the blood-brain barrier several-fold better than current vectors and capsids. In a move somewhat unusual for a startup, the company also has made investments in biomanufacturing because of the relative scarcity of facilities that can manufacture materials of such nuance. To this end, Voyager is collaborating with UMMS’ MassBiologics biomanufacturing entity to create a nonprofit facility to produce good manufacturing practice (GMP)-grade biovectors. The company also recruited Rob Kotin, who co-invented the baculovirus/sf9 (Spodoptera frugiperda 9) manufacturing system while at the US National Institutes of

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Health (NIH), as vice president of production. On February 11, Voyager partnered several of its products with the Genzyme unit of Sanofi (Paris) in return for $100 million upfront and up to $745 million in milestones, plus tiered royalties. Genzyme obtained options to rights outside of the United States for VY-AADC01, VY-FXN01 to treat Friedrich’s ataxia and VY-HTT01 to treat Huntington’s disease, as well as an option to co-commercialize VY-HTT01 in the United States. Genzyme can exercise its options after Voyager completes proof-of-concept testing in humans. The deal includes another, nondisclosed CNS disorder project, but Voyager’s lead monogenic ALS product, VY-SOD101, is excluded. Across the Atlantic, another startup, Lysogene, is using gene therapy to treat a different CNS disorder: Sanfilippo syndrome type A (mucopolysaccharidosis IIIA or MPS IIIA). MPS IIIA is a lysosomal storage disease (LSD) caused by mutations in a gene that encodes N-sulfoglucosamine sulfohydrolase (SGSH). As a result of the defective enzyme, heparin sulfate accumulates in brain cells, causing rapid neurodegeneration, severe and invasive behavioral disorders and mild peripheral symptoms. The Karen Aiach, CEO disease is extremely Lysogene rare, affecting only about 1 in 100,000 births, and patients rarely survive into their twenties. The two main symptoms of MPS IIIA are hyperactivity and sleep disorders. CEO Karen Aiach says that hyperactivity in Sanfilippo children is different from that of typical hyperactive children in that they constantly are moving and shouting, throughout the day and night. Aiach is all too familiar with how difficult the disorder can be on patients and their families because her daughter has the disease. In 2005, when her daughter was born and diagnosed, Aiach was running her own business consultancy specializing in the financial industry following an eight-year stint at accounting firm Arthur Andersen. She and her husband hired a neuroscientist and talked with multiple LSD and gene therapy researchers, as well as biotech companies involved with LSD drug development. They concluded that a gene therapy would be the best approach for two reasons: the disease is monogenic and an enzyme replacement 249

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f e atu r e therapy would not cross the blood-brain barrier. Indeed, she learned that the blood-brain barrier issue was the major reason that drug companies were not interested in targeting the disease. So she decided to set up a gene therapy program in MPS IIIA because it was clear that no one else would. Next, she hired Olivier Danos, now senior vice president of Gene Therapy at BiogenIdec (Cambridge, MA, USA), to lead the program. Danos, who had a laboratory at the Necker Hospital—Enfants Malades (Paris) at the time, focused on determining which vector to use and designing animal studies. They raised funding from family, friends and patient groups to fund an Australian group to perform animal studies using an AAVrh10 to deliver cDNAs encoding SGSH and sulfatase-modifying factor (SUMF1), which activates the catalytic site of SGSH. Palfi says the rh10 serotype is appropriate for MPS IIIA because the vector diffuses to different parts of the brain, which is essential to treat the disease. An initial study of the gene therapy in a mouse model of MPS IIIA resulted in expression of functional enzyme in the mice brains, decreased heparin sulfate storage, improved behavior and increased survival compared to control. Based on those results in 2009, Aiach decided to start Lysogene. Lysogene completed animal toxicology studies that same year and began a fourpatient phase 1/2 clinical trial in France in 2011 (avoiding enrolling subjects with preexisting immunity to the AAVrh10 vector). The company published results from the three-year follow-up last June (Hum. Gene Ther. 25, 506–514, 2014). There were no safety issues related to the gene therapy, the surgery or the post-treatment. In terms of neurocognition, the three oldest patients remained clinically stable and the youngest patient showed some progress, while regression would normally have been expected. Perhaps more importantly, all four patients showed improvement in social interactions, more focused behavior and improvement in sleep. Based on these results, Aiach says regulatory agencies have recommended that the company enroll younger patients and also increase the dose delivered to patients in future studies. Lysogene plans to begin a new trial in 12–20 patients in Europe and the United States by the end of 2015 or early 2016. As a result of the positive trial data, Lysogene was able to raise its series A round. The company has also decided to expand drug development into other undisclosed monogenic LSDs with CNS involveAB ment. 250

Caribou, Editas and Intellia: turning CRISPRs into drugs A bevvy of startups looks to develop CRISPRCas9 technology for therapy. Roughly two decades after clustered, regularly interspaced, short palindromic repeats (CRISPR) were described in the scientific literature (J. Bacteriol. 169, 5429–5433, 1987), Jennifer Doudna and her group at UC Berkeley spun out the first company based on that technology: Caribou Biosciences. Rachel Haurwitz, who was completing her PhD in Doudna’s laboratory at the time, stepped into take the helm as Rachel Haurwitz, CEO CEO of Caribou, and Caribou three years later spun out another company—Intellia Therapeutics (Cambridge, MA, USA)—for moving the gene editing platform into human therapeutics. This is only the first of several companies Caribou plans on spinning out in other sectors, such as agriculture or industrial biotechnology, while Caribou continues working out kinks, according to Haurwitz. Since Caribou’s founding in 2011, much has changed in the CRISPR universe. What started out as a prokaryotic defense system of interest to microbiologists has morphed into arguably the most tractable and user-friendly gene editing system embraced by the broad research community. Two other CRISPR companies have entered the arena: Editas, founded in late 2013 by a group of mostly Cambridge, Massachusetts–based researchers, and Baselbased CRISPR Therapeutics, founded in early 2014 by Emmanuel Charpentier, Doudna’s collaborator on the seminal work illustrating the application of a bacterial immune system to genome engineering (Science 337, 816–821, 2012). CRISPR Therapeutics declined to talk to Nature Biotechnology for this article. Of course, CRISPRs aren’t by any means the first gene editing technology in wide use; zinc fingers and transcription activator–like effector nucleases (TALENs) have already made a splash. But zinc fingers and TALENs rely on a protein–nucleic acid interaction for their target activity, which can be challenging to design and engineer; in contrast, the CRISPRCas9 system is targeted by a guide RNA (combined with the Cas9 nuclease) and works using plain old Watson-Crick base pairing that can be easily designed in the laboratory. And whereas zinc fingers, and to a lesser extent TALENs, became tangled up in patent thick-

ets, CRISPR-Cas9 had been embraced, both in academia and industry, before the battles for IP had begun in earnest (see p. 256). The ease with which CRISPRs can be designed has lowered the bar for people to get involved, says UC Davis’ David Segal, who works with all three gene editing platforms. Particularly for cashstrapped academic laboratories, CRISPR has become the go-to technology. Segal says that he’ll start off most new projects with CRISPR unless there’s a compelling reason not to. This provides an obvious benefit: the number of laboratories using, improving and publishing on CRISPRs is rapidly filling in gaps in knowledge, a benefit to all (in both academia and industry) trying to apply the technology. In fact, the startups in this area have deep benches of scientific advisory members and collaborators—a veritable who’s who in the CRISPR universe—feeding the companies’ insights and in some cases IP. As Intellia’s CEO Nessan Bermingham describes it, it makes no sense to compete with academia; better to partner with them around the development of the technology as they figure out how to optimize the it. These startups involve not only many of the premier laboratories working on CRISPR-Cas9 systems but also the premier venture capital firms investing in biotech as well. Editas CEO Katrina Bosley describes their three investors, Flagship Ventures (Cambridge, MA, USA), Third Rock Ventures (Boston) and Polaris Partners (Boston), as experienced builders of new technologies and modalities. They each have reckoned with the questions facing Editas and the other CRISPR developers,—“deeply important questions everywhere you turn,” she says—from what’s the scientific approach and what’s Nessan Bermingham, the financial strategy CEO Intellia to ethical questions about genome editing and choosing among diverse patient populations with compelling needs who could potentially benefit. Balancing all these risks and opportunities is key, she says. Intellia went after managers with extensive industrial experience, pulling in former pharma executives from Abbott Laboratories (AbbVie), Novartis and Eleven Biotherapeutics to populate their management team. In thinking about applying the technology, Bermingham lists his priorities: delivering the product—and he believes, that he has the talent in-house to do it—and identifying those indications that can derive a therapeutic

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f e atu r e benefit from the level of modification achievable with CRISPR. If a therapeutic application requires 90% homology-directed repair of the cells, for example, with the technology in its current construct, it’s not possible. Bosley also puts delivery as key, and she anticipates working with experts both inside and outside the company, as it may be necessary to deploy a variety of approaches depending on the target and the disease. Off-target effects are commonly cited as an issue with all gene editing technologies in the context of a human therapeutic. With CRISPR, which has a mere 20-base-pair recognition sequence and tolerates mismatches to boot, off-target effects are to be expected. That is one area being addressed by Caribou, according to Haurwitz, who feels that no unbiased experimental approach exists to measure them. The problem, she says, is that if you have an off-target effect that is present in only 0.5% of the population, even using deep sequencing it will be very difficult to see that. And in a situation where several million patient cells will be modified, it’s both meaningful and concerning. But how much of a problem off-target effects really present is debatable among CRISPR researchers. Segal points out that all drugs have off-target effects; drug developers must balance therapeutic effects against their toxicity profiles. That’s the standard set for drugs, they are not expected to have a perfect specificity profile, he says. Furthermore, CRISPR may actually be better when stacked up against traditional gene therapy approaches, at least those that use integrating vectors, explains Derrick Rossi, who has used CRISPR in an ex vivo application in his laboratory at Boston Children’s Hospital. With gene therapy, each cell ends up with a separate and unique genome integration. Hence, with tens of millions of cells transplanted (common for blood stem cells) the number of genome integrants is staggering, says Rossi. With today’s CRISPR technology, knocking out a gene is possible; adding a gene is doable in the laboratory setting, but not yet in a way that could be used in humans, according to Bosley. At Editas, they will be drilling down on specific targets (undisclosed as of yet) to figure out how to make the genomic edit in a way that can go into humans. An obvious place to start, according to all involved, are ex vivo applications, as delivery is a more addressable problem. Hematopoietic stem cells and CAR-T cells are likely in everyone’s sights. In that regard, Intellia and Caribou announced in January a collaboration with Novartis, which will receive exclusive rights to develop programs for engineering CAR-T cells,

as well as some undisclosed targets for ex vivo editing of hematopoietic stem cells. A glimpse of the potential is provided by a recent paper in Cell Stem Cell (15, 643–652, 2014) from Rossi’s laboratory, which uses CRISPR-Cas to edit out two genes required for HIV infection, C-C motif chemokine receptor 5 (CCR5) and b2 microglobulin (B2M) in human hematopoietic stem cells and T cells, respectively. Together with his collaborator, Chad Cowan, Rossi’s team reported Katrine Bosley, CEO 34–48% deletion effiEditas ciency, with minimal off-target mutations (determined by deep sequencing of potential targeted regions) and cells demonstrating the capacity to differentiate. Kleanthis Xanthopoulos, CEO of microRNA company Regulus of San Diego, is “super excited” about CRISPRs, but argues that the technology lacks an efficient delivery system to enable broad application; he doesn’t think that groups involved in CRISPRs have come to grips with that issue. Haurwitz, though, feels that they are not starting at zero, but points out a lot of effort and money has gone into delivery technologies for other RNA modalities and gene therapy platforms. Whether the exact correct answer is available off the shelf is still to be determined, she says, but she feels that those efforts provide some starting points that can be exploited by the CRISPR people. In January, both Intellia and Editas presented their work at a Keystone Conference in January (Precision Genome Engineering and Synthetic Biology, Big Sky, Montana, January 11–16, 2015). Editas reported on a CRISPR-Cas system isolated from Staphylococcus aureus, which has a protein component that is smaller and thus perhaps easier to deliver than the Cas9 from the system most commonly used from Streptococcus pyogenes. A new Cas protein also brings with it a different integration signal, which will expand the number of potential targeting sites. At the same meeting, Caribou reported improvements on their single-guide RNA, “tuning” it to enhance efficiency, and they presented data showing that direct use of Cas9 protein (rather than using nucleic acid vectors) enhances delivery. Atlas Ventures’ Jean-Francis Formela, who sits on Intellia’s board, says that the current bull market in biotech in general can be attributed to the maturation of the industry and its increasing ability to solve technical problems

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and address unmet needs. In the case of CRISPR-Cas, he feels the rate of adoption is a good index of how quickly the technology will evolve and develop. LD Synlogic: smart bugs and smart drugs Engineering microbes to produce drugs in response to specific host environments offers a new therapeutic approach. Synlogic (Cambridge, MA, USA) believes its expertise in molecular engineering—coupled with ongoing discoveries in the microbiome space—will enable it to engineer live organisms that respond with exquisite sensitivity to their surroundings. The company plans to initially develop oral products that release therapeutics in the gastrointestinal (GI) system to treat diseases like diarrhea and metabolic diseases. The bugs are engineered with on/off genetic switches and circuitry that control synthesis of the therapeutic molecule and respond to signals from the external milieu—the ultimate in the right drug at the right time. The company was unveiled last year, raising $29.4 million in a series A round led by Atlas Venture (Cambridge, MA, USA) and New Enterprise Associates (Timonium, MD, USA), and later joined by the Bill & Melinda Gates Foundation (Seattle), which chipped in another $5 million. Synlogic’s foundation rests on work covered by more than a dozen patents and patent applications from Jim Collins, professor Ankit Mahadevia, of biomedical engiacting president of Synlogic neering at Boston University (Boston), and Tim Lu, an associate professor of biological engineering at MIT (Cambridge, MA, USA). Collins and Lu approached the concept of designing therapeutic microbes as if they were creating an electronic circuit with “on” and “off ” switches. Using this approach, they created a number of synthetic biology tools that allow bacteria to sense physiologic conditions, perform a therapeutic action and then deactivate themselves. The capacity to engineer microbes to sense and respond to their environment is unique in the industry, according to Ankit Mahadevia, venture partner at Atlas and Synlogic’s acting president. The ability to create platforms for drug delivery based on targeted body site is an important advantage of the company’s technologies, according to CSO Paul Miller, a microbial 251

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f e atu r e geneticist by training who has led antibacterial research at Pfizer (New York) and AstraZeneca (London). For example, the company could design activation switches that turn on the therapeutic element when the bacteria enters a certain portion of the gastrointestinal tract, as well as a deactivation switch that triggers the bacterial element to terminate either once it leaves that area or the disease state no longer exists. These activation and deactivation switches could be used as a way to treat many diseases of the GI tract, with therapeutic elements added on a disease-specific basis. Synlogic will not disclose the specific genetic elements it is developing, but a look at Collins’ and Lu’s publications provides some hints of what the company might be planning. For example, Lu and his colleagues at MIT published a paper describing the engineering of Escherichia coli with two orthogonal recombinases, each of whose expression is driven by a different environmental input. The researchers combined the recombinases with recombinasetargeted promoters, terminators and output gene modules to create all 16 two-bit Boolean logic functions in the bacteria (Nat. Biotechnol. 31, 448–452, 2013). Lu and colleagues also recently published a paper on the creation of a platform for generating single-stranded DNA in E. coli in response to environmental signals which, when expressed with a recombinase, result in precise mutations of bacterial genomic DNA (Science 346, 1256272, 2014). Collins and his colleagues have created a riboregulator system that uses two distinct environmentally sensitive promoters that independently regulate the transcription of two different RNA molecules, one of which encodes a regulatory protein. When both RNA molecules are transcribed, that regulatory protein is also translated. The group showed that this RNAprotein regulator system can be used to engineer several different environment-triggered functions in E. coli, including the expression of TonB—a protein that facilitates iron import—in low-iron environments, as well as a ‘kill switch’ to kill the bacteria in response to any change in environmental conditions (Proc. Natl. Acad. Sci. USA 107, 15898–15903, 2010). More recently, that same group expanded that work to create a ‘switchboard’ of four riboregulator systems that controlled the expression of four metabolic genes to regulate carbon flow (metabolic flux) in response to quorum-signaling molecules, DNA damage, iron starvation and extracellular magnesium concentrations in single E. coli cells (Proc. Natl. Acad. Sci. USA 109, 5850–5855, 2012). Synlogic’s initial diseases of focus are undisclosed, but Mahadevia says Synlogic currently has about six active programs. Diseases with GI 252

involvement are an obvious starting point for the company. Indeed, the Gates Foundation’s investment will ensure Synlogic advances its technologies to develop new therapies for severe diarrheal diseases in developing countries. In 2012, Collins received $100,000 from the foundation to engineer the yogurt probiotic Lactobacillus gasseri to detect and kill Vibrio cholerae, which causes cholera, in the human intestine. Miller adds that microbiome studies are identifying more and more diseases that do not have a direct GI involvement but could be treated with a gut-delivered bug, such as metabolic diseases. The company will also look at targeting other tissue surfaces. For products that treat chronic diseases in large populations, the company will likely partner with a larger company that has a franchise, whereas Synlogic will be more likely to keep in-house products against diseases that have smaller trial populations and shorter trials. Always an issue with novel therapeutics is the lack of a clear regulatory pathway. To help with this aspect of drug development, Synlogic brought on Alison Silva as COO. She gained experience in regulatory affairs, clinical development and manufacturing in engineered microbial therapeutics while at Cequent Pharmaceuticals (which merged with MDRNA in 2010) and RNA therapeutics biotech Marina Biotech (which closed down in 2012). According to Mahadevia, regulatory agencies asked Cequent very specific questions regarding, for example, time of residence in the gut and transfer of material from experimental bacteria to endemic gut bacteria. He says Synlogic is “following those blueprints” to address concerns that regulators might have AB early in development. Navitor Pharma: refining mTOR drug targeting Selective targeting of a key nutrient signaling pathway could offer treatments safer than rapamycin for a wide range of diseases. Mammalian target of rapamycin (mTOR) is by no means a new target. But as researchers continue to unravel the myriad ways this master regulator is activated and dysregulated in disease, new opportunities are arising to modulate it therapeutically. Navitor Pharma (Cambridge, MA, USA) aims to develop therapeutics that capitalize on these advancements, many of which were made by its scientific founder, David Sabatini, specifically his work on the role of nutrient signaling in the activation of mTORC1, one of mTOR’s two multiprotein complexes. mTORC1 activation could play a role in diseases as diverse as type 2 diabetes and cancer cachexia. The company

is currently in early discovery with hopes of having compounds that modulate mTORC1’s activity in animal studies this year, according to president and CEO George Vlasuk. The story of mTOR inhibitors began in 1970, when bacteria from soil samples from Easter Island, also called Rapa Nui, were found to have antifungal activity. The microorganism, Streptomyces hygroscopicus, produces the macrolide rapamycin (sirolimus). The compound was later found to have potent George Vlasuk, CEO immunosuppressive Navitor and antiproliferative properties as well, and was developed to prevent transplant rejection, for which the compound was approved by the FDA in 1999. Rapamycin is still used for this indication, as well as in drug-eluting stents. Sabatini, who is now a professor of biology at MIT and a member of the Whitehead Institute, was instrumental in the 1994 discovery of mTOR and its role in immunosuppression, namely to form a gain-of-function complex with FK506-binding protein 12 to directly interact with and inhibit mTORC1 (Cell 78, 35–43, 1994). The complex was thought to have no effect on mTORC2, but chronic treatment with the compound does inhibit mTORC2 in certain cell types, such as pancreatic beta cells and skeletal muscle cells. As a result, long-term use of rapamycin—which is essential for transplant patients—can result in type 2 diabetes. In the past decade or so, Sabatini and other researchers have made great strides in elucidating details around the mTOR signaling pathway, and determining differences between mTORC1 and mTORC2. Upstream, mTORC1 is triggered by nutrients, oxygen, energy levels and growth factors. As a result, the activation mTORC1 initiates pathways that lead to macromolecule biosynthesis, cell cycle progression, growth and energy metabolism. Although less is known about mTORC2, it appears to be triggered only by growth factors and leads to cytoskeletal organization and cell survival and cell metabolism pathways. Recent studies have also shown that both mTORC1 overexpression and underexpression can result in disease. In metabolic diseases like obesity and type 2 diabetes, the complex is turned down in the hypothalamus, which reduces satiety; turned up in adipose tissue, muscle and liver cells, which inhibits insulin signaling and leads to insulin resistance; and has sustained activation in

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f e atu r e pancreatic beta cells, which ultimately causes apoptosis. Similarly, mTORC1 overactivation is implicated in neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease and Huntington’s disease; rare mitochondrial diseases such as Leigh’s syndrome, Friedrich’s ataxia, lymphangioleiomyatosis (LAM) and tuberous sclerosis; autoimmune diseases like psoriasis, rheumatoid arthritis, lupus and muscular sclerosis; and various malignancies. Loss of muscle function and health in such diseases as sarcopenia and cancer cachexia is also associated with lower levels of the complex. The upshot is that mTORC1 is critical to many diseases, whereas mTORC2 appears to be critical to normal cellular functions. Navitor believes that by modulating mTORC1 activity, some of these diseases can be mitigated by returning affected cells to homeostasis. Hongbo Chi, a faculty member in the department of immunology at St. Jude Children’s Research Hospital (Memphis, TN, USA), says that such compounds would no doubt be useful in treating many different diseases. However, he is concerned that because mTORC1 signaling is ubiquitous, inhibiting it may cause safety problems by hitting off-target cells. Vlasuk says that in certain cells where mTORC1 is hyperactivated—such as in pancreatic beta cells in type 2 diabetes—the nutrient-sensing arm of the complex’s activation pathway has become dysfunctional owing to an overabundance of nutrients, such as branchedchain amino acids. In response to amino acid availability, a series of distinct groups of protein targets bring mTORC1 to the lysosomal surface, where it becomes activated. Navitor’s strategy is to selectively target this nutrientdependent pathway before mTORC1 interacts with the lysosome, thus returning the complex’s activity to normal without completely shutting down mTORC1, something that happens with rapamycin and newer mTOR kinase inhibitors, according to Vlasuk. Navitor is not disclosing the diseases on which it will be focused. Vlasuk says rare diseases, such as LAM and tuberous sclerosis, would be ideal for the company to develop to proof-of-concept or even later stages of clinical development. For other diseases like type 2 diabetes or Huntington’s disease, the startup would likely need to partner for clinical studies. He adds that Johnson & Johnson (New Brunswick, NJ, USA), which is a strategic investor of Navitor, has an interest in metabolic disease (the pharma does not have an option to any of the biotech’s programs yet). Johnson & Johnson joined Navitor’s $23-million series A round last June through its venture capital subsidiary, Johnson & Johnson Innovation, along

with Polaris Partners (Boston), Atlas Venture (Cambridge, MA, USA), SR One (Cambridge, MA, USA) and The Longevity Fund (San Francisco). Originally founded and seeded as Calorics Pharmaceuticals in 2009 with a focus on caloric restriction mimetics, the company was relaunched as Navitor in 2014 with the licensing of Sabatini’s work around mTOR and mTORC1. AB Emulate—the latest twist on ‘organs on chips’ Connecting multiple in vitro organ systems on a chip through microfluidics. A common cause of attrition in drug development programs is the failure of current preclinical models to predict activity in humans. Inadequate models too often result in unpleasant surprises when a drug makes it to large and expensive late-stage trials (or worse after it gets into the market). Several companies have been attempting to create in vitro cellular models that are more predictive, and the NIH and the Defense Advanced Research Projects Agency (DARPA) have invested heavily in integrating microfluidics with biology. On the back of a $37-million DARPA grant and a $12-million series A round from private investors, Don Ingber, founding director of Harvard’s Wyss Institute of Biologically Inspired Engineering, has spun out a company called Emulate— so-called because the goal is to emulate human biology. The idea of fabricating chips with interconnected compartments seeded with different cell types to model fluid flow and make predictions about drug pharmacokinetics was pioneered by Mike Shuler, a chemical engineer at Cornell University, over a decade ago. Ingber has taken this basic concept, expanded and embellished on it, creating ‘organs on chips’. Under a cooperative agreement with DARPA, Ingber produced an automated platform that aims to support the viability of ten organ chips for one week and to enable coupling between two different organ chips. In 2012, he published a paper (Sci. Transl. Med. 4, 159ra147, 2012) describing a microfluidic device that claimed to reconstitute lung functions and model pulmonary edema. The hope is that by binding together different organs on a chip, emergent properties will begin to emerge. According to Ingber, in work not yet published, their lung chip has been shown to maintain normal pulmonary barrier function for a week; when the lung model is exposed to an antituberculosis drug, the drug is absorbed across the air sac and into flowing medium, mimicking blood. When the medium was

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transferred to a liver chip, the drug induced increases in liver metabolism similar to those observed in humans that produce undesired drug-drug interactions. Work from Ingber’s laboratory at the Wyss has modeled aspects of lung, bone marrow, kidney and gut physiology on the thumb drive–sized chips. Emulate has an exclusive worldwide license from the Wyss institute at Harvard University for all of the organson-chip technology, including first right of refusal on future improvements, according to President and CSO Geraldine Hamilton. The key, she says, is to make the technology robust and reproducible, accessible to more than a handful of people with a certain know-how. One area where there is a lot of interest, she says, is lung and the development of the smallairway disease model, where asthma, chronic obstructive pulmonary disease, viral infection and exacerbations of other respiratory diseases can be studied. But Hamilton also sees applications in safety testing of chemicals, environmental pollutants and cosmetics and personal health. The case for in vitro cosmetic testing is especially compelling in Europe, where animal testing has been banned since 2013. Clive Svendsen, director of the Regenerative Medicine Institute at Cedars-Sinai Hospital in Los Angeles, which invested in Emulate’s series A round, thinks the field of tissue and organ culture is ready for a paradigm shift. He realized when watching an old video of himself pipetting cells into a 24-well culture dish, that not much had changed in 25 years. And with organoids, which he also has been using, it’s not possible to do high throughput. With chips and microfluidics, on the other hand, you can flow drugs Geraldine Hamilton, through, one after the CSO Emulate other, and see what is being released, using a variety of read-outs. Ingber is no novice when it comes to the biotech industry. He was involved in two startups, one on tissue engineering in the 1980s, and another in the 1990s for doing three-dimensional printing of medical devices, long before it became fashionable. As director of the Wyss, which was set up explicitly with the idea of translating inventions, he recruited 40 people with industry experience, as well as experienced entrepreneurs. So far, this has resulted in nine startup companies formed, 19 additional products licensed, and one product on the market. And as its latest success story, Emulate will be able to hit the 253

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f e atu r e ground running. Eighteen people moved out from the Wyss, including Hamilton and CEO James Coon, both of whom have prior experience in biotech startups. Whether organs on chips are the panacea that Emulate and their (as yet to be named) industrial partners hope for remains to be seen. Even if they can replicate certain aspects of organ physiology, they may not replicate other essential characteristics, according to Martin Yarmush, founding director of Harvard’s Center for Engineering in Medicine, who has been working and consulting in this area for 30 years. The critical question is not whether you can replicate the behavior of known compounds, but whether you can predict the behavior of tens or hundreds of unknowns, reproducibly and convincingly. And they are not the only game in town. Organs on chips will have to prove their worth against a number of other systems—cell culture, static tissue models, three-dimensional tissue models—some of which have already been integrated into pharma work flows. LD Scholar Rock: shutting off the TGF-b spigot Niche modulators may provide some needed selectivity when targeting growth factors with pleiotropic activities. No fewer than 20 drug programs targeting transforming growth factor beta (TGF-b) are in clinical trials, and almost that many have failed. This 33-member family of growth factors, which includes bone morphogenetic proteins, growth and differentiation factors, among others, plays a range of roles in human physiology and pathology— cell migration and Nagesh Mahanthappa, remodeling during CEO Scholar Rock development as well as inflammation and immune suppression in various disease states. Because of their ubiquity and importance in disease, they have been a popular target for drug developers—the problem has been that TGF-b targeting is often associated with serious adverse effects. Serial entrepreneur Timothy Springer, a professor of biological chemistry and molecular pharmacology at Harvard Medical School, thinks he has a solution. Setting his sights on the pro-form of the TGF-b, Springer reasons that the release of the factor from particular microenvironments where latent forms are harbored could mitigate side effects on non-target tissues. His idea is being taken forward in Scholar Rock, 254

which pulled in a $20-million series A round of funding last September. The Springer laboratory’s expertise in structural biology is key. In 2011, they published in Nature (474, 343–349) the crystallographic structure of latent TGF-b, which revealed that the peptides are caged in by arms composed of the peptides’ own N-terminal domains. The arms display the ligand-activator binding sites (RGD motifs), in the case of TGF- b1 for av integrin, while keeping the rest of the peptides sequestered in what they call a “straitjacket.” According to Scholar Rock’s CEO Nagesh Mahanthappa, there’s more sequence diversity among the pro-peptide domains within the TGF-b super family, than in the mature growth factor, which supports the belief that targeting latent complexes will provide greater selectivity. Drilling down on structure is Springer’s forte. His group took a similar approach with integrins, and just last year a program he started over 20 years ago finally reached fruition, with the approval of Entyvio (vedolizumab, a mAb against a4b7 integrin) for ulcerative colitis. (Springer had long since lost control of the molecule. The drug package for approval came out of Takeda, which had acquired the molecule when it bought Millennium, which itself had acquired the molecule through a merger with LeukoSite, a company Springer had co-founded in 1993.) According to Mahanthappa, Springer’s decades of work on integrins show that it is possible to identify antibodies that stabilize proteins in different conformations and mediate different outcomes, whether it’s promoting their activation or inhibiting the interaction. Dean Sheppard, director of the Lung Biology Center at UCSF, feels that Springer’s work on mapping epitopes on integrins complements work being done in his own laboratory and elsewhere targeting integrins. Sheppard has a mAb directed against avb6integrin in phase 2 clinical trials with Biogen Idec (STX-100) for lung fibrosis, one of the indications in Scholar Rock’s sites. Combining what can be learned from epitope mapping from different blocking antibodies with what is known from the crystal structure of integrins and now TGF-b1 could increase the repertoire of TGF-b1 activity that can be blocked in different contexts, Sheppard says, which could improve efficacy and safety ratios. Scholar Rock opened its doors in 2013 with seed funding from Springer himself and Polaris Partners (Boston), where Springer holds a seat as scientific advisor. At the time, they had only the crystal structure and the hypothesis that their targeting approach should work. By September 2014, when they secured their series A round from a larger group of venture capitalists, they had assembled the tool kit for preparing the latent protein in sufficient quan-

tities for antibody production, as well as assays for monitoring activation or suppression of activation. And today, Mahanthappa can say that they have antibodies in hand capable of modulating TGF-b1. The company’s complete platform comprises three critical elements: first, antigen design and synthesis (i.e., a recombinant expression system for growth factor complexes); second, cell-based assays that reconstitute proform activation of different TGF-b superfamily members; and third, recombinant antibody discovery technologies to target the TGF-b epitopes of interest. With respect to this third element in particular, the announcement last November that Greg Carven, one of the inventors of the first anti-PD1 antibody (Keytruda, pembrolizumab; Merck, Whitehouse Station, NJ, USA) has joined Scholar Rock, adds to the company’s cachet. The company has two seasoned biotech principals in Mahanthappa and Springer, who as an investor and founder has seats on both the board of directors and scientific advisory board. Mahanthappa got in early at Avila Therapeutics (employee number two) where he served as vice president of corporate development and operations, until its acquisition by Celgene (Summit, NJ, USA) in 2012, and similarly was founding employee at Alnylam Pharmaceuticals (Cambridge, MA, USA). In those two companies, as with Scholar Rock, he saw platforms that could be transferred from target to target. For now, the focus at Scholar Rock will be on targeting TGF-b1 for fibrotic disease and myostatin for musculoskeletal diseases, but Mahanthappa believes the technology can be applied to all the members of the superfamily, as they all have latent forms. Springer’s roots go back to the early days of biotech, where he says he allowed a breakthrough that revealed a new biological insight, rather than technology, lead him into industry. In the case of LeukoSite, he and his cofounders had worked out a three-step model for leukocyte migration though tissue. Now, with Scholar Rock, the possibility of targeting signaling of TGF-b family members through the microenvironments where they are sequestered motivated Springer’s return to life science ventures. Between LeukoSite and Scholar Rock, Springer has been investing his own money and time in ventures, including Selectar Bioscience (Watertown, MA, USA) and Moderna Therapeutics (Cambridge, MA, USA), where he sits on the board of directors, and advising Polaris. And he’s not yet done spinning out companies. He has it in mind to found another company to develop small-molecule antagonists to integrins and, at some future time, develop a drug program to take on malaria. Then, he says, LD he will be done.

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Quartet Medicine: genetics hits a nerve A new slant on tackling neuropathic pain. For too long, medicine has not had good answers for chronic, neuropathic pain. Only 30% of patients experiencing neuropathic pain receive meaningful pain relief from current treatments. Genetics, however, may finally be providing some promising, new approaches, as the relatively new field of pain genetics starts to provide new targets for drug programs. Clifford Woolf, professor of neurology and neurobiology at Boston Children’s Hospital, and a selfdescribed nongeneticist, let genetics be the driver in his research program. Combining an analysis of gene expression changes in multiple preclinical models of neuropathic pain with genotyping of people with post-surgery pain, Woolf ’s team unearthed a previously unknown link to a pathway involved with modulating neuropathic pain. That pathway, the biosynthetic enzymes for tetrahydrobiopterin (BH4), became the foundation for the startup Quartet Medicine—the company name is a play on the word BH4—which was founded in 2013 by Woolf, Atlas Ventures’ Bruce Booth who chairs the board, Kevin Pojasek, who was entrepreneur in residence at Atlas, and French researcher Kai Johnsson of École Polytechnique Fédérale de Lausanne, whose drug screening program provided an initial lead for a potential therapy. The seminal work was described in a 2006 Nature Medicine (12, 1269–1277, 2006) paper, which followed up on an earlier analysis of gene expression in rat peripheral nerve injury models. That earlier work had found that several hundred genes were upregulated after nerve injury, among them two of three enzymes in the pathway for the co-factor BH4 (GTP cyclohydrolase, which catalyzes the rate limiting step and sepiapterin reductase, which catalyzes the final step). BH4, a small metabolite, is involved with the synthesis of multiple signaling molecules, including catecholamines, serotonin and nitric oxide. According to Gavril Pasternak, chair of neurology at Memorial Sloan-Kettering Cancer Center (New York), this finding was somewhat surprising, as BH4 is not a neurotransmitter itself, but it does suggests a common inroad into several pain-associated pathways. Woolf and his group were able to pin down BH4’s role in inducing pain by showing in the rat model that increasing BH4 levels by administering it intrathecally produced pain, whereas blocking its synthesis attenuated pain. Most compelling of all was the finding of a human variant in the rate-limiting enzyme of the BH4 pathway, GTP cyclohydrolase, that correlates with reduced chronic pain in people undergoing back surgery. Patients homozygous for

the haplotype GCH1, roughly 2% of the population, have a lower risk of developing chronic, persistent pain. Carriers have normal pain sensation and normal baseline levels of BH4, but can’t make more BH4 in response to injury or inflammation, leading to better chronic pain outcomes. These observations led them to explore whether they could mimic that Quartet team, Kevin Pojasek, Annika Malmberg and Mark Tebbe phenotype pharmacologically, by reducing BH4 levels in such a way as Pfizer’s venture arms and funds from Woolf ’s to reproduce the phenotype of those few percent institute, Partners Healthcare Innovation, with of people who had a reduced risk of developing the idea of developing nonsulfa drugs identified by Johnsson’s screens. pain, says Woolf. Having solved the crystal structure of the Independently and serendipitously, Johnsson discovered a drug that just happened to act on enzyme, the Quartet team has moved their first one of Woolf ’s targets in the BH4 pathway. program into lead optimization. Their most Johnsson’s laboratory was conducting studies advanced inhibitor demonstrated BH4 reducof approved drugs with unclear mechanisms of tions in subnanomolar concentrations. The action to uncover the cause of unwanted, off- hope is that early in clinical development it will target effects. Using a yeast three-hybrid screen, be possible to see whether their drug is bindhe found that a common sulfa drug, sulfasala- ing to the target of interest and producing the zine, which has been around since the 1940s expected effect. Booth had been following the work coming and used as an anti-inflammatory, inhibits the final step in the BH4 biosynthetic pathway, and out of Woolf ’s laboratory since meeting him not published the results in a 2011 Nature Chemical long after the 2006 paper came out. For him, the Biology (7, 375–383, 2011) paper. Johnsson confluence of human genetics, tractable chemwent on to characterize the inhibitory effects of istry and the possibility of new approaches to a a broader array of sulfa drugs using biochemical, pressing clinical need, coupled with a clear pharmacokinetic/pharmacodynamic readout made cellular and X-ray crystallography tools. So they had a human genetic link to a new this a compelling investment, he says. Pojasek, who has taken on the role of CEO, target and a potential drug. But that was not all. It turns out you can measure the amount has been talking with pharma and although of BH4 in the blood and other compartments, some drug developers have gotten out of pain, so there’s a biomarker that can report out the he feels the pendulum is likely to swing back, as efficacy of an inhibitor, rather than relying on evidenced by both Novartis and Pfizer taking patients reporting outcomes, which is especially a stake in the company. Pojasek has assembled important given the historically poor translation a lean team of researchers and consultants— of pain programs, says Pojasek. This would also most R&D are outsourced—who work out of enable physicians to identify people most likely the Atlas space alongside several other startups. to benefit from the therapy, those producing This puts Quartet in proximity to the investlarge amounts of BH4. This is an exciting pos- ment team as well as several seasoned CEOs and sibility, according to Pasternak. He likens it to heads of development and drug discovery—an going to the doctor to get a throat culture to interesting and supportive ecosystem that Atlas find the right antibiotic. It’s an enormous step has built up over the past few years. And although neuropathic pain is a large forward, he says. The existing sulfa drugs, though, have some unmet need, Pasternak doesn’t see this as a liabilities. Sulfasalazine is a “horse pill,” says panacea. Pain is a difficult symptom to treat Woolf, as people have to take grams doses, and most likely it will require a polypharmacy. and these drugs have variable exposure and In the meantime, the renewed interest by potentially severe side effects. So the company pharma, as well as advances in pain genetics, was set up with a $17-million series A round provides some hope for relief for long-sufferLD led by Atlas Ventures, along with Novartis’ and ing pain patients.

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