Genetically engineered mouse models in drug discovery research.

Genetically modified mouse models have been proven to be a powerful tool in drug discovery. The ability to genetically modify the mouse genome by removing or replacing a specific gene has enhanced our ability to identify and validate target genes of interest. In addition, many human diseases can be mimicked in the mouse and signaling pathways have been shown to be conserved. In spite of these advantages the technology has limitations. In transgenic animals there may be significant heterogeneity among different founders. In knock-out animals the predicted phenotypes are not always readily observed and occasionally a completely novel and unexpected phenotype emerges. To address the latter and ensure that a deep knowledge of the target of interest is obtained, we have developed a comprehensive phenotyping program which has identified novel phenotypes as well as any potential safety concerns which may be associated with a particular target. Finally we continue to explore innovative technologies as they become available such as RNAi for temporal and spatial gene knock-down and humanized models that may better simulate human disease states.

Defects in embryonic development of EGLN1/PHD2 knockdown transgenic mice are associated with induction of Igfbp in the placenta.

The HIF (hypoxia inducible factor) hydroxylases EGNL1/PHD2 has been implicated in embryonic development. Here we knocked down EGNL1 in vivo by injecting one-cell murine zygotes with lentivirus-containing RNAi. Progeny with demonstrated EGLN1 inhibition had elevated EPO production and erythropoiesis in vivo. The partial inhibition of EGLN1 in utero is embryonic lethal in some, but not all mice on gestation day 14, and is associated with defects in placental and heart development, similar to those noted in the EGLN1 knockout mouse. Importantly, the in utero inhibition of EGNL1 varied greatly between the embryo proper and the placenta. Using this as a tool we show that the embryopathic effects are associated with knockdown of EGNL1 and the associated induction of Igfbp1 (insulin-like growth factor binding protein-1) mRNA in the placenta, but not the embryo.

Analysis of the role of the HIF hydroxylase family members in erythropoiesis.

HIF (hypoxia-inducible factor) hydroxylases have been implicated in EPO (erythropoietin) production and erythropoiesis. Here we examined the role of each of the three EGLN family members and the HIF asparaginyl hydroxylase FIH (factor inhibiting HIF) in EPO production. We examined the effect of inhibiting individual as well as combinations of HIF hydroxylases with RNAi. We found that inhibition of EGLN1 (egl nine homolog 1) as well as other members of the EGLN family (EGLN2 and EGLN3) led to accumulative EPO production in vitro. We then knocked down EGNL1 in vivo by injecting one-cell murine zygotes with lentivirus-containing RNAi. Progeny with demonstrated EGLN1 inhibition had elevated EPO production and erythropoiesis in vivo. Among all the in vitro and in vivo studies, no or minimal VEGF (vascular endothelial growth factor) mRNA or protein stimulation resulted from inhibition of EGLN1.

Targeted disruption of murine organic anion-transporting polypeptide 1b2 (Oatp1b2/Slco1b2) significantly alters disposition of prototypical drug substrates pravastatin and rifampin.

Organic anion-transporting polypeptides (OATP) 1B1 and 1B3 are widely acknowledged as important and rate-limiting to the hepatic uptake of many drugs in clinical use. Accordingly, to better understand the in vivo relevance of OATP1B transporters, targeted disruption of murine Slco1b2 gene was carried out. It is noteworthy that Slco1b2(-/-) mice were fertile, developed normally, and exhibited no overt phenotypic abnormalities. We confirmed the loss of Oatp1b2 expression in liver using real-time polymerase chain reaction, Western Blot analysis, and immunohistochemistry. Expression of Oatp1a4 and Oatp2b1 but not Oatp1a1 was greater in female Slco1b2(-/-) mice, but expression of other non-OATP transporters did not significantly differ between wild-type and Slco1b2(-/-) male mice. Total bilirubin level was elevated by 2-fold in the Slco1b2(-/-) mice despite the fact that liver enzymes ALT and AST were normal. Pharmacological characterization was carried out using two prototypical substrates of human OATP1B1 and -1B3, rifampin and pravastatin. After a single intravenous dose of rifampin (1 mg/kg), a 1.7-fold increase in plasma area under the concentration-time curve (AUC) was observed, whereas the liver-to-plasma ratio was reduced by 5-fold, and nearly 8-fold when assessed at steady-state conditions after 24 h of continuous subcutaneous infusion in Slco1b2(-/-) mice. Likewise, continuous subcutaneous infusion at low (8 microg/h) or high (32 microg/h) dose rates of pravastatin resulted in a 4-fold lower liver-plasma ratio in the in Slco1b2(-/-) mice. This is the first report of altered drug disposition profile in the Slco1b2 knockout mice and suggests the utility of this model for understanding the in vivo role of hepatic OATP transporters in drug disposition.

Genetic deletion of the striatum-enriched phosphodiesterase PDE10A: evidence for altered striatal function.

PDE10A is a newly identified phosphodiesterase that is highly expressed by the medium spiny projection neurons of the striatum. In order to investigate the physiological role of PDE10A in the central nervous system, PDE10A knockout mice (PDE10A(-/-)) were characterized both behaviorally and neurochemically. PDE10A(-/-) mice showed decreased exploratory activity and a significant delay in the acquisition of conditioned avoidance behavior when compared to wild-type (PDE10A(+/+)) mice. However, in a variety of other well-characterized behavioral tasks, including the elevated plus maze (anxiety), forced swim test (depression), hot plate (nociception) and two memory models (passive avoidance and Morris water maze), PDE10A(-/-) mice performed similarly to wild-type mice. When challenged with PCP or MK-801, PDE10A(-/-) mice showed a blunted locomotor response in comparison to PDE10A(+/+) mice. In contrast, PDE10A(-/-) and PDE10A(+/+) mice responded similarly to the locomotor stimulating effects of amphetamine and methamphetamine. Our findings suggest that PDE10A is involved in regulating striatal output, possibly by reducing the sensitivity of medium spiny neurons to glutamatergic excitation. These results are discussed in relationship to the hypothesis that PDE10A inhibition presents a novel treatment for psychosis.

E-prostanoid-3 receptors mediate the proinflammatory actions of prostaglandin E2 in acute cutaneous inflammation.

PGs are derived from arachidonic acid by PG-endoperoxide synthase (PTGS)-1 and PTGS2. Although enhanced levels of PGs are present during acute and chronic inflammation, a functional role for prostanoids in inflammation has not been clearly defined. Using a series of genetically engineered mice, we find that PTGS1 has the capacity to induce acute inflammation, but PTGS2 has negligible effects on the initiation of this response. Furthermore, we show that the contribution of PTGS1 is mediated by PGE(2) acting through the E-prostanoid (EP)3 receptor. Moreover, in the absence of EP3 receptors, inflammation is markedly attenuated, and the addition of nonsteroidal anti-inflammatory agents does not further impair the response. These studies demonstrate that PGE(2) promotes acute inflammation by activating EP3 receptors and suggest that EP3 receptors may be useful targets for anti-inflammatory therapy.

Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKB beta.

The serine/threonine kinase Akt/PKB plays key roles in the regulation of cell growth, survival, and metabolism. It remains unclear, however, whether the functions of individual Akt/PKB isoforms are distinct. To investigate the function of Akt2/PKBbeta, mice lacking this isoform were generated. Both male and female Akt2/PKBbeta-null mice exhibit mild growth deficiency and an age-dependent loss of adipose tissue or lipoatrophy, with all observed adipose depots dramatically reduced by 22 weeks of age. Akt2/PKBbeta-deficient mice are insulin resistant with elevated plasma triglycerides. In addition, Akt2/PKBbeta-deficient mice exhibit fed and fasting hyperglycemia, hyperinsulinemia, glucose intolerance, and impaired muscle glucose uptake. In males, insulin resistance progresses to a severe form of diabetes accompanied by pancreatic beta cell failure. In contrast, female Akt2/PKBbeta-deficient mice remain mildly hyperglycemic and hyperinsulinemic until at least one year of age. Thus, Akt2/PKBbeta-deficient mice exhibit growth deficiency similar to that reported previously for mice lacking Akt1/PKBalpha, indicating that both Akt2/PKBbeta and Akt1/PKBalpha participate in the regulation of growth. The marked hyperglycemia and loss of pancreatic beta cells and adipose tissue in Akt2/PKBbeta-deficient mice suggest that Akt2/PKBbeta plays critical roles in glucose metabolism and the development or maintenance of proper adipose tissue and islet mass for which other Akt/PKB isoforms are unable to fully compensate.

Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase.

Prostaglandin (PG)E2 is a potent mediator of pain and inflammation, and high levels of this lipid mediator are observed in numerous disease states. The inhibition of PGE2 production to control pain and to treat diseases such as rheumatoid arthritis to date has depended on nonsteroidal antiinflammatory agents such as aspirin. However, these agents inhibit the synthesis of all prostanoids. To produce biologically active PGE2, PGE synthases catalyze the isomerization of PGH2 into PGE2. Recently, several PGE synthases have been identified and cloned, but their role in inflammation is not clear. To study the physiological role of the individual PGE synthases, we have generated by targeted homologous recombination a mouse line deficient in microsomal PGE synthase 1 (mPGES1) on the inbred DBA/1lacJ background. mPGES1-deficient (mPGES1-/-) mice are viable and fertile and develop normally compared with wild-type controls. However, mPGES1-/- mice displayed a marked reduction in inflammatory responses compared with mPGES1+/+ mice in multiple assays. Here, we identify mPGES1 as the PGE synthase that contributes to the pathogenesis of collagen-induced arthritis, a disease model of human rheumatoid arthritis. We also show that mPGES1 is responsible for the production of PGE2 that mediates acute pain during an inflammatory response. These findings suggest that mPGES1 provides a target for the treatment of inflammatory diseases and pain associated with inflammatory states.

Targeted disruption of the osteoblast/osteocyte factor 45 gene (OF45) results in increased bone formation and bone mass.

We have previously described osteoblast/osteocyte factor 45 (OF45), a novel bone-specific extracellular matrix protein, and demonstrated that its expression is tightly linked to mineralization and bone formation. In this report, we have cloned and characterized the mouse OF45 cDNA and genomic region. Mouse OF45 (also called MEPE) was similar to its rat orthologue in that its expression was increased during mineralization in osteoblast cultures and the protein was highly expressed within the osteocytes that are imbedded within bone. To further determine the role of OF45 in bone metabolism, we generated a targeted mouse line deficient in this protein. Ablation of OF45 resulted in increased bone mass. In fact, disruption of only a single allele of OF45 caused significantly increased bone mass. In addition, knockout mice were resistant to aging-associated trabecular bone loss. Cancellous bone histomorphometry revealed that the increased bone mass was the result of increased osteoblast number and osteoblast activity with unaltered osteoclast number and osteoclast surface in knockout animals. Consistent with the bone histomorphometric results, we also determined that OF45 knockout osteoblasts produced significantly more mineralized nodules in ex vivo cell cultures than did wild type osteoblasts. Osteoclastogenesis and bone resorption in ex vivo cultures was unaffected by OF45 mutation. We conclude that OF45 plays an inhibitory role in bone formation in mouse.

Receptors and signaling mechanisms required for prostaglandin E2-mediated regulation of mast cell degranulation and IL-6 production.

Mast cells are implicated in the pathogenesis of a broad spectrum of immunological disorders. These cells release inflammatory mediators in response to a number of stimuli, including IgE-Ag complexes. The degranulation of mast cells is modified by PGs. To begin to delineate the pathway(s) used by PGs to regulate mast cell function, we examined bone marrow-derived mast cells (BMMC) cultured from mice deficient in the EP(1), EP(2), EP(3), and EP(4) receptors for PGE(2). Although BMMCs express all four of these PGE(2) receptors, potentiation of Ag-stimulated degranulation and IL-6 cytokine production by PGE(2) is dependent on the EP(3) receptor. Consistent with the coupling of this receptor to G(alphai), PGE(2) activation of the EP(3) receptor leads to both inhibition of adenylate cyclase and increased intracellular Ca(2+). The magnitude of increase in intracellular Ca(2+) induced by EP(3) activation is similar to that observed after activation of cells with IgE and Ag. Although PGE alone is not sufficient to initiate BMMC degranulation, stimulation of cells with PGE along with PMA induces degranulation. These actions are mediated by the EP(3) receptor through signals involving Ca(2+) mobilization and/or decreased cAMP levels. Accordingly, these studies identify PGE(2)/EP(3) as a proinflammatory signaling pathway that promotes mast cell activation.

Engineering autoactivating forms of matrix metalloproteinase-9 and expression of the active enzyme in cultured cells and transgenic mouse brain.

Matrix metalloproteinases (MMPs) are hypothesized to play an important role in the pathogenesis of several central nervous system disorders. Increased levels of expression of MMP-9 (gelatinase B) and MMP-2 (gelatinase A) have been observed in Alzheimer's disease, stroke, multiple sclerosis, and amyotrophic lateral sclerosis. This suggests an aberrant regulation of MMPs that could lead to inappropriate expression of MMP activity. To allow us to evaluate the effect of increased levels of active MMP-9 in the central nervous system, mutant forms of the enzyme were designed to autocatalytically remove the pro domain, yielding active enzyme. This was accomplished by modifying residues in the cysteine switch autoinhibitor region of the propeptide. Stable cell lines and transgenic mice that express G100L and D103N autoactive forms of human MMP-9 were developed to study the role of dysregulation of MMP-9 in disease.

Kinase suppressor of Ras (KSR) is a scaffold which facilitates mitogen-activated protein kinase activation in vivo.

While scaffold proteins are thought to be key components of signaling pathways, their exact function is unknown. By preassembling multiple components of signaling cascades, scaffolds are predicted to influence the efficiency and/or specificity of signaling events. Here we analyze a potential scaffold of the Ras/mitogen-activated protein kinase (MAPK) pathway, kinase suppressor of Ras (KSR), by generating KSR-deficient mice. KSR-deficient mice were grossly normal even though ERK kinase activation was attenuated to a degree sufficient to block T-cell activation and inhibit tumor development. Consistent with its role as a scaffold, high-molecular-weight complexes containing KSR, MEK, and ERK were lost in the absence of KSR. This demonstrates that KSR is a bona fide scaffold that is not required for but enhances signaling via the Ras/MAPK signaling pathway.

Glycemic control in mice with targeted disruption of the glucagon receptor gene.

The action of glucagon in the liver is mediated by G-coupled receptors. To examine the role of glucagon in glucose homeostasis, we have generated mice in which the glucagon receptor was inactivated (GR(-/-) mice). Blood glucose levels were somewhat reduced in GR(-/-) mice relative to wild type, in both the fed and fasted state. Plasma insulin levels were not significantly affected. There was no significant effect on fasting plasma cholesterol or triglyceride levels associated with deletion of the glucagon receptor. Glucose tolerance, as assessed by an oral glucose tolerance test, improved. Plasma glucagon levels were strikingly elevated in both fed and fasted animals. Despite a total absence of glucagon receptors, these animals maintained near-normal glycemia and normal lipidemia, in the presence of circulating glucagon concentrations that were elevated by two orders of magnitude.