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Th CRE L R t M MdlAt The CRE Luc Reporter Mouse Model: A tra The CRE-Luc Reporter Mouse Model: A tra The CRE Luc Reporter Mouse Model: A tra li d ti assay ligand acti assay ligand acti H ll D l F d C h K ik E Holly Dressler Fernando Camacho Kyriakos Econ Holly Dressler, Fernando Camacho, Kyriakos Econ Immunology and Inflammation TSU Sanofi Pharm Immunology and Inflammation TSU, Sanofi Pharm C t t G P lit il lit @ Contact: Greg Polites email: greg.polites@s Contact: Greg Polites email: greg.polites@s Meth Abstract and Introduction Meth Abstract and Introduction Th t dt t thi dl ti th f The transgene used to generate this model contains the fo frequency of functional lineages: MAR (matrix attachment r Abstract N bi h b d l d i i h i i b GPCR dhili d frequency of functional lineages: MAR (matrix attachment r expression 6X CRE; a response element represented by C Numerous bioassays have been developed to investigate the interactions between GPCRs and their ligands. R t b d i th AMP l t (CRE) ld ith bi l i f l if expression, 6X CRE; a response element represented by C virus thymidine kinase minimal promoter LUC 2; a luciferas Reporter based assays using the cAMP response element (CRE) coupled with bioluminescence from a luciferase reporter has been used extensively in vitro with high throughput screens (HTS) of large compound libraries We virus thymidine kinase minimal promoter, LUC 2; a luciferas hGH polyA element which contains the human growth horm reporter has been used extensively in vitro with high-throughput screens (HTS) of large compound libraries. We have generated a transgenic mouse model (CRE-Luc) with a luciferase reporter under the control of a synthetic hGH polyA element which contains the human growth horm transgene expression. have generated a transgenic mouse model (CRE-Luc) with a luciferase reporter under the control of a synthetic promoter containing six copies of CRE, which supports real-time bioimaging in whole animals, tissues, or cells of MAR 6XCRE HSV TK L promoter containing six copies of CRE, which supports real time bioimaging in whole animals, tissues, or cells of GPCR ligand activity in a native environment. Assays with the CRE-Luc mouse will be presented to demonstrate MAR 6XCRE HSV TK min L the wide application of this model to GPCR drug development. We have crossed a CRE-Luc line expressing min luciferase in the pancreas with the Akita pancreatic mutant mouse and demonstrated a significant decrease in the The initial screen for luciferase expressing founders was pe luciferase signal that is proportional to the ablated tissue. A chemically induced psoriasis model was generated by th li ti fI i i dt CRE L li dl if it it tit ti l lt ith th it f The initial screen for luciferase expressing founders was pe forskolin and 10mg/kg rolipram followed by bioimaging with the application of Imiquimod to a CRE-Luc line and luciferase intensity quantitatively correlates with the severity of the induced psoriasis Finally we have completed several assays with primary neuronal cells in situ brain slices forskolin and 10mg/kg rolipram followed by bioimaging with measured in 87 of the 112 DNA positive lineages. Tissue ex the induced psoriasis. Finally, we have completed several assays with primary neuronal cells, in situ brain slices, and whole animals to demonstrate the consistency of the luciferase reporter in these different cellular formats and measured in 87 of the 112 DNA positive lineages. Tissue ex lineages. and whole animals to demonstrate the consistency of the luciferase reporter in these different cellular formats and ligand receptor interactions Access to the CRE-Luc mouse model is available through an exclusive licensing St d i th GPCR AMP i li th ligand receptor interactions. Access to the CRE Luc mouse model is available through an exclusive licensing agreement between Sanofi and Taconic. Studying the GPCR cAMP signaling path Introduction Promoter contains 6 copies of The interaction between GPCRs and their extracellular ligands has proven to be an attractive point of interference f th ti t F thi th h ti lid t h d l d bi h i ld di CRE Luc Promoter contains 6 copies of cAMP response element (CRE) for therapeutic agents. For this reason, the pharmaceutical industry has developed biochemical drug discovery assa s to in estigate these ligand GPCR interactions Here e describe the generation and application of a CRE-Luc cAMP response element (CRE) assays to investigate these ligand GPCR interactions. Here, we describe the generation and application of a transgenic mouse model that contains six cAMP response elements (6X CRE) upstream of a luciferase cDNA The In vivo transgenic mouse model that contains six cAMP response elements (6X CRE) upstream of a luciferase cDNA. The transgene enables the specific monitoring of G protein dependent signaling via molecular bioimaging Molecular In vivo transgene enables the specific monitoring of G protein dependent signaling via molecular bioimaging. Molecular imaging techniques can be performed in the intact organism with sufficient spatial and temporal resolution to study Baseline imaging In vitro imaging techniques can be performed in the intact organism with sufficient spatial and temporal resolution to study biological processes in vivo. Furthermore, the CRE-luc mouse can also used a source of cells and tissues to In vitro support parallel native cellular GPCR assays performed in vitro or ex vivo which cam lead to a more realistic profile of ligand and receptor interactions. C dd i if i i i di id l li ih i Compound dosing Luciferase expression varies amongst individual lines with respect to tissue pattern, baseline signal, and induction levels, as expected for a randomly integrated transgene baseline signal, and induction levels, as expected for a randomly integrated transgene Re-imaging Re-imaging Basal Basal Radiance Addition of IVIS bioimaging Radiance Addition of F k li / IVIS bioimaging Whole live animal imaging Fold indu •Mice dosed i.p. with 10mg/kg rolipram Forskolin / Rolipram Simple, quick Limited resolution •Bioimaged pre (basal) and 4 hours po Rolipram Limited resolution Profile of isoprotereno Induced Profile of isoprotereno Induced T t th • Treatment: hours isoproterenol treatment 175 33 44 155 11 187 73 62 Line # isoproterenol treatment, 10MPK ip 175 33 44 155 11 187 73 62 Line # 10MPK, ip The Imiquimod psoriasis model induces thickening of skin erythema and scaling • Imaging at T=0 and 5 hours The Imiquimod psoriasis model induces thickening of skin, erythema, and scaling • Statistically significant • Imiquimod (IMQ) is a ligand for TLR7 and TLR8 and can exacerbate psoriasis in patients with increase in CNS response b li Imiquimod (IMQ) is a ligand for TLR7 and TLR8 and can exacerbate psoriasis in patients with topical treatment, both locally and at distant sites (side effect) over baseline • IMQ-induced psoriasis is mediated via IL-23 and IL-17 IMQ induced psoriasis is mediated via IL 23 and IL 17 • Skin on back of mice was folded and measured using digital calipers Skin on back of mice was folded and measured using digital calipers • Imiquimod group showed a significant increase in skin thickness Imiquimod group showed a significant increase in skin thickness • Increase in skin thickness was completely blocked by Temovate • Increase in skin thickness was completely blocked by Temovate P fil fi t l d it t Profile of isoproterenol and its ant • Compound induced phot changes in luciferase l l i b i li phot o levels in brain slices can be detected and o can be detected, and quantified by 0 = None 1 = Minimal quantified by bioimaging 1 = Minimal 2 = Moderate 3 Severe bioimaging 3 = Severe 4 = Maximum CRE L Li 64 24 h t t t b li • Potential use to CRE-Luc Line 64: 24 hour treatment baseline • Potential use to identify the region identify the region specific expression of • Significant reduction in bioimaging signal is the transgene and Vehicle Significant reduction in bioimaging signal is observed in both temovate and CsA treated drug interaction Vehicle observed in both temovate and CsA treated groups groups ISO + AMN 1um • One animal in CsA treated group is an outlier ISO + AMN 1um One animal in CsA treated group is an outlier Luciferase levels are increased by Gs agon I i i d T t lFl Luciferase levels are increased by Gs agon Imiquimod Total Flux * primary neu 9.0×10 07 * 07 8.0×10 07 9.0 10 c ** •E18, d3 cortical neurons Gs: ADRβ1/2 isoproterenol 6.0×10 07 7.0×10 07 er sec ** neurons •t-test vs DMSOs Gs: ADRβ1/2, isoproterenol I i i d+ 4 0 10 07 5.0×10 07 ns per •t test vs DMSOs •4 hour treatment Imiquimod + Temovate 3.0×10 07 4.0×10 07 otons 4000 11X 5 Temovate 1 0×10 07 2.0×10 07 Pho 4000 *** 5 5 0.0 1.0×10 07 3000 4 4 ehicle uimod ovate d+CsA 3000 ***p<0.0001 s 3 3 4 s Imiquimod + Veh Imiquim d+Temov uimod+C 2000 cps 2 3 cps Imiquimod + Cyclosporin A I uimod+ Imiqui 1000 1 2 Cyclosporin A Imiqu 1000 1 1 Treatment Group CRE Luc Line 64 in an IMQ study time course profile 0 CRE-Luc Line 64 in an IMQ study, time course profile DMSO 10uM [i t l] [isoproterenol] 0 hr (b li ) 48 hr 120 hr 168 hr Luciferase levels are reduced by Gi agonist (baseline) (2 treatments) (5 treatments) (7 treatments) Luciferase levels are reduced by Gi agonist neuronal cells treated with neuronal cells treated with vehicle Gi: CB1 CP55 940 •E18 d3 cortical neurons vehicle Gi: CB1, CP55,940 •E18, d3 cortical neurons •10μM forskolin/ 10μM rolipram 10μM forskolin/ 10μM rolipram •8 hour treatment •t-test vs F/R IMQ 10uM F/ 10uM R IMQ 150000 20% ↓ 30X 3 150000 20% ↓ *p<0 05 2 45% ↓ 39% ↓ ** *p<0.05 180000 p<0.05 2 s 100000 45% ↓ ***p<0.0005 **p<0.005 130000 2 cps cps 80000 p<0.05 p<0.05 50000 c 25000 30000 RLU 50000 15000 20000 R 5000 10000 DMSO F/R 100nM 1uM 10uM 0 h ra h ra h ra h ra 0 DMSO F/R 100nM 1uM 10uM [CP55940] veh Aldara veh Aldara veh Aldara veh Aldara 0 h 48 h [CP55940] 0 hr 48 hr 120 hr 168 hr TEMPLATE DESIGN © 2008 www.PosterPresentations.com i bi i i d lth t nsgenic bioimaging mouse model that can nsgenic bioimaging mouse model that can nsgenic bioimaging mouse model that can ti f GPCR vation of GPCRs vation of GPCRs id Zh P N W dG P lit nomides Zhen Pang Nancy Wu and Greg Polites nomides, Zhen Pang, Nancy Wu, and Greg Polites maceuticals Bridgewater New Jersey USA 08807 maceuticals, Bridgewater, New Jersey, USA 08807 fi ti h lit 3@ il sanofi-aventis.com or [email protected] sanofi aventis.com or [email protected] hods Summary hods Summary ll i l t hi h i bi ti d hi h llowing elements which in combination produce a high regions); to generate position independent From initial studies we have demonstrated the utility of the CRE Luc model to profile compounds in whole animals regions); to generate position independent CRE cAMP repeated six times HSV TKmin; a simplex From initial studies, we have demonstrated, the utility of the CRE-Luc model to profile compounds in whole animals, tissue extracts slices and primary cells in vitro CRE-cAMP repeated six times, HSV TKmin; a simplex se cDNA optimized for mammalian expression and a tissue extracts, slices, and primary cells in vitro se cDNA optimized for mammalian expression , and a mone minigene with the poly A tail to enhance GPCR agonists antagonists mone minigene with the poly A tail to enhance Gs in vitro: microglia, neurons, T cells, in vitro: microglia, neurons, T cells Luc2 hGH poly MAR cardiomyocytes, MEFs, brain slices in vivo: brain, spinal cord Luc2 hGH poly A MAR in vivo: pancreas, brain, spinal cord A erformed by dosing IP with a combination of 5mg/kg Gi i it T ll b i li i it T ll erformed by dosing IP with a combination of 5mg/kg h an IVIS Lumina Detectable levels of luciferase was Gi in vitro: neurons, T cells, brain slices in vitro: neurons, T cells h an IVIS Lumina. Detectable levels of luciferase was xpression profiles were used to select the optimal xpression profiles were used to select the optimal For further model information contact Greg Polites at: [email protected] or [email protected] h i CRE L t i CRE-luc mice are available from Taconic contact: hway using CRE-Luc transgenic mouse [email protected] L if i i t ll db c Luciferase expression is controlled by cAMP CREB CBP CRE Luc reporter mouse model application strategy c cAMP-CREB-CBP CRE-Luc reporter mouse model application strategy Ex vivo in vivo activity Source of cells for Ex vivo tissues or whole body imaging Source of cells for in vitro assays Compound dosing o l Cli i l o In vivo In vitro Assay Dev Target ID Development Clinical Ti h t Tissue homogenates Starting with a variety of in vivo luciferase expression profiles, our goal for the CRE-Luc model was to more efficiently transition GPCR projects through their traditional major hurdle from pre- program to advanced program status CRE Luc Luciferase assay CRE-Luc Luciferase assay Feasibility Feasibility profiles T,B,macs. Pancreas Lung CNS Liver Kidney Adipose e/RLU No-Go e/RLU Microplate reader No Go Go uction Microplate reader Sensitive, accurate m and 5mg/kg water soluble forskolin Better organ resolution Time consuming ost (induction) Specific pilots to Time-consuming pilots to reach l in vivo with line 187 project application l in vivo with line 187 application decision brain 15X *** No-Go 500000 Go 400000 2 P j t 64 11, 16 187 219 229 175 300000 s/cm2 Project application Mlil T li f dl li i 200000 p/s Multiple Tg lines for model applications 100000 The induction of luciferase levels in the pancreas by the GLP1 agonist AVE0010 is reduced baseline isoproterenol 0 The induction of luciferase levels in the pancreas by the GLP1 agonist, AVE0010, is reduced treatment by streptozotocin treatment spinal cord 15X 200000 15X *** • Streptozotocin is toxic to the insulin producing beta cells of the pancreas 150000 2 • Streptozotocin is toxic to the insulin-producing beta cells of the pancreas • AVE10 administered at 0 1 mg/kg sc 100000 /s/cm2 700 STZ increases blood glucose • AVE10 administered at 0.1 mg/kg, sc • Animals administered STZ (200 mg/kg) or vehicle, i.p. 50000 p/ 600 700 dL) STZ increases blood glucose Animals administered STZ (200 mg/kg) or vehicle, i.p. • Bioimaged 4 days post treatment 0 500 600 mg/d baseline isoproterenol 0 treatment Basal 400 500 e (m Control t i t AMN082 ith b i li treatment 300 cos STZ tagonist AMN082 with brain slices 200 Gluc Section with Hippocampus 100 G CH1 DATA(cpm) Section with Hippocampus Induction by 0 0 2 4 CH1_DATA(cpm) 16000 Forskolin 50uM Induction by AVE0010 0 2 4 Days 14000 15000 16000 photo AVE0010 Days STZ (day 4) blocks the induction of 1 12000 13000 14000 photo STZ (day 4) blocks the induction of luci by AVE0010 lice 10000 11000 12000 1.93x luci by AVE0010 1400 n Sl 8000 9000 Induction by 1200 ion Control 6000 7000 AVE0010, 4 days 800 1000 uct STZ 0 1440 2880 4320 5760 7200 8640 10080 11520 12960 14400 post STZ or vehicle 600 800 indu baseline Vehicle (DMSO) vehicle treatment 400 600 ld i 18000 20000 Vehicle (DMSO) treatment 200 400 Fol 2 14000 16000 18000 0 Before After ce 2 10000 12000 STZ Control Before After STZ treatment St d f CRE L I 2 Akit i Slic 4000 6000 8000 STZ treatment Study of CRE-Luc Ins2 Akita mice ISO 1um 0 2000 4000 ISO 1um 0 0 1440 2880 4320 5760 7200 8640 10080 11520 12960 14400 • Ins2 Akita is an autosomal dominant mutation that causes early onset hyperglycemia ists ADβR (isoproterenol) and dopamine in • Ins2 Akita is an autosomal dominant mutation that causes early onset hyperglycemia ists ADβR (isoproterenol) and dopamine in • CRE-Luc crossed with Ins2 Akita to determine correlation of βcell function in this T1DM model uronal cells • 8-week old mice were treated with GLP1 agonist (0.1mpk, sc) imaged at 4 hr on day 2 *P < 0.05, Akita/+ vs WT Two-way ANOVA model Akita/+ Akita/+ Akita/+ Akita/+ WT Gs: DRD •E14, d4 striatal neurons s s a 600 Two-way ANOVA model. Akita/+ Akita/+ Akita/+ Akita/+ WT Gs: DRD, dopamine •t-test vs media •5 hour treatment 500 3X dopamine •5 hour treatment 400 on WT 50 ** 3X 300 uctio WT Akita/+ 00 50 * * p<0.01 200 indu Akita/+ 00 50 100 200 Fold * 00 50 00 0 100 F 50 00 0 Male Female Females Males 50 00 • Decreased CRE-Luc induction by the GLP1 agonist (0 1 mpk sc 4 hrs) was observed in the highly Male Female 50 00 50 • Decreased CRE-Luc induction by the GLP1 agonist (0.1 mpk, sc, 4 hrs) was observed in the highly diabetic male mice di 3M 10 M 0 50 diabetic male mice. media 3uM 10uM [d i ] The ADRβ3 receptor agonist CL316 243 induces luciferase expression in [dopamine] The ADRβ3 receptor agonist CL316,243 induces luciferase expression in adipose lung and small intestines ts for CB1 and mGluR7 in primary cortical adipose, lung, and small intestines ts for CB1 and mGluR7 in primary cortical h forskolin plus rolipram •CL316 243(1mg/kg) was administered by i p injection h forskolin plus rolipram CL316,243(1mg/kg) was administered by i.p. injection •Bioimaged 4 hours post dosing then tissue was harvested Gi mGluR7 AMN082 f f f f Bioimaged 4 hours post dosing, then tissue was harvested Gi: mGluR7, AMN082 •E18, d3 cortical Before After treatment Before After treatment neurons •10μM forskolin 10uM F •10μM forskolin •4 hour treatment 10uM F •t-test vs F 78x 59x 95x Bioimaging results 35000 ns * * p<0 005 78x 59x 95x 38x 34x 30000 ns 9X p<0.005 34x 25000 40% 38% 20000 40% 38% ** ** Line 31 Line 175 15000 20000 ** ** Tissue fold-induction induced level fold-induction induced level Line 31 Line 175 10000 15000 Tissue fold-induction induced level fold-induction induced level Adipose 22.3 58.3 20.0 10.3 L i 5000 10000 Adipose 22.3 58.3 20.0 10.3 Intestine 13.3 7.8 2.7 2.3 Luci enzyme i ti 5000 Pancreas 7.5 1.5 0.9 0.5 assay in tissue extracts media DMSO F 1nM 10nM 100nM 1uM 0 Spleen 10.4 0.5 1.6 0.8 L 35 9 20 8 49 5 1702 5 extracts mediaDMSO F 1nM 10nM 100nM 1uM [AMN082] Lung 35.9 20.8 49.5 1702.5 Brain 07 100 5 10 89 3 [AMN082] Brain 0.7 100.5 1.0 89.3
