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“The potential use of RNA
interference to down-regulate the
BMP-antagonist Noggin and increase
bone formation during distraction
osteogenesis.”
Ana Cristina F. Bassit
Marie-Hélène Gaumond
Pierre Moffatt
Reggie Hamdy
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Distraction Osteogenesis (DO)
• Unique surgical technique that stimulates bone formation, most frequently used to promote limb lengthening through slow and progressive distraction after osteotomy.
• The great challenge is to reduce the consolidation phase and prevent the complications related to the maintenance of the external fixator for a long period.
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Bone morphogenetic proteins - BMPs
1965 – Urist demonstrated that the implantation of demineralized bone matrix could induce ectopic bone formation .
Bone morphogenetic proteins (BMPs) were identified as signaling molecules that participate in the skeleton development during embryonic phase.
BMPs also play a key role in bone healing and have been used as potent osteoinductive growth factors.
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BMPs decrease time for fracture healing and increase bone regeneration, but the use of exogenous BMPs is still controversial.
We considered manipulating the BMP-antagonist Noggin through RNA silencing to increase endogenous BMPs levels.
Bone morphogenetic proteins - BMPs
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BMP
antagonist
Noggin
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shRNA pathway
Rao, DD et al. Adv Drug Deliv Rev. 2009
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Alignment
mouse
rat
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Max score = Total score Ident. = 97%
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Sigma Mission shRNA
#1 – (165-185) shRNA – N185 – GD # 609
#2 – (420-440) shRNA – N420 – GD # 610
#3 – (137-157) shRNA – N157 – GD # 611
#4 – (78-98) shRNA – N98 – GD # 612
#5 – (354-374) shRNA – N374 – GD # 613
noggin NM 008711 (699bp)
#2 #3 #4 #5 #1
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pLKO.1 Puro-Noggin
GD610 (pLKO.1-puro-Noggin TRCN0000066294 (N420))
7084 bp
ampi
puropart of Env GP160 glycoprotein
TRCN0000066294 (N420)
puro
PPT
partial U3
SIN/3'-LTR
R
U5'
SV40pA
pUCori
HIV 5'-LTR
SD
Psi
5'GAG D3rdG
RRE
pLKO-REV
pLKO-FW D puroFW D
puro-REV
hPGK
RSV
U6
bla promoter
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• These vectors also encoded a drug resistance gene inactivating puromycin for posterior selection of the cells
• After bacteria amplification, plasmids were purified (QIAGEN® Plasmid Purification kit).
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Production of lentiviruses
• The production of lentiviruses expressing the shRNAs was realized by transfection of HEK cells, with X-tremeGene 9 DNA Transfection Reagent - Roche®.
• Seventy two hours after transfection, the media was collected and lentiviral particles were concentrated by ultracentrifugation.
pLKO.1-puro-NON TARGET
7085 bp
ampi
puropart of Env GP160 glycoprotein
puro
PPT
partial U3
SIN/3'-LTR
R
U5'
SV40pA
pUCori
HIV 5'-LTR
SD
Psi
5'GAG D3rdG
RRE
NON TARGET shRNA
pLKO-REV
pLKO-FW D puroFW D
puro-REV
hPGK
RSV
U6
bla promoter
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Infection with lentiviruses
UMR106 rat osteosarcoma cells were seeded in 12-well plates and infected with the Noggin and nontarget shRNA lentiviruses.
Followed by amplification
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Selection with puromycin
+ puromycin
Added 2.5 µg/mL of puromycin to media.
UMR – Non-infected, no puromycin (ctrl)
UMR- infected
UMR – Non-infected, plus puromycin
+ puromycin
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Cells and conditioned media were collected and processed for qRT-PCR and Western blotting to analyze Noggin mRNA expression and protein production and secretion.
UMR-106 cells infected with lentivirus
amplification
12-well plates were seeded at 300 000 cells/well
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• The lentiviruses expressing shRNA 609 and 610 caused a decrease of approximately 50% of Noggin gene expression.
Noggin gene expression
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0
0.0002
0.0004
0.0006
0.0008
0.001
0.0012
0.0014
0.0016
0.0018
NT 609 610 611 612 613
2-d
CT
shRNA sequences
Noggin Gene Expression
Noggin gene expression
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Protein Expression
• Western blotting analysis showed an approximate 55% decrease in secretion of Noggin in the media of cells infected with shRNA 609 and 610.
• Also, Noggin cellular expression was reduced to less than one fifth as compared to NT-infected cells.
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Ponceau
2013-09-30
175
80
58
46
30
25
17
7
{
1 2 3 4 5 6 5 6 WT 1 2 7 8 9 10 11 12 WT
{ { { { { { {
10µl 10µl 10µl 10µl 10µl 10µl 10µl 10µl 10µl 10µl 16µl 10µl 10µl 15µl 15µl16µl5µl 5µl
Detection
40 min
exposition
2013-10-01
Detection
1 hour
exposition
2013-10-01
NT NT610609 610 611 612 613
1- NT 1/10 3- 609 1/10 5- 610 1/10 7- 611 1/10 9- 612 1/10 11- 613 1/10
2- NT 9/10 4- 609 9/10 6- 610 9/10 8- 611 9/10 10- 612 9/10 12- 613 9/10
175
80
58
46
30
25
17
7
175
80
58
46
30
25
17
7
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Detection
20 min
exposition
2013-10-23
Detection
40 min
exposition
2013-10-23
175
80
58
46
30
25
17
7
Ponceau
2013-10-21
175
80
58
46
30
25
17
7
175
80
58
46
30
25
17
7
NT 609 610 610 NT 609 610 610
Media Cells
Media Media Cells 6.25µl 25µl
175
80
58
46
30
25
17
7
NT 609 610 610 NT 609 610 610
Media Cells
6.25µl 25µl
Ponceau
2013-10-21
Cells
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• Development of a DO external fixator for rats.
• In vivo studies: injection of shRNA will be done
at the distraction site.
• Use of nanoparticles as an alternative siRNA delivery system.
Future research and clinical application
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Thank you! Thank you