Th CRE L R t M M d l A tThe CRE Luc Reporter Mouse Model: A traThe CRE-Luc Reporter Mouse Model: A traThe CRE Luc Reporter Mouse Model: A trali d tiassay ligand actiassay ligand actiy g
H ll D l F d C h K i k EHolly Dressler Fernando Camacho Kyriakos EconHolly Dressler, Fernando Camacho, Kyriakos EconImmunology and Inflammation TSU Sanofi PharmImmunology and Inflammation TSU, Sanofi Pharmgy ,
C t t G P lit il lit @Contact: Greg Polites email: greg.polites@sContact: Greg Polites email: greg.polites@s
MethAbstract and Introduction MethAbstract and Introduction Th t d t t thi d l t i th fThe transgene used to generate this model contains the fofrequency of functional lineages: MAR (matrix attachment r
AbstractN bi h b d l d i i h i i b GPCR d h i li d frequency of functional lineages: MAR (matrix attachment r
expression 6X CRE; a response element represented by CNumerous bioassays have been developed to investigate the interactions between GPCRs and their ligands. R t b d i th AMP l t (CRE) l d ith bi l i f l if expression, 6X CRE; a response element represented by C
virus thymidine kinase minimal promoter LUC 2; a luciferasReporter 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 hormreporter has been used extensively in vitro with high-throughput screens (HTS) of large compound libraries. Wehave 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 g p
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
Lg y y p
the wide application of this model to GPCR drug development. We have crossed a CRE-Luc line expressing minluciferase in the pancreas with the Akita pancreatic mutant mouse and demonstrated a significant decrease in the
The initial screen for luciferase expressing founders was peluciferase signal that is proportional to the ablated tissue. A chemically induced psoriasis model was generated by th li ti f I i i d t CRE L li d l if i t it tit ti l l t ith th it f The initial screen for luciferase expressing founders was pe
forskolin and 10mg/kg rolipram followed by bioimaging withthe 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 exthe 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 thgligand 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 pathg
IntroductionPromoter contains 6 copies ofThe 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 l i d t h d l d bi h i l d di CRE LucPromoter 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 aCRE-LuccAMP 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 vivotransgenic 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 vivotransgene 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 vitroimaging 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
ase e ag g In vitrog p ,
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 d d iif i i i di id l li i h i
Compound dosingLuciferase expression varies amongst individual lines with respect to tissue pattern, p g p p
baseline signal, and induction levels, as expected for a randomly integrated transgenebaseline signal, and induction levels, as expected for a randomly integrated transgeneRe-imagingRe-imaging
BasalBasal
RadianceAddition of IVIS bioimaging
RadianceAddition of
F k li /IVIS bioimagingWhole live animal imaging Fold indu
•Mice dosed i.p. with 10mg/kg rolipramForskolin /Rolipram
g gSimple, quickLimited resolution •Bioimaged pre (basal) and 4 hours poRolipram Limited resolution
Profile of isoproterenoInduced
Profile of isoproterenoInduced
T t t h• Treatment: hoursisoproterenol treatment
175 3344 155 11 1877362Line #isoproterenol treatment, 10MPK ip175 3344 155 11 1877362Line # 10MPK, ip
The Imiquimod psoriasis model induces thickening of skin erythema and scaling • Imaging at T=0 and 5 hoursThe Imiquimod psoriasis model induces thickening of skin, erythema, and scaling g g
• 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 baselinep , y ( )
• IMQ-induced psoriasis is mediated via IL-23 and IL-17IMQ induced psoriasis is mediated via IL 23 and IL 17• Skin on back of mice was folded and measured using digital calipersSkin on back of mice was folded and measured using digital calipers• Imiquimod group showed a significant increase in skin thicknessImiquimod 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 f i t l d it tProfile of isoproterenol and its ant
• Compound induced
photchanges in luciferase l l i b i li phot
olevels in brain slices can be detected and ocan be detected, and quantified by
0 = None1 = Minimal quantified by
bioimaging1 = Minimal2 = Moderate3 Severe bioimaging 3 = Severe4 = Maximum
CRE L Li 64 24 h t t t b li• Potential use toCRE-Luc Line 64: 24 hour treatment baseline• Potential use toidentify the regionidentify the region specific expression of • Significant reduction in bioimaging signal is p pthe transgene and Vehicle
Significant reduction in bioimaging signal is observed in both temovate and CsA treated g
drug interactionVehicle observed in both temovate and CsA treated groupsgroups
ISO + AMN 1um• One animal in CsA treated group is an outlier ISO + AMN 1umOne animal in CsA treated group is an outlier
Luciferase levels are increased by Gs agonI i i d T t l Fl Luciferase levels are increased by Gs agonImiquimod Total Flux
* primary neu9.0×1007*
07
8.0×1007
9.0 10
c ** •E18, d3 corticalneurons Gs: ADRβ1/2 isoproterenol6.0×1007
7.0×1007
er s
ec **neurons•t-test vs DMSOs
Gs: ADRβ1/2, isoproterenolI i i d + 4 0 1007
5.0×1007
ns p
er
•t test vs DMSOs•4 hour treatment
Imiquimod +Temovate 3.0×1007
4.0×1007
oton
s
400 011X
5Temovate
1 0×1007
2.0×1007Pho
400 0 * * * 55
0.01.0×1007
300 0 44
ehicl
e
uim
od
ovat
e
d+CsA 300 0
* * * p<0 .0001
s 334
sImiquimod +Ve
h
Imiq
uim
d+Te
mov
uim
od+C
2000cps
23
cpsImiquimod +
Cyclosporin A
I
uim
od+
Imiq
ui
1000 12Cyclosporin A
Imiq
u
1000 11
Treatment Group
CRE Luc Line 64 in an IMQ study time course profile 0CRE-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 agonistneuronal cells treated withneuronal cells treated with
vehicleGi: CB1 CP55 940•E18 d3 cortical neurons
vehicleGi: CB1, CP55,940•E18, d3 cortical neurons
•10µM forskolin/ 10µM rolipram10µM forskolin/ 10µM rolipram•8 hour treatment•t-test vs F/R
IMQ 10uM F/ 10uM RIMQ
150000 20%↓30X3150000 20%↓
*p<0 05245%↓39%↓
**
*p<0.051 8 0 0 0 0
p < 0 . 0 5
2s10000045%↓***p<0.0005
**p<0.0051 3 0 0 0 0 2
cpsp
cps8 0 0 0 0
p < 0 . 0 5 p < 0 . 0 5
50000
c
2 5 0 0 03 0 0 0 0
RLU
500001 5 0 0 02 0 0 0 0R
5 0 0 01 0 0 0 0
DMSO F/R 100nM 1uM 10uM0
h ra h ra h ra h ra
05 0 0 0
DMSO F/R 100nM 1uM 10uM [CP55940]
veh
Aldara veh
Aldara veh
Aldara veh
Aldara
0 h 4 8 h [CP55940]0 h r 4 8 h r 1 2 0 h r 1 6 8 h r
TEMPLATE DESIGN © 2008
www.PosterPresentations.com
i bi i i d l th tnsgenic bioimaging mouse model that cannsgenic bioimaging mouse model that cannsgenic bioimaging mouse model that can ti f GPCRvation of GPCRsvation of GPCRsid Zh P N W d G P litnomides Zhen Pang Nancy Wu and Greg Politesnomides, Zhen Pang, Nancy Wu, and Greg Polites
maceuticals Bridgewater New Jersey USA 08807maceuticals, Bridgewater, New Jersey, USA 08807, g , y,fi ti h lit 3@ ilsanofi-aventis.com or [email protected] aventis.com or [email protected]
hods Summaryhods Summaryll i l t hi h i bi ti d hi hllowing 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 animalsregions); 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 vitroCRE-cAMP repeated six times, HSV TKmin; a simplex
se cDNA optimized for mammalian expression and atissue 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 antagonistsmone 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
g , , ,cardiomyocytes, MEFs, brain slices
g , ,in vivo: brain, spinal cord
Luc2 hGH poly A
MARy y , ,
in vivo: pancreas, brain, spinal cord, p
A p , , p
erformed by dosing IP with a combination of 5mg/kg Gi i it T ll b i li i it T llerformed 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 optimalxpression 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 iCRE-luc mice are available from Taconic contact:
hway using CRE-Luc transgenic [email protected]
L if i i t ll d bc
Luciferase expression is controlled bycAMP CREB CBP
CRE Luc reporter mouse model application strategyc cAMP-CREB-CBP
CRE-Luc reporter mouse model application strategyEx vivo
in vivo activitySource of cells forEx vivo
ytissues or whole body imaging
Source of cells for in vitro assaysCompound dosingo
l
y
Cli i l
Co pou d dos go
In vivoIn vitroAssay DevTarget ID Development Clinical
Ti h tTissue 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 LucLuciferase assay CRE-LucLuciferase assay
FeasibilityFeasibilityprofiles T,B,macs. PancreasLungCNS Liver Kidney Adipose
e/RLU p
No-Goe/RLU
Microplate reader No GoGouction Microplate reader
Sensitive, accuratem and 5mg/kg water soluble forskolin
,Better organ resolutionTime consumingost (induction) Specific
pilots toTime-consuming
pilots to reach l in vivo with line 187 project
applicationl in vivo with line 187
applicationdecision b r a in 1 5 X
***No-Go
500000
Go400000
2
P j t64 11, 16187 219229 175300000
s/cm
2
Project application
M l i l T li f d l li i200000p/
s
ppMultiple Tg lines for model applications100000
The induction of luciferase levels in the pancreas by the GLP1 agonist AVE0010 is reducedbaseline isoproterenol0
The induction of luciferase levels in the pancreas by the GLP1 agonist, AVE0010, is reduced t r e a tm e n t
by streptozotocin treatments p in a l c o r d1 5 X y p
200000 1 5 X***
• Streptozotocin is toxic to the insulin producing beta cells of the pancreas150000
2
• Streptozotocin is toxic to the insulin-producing beta cells of the pancreas• AVE10 administered at 0 1 mg/kg sc
100000
/s/c
m2
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/
600700
/dL)
STZ increases blood glucoseAnimals administered STZ (200 mg/kg) or vehicle, i.p.• Bioimaged 4 days post treatment
0 500600
mg
/dg y p
baseline isoproterenol0
t r e a tm en tBasal
400500
se (m Control
t i t AMN082 ith b i lit r e a tm en t
300cos
STZtagonist AMN082 with brain slices 200
Glu
c
Section with Hippocampus 100
G
CH1 DATA(cpm)
Section with HippocampusInduction by
00 2 4CH1_DATA(cpm)
16000Forskolin 50uM Induction by
AVE00100 2 4
Days140001500016000
photoAVE0010 Days
STZ (day 4) blocks the induction of
1 120001300014000photo STZ (day 4) blocks the induction of
luci by AVE0010
lice
100001100012000
1.93x luci by AVE00101400
nSl
80009000 Induction by 1200
tion Control
60007000 AVE0010, 4 days
8001000
uct STZ
0 1440 2880 4320 5760 7200 8640 10080 11520 12960 14400 post STZ or vehicle 600
800
ind
u
baseline CH1_DATA(cpm)Vehicle (DMSO)vehicle
treatment 400600
ld i
1800020000
Vehicle (DMSO) treatment200400
Fol
2 140001600018000
0Before After
ce 2
1000012000
STZControlBefore After
STZ treatment
St d f CRE L I 2 Akit i
Slic
400060008000 STZ treatment
Study of CRE-Luc Ins2 Akita miceISO 1um 020004000
ISO 1um 00 1440 2880 4320 5760 7200 8640 10080 11520 12960 14400
• Ins2Akita is an autosomal dominant mutation that causes early onset hyperglycemiaists ADβR (isoproterenol) and dopamine in
• Ins2Akita is an autosomal dominant mutation that causes early onset hyperglycemiaists ADβR (isoproterenol) and dopamine in • CRE-Luc crossed with Ins2Akita to determine correlation of βcell function in this T1DM modeluronal cells • 8-week old mice were treated with GLP1 agonist (0.1mpk, sc) imaged at 4 hr on day 2g ( p ) g y
*P < 0.05, Akita/+ vs WTTwo-way ANOVA modelAkita/+ Akita/+ Akita/+ Akita/+WT
Gs: DRD •E14, d4 striatal neuronss s a
600Two-way ANOVA model. Akita/+ Akita/+ Akita/+ Akita/+WT
Gs: DRD, dopamine
•t-test vs media•5 hour treatment
500
3X
dopamine •5 hour treatment400
on WT
50 * *3X
300uctio
WT
Akita/+
0050
* * p<0 .01 200indu
Akita/+
0050
p
100
200
Fold
*
005000
0
100F
5000 0
Male FemaleFemales Males
5000
• Decreased CRE-Luc induction by the GLP1 agonist (0 1 mpk sc 4 hrs) was observed in the highlyMale Female
500050 • Decreased CRE-Luc induction by the GLP1 agonist (0.1 mpk, sc, 4 hrs) was observed in the highly
diabetic male micedi 3 M 10 M
050 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 intestinests for CB1 and mGluR7 in primary cortical adipose, lung, and small intestinests for CB1 and mGluR7 in primary cortical h forskolin plus rolipram •CL316 243(1mg/kg) was administered by i p injectionh 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 treatmentneurons•10µM forskolin
10uM F•10µM forskolin•4 hour treatment10uM F•t-test vs F
78x 59x 95xBioimaging results
35000 ns **p<0 00578x 59x 95x 38x 34x
30000 ns9Xp<0.005 34x
25000 40%38%20000
40%38%*** * Line 31 Line 175
1500020000 * ** *
Tissue fold-induction induced level fold-induction induced levelLine 31 Line 175
1000015000 Tissue fold-induction induced level fold-induction induced level
Adipose 22.3 58.3 20.0 10.3L i5000
10000 Adipose 22.3 58.3 20.0 10.3Intestine 13.3 7.8 2.7 2.3Luci enzyme
i ti5000 Pancreas 7.5 1.5 0.9 0.5assay in tissue extracts
mediaDMSO F 1nM 10nM100nM 1uM0 Spleen 10.4 0.5 1.6 0.8
L 35 9 20 8 49 5 1702 5extracts
mediaDMSO F 1nM 10nM100nM 1uM[AMN082]
Lung 35.9 20.8 49.5 1702.5Brain 0 7 100 5 1 0 89 3[AMN082] Brain 0.7 100.5 1.0 89.3
