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Gsalpha receptor linked diseases
and AAV vectors:McCune Albright
SyndromeDocenti:Prof.ssa Isabella Saggio
A.A. : 2011 / 2012
Studenti:Tanino Albanese
Francesca FerrandinoPaola Martufi
Rosanna MatteraValentina Sofia
Terapia GenicaCorso di :
Desease: McCune Albright syndrome
Clinical features: café-au-lait skin pigmentation precocious puberty polyostotic fibrous dysplasia of
bone (FD):• Normal bone replaced by
fibrous tissue• Pain, fracture • Deformity in: ribs, long bones, and
cranio.
Estimated prevalence range:
1/100,000 and 1/1,000,000
Molecular causes
IL-6
IL6
Fos
FGF-23
Gsα-GTP
cAMP
Gsαβγ-GDP
Is a cytokine involved in the differentiation of osteoclasts
Is a growth factor that is involved in
early osteogenic differentation
* *384 aa
GTP biding domain
R201C; R201H; R201S.
Q227K; Q227R.
1aa
POSSIBLE MUTATION
Helix domain GTP biding domain
Gsα protein structure
Hydrolysis of GTP
Auto inactivation
RGS protein:inibihition of Gsα thought GTPase
stimulation.
Regulator of G-protein
Signaling (RGS proteins)
SNX13 (RGS-PX1):• Stimulate GTPase activity +•Specific interaction with Gsα +
+• Multidomain: is a vescicular
traffic stimulator -(function loss by mutation) +
• Size 3,474 bp +
RGS-2:• Stimulate GTPase activity
+• Interact also with Gqα -• Able to bind Adenilate
Cyclase +• Multiactivity: translation regulation by bind of eIF2B
-(function loss by mutation)
+• Little size (1,3 kb) ++
Candidates:
Objectives
Overexpression of RGS-PX1 to rescue Gsα turn-off mechanism
ADVANTAGES : No cytotoxicity Infection of both dividing and
non dividing cells Low immune responces Long-term transgene
expression
DISAVANTAGES: Small size Inefficiency of purified AAV Variable transduction
efficiency
ITR GFPFLAG-RGS-PX1
EF1α
ITR IRES
AAV2 TRANSDUCTIO
N
167 pb 172 pb24+3474pb 714pb79pb
Detailed experimental planBone Marrow Stromal Cells (BMSCs)
trasduced with GsαR201C lentiviral vector.
AAV2-FLAG-RGS-PX1-GFP vector
AAV2-GFP vector
GFP
FLAGRGS-1GFP
BMSCscontrol
BMSCsFD
GFP
Mock Mock
FLAGRGS-1GFP
In vitro model for FD:
Vectors:
ITR GFPFLAG-RGS-PX1eIF1α ITRIRES
Transduced cells separatedthought FACS-SORT using
GFP
Transgene expressionRT-qPCR for RGS-
PX1Primer for RGS-PX1 wtPrimer for FLAG-RGS-PX1
RGS-
PX1
mRN
A re
lativ
e ex
pres
sion
1
0BMSCs
controlBMSCs-FD
mockBMSCs-FDAAV-GFP
BMSCs-FDFLAG-RGS-PX1
*data for normal BMSCs not showed
FLAG-RGS-PX1
β-actin BMSCs
controlBMSCs-FD
mockBMSCs-FDAAV-GFP
BMSCs-FDFLAG-RGS-PX1
Western Blot for RGS-PX1
Normal BMSCs
BMSCs FD model
Flu
ore
scen
ce
Act
ivit
ymock RGS-PX1 mock RGS-PX1
FACS for EGF-Receptor
Side effect: need to analyse of RGS-PX1 contribute on membrain EGFR level.
Rescue of FD cells: molecular analysis
RT-qPCR for cFOS and IL6
Primer for IL-6Primer for cFOS
mRN
A re
lativ
e ex
pres
sion
1
0BMSCs
controlBMSCs-FD
mockBMSCs-FDAAV-GFP
BMSCs-FDFLAG-RGS-PX1
*data for normal BMSCs not showed
FD patient
s
cFOSIL-6
BMSCscontrol
BMSCs-FDmock
BMSCs-FDAAV-GFP
BMSCs-FDFLAG-RGS-PX1
Western Blot for FOS and IL6
Normal BMSCs
BMSCs FD model
cAM
P/F
GF
23
leve
lsmock RGS-PX1 mock RGS-PX1
FGF-32 levelcAMP level
ELISA for cAMP and FGF23
Histological analysis
Non treatedBMSCs
TreatedBMSCs
Non treated
FD-BMSCs
TreatedFD-cells
?Induced Differenziation
Recovery of normal capacity of differenziation
In vivo model: Diffenziation in a Xenograph mouse
Matrix
Hematopoieticcells
Normal bone
Mastocyte
AdipocyteNormal BMSCs
and Transfected FD
Untransduced FD-BMSCs
Matrix
Fibrous tissue
Proper Ossicle
Dysplasic tissue
• Block of activity thought mutagenenesis of PX-domain : loss of membrain localizzation
• Choose of another RGS protein
Pitfall and Solution
Protection of EGFR stimulate cell proliferation
• Use a leak promoter for expression of GsαR201C
• Use expianted FD cells as model
Gsα iperexpressed in LV-GsαR201C induced model cell interfere with RGS2-PX1 activity
• Choose of another RGS protein with GAP activity (for ex. RGS2)
RGS-PX1 do not give awaited rescue
contanct the vendor
about 75 €
about 375 €
about 920 €
about 473 €
about 280 €
Contact the vendor
Materials and costs
BioVision
GenScript
ISO
Life tecnologies
pAAV-IRES-GFP Expression Vector
Western blot
cAMP Activity Assay Kit
Fibroublast Growth Factor 23 ELISA KIT
Antibodies anti-FGF23
Antibodies anti-cAMP
Mice