click to enlarge Knockout rat from Sigma-Aldrich. (All figures: Sigma-Aldrich)

Due to the escalating cost of drug development and the steady decline of marketed product approval, emphasis in the drug discovery industry has long focused on improving the rate of drug attrition. This ratio is estimated at one drug making it to market for every 10,000 compounds that fail along the way.

The “fail early, fail often” paradigm often applied to drug discovery focuses on making improvements to the preclinical stage of development. In vitro and in vivo models are used to predict how compounds will behave in humans in terms of efficacy, pharmacokinetics, and safety. The findings from these studies are typically used to seek approval from the FDA to conduct human clinical studies.

Given that animal models are currently the gold standard for predicting human responses, over the past five years extra effort has been given to the development of more predictive models. This is especially important considering that retrospective industry experience estimates that rodent toxicology studies predict only 43% of human toxicities; non-rodent studies predict 63%; and the combination of both predict about 71% of human toxicities.⁴ There is clearly room for improvement.

Given this industry challenge, Sigma-Aldrich set out to design and develop a suite of knockout rat models that lack the key drug transporters Mdr1a, Mrp1, Mrp2, and Bcrp. By measuring the efflux ratio of the test substrate or substance applied to these rats, these models will be able to determine which transporters potential therapeutics interact with, as well as potential mechanisms of the observed toxicity. It is envisioned that these models will offer an improved alternative to existing mouse knockouts of these genes.

click to enlarge Figure 1. ZFN pairs are designed to target sequence and injected into one-cell embryos. Embryos are transferred into foster mothers. Resulting pups are genotyped and founders identified.

Due to their physiology and more human-like responses to xenobiotics, it is commonly accepted that rats are superior to mice for many ADME-Tox applications. In addition, their larger size facilitates surgical, cell biology, and physiological experimentation. This is particularly important when studies require dissection or isolation of specific cell types and organ structures, as is the case with pharmacokinetics. This, combined with the fact that most historic toxicology data has been collected in the rat, makes them the preferred model organism for determining drug metabolism and early toxicology profiles.

Unfortunately, due to a lack of tools for genome engineering, desirable mutant rats have been hard to come by.

Zinc finger nuclease-mediated gene targeting in the rat opens up a whole new world of possibilities for the development of more predictive models of drug metabolism in humans.

Sigma injected ZFNs into one-cell rat embryos to introduce site-specific modifications on the chromosome, leading to the disruption of gene function (figure 1). Similar to creating a transgene, knockout founders were obtained in 6 weeks from the time of injection. These were then bred to establish colonies with each of the following genes disrupted, Mdr1a, Mrp1, Mrp2, and Bcrp. With regard to genotoxicity assessment, this technique was used to make a deletion within exon 3 of the rat Tp53 gene, resulting in the creation of heterozygous (+/–) and homozygous null (–/–) rats. The tissues of the knockout rats express no p53 protein (figure 2), and ongoing characterization will demonstrate the spectrum and incidence of tumor formation and validate them as models for early carcinogenicity screening. The Tp53 knockout rat will complement the current murine bioassay for carcinogenicity by reducing the latency period for the detection of carcinogens. This has the potential to reduce the length and cost of carcinogenicity assays, increase the accuracy of the results, and reduce the number of animals needed to yield reproducible, physiologically relevant data. ZFN construction and preparation.

The ZFN expression plasmids were obtained from Sigma’s CompoZr product line. The CompoZr system was employed to create gene-specific ZFN pairs. mRNA was prepared from the constructs, using MessageMax (Epicentre Biotechnology, Madison, Wisc.) or mMessage Machine (Ambion, Austin, Tx.). The RNAs were purified, quantified, combined at a 1:1 ratio, and either transfected into tissue culture cells—for validation—or injected into single cell embryos—for generating mutant animals.

click to enlarge Figure 2. Western blot using cytoplasmic lysates from both wild-type Sprague Dawley and Tp53 homozygous knockout tissues.

ZFN validation
To confirm ZFN activity, mRNA pairs were transfected into rat C6 cells using a Nucleofector (Lonza, Basel, Switzerland). Twenty-four hours later, genomic DNA was prepared using QuickExtract (Epicentre Biotechnology). PCR amplification of the region surrounding the target site was performed and the amplicons were denatured, renatured, and treated with an endonuclease S to cleave mismatches in the dsDNA (Transgenomic Inc., Omaha, Neb.). The reaction results were analyzed using polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Inc, Hercules, Calif.) and visualized with ethidium bromide staining.

Embryo microinjection
CD IGS (Charles River, Wilmington, Mass.) rats were housed in static cages receiving 12 hours of light and 12 hours of darkness. Three to four week-old females were injected with PMS 48 hours prior to hCG injection. One-cell stage fertilized eggs were harvested 10 to 12 hours after hCG injection, and used as recipients for microinjection of ZFN mRNAs targeting either Mdr1a, Bcrp, Mrp1, Mrp2, or Tp53. Injected eggs were transferred to pseudopregnant females (0.5 dpc).

Founder identification
Tail or toe biopsies were used for genomic DNA extraction and analysis. Founders identified with the nuclease S mutation detection assay were also screened for the presence of large deletions by PCR amplification of the locus and gel electrophoresis. Distinct bands with a molecular weight lower than that expected for wild-type alleles were gel purified and sequenced.

Gene expression analysis
F2 wild-type and homozygous mutant littermates were sacrificed at 5 to 9 weeks of age. Tissues were harvested and used immediately, or stored in RNAlater (Ambion) at -20°C. Total RNA was prepared using GenElute Mammalian Total RNA Miniprep kit (Sigma), and treated with DNAseI from New England Biolabs in Ipswich, Mass. RT-PCR was carried out using the SuperScript III kit (Invitrogen Corporation; Carlsbad, Calif.) and the products were analyzed by agarose gel electrophoresis with ethidium bromide. Distinct bands were gel purified and sequenced.

Discussion
These five new animal models carry site-specific deletions in the well-established drug transporters: Mdr1a (P-glycoprotein), Mrp1 (Multiple drug resistance-associated protein 1), Mrp2 (Multiple drug resistance-associated protein 2), Bcrp (Breast cancer resistance protein) and Tp53 (Tumor protein 53). Until now, the availability of relevant, genetically modified rats was limited. Knockout mice are readily available, but are challenging to use in ADME-Tox applications due to their size and physiology. The knockout rat models offer an improved platform that can reduce drug development costs by providing a more human-like model, while simultaneously decreasing time to market. This enables researchers to go back and address issues much sooner than they can today, hopefully improving attrition rates.

About the Author
Kristen Bettinger joined SAGE Labs in 2009 and her role as product manager makes her the key liaison to the R&D community, defining and commercializing preclinical research models. Phil Simmons leads the marketing and business development functions for SAGE Labs. Dr. Cui joined SAGE Labs in 2009 and manages a dedicated team of specialists focused on creating and producing next-generation transgenic research models using CompoZr ZFN and RNAi technologies. Dr. Weinstein is the current Director of SAGE Labs. Prior to his current assignment, he managed scientific operations for the RNAi business within Sigma-Aldrich.

References
1. Zan Y, et al. Production of knockout rats using ENU mutagenesis and a yeast-based screening assay. Nat Biotech. 2003;21:645-651.
2. Katida, K. et al. Transposon-tagged mutagenesis in the rat. Nat Meth. 2007:4:131-133.
3. Geurts , A. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science. 2009;325:433.
4. Olson H, Betton G, Robinson D, et al. Concordance of the toxicity ofpharmaceuticals in humans and in animals. Regul Toxicol Pharmacol. 2000; 32:56-67
5. Vlaming MLH, et al. Carcinogen and Anticancer Drug Transport by Mrp2 in vivo: Studies using Mrp2 knockout mice. J Pharmacol Exp Ther. 2006;318:319-327.

This article was published in Drug Discovery & Development magazine: Vol. 13, No. 6, July/August, 2010, p. 21-23.