Gs-alpha and G coupled receptor
diseases and gene therapy, lv and
zinc finger
Tania Battisti
Eloise Boldrini
Ilaria Caserta
Fabiola De Mattia
Ilaria Genovese
Terapia Genica
Docente: Prof.ssa Isabella Saggio
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
GPR

β
GDP
α
GTP
AC
Signalling Pathway
Objective: Why and What?
WHY GENE THERAPY?
Actual treatments:
 Surgery and
endocrine therapy
 Radiotherapy
WHAT CAN I DO?
 Avoid GH-hypersecretion
 Recovery Gsα-GTPase
activity
 Rescue WT genomic
GNAS sequence and WT
phenotype
GENE THERAPY
ZINC FINGERS NUCLEASE
WHAT IS ZINC FINGER NUCLEASE?
Recognition
DS Clivage
(DSB)
HDR with
esogenous
episomal donor
Wt 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
Transducted
cells
mRNA ZFN
Arg201
gsp oncogene-inducible
GH4C1 cell line
Donor Plasmid
Transducted
cells
Expected Results
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
CEL-1 Assay
Expected Results
BIOLOGICAL EFFECTS
 cAMP level
 Pit-1 /GH level
Control
pZNFArg201+
Arg201+
pZNF
cAMP Assay
mRNA expression
Control
pZNFArg201+
Pit-1
GH
Expected Results
BIOLOGICAL EFFECTS
 Biomarker Levels
In situ hybridization for human PTTG in normal pituitary gland
and in GH-secreting pituitary adenoma
Xun Zhang et al, “Journal of Clinical Endocrinology and Metabolism”, 1999
D.G.Morris et al, “European Journal of Endocrinology” (2005)
Ribeiro-Oliveira et al., “Endocrine related Cancer” 2008
rt-PCR
Immunofluorescent
Western blotting
Animal Model
CMV
RSV
CMV
GAG
REV
PRO
POL
RRE
Ψ
PolyA
VSV-G
PolyA
PolyA
Roche et al, 2004
293 T cells
Tropism
48 H
RCR-assay
Animal Model
Titration
ELISA (p24)
Transduction Efficiency
rt-PCR
FACSsort
EMBRYO
Stable mutated mice
population
F1
(4 month)
ex vivo experiments
Animal Model:
GsαR201C mouse
(Pituitary Adenoma)
Treatment:
ZFN – Donor Plasmid(Arg201)
injection
Arg201
iPS Mouse
Trasduced iPS
sequencing
in vivo experiments
Stereotaxic reinjection of
transducted epithelial stem cells
Mice GsαR201C transducted with ZFN and DNA plasmid (control)
Mice GsαR201C transducted with ZFN and Donor Plasmid (Arg201)
 PCR, direct
GsαR201 Levels
 Molecular Analysis
sequencing
cAMP Levels
 ELISA
GH Levels
 Morphological Analysis  Immunoistochemistry
 Pathological Analysis
 MRI
GsαR201
GH
Future prospectives
Follow up: after 2 and 5 months: stabilized adenoma
after 12 months adenoma: regression
Human ex vivo experiment
Fase I clinical trial (20-80 people)
Materials and costs
Zinc 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