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

Genome Editing for R&D and Therapeutics

  • The ability to edit the human genome in a targeted and specific way allows correcting genes with harmful mutations, producing therapeutic proteins, and custom designing cell lines to model human diseases, such as cancer. Many feel that gene editing promises to write the next chapter in biomedicine.

    Science described new gene editing technologies as “genomic cruise missiles” and named them as runners up to the scientific discovery of the year for 2012 (which went to the Higgs Boson).

    Added to technologies in place for the last decade, a number of other powerful new techniques are putting DNA modification into the hands of even nonexperts. Which will win out as the premier tool is something no one yet knows. Each has its advantages and disadvantages. What is clear is that readily modifying human and other genomes within a wide range of cells is now a reality.

  • ZFN Technology

    Click Image To Enlarge +
    A pair of zinc finger nucleases (ZFNs) bound to DNA. ZFNs are able to recognize their intended gene targets through their engineered ZFP DNA-binding domains. [Jeff Miller, Ph.D./Sangamo]

    Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate highly specific targeted genomic editing by creating double-stranded breaks in DNA at user specific locations. “You can use ZFN technology to create your choice of stable, genetically engineered cell lines or organisms harboring gene deletions, additions, or other modifications with exquisite specificity,” says Greg Davis, Ph.D., principal R&D scientist for ZFN technology at Sigma-Aldrich.

    The company is focusing a segment of their business on providing ZFN products and ZFN custom services. They became the exclusive licensee and distributor of such research reagents under an agreement established in 2007 with Sangamo Biosciences.

    According to Dr. Davis, slicing both strands is important for site-specific mutagenesis applications because the break stimulates the cell’s natural DNA-repair processes, i.e., homologous recombination and nonhomologous end joining (NHEJ). Harnessing nature’s toolbox for repair allows generation of precisely targeted in vitro or in vivo genomic editing with targeted gene deletions (knockouts), integrations, or other modifications.

    “Via the NHEJ pathway, ZFNs have enabled creation of knockout cells and animals at high frequency without the need for drug selection or exogenous DNA templates. This has greatly reduced timelines for isolating modified cells and animals,” he explains.

    Using the technology, the company has created knockout and SNP insertion cell lines in their oncology and breast cancer panels. Additionally, the Pathway and Cytoskeletal Marker cell lines comprise a suite of genes tagged at the endogenous locus that allow an investigator to monitor protein expression and localization.

    A large part of Sigma’s ZFN technology is devoted to custom service for its clients. Kevin Kayser, Ph.D., director of the CHOZN® platform for SAFC, notes, “Not everyone wants to be a gene-editing expert. Sometimes it is more efficient to outsource that. We provide off-the-shelf specific ZFNs to target genes for human, mouse, and rat studies in the form of cell lines and animals.”

    SAFC has taken the ZFN technology a step further by utilizing it to create CHO cell lines for use in biopharmaceutical applications. “These CHOZN cell lines were purpose built to enable biopharmaceutical producers to advance their molecules in the clinic faster, while adhering to their stringent regulatory expectations,” says Dr. Kayser.

    SAFC currently provides both glutamine synthetase and dihydrofolate reductase knockout CHO cell lines to biopharmaceutical customers, as they represent the two most commonly used metabolic selection systems in the industry, according to Dr. Kayser.

  • ZFN Therapeutics

    Translating technology to patient treatment is the ultimate goal of many gene editing pursuits. Sangamo BioSciences is providing encouraging early data on the utility of ZFN therapeutics. They are now in a Phase II and two Phase I/II clinical trials to evaluate SB-728-T, a ZFN-modified T-cell product for the treatment of HIV/AIDS.

    Philip D. Gregory, D.Phil., vp of research, and CSO, describes the company’s approach. “HIV-1 gains access to target cells by sequentially binding the viral pg120 Env protein to the CD4 receptor and a chemokine co-receptor. The major co-receptor is CCR5 that is expressed on key T-cell subsets. It is relatively common for Western Europeans to have a 32-base pair deletion that confers resistance to HIV-1 infection and AIDS in homozygotes.

    “We are utilizing our ZFN-mediated gene disruption technology to disrupt the CCR5 gene in cells of patients’ immune systems. This effectively creates an immune system within an immune system. The patient now has a population of cells that are permanently resistant to HIV and opportunistic infections and thereby mimics the immune system of those that carry the natural mutation.”

    Sangamo is also employing its ZFN platform to examine curative treatment for a range of monogenic diseases such as hemophilia and lysosomal storage disorders like Gaucher and Fabry diseases.

    “ZFN technology is perfect for the goal of personalized medicine, especially for development of potentially curative approaches to single-mutation diseases. An example is our program that targets hemophilia in which the liver fails to produce sufficient amounts of coagulation factor IX due to a single mutation,” says Dr. Gregory.

    “We designed a ZFN approach in mice that could induce gene targeting in the liver to allow delivery and correction of the mutated gene. This strategy succeeded in the stable liver-specific production of human Factor IX protein to 100% of normal circulating levels.”

    The study was performed in collaboration with the laboratory of Katherine A. High, M.D., director of the Center for Cellular and Molecular Therapeutics at The Children’s Hospital of Philadelphia.

    According to Dr. Gregory, “All technologies share the common goal of targeting the genome for therapeutics. This represents a huge paradigm shift for how medicine is performed. ZFN technologies are leading the field.”


    One of the new kids on the block is a tool that promises easy access to gene editing by even nonexperts in the field. TALENs (transcription activator-like effector nucleases) work much like ZFNs to home in on and cut specific genomic DNA sequences. But unlike ZFNs that bind to groups of three base pairs, TALENs binds to individual nucleotides, allowing their use anywhere on the genome.

    Cellectis believes its TALEN™ technology will greatly speed up the process. “We jumped into this technology initially because we could see that it provides a very efficient and rapid means to perform gene editing,” indicates Philippe Duchateau, Ph.D., CSO.

    “In only about one week, one can design and begin implementing gene-specific modifications. Additionally, our TALEN technology has a number of advantages over other technologies. They are less costly to produce, are really efficient tools, and may be designed to target virtually any DNA sequence. Genome editing is no longer a matter for the specialists.”

    Dr. Duchateau also notes recent improvements enhancing efficiency of the technology.

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