LIFE SCIENCE TECHNOLOGIES
Not long ago, a clinically relevant and genomics-based model of disease meant an animal, but today's models also include cell lines and even computer-based simulations. Still, bioengineered mice and rats make up many of the models, and these genetically modified organisms provide increasingly accurate representations of how drugs treat human diseases. To get the most from genomics-based models for drug research, scientists can now combine information from bioengineered organisms, genomically modified cell lines, and computational models.
By Mike May
Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.
The lab mouse represents an icon of clinical research. A PubMed search of mouse model pulls up more than 150,000 articles, which indicates the pervasive use of this clinical tool in modeling diseases. Moreover, groups of scientists keep developing new ways to use mice in basic and medical research.
For example, two consortia—the International Knockout Mouse Consortium (IKMC) and the International Mouse Phenotyping Consortium (IMPC)—hope to improve bioengineered mouse models. First, the IKMC, based in laboratories in Europe and North America, is making individual mutant embryonic stem cell lines in which one of the mouse's roughly 20,000 protein-coding genes has been knocked out. So far, the IKMC has created more than three-quarters of these cell lines. As a next step, the IMPC—composed of 10 research institutions around the world—will produce knockout animals for each gene and analyze the phenotypic changes. Although the list of phenotypic tests remains under development, it will likely range from basic blood work to behavioral assessments. The IMPC plans to phenotype all 20,000 knockout mice by 2020.
To breed the cohorts of mice needed for this phenotyping project, the IMPC turned to some powerhouses in breeding, including Charles River Laboratories in Wilmington, Massachusetts. "The repository of knockout-mouse lines and the corresponding phenotypic data will lead to a global availability of novel mouse models across many areas of disease," says Iva Morse, corporate vice president, Charles River Genetically Engineered Models and Services. "Because of the IKMC and IMPC's efforts, researchers will be able to search for specific genes, examine the primary phenotype screens, and then get the animals for further testing."
Once all of the knockout mice are phenotyped, researchers will be able to use a database to review some of the fundamental anatomical, biochemical, and physiological impacts of each gene as well as possible disease-related factors. This phenotypic database should free researchers and resources to pursue more complex questions, such as how genes interact with environmental factors.
Rather than only working with existing mouse strains, some researchers are developing new ones since many of the existing strains have little genomic variation. "The standard lab mouse is not exactly what we thought in terms of diversity," says John French, leader of the host susceptibility group at the National Institute of Environmental Health Sciences in Research Triangle Park, North Carolina. He explains that inbreeding has created some identical regions in the genomes of lab mice. For example, rather than having gene variants at a specific locus, inbred mice often all have the same allele in that spot. Consequently, mice from one line can be very similar to each other but extremely different from other mouse lines. For example, French describes using 18 strains of inbred mice to study the physiological impact of benzene exposure on biochemical processes that modify foreign compounds, such as drugs or poisons. Some characteristics—such as the amount of benzene in the blood after the same level of exposure—varied by as much as 36-fold between the strains. French notes that benzene exposure can lead to leukemia in both mice and humans, and this variance between inbred strains may be useful for risk assessment, because some strains may show higher incidence of disease than others.
Other experts also worry about the lack of diversity in traditional mouse lines. "The way rodent models have been used in drug development and toxicity testing only surveys a limited scope of the genetic diversity that's available," says Gary Churchill, a biostatistician at The Jackson Laboratory in Bar Harbor, Maine. "To assess if a drug will have dangerous side effects in a subset of the human population, we must sample broad genetic diversity in the model system."
To meet that need, Churchill and his colleagues created the Diversity Outbred (DO) Mouse population, which represents 45 million single nucleotide polymorphisms (SNPs) and 4 million copy number variations. As Churchill says, "We've captured mouse genetic diversity." In comparison, the most commonly used strains of mice—roughly 100 of them—only include about 12 million SNPs.
When studying a drug's impact, Churchill says, it will probably take 100-200 DO animals to explore the potential for diverse effects. Using this many animals should cover all of the SNPs and copy number variations, which will indicate how the drug would perform when encountering various genotypes.
During drug development, testing a new compound for toxicity is vital. However, Alison Harrill, a research investigator at The Hamner Institutes for Health Sciences in Research Triangle Park, North Carolina, says, "Drug safety is becoming a key bottleneck." When taking a new compound from the discovery stage through to regulatory approval, toxicity testing can take so much time and effort that it slows down the entire process. Moreover, some toxicity issues only emerge after a drug has gained regulatory approval. For instance, Harrill says, "Hepatotoxicity issues [have been found to] exist in about one-third of drugs pulled from the market."
In hopes of revealing toxicities at the research stage, Harrill works with DO mice because, she says, they provide a "better estimate of human responses and identify genetic variants that might increase the risk of an adverse drug reaction." She also notes that this strategy can be used to rescue drugs that have failed toxicity testing in previous clinical trials and allow researchers to develop screens that predict which patients would respond safely to the drugs and which ones would not.
Mouse models of diseases can also help researchers find new drug targets. For diabetes, for instance, scientists at Lexicon Pharmaceuticals in The Woodlands, Texas, searched the company's collection of knockout mice for animals that have altered glucose control. According to Brian Zambrowicz, chief scientific officer, they found that knockout mice lacking the gene for either sodium-dependent glucose transporter 1 (SGLT1) or SGLT2 exhibited a "much improved oral glucose tolerance," or the ability to maintain balanced blood levels of glucose after eating. Because type II diabetes causes glucose levels to rise dangerously high in the blood, Zambrowicz and his colleagues hoped that a compound that blocks SGLT1 and 2 might treat the disease. The Lexicon scientists then developed cell-based assays to screen for compounds that inhibit these transport proteins and discovered LX4211, a compound that inhibits both transporters. One way this compound helps to maintain a balanced level of blood glucose is through its effects on SGLT2, which participates in the absorption of glucose by the kidney. Blocking this protein increases the amount of glucose that gets excreted in urine. In addition, LX4211 triggers the release of glucagon-like peptide-1 and peptide YY in the gastrointestinal track, and this mechanism provides further glucose regulation.
Genomic tools can also be used to reveal how a drug is metabolized. For example, Xavier de Mollerat, senior scientist at Life Technologies in Carlsbad, California, says that a scientist who suspects that a drug's metabolism depends on a specific receptor can use small interfering RNA (siRNA) to knock down, or inhibit, the receptor's expression and measure the drug's distribution before and after treatment. If the measurements differ, the receptor is involved. Researchers can also use this technique to make a transient animal model of a disease. If the lack of one or more genes is known to be involved in a disease, a researcher can use siRNA to knock down the gene(s). For example, Invivofectamine 2.0 from Life Technologies can simultaneously silence up to four genes with just one application of siRNA. As explained by de Mollerat, "This technology's lipid delivery system is designed to use siRNA in vivo. You inject the siRNA, and it knocks down its targets for weeks."
This technology also only requires a few steps. A scientist combines the desired siRNA with the Invivofectamine 2.0 reagent, performs a couple of simple processes, such as an incubation step, and then injects the mixture into the animal's blood stream or to a more specific location if desired. According to de Mollerat, the siRNA will knock down the gene's protein expression by 80-90 percent. "If you know the sequence, you can target it," de Mollerat says.
De Mollerat says that similar technology might work with messenger RNA (mRNA). Then, a researcher could compare knocking down a gene with siRNA to overexpressing it with mRNA. That would provide further information about a gene's function in general or how it participates in a disease.
Although mice have been the main bioengineered animal used for disease modeling in the past and will continue to be abundant in the future, genomically modified rats will also become an increasingly common tool. The numerous and extensive history of existing mouse lines provide good reasons to stick with this rodent, but the rat model offers upsides of its own. Just the larger size of rats can enhance certain aspects of drug research. For example, when scientists need a large amount of blood, such as for testing a compound's absorption, distribution, metabolism, and excretion (ADME), it is easier to obtain the necessary quantity from bigger animals. In addition, the physiology of liver metabolism in rats resembles that of humans more closely than mice do.
Consequently, bioengineered rat models will likely become more widely available in the next few years. For example, Charles River Laboratories arranged to distribute rat models of cancer related to the genes p53 and BCRP from Transposagen Biopharmaceuticals in Lexington, Kentucky. As Morse explains: "New methods for manipulating the rat genome further advance functional genomics and allow us to make novel models of human disease." Even Pfizer, headquartered in New York, New York, is providing a genetically engineered rat model of diabetes to Charles River for distribution.
Scientists are continually expanding the catalog of diseases modeled in rats. One of the most exciting new advances arose from a collaboration between scientists at Sigma Life Science in St. Louis, Missouri, and Autism Speaks, an advocacy group. As Edward Weinstein, director of Sigma Advanced Genetic Engineering Labs, explains, "We thought that we could use rats to make some impact on autism." So he and his colleagues worked with Autism Speaks to help them "connect to the community and understand what the best models to make would be," Weinstein says.
Through this collaboration, Sigma scientists learned that a range of genes—including MECP2, FMR1, NLGN3, and others—contribute to autism. Consequently, Weinstein and his colleagues developed knockout rats for many of these genes.
"We hope that using a higher level organism—a rat instead of a mouse—will help unravel some of [the genomic complexity of autism]," explains Weinstein.
Creating models for clinical research does not solely depend on animal use these days. When conducting drug research, scientists often turn to cells for modeling as soon as possible, and Sigma Life Science offers a range of disease-model cell lines.
In particular, Sigma makes many cell lines for cancer research. Brad Keller, product manager at Sigma Life Science, says, "Genetically engineered cell lines for breast cancer are our biggest offering." He adds that they also have cell lines for colorectal and lung cancer. "Each cell line is specific," says Keller. For example, a cancer cell line will include a mutated gene discovered in patients with the disease. Moreover, these cell lines are created from human cells, which makes any experimental results more relevant to human disease.
Beyond cell lines, some projects involve computer simulations. For instance, the Broad Institute in Cambridge, Massachusetts, developed The Connectivity Map (cmap), which uses computer algorithms and genome-wide expression data to explore the connection between diseases, genes, and drugs. To expand cmap, researchers developed the L1000 assay, which runs on the FLEXMAP 3-D system from Luminex in Austin, Texas. This assay can screen about 1,000 genes per sample. "The assay makes genome-wide transcriptional profiling of compound treatments possible at library scale for the first time," says Matt Grow, Luminex's manager of strategic development.
In addition, Colin Hill, chief executive officer at GNS Healthcare in Cambridge, Massachusetts, points out that computer models "are being seen more and more as an important alternative to animal models." As an example, the National Cancer Institute of the U.S. National Institutes of Health came to Hill's company for the analysis of data from genetically modified mouse models of nonsmall cell lung cancer. In short, Hill's machine learning technology is searching the data—from imaging, genes, pathology, and more—for connections that could reveal mechanisms behind this cancer.
By combining animal models, cell lines, and simulations, researchers are now able to develop a wide ranging toolkit for modeling diseases. This breadth of models then allows for more in-depth testing of new drugs in a wider range of genomic variations.
National Cancer Institute
National Institute of Environmental Health Sciences
Sigma Life Sciences
The Hamner Institute for Health
The Jackson Laboratory
Note: Readers can find out more about the companies and organizations listed by accessing their sites on the World Wide Web (WWW). If the listed organization does not have a site on the WWW or if it is under construction, we have substituted its main telephone number. Every effort has been made to ensure the accuracy of this information. Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.
Mike May is a freelance writer and editor for science and technology.
|This article was published as a special advertising feature in the 24 February 2012 issue of Science magazine.|
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