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Mar 1, 2013 (Vol. 33, No. 5)

Genome Editing for R&D and Therapeutics

  • “Although TALENs have exquisite programming specificity, one problem is their sensitivity to methylation, a ubiquitous DNA modification. We overcame this major bottleneck by using a combination of biochemical, structural, and cellular approaches and have developed an efficient and universal method to overcome this limitation. Moreover, by coupling end-processing enzymes with nucleases, we improved even further targeted gene disruption in a variety of cell types.”

    In addition to tools and services, Cellectis is also focusing on therapeutic applications based on gene editing. They are targeting cancer with a strategy of adoptive immunotherapy that engineers immune T cells to make them recognize and kill cancer cells. They are also pursuing diabetes in collaboration with Novo Nordisk.

    “There is a huge market for companies wanting to model their test drugs. We are also utilizing our TALEN technology to prepare tailor-made cell lines. Since our TALENs can be rapidly designed and synthesized, this is a significant advantage for creating these cell lines with both speed and precision. Clearly, we are just at the beginning stages of this powerful technology,” says Dr. Duchateau.

  • Novel Gene Editing Approach

    Transposagen offers a technology that they describe as the first footprint-free gene editing platform. Jack Crawford, vp of business development, explains: “The problem with recombinases and viral platforms is that they incorporate extraneous DNA into a genome. This can be a significant hurdle when using these technologies for human therapeutic applications.

    “We wanted a system that did not add extra sequence, so we designed our Footprint-Free™ gene editing system that uses a combination of our XTN™ site-specific nuclease technology, which is a customized TALEN, and our piggyBac™ DNA modification system. The latter is a DNA transposon, a mobile genetic element that can quickly and easily be removed from the genome in a footprint-free manner upon expression of a transposase enzyme.”

    Transposagen’s CEO, Eric Ostertag, M.D., Ph.D., says, “We are excited that we can provide this footprint-free gene editing platform. Clinical trials for a variety of cancers are about to begin in the U.S. and Australia using our piggyBac technology, and the Footprint-Free gene editing technology has been used to correct a variety of congenital liver diseases in animal models. We are working to increase the efficiency of the system in order to speed up the gene editing process. This is especially important in the arena of cancer treatment where a patient cannot always wait several months for treatment.”

  • Adeno-Associated Virus

    Click Image To Enlarge +
    Horizon’s Genesis system carries out gene editing with rAAV vectors.

    An alternative approach to endogenous gene-editing is the recombinant adeno-associated virus (rAAV) Genesis™ system from Horizon Discovery. Clearly differentiated from nuclease systems by not requiring DNA breaks, Genesis uses the cells’ natural homologous recombination machinery. Horizon claims its rAAV technology provides great flexibility in being able to sequence-mutate genes as easily as it can delete them, without causing the off-target alterations or sequence errors that can occur with nucleases, and with sensitivity at single base-pair resolution.

    “While Horizon is relatively new to the commercial gene-editing arena, we are fast catching up. This is because of rAAV’s precision, but also because we are bringing significant advances in efficiency and throughput,” says Chris Torrance, Ph.D., CSO and founder of the company.

    “We are also experts in translating this technology into better understanding gene function, how DNA mutations cause disease, and accelerating the process of novel target identification and drug discovery. Developing such tools and services is unique among gene-editing companies, but has allowed us to reach out to the wider research community, where it is still surprisingly underappreciated that gene editing is now an accessible and robust technology.”

    Horizon has utilized Genesis to create more than 500 X-MAN™ (gene-X mutant and normal) genetically defined isogenic disease model cell lines. “These and many more disease models are needed to study cancer biology and support more efficient drug discovery, because genomics advances tell us that cancer is essentially hundreds of orphan diseases, comprised of multiple subtypes driven by distinct genetic features,” says Dr. Torrance.

    “Cellular disease models (i.e., ‘patients-in-a-test-tube’) will greatly facilitate many aspects of novel targeted drug discovery, especially the stratification of patients into more focused clinical trials where they have the best chances of responding.

    “Horizon is passionate about spreading the word on genome editing. We have already entered into many collaborations, including forming more than 30 centers of excellence with small and large nonprofit organizations, providing them free training in rAAV gene editing, and plan to launch ready-to-edit kits in the future. We are finding scientists very receptive to the technology, and we also get to expand our disease knowledge base and range of disease models. It is a win-win situation.”

  • RNA-Based Editing

    Click Image To Enlarge +
    Factor Bioscience relies on RNA-based gene-editing and reprogramming methods to model human diseases in vitro and in vivo. Image shows human neurons, tagged with a green fluorescent marker, that were derived from the skin cells of an adult patient using Factor’s technology.

    Two major challenges with the introduction of zinc finger or TALEN nucleases are getting a high enough level of expression and persistence of the introduced DNA constructs, notes Matt Angel, Ph.D., CEO, Factor Bioscience. “The use of synthetic RNA to express proteins is often overlooked. Our company is developing research tools and therapeutics using our RNA-based gene-editing and reprogramming technologies.”

    According to Dr. Angel, the idea is to deliver an RNA that encodes a gene-editing protein, such as a TALEN, that is targeted to a specific gene. “When our RiboSlice™ molecules are delivered, cells translate them into TALENs that subsequently create either nicks or double-strand breaks in the cell’s DNA. This leads to the disruption or targeted insertion of a specified gene, if we co-deliver the desired gene DNA template.”

    The company recently received two Small Business Innovation Research grants aimed at developing treatments for Alzheimer’s disease.

    “Good research models for the study of Alzheimer’s disease are lacking. The efficiency of RiboSlice provides a way to generate complex, well-defined mutations and do so very rapidly. Our technologies allow us to insert or delete genes, introduce defined mutations, and control cell type. Our humanized rodent models will allow the pharmaceutical industry to screen drug candidates in a new way,” explains Dr. Angel.

    Factor is also utilizing its technologies for integration-free reprogramming and directed differentiation to create a library of modified human neural cells that exhibit specific mutations found in Alzheimer’s disease.

    “These cells can be utilized for high-content screening of therapeutic candidates and represent the first isogenic library of cells containing defined mutations for screening drugs to treat or prevent Alzheimer’s disease,” he points out.

    Dr. Angel says there are many other applications envisioned by the company. “We are currently transitioning from the discovery phase to preclinical studies. We are interested in connecting with other partners as we envision that this technology can be utilized in a wide variety of genetic diseases. For example, in type 1 diabetes, one needs cells with a normally functioning insulin gene. Our technologies can create these cells efficiently.”


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