ingegneria animale applicazioni
DESCRIPTION
CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL FARMACO AA 2010-11 Adriana Maggi LEZIONE 13. INGEGNERIA ANIMALE APPLICAZIONI. I SISTEMI REPORTER POSSONO ESSERE UTILIZZATI PER LA GENERAZIONE DI ANIMALI IN CUI SI POSSANO MISURARE EVENTI MOLECOLARI SPECIFICI IN TEMPO REALE?. - PowerPoint PPT PresentationTRANSCRIPT
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INGEGNERIA ANIMALEAPPLICAZIONI
CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL FARMACO AA 2010-11
Adriana MaggiLEZIONE 13
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I SISTEMI REPORTER POSSONO ESSERE UTILIZZATI PER LA GENERAZIONE DI ANIMALI IN CUI SI POSSANO MISURARE EVENTI MOLECOLARI SPECIFICI IN TEMPO REALE?
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IN VIVO IMAGING
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MULTI-MODALITY MOLECULAR IMAGING IN LIVING SMALL ANIMALS
Functional Magnetic Nuclear Resonance
Optical imagingBioluminescence
Ultrasound
MicroPETPositron emission tomography
Nuclear imaging
Micro SPECTsingle-photon emission
computerized tomography
2/06
Fluorescence
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LA GENERAZIONE DI UN TOPO REPORTER
La scelta del:
sistema di ingegneria animale
reporter bioluminescenza, PET, NMR
costrutto
reporter
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Reporter bioluminescenti e fluorescenti
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I geni reporter
Monomerico, non richiede substrato, assente nei mammiferi, varianti con diversa l di fluorescenza. Bassa sensibilità per mancanza di amplificazione
fluorescent proteins (GFP, varianti RFP, BFP)
Utilizzabili per PETDifficoltà nel generare i ligandi radiomarcati
Ligandi per sostanze radiomarcate
Alta attività specifica, mancanza di attività endogena (basso bkg) Richiede l’aggiunta di cofattore, O2, ATP.
Luciferasi (Lucciola)
Utile per studi di tipo anatomico-funzionale
Ben caratterizzata, stabile, rilevazione automatizzabile
B-Galattosidasi (Batterica)
GENE
Presenza di attività endogena in cellule di mammifero, enzima tetramerico (risposta non lineare)
Applicabile a studi di bioluminescenza in vivo
Applicabile a studi di fluorescenza in vivo
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LA GENERAZIONE DI UN TOPO REPORTER
La scelta del:
sistema di ingegneria animale
reporter bioluminescenza, PET, NMR
costrutto
reporter
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Studio della funzione di promotore
reporter gene reporter gene
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I sistemi reporter nello studio di rilascio/sintesi di trasduttori del segnale (complementazione)
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I sistemi reporter nello studio di interazioni tra proteine
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INSULATOR( MAR )
INSULATOR
( MAR )
lightluciferin + ATP = oxyluciferin + AMP +
TKERE 2x
firefly luciferase
+/- +/-
ERE-Luc reporter mouse
The ERE-Luc reporter mouse: a model to study of ER transcriptional activity
Ciana et al., 2001
ERE
kinase-dependent activation
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i.p. of D-luciferin
20 min.
Evaluation of ER transcriptional activity
5 min. after the acquisition
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18.516.514.513.5 15.5
ERE-Luc mouse
ER is transcriptionally active at day 14.5 pc
dpc (day post conception)
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ERE-Luc mouse
P118.516.514.513.512.5dpc (day post conception) post-natal
day 1
endoderm mesoderm ectodermimaging IHC
A B C D
E
F
G
H
C – intestine 20xD – bone 40xE – heart 40xF – forebrain 10xG – moustache 40x H – skin 20x
16.5 dpc 16.5 dpc
GP Rando, 2007
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ERE-Luc mouse bone
0
2
4
6
thymus
01
23
4
brain
0
5
10
15
20
P E M D2
intestine
0
48
1216
bone
0
2
4
6
thymus
01
23
4
brain
0
5
10
15
20
LUCI
FERA
SE A
CTIV
ITY
(RLU
)
P E D
intestine
0
48
1216
liver
0
10
20
P E M D2
ovaries
0
2
4
uterus
13579
0
6
12
liver
0
10
20
P E D2P E D
ovaries
0
2
4
uterus
13579
0
6
12 hypothalamus
M D P E
Estr
adio
l(pg/m
l)
1020
30
40
50
day 1 day 2 day3 day 4
SESTRUS
E2
(p
g/m
l)
1020304050
M D P E
Luciferase activity in adult, cycling females
Ciana et al, Nature Ned., 2003
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Suckling Mice
Pregnancy &
EmbryoDevelopment
13.5
14.5
15.5
16.5
17.5
18.5
19.5
12.5
DAY 18.5
DAY 16.5
DAY 13.5
DAY 14.5
DAY 15.5
DAY 1
DAY 10
Immature Mice
Adult Mice
LIFE
CYCLE
ERE-Luc mice to understand ER involvement in mammals physiopathology
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THE COMPLEXITY OFESTROGEN ACTION
ERE
G-protein,
IP3K...
SP1NFKBAP1
THE COMPLEXITY OF ESTROGEN TARGETS
• REPRODUCTIVE SYSTEM- male and female gonads- hypothalamus and pituitary
• SKELETAL SYSTEM• VASCULAR SYSTEM
- endothelium- smooth muscle cells
• RESPIRATORY SYSTEM• IMMUNE SYSTEM• NERVOUS SYSTEM
- central- peripheral
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CNS
cardiovascular
reproductive
bone
ERE
SP1NFKBAP1
tissue-specificcoregulators
growthfactors P
ER COMPLEXITY OF ACTION and A NEW CLASS OF DRUGS: Selective Estrogen Recepor Modulators
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ESTROGEN REPLACEMENT THERAPY
The efficacy of SERMs on estrogen receptor transcriptional activity was measured in a model
of surgical menopause (ovx mice)
SERMs ability to replace the natural hormone was evaluated by comparison with ER activity in
healthy, cycling mice
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Reproductive organs(mammari glands and vagina)
Muscle-Skeletal System
Hepatic area
Measuring bioluminscence in the ERE-Luc reporter mouse
Thymic area
Intestine
Bioluminescence after6h treatment with
15b-estradiol (50ug/kg)
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pellet days of treatment
CONTROLS
REFERENCE DRUG
DRUG OF INTEREST
Manual
In vivo analysis of photon emission
Automatic
Rando et al. 2009
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REPORTER MICE TO STUDY DRUG ACTION “IN VIVO”
0
5
10
15
20 UTERUS
RLU
0
500000
1000000
1500000
2000000
AUC
VAGINA
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2110000
40000
70000
100000
VAGINA
Days
Phot
on e
miss
ion
p/s/
cm2/
sr
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 210
100000
200000
300000
400000
CHEST
Days
Phot
on
emis
sion
p/
s/cm
2/sr
0
2000000
4000000
6000000
8000000
AUC
CHEST
0
100
200
300
400
500 LIVER
RLU
VehiclePCUD 3mg/kgBZA 10mg/kgBZA 10mg/kg + PCUD 3mg/kgRaloxifene 10mg/kg
Luciferase enzymatic activityPhoton emission
Biserni et al, in preparation
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0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2110000
40000
70000
100000
REPRODUCTIVE AREA
Days
Phot
on e
mis
sion
p/
s/cm
2/sr
0
500000
1000000
1500000
2000000
AUC
°**
VehiclePCUD 3mg/kgBZA 10mg/kgBZA 10mg/kg + PCUD 3mg/kgRaloxifene 10mg/kg
Effects of SERMs on ER activity – in vivo imaging
CHRONIC treatment (21 days)
Biserni et al, in preparation
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Effects of SERMs on ER activity – in vivo imaging
CHRONIC treatment (21 days)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2120000
40000
60000
80000
100000
TAIL
Days
Phot
on e
mis
sion
p/
s/cm
2/sr
0
500000
1000000
1500000
2000000
AUC
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2120000
40000
60000
80000
100000
LIMBS
Days
Phot
on e
mis
sion
p/
s/cm
2/sr
0
500000
1000000
1500000
2000000
AUC
VehiclePCUD 3mg/kgBZA 10mg/kgBZA 10mg/kg + PCUD 3mg/kgRaloxifene 10mg/kg
Biserni et al, in preparation
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limb
0
100
200
300
400
500
AU
C
tail
0
50
100
150
200
250
AU
C
limb
0
50
100
150
200
250
cts/c
m2 s
(vs c
yc=
100%
)
tail
0
50
100
150
200300400500600
cts
/cm2 s
(vs c
yc=
100%
)
LIMB TAIL
ACUTE
CHRONIC
ovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAMovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAM
Rando et al, Mol. Endocrinol. 2010
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hepatic
0
100
200
300
500150025003500
AU
C
abdomen
0
50
100
150
200
250
AU
C
hepatic
0
100
200
300
400500
100015002000
cts/c
m2 s
(vs
cy
c=
100
%)
abdomen
0
50
100
150
200
250
cts
/cm2 s
(vs c
yc=
100%
)
HEPATIC AREA ABDOMENACUTE
CHRONIC
ovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAMovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAM
Rando et al, Mol. Endocrinol. 2010
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SERCHING FOR NOVEL MODALITIES TO MEASURE THE EFFICACY OF SERMs
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 210
100000
200000
300000
400000
CHEST
Days
Phot
on e
mis
sion
p/
s/cm
2/sr
AUC
N° peaks, amplitude, frequency
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0
3
6
9
12
0
2
4
6
8
0
3
6
9
12
0
2
4
6
8
Peak
s/21
dPe
riod
(d)
GENITAL AREA SKELETAL AREA
***
*
*
* ****
2
3
4
5
6
2
3
4
5
6
Ampl
itude
0
50
100
150
200
250
0
100
200
300400600800
**
* ***
050
100150200300450600
0
50
100
150
200
***
*
**
D
A
B
C
E
AUC
Pote
ncy
ovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAMovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAM
Rando et al, Mol. Endocrinol. 2010
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PHENETICS OF DRUG ACTION
THE APPLICATION OF AGGLOMERATIVE HIERARCHICAL CLUSTERING
(AGGLOMERATIVE NESTING version 1.02 )
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2
4
6
8
0.1
1
10
100
1000
10
100
1000
10000
**p<0.01
0 2 4 6 81
10
100
2 4 6 8 0.1 1 10 100
**p<0.01
10 100 1000 10000
**p<0.01
peak number period amplitude AUC
per
iod
amp
litu
de
AU
Cp
ote
ncy
a
period 0.04
amplitude 0.05 <0.01
AUC 0.03 0.05 0.96
potency 0.10 0.05 0.76 0.46
peaks number
period amplitude AUC
b
DEGREE OF FUNCTIONAL CORRELATION AMONG THE PARAMETERS SELECTED
Rando et al, Mol. Endocrinol. 2010
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Space-temporal analysis of drug action in living animals
clustering data to generate novel families of compounds
Genital area
Skeletal area
ovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAMovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAMRando et al, Mol. Endocrinol. 2010
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Genital area
Skeletal area
Reverse Medicinal ChemistryA
B
C
Cl
ovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAMovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAM
Rando et al, Mol. Endocrinol. 2010
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CONCLUSION 1
Adding the time dimension to the study of drug activity leads to a novel ability to define drug efficacy
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 220
2000
4000
6000
8000
LIMBGENITALAREAHEPATIC AREA
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 220
20000
40000
60000
80000
LIMBGENITAL AREAHEPATIC AREA
INTACT
OVX
Biserni et al. in preparation
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TISSUE SPECIFIC EFFECT OF OVX ON THE AMPLITUDE AND FREQUENCY OF ER ACTIVITY
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CONCLUSION 2
The possibility to measure in vivo the activity of estrogenic compounds on their target, may lead to the identification of novel and more efficacious therapies for the post-menopause
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Funding: EU Strep EWA EWA LSHM-CT-2005-518245
EU IP CRESCENDO LSHM-CT-2005-018652
EU NoE DIMI LSHB-CT-2005-512146
NIH RO1(AG027713)
University of MilanCenter of Excellence on
Neurodegenerative Diseaseas
Collaborators at Milan University: Paola Campadelli
David Horner
Paolo CianaElisabetta Vegeto
Gianpaolo RandoValeria BenedusiSara Della TorreCristina VantaggiatoCristian IbarraBalaji RamachandranAndrea BiserniMonica RebecchiClara Meda
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“The whole is more than the sum of its parts “Aristotle (384 BC – 322 BC)Methapysics
The real impact of molecular engineering on drug discovery
MODERN PHARMACOLOGY NEEDS TO REVISIT ANIMAL
MODELS