REPRINT FROM JANUARY 17, 2019 MELETIOS VERRAS/ISTOCK/GETTY IMAGES PRODUCT R&D MODALITY MOVES AT VERTEX By Lauren Martz, Associate Editor In licensing a suite of newly discovered gene editing tools, pulmonary delivery of mRNAs and develop mRNA therapies Vertex Pharmaceuticals Inc. is broadening its options for solving for CF. Vertex paid $40 million up front including $20 million in problems that its core small molecule technology can’t address. cash and a $20 million convertible note. Moderna also is eligible On Jan. 3, Vertex announced a deal with Arbor Biotechnologies for up to $275 million in milestones and royalties. Inc. that gives it access to novel CRISPR endonucleases — the A Vertex spokesperson told BioCentury the collaborations with workhorse enzymes of the technology — allowing the company CRISPR, Moderna and Arbor are part of the company’s ongoing to sidestep the complex IP landscape surrounding the more research into gene-based therapies, but did not disclose whether commonly used CRISPR-Cas9 (see “Plenty of CRISPR Pie.”) Vertex has internal discovery programs in this area. The same day, Arbor researchers published aScience study that Vertex’s small molecule approach in CF may eventually identified four new Type V CRISPR endonucleases — Cas12c, cover about 90% of patients by using combinations of CFTR Cas12g, Cas12h and Cas12i — with unique features that apply potentiators and correctors. Potentiators help open the mutated to diagnostics, base editing and high-specificity cleavage of a protein channel at the cell surface to increase chloride flow, and broad range of target sites. correctors help the mutated protein form the right shape to The partnership is the third in just over three years that indicate traffic to the cell surface. Vertex is branching out the modality base of its early pipeline. The company’s approved therapies — Symdeko tezacaftor/ “It’s a high order priority for us to allocate capital outside the ivacaftor, Orkambi lumacaftor/ivacaftor and Kalydeco ivacaftor company to do collaborations to give us opportunities with — are in several Phase II and Phase III trials studying triple- more modalities and more medicines for diseases that we would therapy combinations with Vertex’s investigational treatments be interested in,” COO and EVP Ian Smith told investors on the to address the 90% of CF patients who express some form of the company’s 3Q earnings call last year. CFTR channel. In October 2015, Vertex partnered with CRISPR Therapeutics Vertex told BioCentury that gene editing is one of several AG to gain rights to up to six CRISPR-Cas9-based therapies strategies it is using to address the 10% of patients who don’t for cystic fibrosis, sickle cell disease and other indications for make the CFTR protein. For example, the mRNA approach $105 million up front, including $75 million in cash and a $30 aims to produce functional copies of the CFTR channel. million equity investment. CRISPR is eligible for $420 million With gene editing, the goal would be to correct the CFTR in milestones, plus royalties, for each of the six. mutations in the lung epithelial cells that line the airways. Vertex In July 2016, Vertex added mRNA therapeutics to its repertoire declined to say whether it expects to ultimately extend these through a deal with Moderna Therapeutics Inc. to explore approaches to the full patient population. “Vertex is committed BIOCENTURY INNOVATIONS ©2019 BIOCENTURY PUBLICATIONS, INC. to bringing effective treatments to all patients with CF,” the homing, engineered form of Cas13d could edit pathological tau company told BioCentury in an emailed statement. isoforms responsible for frontotemporal dementia. The deal with Arbor gives Vertex an array of gene editing tools Arbor said its deal with Vertex allows it to realize the translational with different properties, to complement the existing pipeline. potential of the newly discovered isoforms. Vertex told BioCentury the deal includes a $30 million upfront “We’re excited to see how the newly published Cas12 family payment to Arbor, a $15 million convertible note and mid- enzymes can potentially treat serious human diseases such as single digit royalties. cystic fibrosis,” said an Arbor spokesperson. The partners will work together to discover proteins and tools All four new enzymes have a collateral cleavage property ideal that Vertex may use to develop new therapies, with a focus for diagnostic applications; Cas12h is about 33% smaller than on discovering novel programmable DNA endonucleases or Cas9 — an advantage for delivery, Cas12i creates double- nickase and transduction approaches. Most of the discovery stranded DNA nicks for base editing, and Cas12c offers a broad activities will be conducted by Arbor and funded by Vertex. range of target sites (see Figure: “CRISPR Enzyme Lineup”). Under the terms of the deal, Vertex has an exclusive license to In the Science study, the Arbor team demonstrated that the new develop gene editing therapy products for five diseases including endonucleases join Cas13, Cas14 and other Cas12 family members CF, using CRISPR DNA endonucleases discovered through the as diagnostic tools with collateral cleavage properties. When collaboration. Cas12g encounters a target RNA sequence, and when the others encounter a target DNA sequence, they cleave the target sites then ENZYME DIVERSITY indiscriminately cleave RNA and/or single-stranded DNA. Arbor emerged from stealth mode last year with $15.6 million Two companies, Mammoth Biosciences and Dahlia Biosciences in series A funding, a paper highlighting a novel CRISPR Inc., have disclosed technologies to create DNA- and RNA-based enzyme, and a novel biodiscovery platform. Arbor’s scientific CRISPR diagnostics that rely on a combination of enzymes with founders include Feng Zhang, one of the pioneers of CRISPR- collateral cleavage activity to detect DNA or RNA sequences. based gene editing, and David Walt, a co-founder of Illumina Inc. and Quanterix Corp. The paper also identified Cas12h, which “represents the smallest double-stranded DNA targeting system discovered to date,” said Zhang, a core institute member at the Broad Institute of MIT an Arbor spokesperson. and Harvard and co-founder of Editas Medicine Inc., sits on the opposite side of the CRISPR-Cas9 patent dispute from While the most commonly used Cas9 orthologs are in the 1,300 CRISPR Therapeutics, which joins the University of California amino acid range, Cas12h is between 870 and 933 amino acids in disputing the inventorship of the technology. long. According to Arbor, the smaller size may enable a wider range of delivery options for gene editing. But Arbor’s technology is one of several discoveries that offer paths around that IP dispute, which focuses on CRISPR-based Cas12h is also able to target sites in the genome with a different gene editing using Cas9. Several other CRISPR enzymes have protospacer adjacent motif (PAM) sequence than those targeted been identified that expand the possible applications and offer by Cas9. A PAM is a short nucleotide sequence required at a different editing properties from Cas9 (see “Cutting Edge target site for the CRISPR endonuclease to bind. Diagnostics”). The team also demonstrated that Cas12i is the first example Using its protein biodiscovery platform to mine bacterial of a Type V enzyme with predominant double-stranded DNA genomes, Arbor is expanding the collection of CRISPR nickase activity. enzymes. The company has not disclosed immediate plans to The DNA nickase preferentially cuts the DNA strand that is non- develop therapies in-house. complementary to the guide RNA. Creating double-stranded The newly described enzymes add to Cas13d, the first enzyme breaks requires that paired nickases each hit the correct site, Arbor disclosed upon its launch. Cas13d is small compared driving down off-target double-stranded edits. with other Cas enzymes at just over 900 amino acids, and has Other benefits of nickases are increased gene insertion accuracy properties well suited for diagnostics. A group at Salk Institute due to the long overhangs created by the nicks, and utility in base for Biological Studies discovered in parallel that a nucleus- editing, which involves using deactivated Cas9 or Cas nickases plus deaminase enzymes to convert one target base to another. BIOCENTURY INNOVATIONS ©2019 BIOCENTURY PUBLICATIONS, INC. CRISPR ENZYME LINEUP The original CRISPR-based gene editing technology was based on the has a minimal PAM sequence of either one (TN) or two (TG) nucleotides, endonuclease Cas9, which sits at the center of the disputed IP. An assortment creating fewer restrictions on target sites than enzymes with more complex of alternative enzymes have since been discovered. Vertex Pharmaceuticals PAMs. The figure includes PAM sequences, which can vary by species of the Inc. has partnered with Arbor Biotechnologies Inc. to access its CRISPR toolkit, most commonly used orthologs. which includes five enzymes the startup discovered: Cas12c, Cas12g, Cas12h, The type of cut impacts the types of repair mechanisms used. Staggered cuts Cas12i and Cas13d. used by Cas12a improve homology-directed repair efficiency needed for gene Each of the enzymes has different features, lending to use in gene knockdown insertion, while Cas12i's nicks, which are created by preferentially cutting only or correction, base editing, or diagnostics. The enzyme properties vary by the one of the DNA strands, triggers base excision repair ideal for editing a single type of nucleic acid it targets, size, protospacer adjacent motif (PAM), type of nucleotide position. cut and collateral cleavage capability. With the exception of Cas9, the CRISPR endonucleases have a collateral The figure shows the landscape of reported Cas enzymes with translational cleavage property useful for diagnostics. The enzymes can indiscriminately relevance. Cas enzymes (green circles) cleave a target DNA or RNA site cleave ssDNA and/or RNA after cleaving the target site. determined by a guide RNA, which is made up of a crRNA and sometimes Cas14 targets sites on single stranded DNA (ssDNA), which may be useful for a tracrRNA.