Gs-alpha and G coupled receptor diseases and gene therapy, lv and zinc finger Tania Battisti Terapia Genica Eloise Boldrini Docente: Prof.ssa Isabella.

Gs-alpha and G coupled receptor diseases and gene therapy, lv and zinc finger

Tania Battisti Terapia GenicaEloise Boldrini Docente: Prof.ssa Isabella SaggioIlaria CasertaFabiola De MattiaIlaria Genovese

Anno Accademico 2012/2013

GH-secreting Adenomas

Clinical features:•Hyperplasia•GH hypersecretion•GHRH receptor overexpression•Endocrine syndromes: Acromegaly

GNAS mutation

Arg201 > Cys

A. Spada et al., MCE 1998

Signalling Pathway

AC

GPR

β γ

GDPα

GTP cAMP CREBPKA

Signalling Pathway

ZINC FINGERS NUCLEASE

GENE THERAPY

WHY GENE THERAPY?

Objective: Why and What?

Actual treatments: Surgery and

endocrine therapy Radiotherapy

Avoid GH-hypersecretion Recovery Gsα-GTPase

activity Rescue WT genomic GNAS sequence and WT

phenotype

WHAT CAN I DO?

WHAT IS ZINC FINGER NUCLEASE?

Recognition DS Clivage (DSB)HDR with esogenous episomal donorWt sequence rescue

WHAT IS ZINC FINGER NUCLEASE?WHY ZINC FINGER NUCLEASES?

Advantages:

Specific recognition of long target sequences Works in all type of cells Long-term expression Use endogenous mechanism: HDR Safety No drugs selection required for screening

Disadvantages:

Low efficiency in vivo

Off target problems

High Costs

Long time procedure

in vitro experiments

Human GH-secreting adenoma primary cells

gsp oncogene-inducible GH4C1 cell line

mRNA ZFN

Donor Plasmid

Arg201

Transducted cells

Transducted cells

Expected Results

CEL-1 Assay

ZFN EFFICIENCY PCR genomic DNA and CEL-1 assay Diluition cloning Extract DNA from clones PCR genomic DNA and CEL-1 assay Identify positive clones and sequence DNA

Expected Results BIOLOGICAL EFFECTS

cAMP level Pit-1 /GH level

pZNFArg201+ Control pZNFArg201+

cAMP Assay

mRN

A e

xpre

ssio

n

Pit-1 GH

ControlpZNFArg201+

Expected Results

Ribeiro-Oliveira et al., “Endocrine related Cancer” 2008

BIOLOGICAL EFFECTS

Biomarker Levels

rt-PCRImmunofluorescentWestern blotting

D.G.Morris et al, “European Journal of Endocrinology” (2005) Xun Zhang et al, “Journal of Clinical Endocrinology and Metabolism”, 1999

In situ hybridization for human PTTG in normal pituitary glandand in GH-secreting pituitary adenoma

Roche et al, 2004

Animal Model

Ψ-

CMV GAGRREPOLPRO

PolyA

RSV REV PolyA

CMV VSV-G PolyA Tropism

293 T cells48 H

RCR-assay

Animal Model

Transduction Efficiency

rt-PCRFACSsort

Stable mutated mice population

F1

(4 month)

TitrationELISA (p24)

EMBRYO

ex vivo experimentsAnimal Model:GsαR201C mouse

(Pituitary Adenoma)Treatment:

ZFN – Donor Plasmid(Arg201) injection

iPS Mouse +

Trasduced iPS

Arg201

sequencing

in vivo experiments

Mice GsαR201C transducted with ZFN and DNA plasmid (control) Mice GsαR201C transducted with ZFN and Donor Plasmid (Arg201)

Stereotaxic reinjection of transducted epithelial stem cells

Molecular Analysis

Morphological Analysis

Pathological Analysis

GsαR201 LevelscAMP LevelsGH Levels

PCR, direct sequencing ELISA

MRI

GsαR201

GH

Immunoistochemistry

Future prospectives

Follow up: after 2 and 5 months: stabilized adenoma after 12 months adenoma: regression

Human ex vivo experimentFase I clinical trial (20-80 people)

Materials and costsZinc finger plasmid and mRNA 6,440 €

cAMP Assay 1000 €

Cloning 1000 €

Lentivector Production and Injection

4,500 €

ELISA 1000 €

Immunoistochemistry 1000 €

Stabulation 800 € -month

References- Castro M. et al: ”Gene therapy for pituitary tumors: from preclinical models to clinical implementation” Frontiers in Neuroendocrinology (2003) 24: 62-77

- Lania A, Mantovani G, Spada A:” Genetics of pituitary tumors: Focus on G-protein mutations” Experimental Biology and Medicine (2003) 228:1004-1017

- Lania A, Spada A: “ G-protein and signaling in pituitary tumors” Hormone research (2009) 71:95-100

- Pertuit M. et al : “ Signalling pathway alteration in pituitary adenomas: involvement of Gsa, cAMP and mitogen-activated protein kinase” Journal of Neuroendocrinology (2009) 21:869-877

- Roche C. et al:“Lentiviral vectors efficiently transduce in human gonadotroph and somatotroph adenomas in vitro. Targeted expression of transgene by pituitary promoters” Journal of Endocrinolgy (2004) 183:217-233

- Spada A, Lania A, Ballarè E: “G protein abnormalities in pituitary adenomas” Molecular and Cellular Endocrinology (1998) 142:1-14

- Technical Bullettin “CompoZr Custom Zinc Finger Nuclease (ZFN)” Sigma-Aldrich

- Fyodor D. et al: “Genome editing with engineered zinc finger nucleases” Nature Review (2010) Vol. 11

- Urnov FD, Rebar EJ, Holmes MC, Zang SH, Gregory PD: “Genome editing with engineered zinc finger nuclease” Nature review Genetics (2010) Volume II

-Vandeva S. et al: “The genetics of pituitary adenomas” Best Practice & Research Clinical Endocrinology & Metabolism (2010) 24:461-476

References- M. Pertuit, D. Romano, C. Zeiller, A. Barlier, A. Enjalbert, and C. Gerard, “The gsp Oncogene Disrupts Ras/ERK-Dependent Prolactin Gene Regulation in gsp Inducible Somatotroph Cell Line” Endocrinology, April 2011, 152(4):1234 –1243

- Xun Zhang et al, “Pituitary Tumor Transforming Gene (PTTG) Expression in Pituitary Adenomas”, Journal of Clinical Endocrinology and Metabolism, 1999

- Antoˆ nio Ribeiro-Oliveira Jr et al, “Protein western array analysis in human pituitary tumours: insights and limitations”, Endocrine-Related Cancer (2008) 15 1099–1114

- Damian G Morris et al, “Dif ferential gene expression in pituitary adenomas by oligonucleotide array analysis”, European Journal of Endocrinology (2005) 153 143–151