Focus Feature on CRISPR: Deals advance CRISPR technology

Companies and academic institutions work together to unlock cellular reprogramming, single cell RNAseq-linked screening, stem cell-derived therapies for diabetes, engineered mammalian cell lines and more

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The months of August and September have brought our team at DDNews a full plate of licensing agreements and collaborations focused around CRISPR technologies, with one strategic acquisition to spice things up. In addition, we have a duo of granted patents, a $110-million funding round for Synthego’s genome engineering platform capabilities and an effort to correct sickle cell-causing mutations with the CRISPR/Cas9 system to round out this month’s focus feature on CRISPR.
To start us out, in September, San Diego-based Fate Therapeutics divulged that it has exclusively licensed intellectual property from the J. David Gladstone Institutes which covers the generation of induced pluripotent stem cells (iPSCs) using CRISPR-mediated gene activation. This new approach for inducing pluripotency uses CRISPR to directly target a specific location of the genome and activate endogenous gene expression, and it doesn’t rely on established methods of cellular reprogramming that require the transduction of multiple transcription factors. The discovery was made by a team of scientists led by Dr. Sheng Ding, a senior investigator at Gladstone and a scientific founder of Fate Therapeutics.
“Fate Therapeutics was founded on a commitment to innovation in the field of iPSC technology, and we will continue to invest in exciting new technologies that extend our dominant leadership position in the development of off-the-shelf, iPSC-derived cell products,” said Scott Wolchko, president and CEO of Fate Therapeutics. “Dr. Ding was instrumental in successfully pioneering the use of small molecules to generate iPSCs and the building of our iPSC product platform, and we look forward to advancing this novel CRISPR gene activation approach for cellular reprogramming.”
CRISPR can precisely edit the genome by targeting a unique sequence of DNA, but in this case Ding repurposed CRISPR to enable target gene activation, allowing regulation of endogenous gene expression. His research team showed that targeting a single location of the genome using CRISPR genome activation could trigger iPSC generation. The findings were published in an article entitled “CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency,” in the journal Cell Stem Cell in January 2018.
Fate Therapeutics is using clonal master iPSC lines to overcome the complexity, heterogeneity and substantial costs associated with sourcing cells from a patient or an allogeneic donor. Instead, iPSC-derived cell products can be consistently and repeatedly mass produced and delivered in an off-the-shelf manner, significantly reducing the cost of, and time to, patient treatment.
Fate Therapeutics says it has built a dominant intellectual property position broadly covering iPSC technology and iPSC-derived cell products. Its proprietary portfolio includes compositions and methods for generating iPSCs, including engineering their biological properties using CRISPR and other nucleases, and for producing genetically edited cells of the hematopoietic lineage, including natural killer cells and T cells, from iPSCs. Fate Therapeutics’ iPSC product platform is supported by over 100 issued patents and 100 pending patent applications.
Building a single cell RNAseq-linked CRISPR screening platform
Meanwhile, Horizon Discovery of Cambridge, U.K., mentioned back in August that it had entered into a collaboration with a global pharma partner to co-develop and apply a novel cutting-edge research tool for target identification and validation. As part of the collaboration, Horizon will lead the development and application of single-cell RNAseq-linked pooled CRISPR screening, using a key discovery paradigm of the pharma partner. Horizon will also use the development process, acquired infrastructure and optimization data in order to further develop and improve these tools to offer to future partners, as part of its research services operations.
Functional genomic screening with CRISPR/Cas9 technology has provided a powerful and precise way to identify and validate novel drug targets, and to elucidate unknown drug mechanisms. The depth of biological insight possible from Horizon’s single-cell RNAseq-linked CRISPR screening platform will reportedly allow researchers to ask far more complex experimental questions and speed up the time from discovery to validation by integrating the effect of gene editing with complex phenotypic signature mapping. With this technology, pathways, biomarkers and cell function can all be studied and revealed with ultra-rich data.
Horizon’s pooled format screens offer researchers access to highly robust whole-genome level analyses said to yield outstanding data quality. While this approach has proven a potent research tool, it is currently challenging to adequately multiplex the analysis from these screens to evaluate complex biological phenomena. Coupling pooled screening to single-cell RNAseq allows the opportunity to address the impact of CRISPR-based gene modification on a global transcriptomic level, at single-cell resolution. This is intended to enable customers to address critical gaps in target identification and validation as they work to develop novel and more effective drug therapies.
“Pooled CRISPR/Cas9 knockout screens have rapidly become an important tool in novel drug target identification and validation. Horizon continues to innovate in this important area, having launched our CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screening service in 2017, and through the development of RNAseq-linked CRISPR screening,” commented Terry Pizzie, CEO of Horizon Discovery. “The co-development of this tool with a major pharma partner provides a substantive advance to Horizon’s already world-leading screening capabilities, offering our customers cutting-edge solutions not available elsewhere.”
The hopeful destruction of diabetes
Also in September, Zug, Switzerland-based CRISPR Therapeutics and San Diego-based ViaCyte announced their collaboration focused on the discovery, development and commercialization of gene-edited allogeneic stem cell therapies for the treatment of diabetes. Decades of clinical data with islet transplants indicate that beta cell replacement approaches may offer curative benefit to patients with insulin-requiring diabetes. ViaCyte has pioneered the approach of generating pancreatic-lineage cells from stem cells and delivering them safely and efficiently to patients.
PEC-Direct, ViaCyte’s lead product candidate currently being evaluated in the clinic, uses a non-immunoprotective delivery device that permits direct vascularization of the cell therapy. This approach has the potential to deliver durable benefit, but because the patient’s immune system will identify these cells as foreign, PEC-Direct will require long-term immunosuppression to avoid rejection. As a result, PEC-Direct is being developed as a therapy for a subset of patients with type 1 diabetes at high risk for acute complications.
CRISPR gene editing offers the potential to protect the transplanted cells from the patient’s immune system by ex-vivo editing of immune-modulatory genes within the stem cell line used to produce the pancreatic-lineage cells. The speed, specificity and multiplexing efficiency of the CRISPR system are said to make it ideally suited to this task. CRISPR Therapeutics is pursuing a similar approach for its allogeneic CAR-T programs, and has established significant expertise in immune-evasive gene editing. The combination of ViaCyte’s stem cell capabilities and CRISPR’s gene editing capabilities has the potential to enable a beta-cell replacement product that may deliver durable benefit to patients without triggering an immune reaction.
“We believe the combination of regenerative medicine and gene editing has the potential to offer durable, curative therapies to patients in many different diseases, including common chronic disorders like insulin-requiring diabetes. ViaCyte is a pioneer in the regenerative medicine field, and has built a compelling clinical program, robust manufacturing capabilities and assembled a strong intellectual property position. Partnering with ViaCyte will allow us to accelerate our efforts in regenerative medicine, an area that we believe will provide a variety of longer-term opportunities for our company,” noted Dr. Samarth Kulkarni, CEO of CRISPR Therapeutics.
Under the terms of the agreement, CRISPR Therapeutics and ViaCyte will jointly develop an immune-evasive stem cell line as a first step on the path to an allogeneic stem-cell derived product. Upon successful completion of these studies and identification of a product candidate, the parties will both assume responsibility for further development and commercialization worldwide. Upon execution of the agreement, ViaCyte will receive $15 million from CRISPR Therapeutics, which at CRISPR Therapeutics’ election may be paid in either cash or CRISPR Therapeutics stock. ViaCyte also has the option, under certain circumstances, to receive an additional $10 million from CRISPR Therapeutics in the form of a convertible promissory note.
“Creating an immune-evasive gene-edited version of our technology would enable us to address a larger patient population than we could with a product requiring immunosuppression. CRISPR Therapeutics is the ideal partner for this program, given their leading gene-editing technology and expertise and focus on immune-evasive editing. We are thrilled to have the opportunity to partner with CRISPR Therapeutics on what we believe could be a transformational therapy for patients with insulin-requiring diabetes,” said Dr. Paul Laikind, CEO and president of ViaCyte. “We also believe that this approach may have many other applications which we and CRISPR Therapeutics may explore in the future.”
Making mammalian cell lines
In other licensing news, Oxford Genetics disclosed back in August that they have secured a multimillion-pound contract with a leading global ecommerce provider of reagents and tools to the research and clinical community. As part of the agreement, Oxford Genetics will leverage its high-throughput automated genomic engineering platform for CRISPR modification of mammalian cell lines.
Headquartered in Oxford, U.K., Oxford Genetics is a synthetic biology company dedicated to developing and delivering technologies and services to its customers that will help to change the gene-editing industry. The company says its use of automation allows it to develop new and innovative solutions, the latest of which is the mammalian CRISPR cell line engineering platform.
“This contract highlights Oxford Genetics’ commitment to providing the highest-quality cell line engineering services to its global customer base and continuing to add value to their operations,” said Ryan Cawood, CEO of Oxford Genetics. “We have moved away from manual processing, which is the norm in this market, in favor of automated, scalable platforms. This approach means we are well positioned to deliver the large number of custom-engineered cell lines per year that the global market is forecast to need. We are excited about this project which will see us significantly expand our product portfolio and continue to deliver innovative products and solutions to the pharmaceutical industry.”
Accelerating gene-editing technology
And finally we come to the acquisition by Boulder, Colo.-based Inscripta, which was announced in September. Inscripta acquired Solana Biosciences, a life-sciences company founded by Illumina veterans. The companies say in a press release that Inscripta and Solana “joined forces to accelerate the commercialization of Inscripta’s suite of gene-editing technology tools. Inscripta’s acquisition of Solana assembles an elite life-sciences product development and manufacturing pipeline for precision gene editing.”
“Inscripta is building the tools to take on the next frontier of gene-editing discoveries. The addition of the Solana team brings world-class scientific product development, manufacturing and operations experience to further enable Inscripta to provide the best gene-editing tools to both commercial and academic researchers,” said Kevin Ness, CEO of Inscripta.
Inscripta provides a full suite of CRISPR-based genome-editing and tracking technologies to researchers, allowing genome editing of living cells to be simple, efficient and robust. The company has also developed a unique family of CRISPR enzymes (MADzymes) and is creating additional enzymes, reagents, instruments and software with improved capabilities for multiple genome-editing applications.
Inscripta has introduced two unique CRISPR enzymes, MAD7 and MAD2, as part of the company’s own MADzyme family. Recently, the U.S. Patent and Trademark Office granted Inscripta patents covering the use of both of the MADzymes in editing systems in multiple cell types, including microbes, plants and mammalian systems. The company introduced its MAD7 enzyme to commercial and academic researchers with no upfront licensing fees or “reach-through royalties” on products made using the technology. This approach was reportedly the first step in the company’s path to reshape forward genome engineering and make it more accessible for the research and commercial community.
“Solana is passionate about creating and introducing innovative tools to the life-sciences industry. We are excited to join a team of top scientists and developers at Inscripta to further empower researchers in the emerging gene-editing field,” mentioned Tom Rosso, co-founder of Solana Biosciences and former vice president of operations at Illumina. “Together, our team will design, build and commercialize a new suite of tools that will revolutionize life sciences.”
Following the acquisition, Rosso will become vice president of operations at Inscripta and lead the process development, technology transfer, manufacturing and operations teams at the company. In addition, Inscripta will expand operations and obtain a new office in the life-sciences hub of San Diego.
“From my days at Illumina, I know the people behind Solana, and I can say that they will bring unparalleled expertise and experience to Inscripta, helping the company grow, diversify and scale its operations to meet the rapidly advancing needs of the gene-editing industry,” enthused John Stuelpnagel, chairman of Inscripta’s board of directors, as well as co-founder and first CEO of Illumina. “With the addition of Solana’s talent and capabilities, I am confident that Inscripta will fundamentally transform genome writing, just as Illumina did for genome reading.”

Genetics Research granted patent for nucleic acid enrichment method
WAKEFIELD, Mass.—Genetics Research announced in early October that the U.S. Patent and Trademark Office (USPTO) has granted the company U.S. Patent No. 10,081,829, entitled “Detection of targeted sequence regions.”
The patent covers a novel method for enriching sequence specific nucleic acids called Negative Enrichment, in which Cas endonuclease complexes are used to protect targeted sequences of interest in the presence of exonucleases. The exonucleases substantially degrade all of the nucleic acid in a sample except for the nucleic acid of interest, leaving the nucleic acid of interest isolated and amenable to analysis.
Tony Shuber, president and chief operating officer of Genetics Research, said: “Our Negative Enrichment platform is agnostic to the size of the targeted sequence of interest, and our research team has demonstrated the ability to apply our Cas9-associated Negative Enrichment platform to multiple, sequence-specific applications. These include long DNA analysis and short fragment enrichment, such as circulating cfDNA. We have also taken advantage of the specificity associated with Cas9/sgRNA complexes and can demonstrate the ability to achieve single-base discrimination by Negative Enrichment.”
This is the first patent that the USPTO has granted the company on its Negative Enrichment platform—Genetics Research has 32 additional patents pending.
“I am confident that the proprietary technology we have developed at Genetics Research represents the next generation of target enrichment technologies and will have a significant impact on the research and clinical space,” said Thomas A. Shields, chairman, CEO and a principal investor in Genetics Research.

EPO grants ToolGen a patent on Cas9 editing system
SEOUL, South Korea—ToolGen publicized in July that the European Patent Office (EPO) has issued a decision to grant ToolGen a European patent covering a CRISPR/Cas9 genome-editing system adapted for mammalian cells.
Jongmoon Kim, CEO of ToolGen, noted, “We are committed to developing transformative therapeutics and agricultural products using our CRISPR/Cas9 genome-editing system. This patent grant from the EPO begins to lay the groundwork for our intellectual property in Europe and further validates our research efforts. With this patent in hand, we intend to pursue additional IP applications and licensing partnerships worldwide.”
The European patent (number EP 2912175 B1) that was granted is entitled “Composition for cleaving a target DNA comprising a guide RNA specific for the target DNA and Cas Protein-encoding nucleic acid or Cas protein, and use thereof.”
In addition to Europe, ToolGen is pursuing patent rights worldwide and has issued patents in South Korea and Australia. ToolGen has licensing partners for these patent rights in agriculture and clinical research.
ToolGen has one of the earliest filing dates for the use of CRISPR/Cas9 in mammalian systems, which is significant in first-to-file patent systems such as that of the EPO. ToolGen’s European patent is based on a patent application filed by ToolGen on Oct. 23, 2012, and would cover key European countries including Austria, Denmark, France, Germany, Italy, the Netherlands, Spain, Sweden and the United Kingdom.

Synthego receives $110M in Series C funding
REDWOOD CITY, Calif.—In late October, Synthego disclosed a $110-million Series C financing round led by Founders Fund, with additional investment from existing investors 8VC and Menlo Ventures. This brings Synthego’s total funding to $160 million. The new funds further Synthego’s stated mission to accelerate CRISPR capabilities for rapid and precise genome engineering in applications such as cell and gene therapies.
“This new funding allows us to expand our reach and build out our full stack platform capabilities at a perfect time,” said Paul Dabrowski, co-founder and CEO of Synthego. “Biological medicines are on the cusp of a revolution with the coming curative cell and gene therapies, and we are proud to support this industry.”
Championing a mission of CRISPR access for all researchers, Synthego has grown rapidly since its prior funding round. The company’s industry-leading genome engineering platform capabilities and CRISPR products have reportedly accelerated production capacity and commercial expansion to thousands of key customers since its Series B financing and commercialization only 22 months ago.
With this new round of funding, Synthego will double down on its Engineered Cells and CRISPRevolution product lines, further expand its market in Europe and Asia and continue extending its full stack genome engineering platform to enable access to new capabilities for scientists.
“The precision of genome editing gives it the potential to develop an entirely new class of therapies for human disease. The headlines around genome editing make it sound simple, but the reality is that bringing laboratory discoveries to humans is a complex process requiring specialized expertise, reagents and commitment,” points out Dr. Matthew Porteus, a professor at Stanford University, physician at the Lucille Packard Children’s Hospital and pioneer of cell-based therapies.
“Synthego has brought an energetic and innovative approach to the genome-editing field which has accelerated the work of researchers throughout academia and the private sector,” added Porteus. “Moreover, the engineering background they bring to the field means that they have developed solutions to problems that biologists and geneticists might not have come up with. They have been highly collaborative in their work and the work in my research program is substantially farther along because of that collaboration than it would have been otherwise.”
Synthego also announced that Porteus will join its advisory board. Porteus is also a scientific co-founder of CRISPR Therapeutics, a company focused on developing gene-editing based therapeutics. He joins industry leaders Sir Andrew Witty, CEO of Optum, and Dr. Jennifer Doudna, co-inventor of CRISPR, on the advisory board.
“Our vision is a future where cell and gene therapies are ultimately as accessible as vaccines, so that everyone can benefit from next-generation cures,” noted Dabrowski. “Synthego will continue to innovate to help researchers redefine the boundaries of transformative medicines.”

Correcting sickle cell mutation with CRISPR/Cas9
CORALVILLE, Iowa—In a study published in Nature Medicine back in August, researchers at Integrated DNA Technologies (IDT) and the laboratory of Dr. Matthew Porteus at Stanford University described a novel Cas9 mutant that shows improved specificity and maintains high activity when used in the medically relevant ribonucleoprotein (RNP) format. Potential for medical use of the new mutant enzyme was demonstrated in human hematopoietic stem and progenitor cells (HSPCs), where it was able to correct the mutation in the beta-hemoglobin gene responsible for sickle cell disease.
Several groups have previously described Cas9 mutants with improved specificity; however, all show significantly reduced activity when used in the clinically relevant RNP format. These previous mutants were developed using intelligent design based on known protein crystal structures. IDT scientists used an unbiased method to screen approximately 250,000 random Cas9 mutants, in order to identify those rare mutants that improved specificity without compromising activity.
After several rounds of selection a single mutant emerged: Alt-R HiFi Cas9 nuclease, which provides the desired high on-target, low off-target characteristics. In the published study, a collaborative team from IDT and Stanford demonstrated the robust on-target editing and minimal off-target cleavage achieved by HiFi Cas9 in several therapeutically relevant loci in hard-to-edit HSPCs. They also showed HiFi Cas9-mediated correction of the sickle cell disease-causing p.E6V mutation in patient-derived HSPCs.
“Previous attempts at improving Cas9 specificity characterized the mutants using plasmid-based methods that result in sustained overexpression of the Cas9 protein, which increases off-target activity and is not ideal for medical applications. This sustained overexpression, however, rescued function of the mutants that otherwise showed low activity when used in the more transient RNP format,” said Dr. Mark Behlke, chief scientific officer at IDT and a co-author of the study. “We specifically performed a broad screen to identify a mutant that performs well when used at the lower protein levels achieved with RNP delivery, maximizing safety and further reducing unwanted side effects. Prof. Porteus demonstrated utility using the new system to correct the SCD mutation in normal human blood-forming stem cells while minimizing known off-target activity. We anticipate significant interest in use of the new Cas9 mutant in translational medical applications.”
The novel HiFi Cas9 nuclease is now commercially available as Alt-R HiFi Cas9 Nuclease V3.

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