2015/2016 – Group 2:

Justine Habault, Federica Caldarelli, Silvia Gasparini and Walter Di Donato

Gsα receptor

AdenovectorProfessor:

Isabella Saggio

Tutors:

Mattia LaTorre, Romina Burla

GENE THERAPY PROJECT:

A NOVEL STRATEGY FOR THE TREATMENT OF

FIBROUS DYSPLASIA

Fibrous Dysplasia (FD)

• Genetic, noninherited disease

• Missense mutation in the GNAS complex locus (R201C and R201H) encoding G protein’s α

subunit (Gαs)

• The disease-causing mutations occur post-zygotically (somatic mosaic disorder)

• Affects skeletal stem cells causing dysfunctional osteoblasts

• May affect one (monostotic form) or several bones (polyostotic form)

Atlas of Genetics and Cytogenetics in Oncology and Hematology

No existing cure Severe manifestations, pain Can evolve into sarcoma

Gsα-receptor signaling pathway

Wt Gsα: normal

GTPase activity

Physiological cAMP

level

Normal PKA activity

Normal bone

formation

Mutated Gsα:

GTPase activity:

constituvely active Gsα

cAMP level

PKA activity

Potential therapeutic targets:

• GNAS mutations: single-nucleotide

Gene-correction should be very specific (high risk of off-target)

• cAMP/PKA pathway: universal pathway

Necessity for a very specific therapeutic strategy (high risk of

side effects)

Abnormally

differentiated osteoblasts

VSGsα mutated pathway

Mutation:

decreased

GTPase activity

Increased cAMP

concentration

Increased

PKA activity

Increased CREB

phosphorylation

Higher PKA activity

• Lack of mineral deposition

• Upregulation of osteogenic markers (ALP, c-fos…) + IL-6

Our choice: PKIγ, a specific PKA C-subunit inhibitor

• PKI: Protein Kinase Inhibitor

- 3 isoforms (PKIα, β and γ)

• PKIγ

- Short peptide (7,7kDa)

- Specifically inhibits PKA C-subunit (catalytic activity)

- Endogenously expressed in BMSCs (low imunogenicity)

- PKIγ persistent overexpression leads to downregulation of osteogenic markers

- No evidence that it is involved in FD (Its pattern of expression is not altered by PKA signaling)

Dalton G. D., Dewey W. L. (2006).

Effect of PKIγ on PKA activity (A) and CREB phosphorylation (B)

Chen et al, JBC (2005)

(B)(A)

Structure of PKI and its inactive mutant PKI4A (A) and their effect on CRE-luciferase activity (B)

Iglesia-Bartolome and al, (2015)

(B)(A)

Our proposal Restore PKA normal catalytic activity by using PKIγ under the control of a CRE: restore a normal

osteogenesis

PKIγ cDNA

(231bp)

• CRE: cAMP Response Element Enhancer that directly responds to cAMPendogenous level

• CAMV 35S: minimal promoterNot strong enough to induce the transgene expression itselfExpression directly dependent to the CRE

C

V

C

V

C

V

C

VNormal BMSC :

Physiological level of cAMP

Low expression of the CRE-induced

PKI

Diseased BMSC:

Higher level of cAMP

High expression of the CRE-induced PKI at first

When PKA activity is sufficiently decreased, lower

expression of CRE-induced PKI

CRE

(8bp)

CAMV35S

(345bp)

Total size of the construct = 584pb

What system of delivery ?• The Adeasy system combined with an ameliorated PiggyBac system

Combines the advantages of :

- Adenovectors: safe and well-tolerated, high efficiency of Ad cell/ nucleus entry process, transduction of both dividing and non-dividing cells

- Adeasy system: Rapid and efficient, preparation of high stock of purified viruses, insert size up to 7,5kb

- PiggyBac system: Stability of transgene expression, robust and highly efficient transposition

Specificity of the system: Transposase and transposon delivered in the same plasmid Self-inactivation of the transposase induced by the lost of pA signal (shared with the GOI) HSV-tk/ganciclovir based negative selection to eliminate the rare transposase-expressing cells

Adapted from

Chakraborty and al. (2015)

eTotal size insert

6,905bp

711bp 13bp 13bp278bp584bp584bp 575bp1782bp72bp 13bp500bp 500pb

CRE

8pb

CAMV35S

345pb

PKIγ cDNA

231pb

CMV

72pb

eGFP

714pb

Linearize with Pme1

Transform into E.coli

Select with kanamycin

Backbone vector

Transfect 293 cells

Follow transfection with GFP

Harvest the viruses in 14-20 d

Linearize with Pac1

Shuttle vector

The AdEasy System

• Minimum of enzymatic manipulations, employing homologous recombination in bacteria cells

• Very fast and efficient generation of recombinant adenoviruses

AdEasy system protocol

Luo and al. (2007)

• Overview of the system ΔE3ΔE1

RITROri

pAdEasy-1

33,414bp

Amp

RITR

pShuttle

6,625bp

Ori

Kan

LITR

Insert

Insert

The AdEasy System

TIMING

• Step 1 : 6-15 d

cloning insert into a shuttle

vector

modify the fiber domain on

Ad genome

• Step 2 : 10-30 d

generating the initial stocks

of adenoviruses

stepwise amplification for

high-titer adenoviruses

determining viral titers

• Cloning of the construct into a shuttle vector :

4 different conditions for our experiments :

1) Mock

2) Vector GFP: AdShuttle (GFP alone)

3) Vector PKI4A: AdShuttle-PKI4A (GFP+ CAMV35S-CRE-PKI4A

construct mutant version of PKI)

4) Vector PKIγ: AdShuttle-PKIγ (GFP + CAMV35S-CRE-PKIγ construct)

• Modify the Ad fiber domain on the backbone vector :

Ad5 fiber gene will be replaced by a chimeric Ad5 fiber tail domain and

Ad35 fiber shaft and knob domains with increased tropism for hMSCs

TAIL SHAFT KNOB

Ad5 Ad35

A simplified system for generating recombinant

Adenoviruses He and al., 1997.

pAdEasy-1/F35

ΔE3ΔE1

RITROri

Experimental setup and objectives of the project

Adenovectors

generation

Adenovectors

amplification

In vitro

experiments

In vivo

experiments

• Proof of concept that PKI can serve as a therapeutic strategy for FD:

- In vitro (hMSCs)

- In vivo (mouse model of xenograft)

• Show that AdPKI-F35 is the right vector to efficiently deliver PKI to target cells

In vitro experiments (hMSCs)

• Infection of hBMSCs with the different vectors 4 types of cells:

Wt BMSCs

Cells from FD-donor

Mutant BMSCs2) Selection of CD63+/CD146+ cells

BMSCs from

FD-donor

BMSCs from

healthy donorCells from healthy donor

3) Single-colony isolation

+ Genotyping (R201C and R201H)

Wt BMSCs from

FD-donor

= wt

Mutant BMSCs

= mt

Mosaic of wt and

mutant BMSCs

= mosaic

Wt BMSCs from

healthy donor

= CL-

4) Expansion and

infection with the viruses

5) Addition of ganciclovir +

Selection of GFP+ cells

(5 days later)

1) Isolation of cells from donors

Is the transduction efficient ?

% o

f tr

an

sd

ucte

dce

lls0

20

40

60

80

100

Mock GFP

PKI4A PKIγ

BMSCs from FD-donor

MOI=1

• Serial MOI trials

• Transduction : GFP

Cell death: PI + Annexin V

The results should be identical

whatever the cell type and vector

used

We will choose an optimal MOI

for further experiments

• Measurement of number of copy/genome: qPCR

copy number PiggyBac Kit (SBI)

We expect to have about one copy/genome

• Measurement of residual transposase

expression: mOrange expression as a proxy

We expect the transposase to have no residual

activity

Are the integration and

transposase inactivation efficient ?

0

5

10

15

20

25

30

P0 P0+5d P1 P2 P3

% m

Ora

nge

ce

lls

(Re

altiv

eto

GF

P+

ce

llsa

t P

0)

BMSCs from FD-donor

Is the strategy working ?

• PKIγ expression

PKIγ mRNA expression (RT-PCR)

CL- Wt Mt Mosaic

Fold

Incre

ase

Mock GFP

PKI4 PKIγ

PKIγ protein expression (Western Blot)

PKIγ

β-Actin

Mock GFP PKI4 PKIγCL-

PKIγ

β-Actin

Mock GFP PKI4 PKIγCL-

Mosaic cells Mt cells

PKIγ expression should increase in PKIγ treated FD cells

This global increase should be higher in mutant pure cultures

• Functional assay: PKA activityA

bso

rba

nce

(4

50

nm

)

0 1 2 7 14

Time

Mt cells

mock and PKIA4

Mt cells

PKIγ

CL- and Wt cells

All vectors

Mosaic cells

mock and PKIA4

Mosaic cells

PKIγ PKA activity should be normalized in FD cells

There should no effect or only

a slight decrease in normal cells

PKA assay

• Effect on FD markers levels

CL- and wt (+/- PKIγ)

Mt Mosaic Mt + PKIγ Mosaic + PKIγ

Fo

ldch

an

ge

ALPIL-6cAMP

ELISA assay

CL- and wt (+/- PKIγ)

Mt Mosaic Mt + PKIγ Mosaic + PKIγ

c-fos

RT-PCR

We expect a normalization of ALP, c-fos and IL-6 levels

We expect no change in cAMP level

Calcium

deposition

Day 1

Day 14

Day 21

A B C

A: Normal BMSCs

B: FD BMSCs

C: FD BMSCs + PKIγ

Cultured in osteogenic medium

PKIγ-transduced FD

cells should normally differentiate

Is the strategy working ?

• Effect on in vitro differentiation

Fo

ldch

an

ge

Is the system safe ?

• Control of insertion sites by Splinkerette-PCR

• Cytotoxicity measurement by FACS analysis

There should be no effect on cell death

• Proliferation assay

There should be no effect on cell proliferation

• Effect on other essential cellular pathways

There should be no dysregulation of other

signaling pathways

Flanking sequences identified by searching the draft human genome

PCR and sequencing

First and second round PCR

Ligation

Annealed splinkerette

oligonucleotides

PiggyBac-mediated insert

0

10

20

30

40

50

60

70

0 1 7 14

Normal BMSCs FD BMSCs + Mock

FD BMSCs + PKI4A FD BMSCs + PKIγ

Cell

de

nsity

(1x1

0^5

/ml)

Cell proliferation analysis (BrdU incorporation assay)

0

5

10

15

20

25

30

35

0 1 7 14 dayshBMSCs from

healthy donorhBMSCs from healthy

donor + PKIγ

hBMSCs from FD

donor + PKIγ

Microarray analysis

Splinkerette-PCR

Xenograft model of SCID mice• Xenograft of hBMSCs in immunocompromised mice to evaluate AdShuttle-PKIγ potential to

regenerate a complete ectopic ossicle

Why using this model ?

• In vivo: Adenoviruses too immunogenic (IL-6 response)

• Ex vivo: Need for preliminary experiments on mice cells, need for

a model that allow a direct comparison of the different conditions

hBMSCs from

healthy donor

Mock treated

GFP

PKI4A

PKIγ

hB

MS

Cs

fro

mF

D-d

on

or

HA/TCP carrier

(10^4 cells)

poorly mineralized

woven bone

fibrous tissue

HA/TCP Carrier

normal lamellar bone

normal hematopoietic

marrow

FD ossicle

Normal ossicle

Cured ossicle

Histology

n=6

Future experiments

Mouse model

characterization for

PKA activity

Ex vivo experiments

In vivo

experiments

Use 3rd generation

adenoviruses

Clinical trial

Only if ex vivo is

not efficient

Repeat the

experiments in mouse

cells (GsαR201C and

PKA mouse models)

Potential pitfalls and solutions

• Trouble to isolate or expand single-clones- Try other growth media- Try BMSC-LV-Gs201C cell model

• Trouble to efficiently transduce BMSCs- Try another adenovector with RGD-modified domain

• GFP toxic effect- Use another reporter gene (HrGFP)

• Insertional mutagenesis- Use of a suicide gene

• PKI efficiency issues- Increase the number of CRE copies- Try another isoform (PKIα affinity=6-fold higher)- Try a strong promoter (such as CRE)

• PKI toxicity issues (it causes cell death, or trigger too strong effects) or interfers with other pathways

- Try a synthetic PKI

• cAMP accumulation in the cell due to lower PKA activity- Try to combine our system with PDE4 to degrade cAMP

Timescale, materials and cost of the project• http://www.taconic.com

• http://www.scienceexchange.com

• https://www.promega.com

• http://www.sigmaaldrich.com

• http://www.abcam.com

• http://www.qiagen.com

• http://www.merckmillipore.com

• http://www.antibodies-online.com

• http://www.thermofisher.com

• http://www.genomics.agilent.com

• http://www.abcam.com

• http://www.thermofisher.com

• http://www.transposagenbio.com

• http://www.integratedsci.com

• http://www.transposagenbio.com

This project has a timescale of 2 years and a total cost of 30.000 €

• NOD-SCID Mouse (one male, one female): 188€

• MicroArray Analysis: 695€

• cAMP assay (c-AMP-Glo™ assay): 299€

• Alizarin Red S staining: 65€

• Annexin V-FITC Apoptosis Detection Kit: 505€

• RT-qPCR analysis QuantiTect SYBR Green PCR kit: 417€

• BrdU Cell Proliferation Assay Kit: 339€

• Alkaline Phosphatase ELISA Kit: 680€

• PKIγ antibody: about 300€

• AdEasy Adenoviral Vector Systems: 357€

• Functional Assay of PKA activity kit: 567€

• IL-6 ELISA Kit: 450€

• Super piggyBac transposase expression vector: 350€

• PiggyBac™ splinkerette PCR Kit: 399€

• PiggyBac copy number Kit: 522€

• Cell culture: 2 000€

• Animal facility: 5 000€

References• Riminucci M, Saggio I, Robey PG, Bianco P. Fibrous dysplasia as a stem cell disease. J Bone Miner Res. 2006; 21(Suppl 2):P125–131. [PubMed: 17229001]

• Dalton G. D., Dewey W. L. (2006). Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase

function. Neuropeptides 40, 23–34. 10.1016/j.npep.2005.10.002

• Development of an adenoviral vector system with adenovirus serotype 35 tropism; efficient transient gene transfer into primary malignant hematopoietic cells, Nilsson and al.

(2004); The Journal of Gene Medicine

• Constitutive expression of GsαR201C in mice produces a heritable, direct replica of human fibrous dysplasia bone pathology and demonstrates its natural history, Saggio and

al. (2014); JBMR

• Skeletal progenitors and the GNAS gene: fibrous dysplasia of bone read through stem cells, Riminucci and al. (2010); Journal of Molecular Endocrinology

• Transfer, Analysis, and Reversion of the FibrousDysplasia Cellular Phenotype in Human Skeletal Progenitors, Piersanti and al. (2010); JBMR,

• Robey and al. (2015); Methods Molecular Biology

• Self-Renewing Osteoprogenitors in Bone Marrow Sinusoids Can Organize a Hematopoietic Microenvironment, Sacchetti and al. (2007); Cell

• Osteoclastogenesis in fibrous dysplasia of bone: in situ and in vitro analysis of IL-6 expression, Riminucci and al. (2003); Bone

• Skeletal progenitors and the GNAS gene: fibrous dysplasia of bone read through stem cells, Riminucci and al. (2007); Journal of Molecular Endocrinology

• A simplified system for generating recombinant adenoviruses, Tong-Chuang and al. (1997); Medical Sciences

• A protocol for rapid generation of recombinant adenoviruses using the AdEasy system, Luo and al. (2007); Nature Protocols

• Protein kinase inhibitor peptide (PKI): A family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein-kinase function. Dalton and al. (2006);

Neuropeptides

• Downregulation of cAMP-dependent protein kinase inhibitor is required for BMP-2-induced osteoblastic differentiation. Zhao and al. (2006), IJBCB

• Inactivation of a Gαs–PKA tumour suppressor pathway in skin stem cells initiates basal-cell carcinogenesis. Iglesia-Bartolome and al (2015), Nature Cell Biology

• Endogenous Protein Kinase Inhibitor Terminates Immediate-early Gene Expression Induced by cAMP-dependent Protein Kinase (PKA) Signaling. Chen and al, (2005); JBC

• An Activator of the cAMP/PKA/CREB Pathway Promotes Osteogenesis from Human Mesenchymal Stem Cells, Kim and al (2012); Journal of Cellular Physiology

• Osteoblast-like Cells Complete Osteoclastic Bone Resorption and Form New Mineralized Bone Matrix In Vitro, Mulari and al. (2004); Calcified tissue international

• Inhibition of interleukin-6 receptor directly blocks osteoclast formation in vitro and in vivo, Axmann and al. (2009); Athritis Rheum

• Fibrous Dysplasia as a stem cell disease, Riminucci and al. (2009); Journal and bone and mineral research

• The nature of fibrous dysplasia, Feller and al.(2009); Head and Face Medicine

• PiggyBac Transposon plus Insulators Overcome Epigenetic Silencing to Provide for Stable Signaling Pathway Reporter Cell Lines, Mossine and al, Plos One (2013)

Thank you for your attention