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

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

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.

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]

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.

“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

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.”

Horizon’s Genesis system carries out gene editing with rAAV vectors.

RNA-Based Editing

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.”

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.

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