David Smoller — targeted knockout rats

Dr. David Smoller, President of the Research Biotech Business Unit at Sigma-Aldrich, speaks to Pharmaceutical Technology Europe about the creation of the first targeted knockout rats, which will enable the development of new human disease models. The rats were created using Sigma-Aldrich's CompoZr zinc finger nuclease (ZFN) technology. More about this development can be read here.

Q1: Although knockout mice have been used in scientific studies for a while, it has taken years for researchers to create knockout rats. What have been the main difficulties?
Rats are often the preferred choice of disease researchers to conduct testing; rats are a closer physiological model to human disease than mice and are often used to test the efficacy of drugs or treatments for diseases. Researchers began creating knockout mouse models in the 1990s through embryonic stem cell manipulation, which does not work well in rats. This technique established knockout mice as an industry standard for disease modeling despite the inherent drawbacks of the mouse as a research model. Each species has unique barriers and bottlenecks for genetic manipulation; mouse genomes were simply easier to target and manipulate than rat genomes with the technology available at the time. Now, with CompoZr ZFNs, Sigma-Aldrich, along with its academic and corporate partners, has developed a technique to quickly and easily induce targeted gene knockouts in rats. Although individual, untargeted knockout rats have been created before, Sigma-Aldrich will be able to produce targeted knockout rats in a very short period of time with this ZFN technology.

Q2: After so many other researchers have failed to create knockout rats, what made this particular technique succeed?
A handful of knockout rats have been available to researchers for several years, but the main issue limiting their use for research was the practicality and efficiency of producing knockouts with previous techniques, which were untargeted, costly and time consuming. That’s why our ZFNs are so significant. ZFNs are a class of engineered DNA-binding proteins that facilitate targeted editing of the genome within a living cell by creating double-strand breaks in DNA at user-specified locations. Double-strand breaks stimulate the cell’s natural DNA-repair processes, both homologous recombination and nonhomologous end joining, to induce site-specific genetic changes. Previous genetic engineering technologies, including nuclear transfer or the manipulation of embryonic stem cells, could not precisely target or modify a specific region of the genome. Using CompoZr ZFNs, researchers can — for the first time — generate precisely targeted genomic edits, creating cell lines or organisms with user-specified gene deletions, insertions or modifications. These genomic changes are permanent and heritable. The technology has already been widely demonstrated for use in a number of organisms, including invertebrates, zebrafish, mice, cell lines and now rats.

Q3: Why was zinc finger nuclease (ZFN) technology so crucial?
ZFN technology provides researchers with the ability to target and precisely manipulate the genome of eukaryotic organisms to produce permanent and heritable changes. This technology enables scientists to build entirely new animal models and grow cell lines or entire organisms faster and more efficiently than with conventional technology.

Q4: How have researchers across the globe responded to the breakthrough?
Those familiar with ZFN technology were happy to see it come to market last year via our CompoZr ZFN platform. Those same researchers are even more excited to see its potential for a very practical application: knockout rats for disease research. Researchers in the pharmaceutical and biotech industries in particular, who know the current limitations in both targeted gene editing and biological models for disease research, have been eager to see how well our knockout models perform. I think the greater scientific community has been interested to see where else this technology and technique can be applied, whether in multiple knockout models, gene knock-ins or the development of disease models in higher-order species.

Q5: What benefits does the knockout rat offer over the knockout mouse?
Rats are physiologically similar to humans, making them ideal subjects for modeling human disease and for drug-evaluation studies. ZFN-mediated genome editing is being used to generate novel animal models of disease that more closely mimic human disease, as well as to provide more realistic data on the potential toxicity of new drug compounds. ZFN technology bypasses the current need to conduct cumbersome gene targeting experiments involving nuclear transfer or embryonic stem cells and enables the rapid engineering of animal models. Using CompoZr, scientists can create targeted knockout cell lines and animals in less than half the time of other methods — as little as 1 month for cell lines and 4 months for animal models.

The world’s first targeted knockout rat (left) was created using CompoZr zinc finger nuclease technology to knock-out the green fluorescent protein (GFP) gene in the GFP rat (right).

Q6: What is the next step for the consortium involved in the creation of the knockout rat?
Sigma-Aldrich announced the launch of the Sigma Advanced Genetic Engineering (SAGE) Labs in August of 2009. The primary focus of this will be to develop knockout rats with targeted deletions of genes associated with neurological, cardiovascular and inflammatory diseases and for toxicology research. Current animal models in these areas, particularly the now-dominant mouse model, often fail to accurately mimic human disease expression and toxicology response. Our goal is to create knockout models that provide a more faithful human biological model and also have wide research and commercial appeal. Our research partners at the Medical College of Wisconsin (WI, USA), led by Dr. Howard Jacob, are exploring further ZFN-mediated gene editing by attempting multiple knockouts in a single organism. Meanwhile, Open Monoclonal Technology (CA, USA), led by Dr. Roland Buelow, is attempting to develop a fully human monoclonal antibody platform based on transgenic rats, and our partners at Sangamo (CA, USA) are exploring other uses of ZFN technology, including its use in a clinical trial for HIV treatment.

Q7: Although they are a necessity in research, animal models of diseases raise a number of ethical questions. How do the researchers involved in the knockout rat research respond to these concerns?
Research animals fulfil a crucial role in biological research and the understanding of human diseases, and we hope that the knowledge our research models provide will help create better human therapeutics. That said, Sigma-Aldrich is committed to ethical research; we follow proper laboratory practices and we look to the “Guide for the Care and Use of Laboratory Animals,” which is supported by the US National Institutes of Health and the National Research Council. We ensure that every employee understands the importance of humane care and animal welfare through rigorous orientation and training sessions. We can make a strong case that this approach is a positive development for the field. First, the knockout technique using CompoZr ZFNs actually reduces the number of animals required to develop a viable model. And second, since rats are better models of human diseases, the use of knockout rats for disease research may also reduce the number of animals required for testing.

Q8: What kind of future do you predict for the knockout rat?
Transgenic animal sales and services are a roughly $1 billion field. Initially, we will be a small part of that, but we believe there is significant demand for superior human disease models, especially in the field of toxicology testing, and we expect the strong initial demand to grow when the research community sees how well these models perform. We see a great future for this technology and knockout rats, especially as our genetic understanding of disease grows every day.

Q9: And for ZFN technology?
Sigma-Aldrich, in concert with our academic and corporate partners, is continually exploring new research applications for the CompoZr ZFN technology. One exciting application is generating gene knockouts in other animals for disease research. Because the technique makes direct changes to an early embryo and does not rely on embryonic stem cell manipulation, we have every reason to expect ZFNs will be applicable for other research animals as well. We are also exploring the use of ZFNs for the targeted knock-out of multiple genes within the same animal. The ability to perform multiple-gene deletions will allow us to address more complex genetic diseases and could be a boon for the biotech industry.