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