By Kevin Kayser
The field of targeted genome editing has advanced rapidly since the recent advent of Zinc Finger Nuclease (or ZFN) technology. ZFNs are custom-engineered fusion proteins that bind DNA in a targeted manner, creating double-stranded breaks at precise locations, which enable insertions, deletions, or modifications of the genome. The result is a reliable method for creating genetically modified cell lines and model animals that is both faster and more accurate than traditional methods.
Founded in the mid-1990s to exploit patents licensed from three academic institutions — Johns Hopkins University, the Massachusetts Institute of Technology (MIT), and the Scripps Research Institute — Sangamo Biosciences developed the ZFN platform into the robust technology that is used today. Through a series of acquisitions and licenses, the company captured the intellectual property in this field and used this to dramatically advance the science. In recent years, Sangamo has turned its focus to the use of Zinc Finger Protein (ZFP) technology in therapeutic areas.
Genetic modification in a living cell or organism can give researchers important insights into genetic diseases and pathway biology, but it has proven to be a major challenge for researchers. ZFN technology has proved sufficiently robust for use in gene editing and has the potential to be a powerful tool.
How Does It Work?
A Zinc Finger Nuclease is a chimeric protein composed of a ZFP fused to the FokI endonuclease. ZFPs are relatively small, at about 30 amino acids, and take their name from the fact that the protein is complexed to one or more zinc atoms, which act to hold the 3-D structure of the folded protein in place. These complexes or modules can be engineered to bind to a specific sequence of three DNA nucleotide bases. So everywhere there is the module’s target sequence, say ATT or CGA (Chorionic gonadotropin alpha), the Zinc Finger will bind. As there are only four bases in the structure of DNA, statistically there will be many points in an organism’s genome where this sequence appears. Therefore, the ZFP module could bind in several places throughout the genome. A dramatic increase in specificity is achieved when several modules are joined together. Four connected modules, for example, will only bind to a specific 12 nucleotide base sequence, which is far less likely to be repeated in the genome. When this DNA binding domain is fused with an endonuclease, you have a functional ZFN, which is able to introduce a break at a precise location in the target DNA strand. If a matched pair of ZFNs targeting opposite strands of a DNA sequence at the same locus is introduced into a cell, then both strands of DNA can be cut, resulting in a double stranded break (DSB).
Once the DNA has been cut, the next step in the targeted genome editing process is to repair the DNA. This is achieved by relying on one of the cell’s natural biological processes to repair DSBs, either nonhomologous end-joining (NHEJ) or homology-dependent repair (HDR). NHEJ is the simplest. It does not utilize any form of repair template, and therefore, the repair process is often imperfect. The end result is commonly a mutated DNA sequence that can effectively knock out expression of a gene. With HDR, if a repair template is introduced into the cell along with the ZFNs, HDR can be utilized by the cell to integrate exogenous DNA into the genome.
ZFN mutagenesis has many advantages over alternative methods such as chemical mutagenesis or gamma irradiation where mutations are unpredictable. ZFNs both accurately and rapidly enable targeted modifications in any area of the genome, with the edits being introduced in a single transfection experiment. The speed is impressive — gene knock-out or knock-in cell lines can be created in as little as two months, with single or biallelic edits occurring in up to 20% of the transfected population. Importantly, the mutations are both permanent and heritable, and the technique has proved effective in a variety of cell types.
Customized ZFNs are now being created based upon customer requests within a couple of months. These validated pairs of ZFNs are designed to create DNA strand breaks at the desired genomic location in a specified cell line or organism. Custom ZFN pairs, where the researcher defines the target site in their gene of interest, are available, and costs depend on whether or not the product will be used for research or commercial applications. This ZFN pair can then be used to create the desired genotype/phenotype in any cell line with the same ZFN target sequence.
In addition to the custom ZFNs, so-called “Knock-Out” ZFNs are also now available. These “off-the-shelf” ZFNs are target genes that are commonly studied and are predesigned and manufactured. For researchers interested in transgene expression studies, Targeted Integration (TI) Kits are now commercially available and allow users to rapidly integrate the gene they are interested in into a single location on human chromosome 19, from where it will be stably expressed. Unlike other methods for transgene expression in human cells, the kit allows scientists to study the gene in the cell line of their choice by providing stable and uniform protein expression, without the need to engineer cell lines that have transgene landing pads.
The potential of ZFNs is vast, and the number of research publications in the field has increased dramatically in recent years. While the earliest of these focused squarely on the technology itself and its theoretical potential, more recent publications have focused on practical applications of the technology.
One early technology-based paper, from Luigi Naldini of the Fondazione San Raffaele del Monte Tabor in Milan, Italy, described the utility of ZFNs in human genome editing, exploiting the infectivity of a form of lentivirus vector to give efficient delivery to a variety of different cell types, including stem cells. This was an excellent illustration of the potential of using ZFNs in gene editing.
Now that the power of ZFNs in genome editing is clear, increasing numbers of real-world applications have been revealed. Researchers have been using the technology to better understand the biology of the systems they are studying. For example, by knocking out a gene, its activity and normal role in both development and behavior can be studied. The potential of “knock-ins” — where genes are inserted — is also becoming clear. For example, a recent paper from David Allis at the Rockefeller University in New York highlighted the use of TI by tagging a particular gene so the genomewide profiles of variations of that gene could be determined in mammalian embryonic stem cells and neuronal precursor cells.
It’s not only academia that is embracing ZFN technology. Biopharmaceutical companies have also been using ZFNs in their research. Eli Lilly and Pfizer have presented examples where ZFNs were used to create gene knock-out cell lines. The speed and accuracy in creating robust cell lines is a particular advantage for industry, where companies are trying to reduce the overall timelines from initial discovery to clinical trials. Pfizer, for example, recently announced at IBC’s Cell Line Development and Engineering conference that it could reduce the time from transfection to the start of toxicology studies by up to eight weeks by using a ZFN knock-out cell line.
Creating off-the-shelf cell lines with specific knock-outs that mimic disease states are also being developed. The BRCA1 gene is a good example; some mutations of this gene are associated with a high risk of breast cancer, so a breast cancer cell line with this gene knocked out would be important. Similarly, point mutations in p53, also associated with cancers, are being developed. These cell lines will allow diseased cells to be compared with a parental cell line, which differs only in the site of the mutation.
Another development is the creation of cell lines that contain fluorescent fusion tags. When these are fused to genes in the cell, they can be used to identify the state of the cell. In other words, they make it very simple to spot whether the cell is responding to a particular stimulus or drug molecule using a color change. These will be particularly important in high-throughput screening processes, as no complex analytical methods are required to pick out cells where the stimulus has had an effect. This ability to create reporter tags of endogenous genes and make them quantitative will enable a variety of different screening options or differential analyses to be carried out.
The technology is also being used to create transgenic animals. Knock-out mice, for example, have been important research tools for the past 20 years and were created by modifying mouse embryonic stem cells. A knock-out rat would be useful in biomedical research, as rats are generally better models for human disease than mice. However, it had proved impossible to make a knock-out rat the same way because of its increased complexity. An important advance in trangenics took place just last year with the creation of the first knock-out rat through the use of ZFNs.
In collaboration with Howard Jacob at the Medical College of Wisconsin, Sangamo and Open Monoclonal Technology, Sigma Advanced Genetic Engineering scientists managed to knock out an inserted reporter gene and two native rat genes. Importantly, there was no effect on any other genes, and the changes were heritable as they were passed on to the rats’ offspring. A knock-out rat can be bred from scratch in about four months, compared to the year or more it takes to create a line of knock-out mice using embryonic stem cell technology.
The knock-out rats are already attracting a good deal of attention from the research community, and the Michael J. Fox Foundation is funding the creation of six different knock-out rats, each with the deletion of a gene thought to be involved in Parkinson’s disease. The hope is the modified rats will give insights into the molecular basis of the disease. Founder rats of five of these have already been created, and large colonies of each knock-out line are now being bred. The first of these should be available for customers by the end of this year, with the remainder following in early 2011.
ZFN technology also has huge potential in cell line engineering for biopharmaceutical manufacturing. Biological drugs already represent about 10% to 12% of the total global pharma market of about $825 billion, and revenues from these types of drugs are growing three to four times as quickly as traditional small molecule drugs. Companies are looking at more efficient and cost-effective methods for manufacturing, and ZFNs can help.
Chinese Hamster Ovary (CHO) cell lines harboring genetic modifications in specific genes of interest are also being created. These new cell lines will enable cleaner and faster selection methods as well as more robust growth, productivity, and product quality characteristics. With the use of ZFNs, several genetic loci can be altered in the same cell line. Through this trait stacking, or engineering several desirable characteristics into a single host cell, ‘ideal’ cell lines can be developed for biotherapeutic manufacturing. These cell lines can lead to real economic benefits to the biopharma manufacturer by producing better quality products more quickly and efficiently.
In a few short years, ZFNs have made a significant impact on the scientific community. Their ability to make targeted, heritable changes to genes has already led to quicker development of knock-in and knock-out cell lines for drug discovery programs and more productive cell lines for manufacturing. In the future, there is the potential to improve drug safety by developing better cell lines and animal models for toxicology and drug metabolism studies. The knock-out rat is proof positive of the technology’s potential in creating model animals. This technology will also be able to facilitate, in livestock, the improvement of traits that will improve the nutritional value of foods. It’s only the beginning of the story, and it is already clear that ZFNs have the potential to improve the quality of life.
About The Author
Dr. Kevin Kayser is the manager of the Cell Culture Engineering group at SAFC and an adjunct professor at Saint Louis University (St. Louis, MO). He is responsible for the oversight of a team of scientists developing new products, services, and platform technologies for SAFC.