gene silencing using polypurine reverse hoogsteen hairpins
DESCRIPTION
Seminar led by Carles Ciudad, PhD Recently, we developed an alternative type of molecules to decrease gene expression named Polypurine Reverse-Hoogsteen Hairpin (PPRH). PPRHs are DNA molecules formed by two antiparallel polypurine strands linked by a pentathymidine loop that allows the formation of intramolecularHoogsteen bonds between both strands. These hairpins bind polypyrimidine targets in the DNA via Watson-Crick bonds. Concretely, there are two types of PPRHs capable of decreasing gene expression, that differ in the location of the target sequence and their mechanism of action: Template-PPRHs, which bind to the template strand of the dsDNA (de Almagro et al., 2009), and Coding-PPRHs (de Almagro et al., 2011), which bind both to the template strand of the dsDNA and the mRNA. We analyzed important properties- stability and immunogenicity- of these molecules for their potential therapeutic approach. Stability experiments performed in different types of serum (human and murine) and in human prostate cells (PC3) revealed that PPRHs half-life is much longer than that of siRNAs, its main competitor. The activation of the innate immune response was evaluated analyzing the levels of the transcription factor IRF3, the cleavage of the proteolytic enzyme Caspase-1, and the expression levels of several pro-inflammatory cytokines: type-I interferons, TNFa, IL-6, IL-8, IL-1b, IL-18 and IL-33. These determinations indicate that PPRHs do not activate the immune response, unlike siRNAs, and therefore are suitable for in vivo administration. In this regard, we decided to further explore the in vitro and in vivo effect of PPRHs in cancer, choosing survivin as a target for its implication in apoptosis, mitosis and angiogenesis, and its overexpression in different tumors. We designed and tested several PPRHs against survivin. After an in vitro screening, including cytotoxicity, apoptosis, mRNA and protein levels, we chose the most effective one for in vivo studies. We conducted two types of administration, namely intratumoral and intravenous, in a xenografted model of prostate cancer cells (PC3). The results showed that the chosen Coding-PPRH proved to be effective in decreasing tumor volume and weight. These findings represent the proof of principle of PPRHs as a new silencing tool for cancer gene therapy.TRANSCRIPT
GENE SILENCING USING POLYPURINE REVERSE HOOGSTEEN HAIRPINS
Carles J. Ciudad, Laura Rodríguez, Xenia Villalobos, Núria Mencia, Jeanne Prévot, Carlota Oleaga and Veronique Noé
Department of Biochemistry and Molecular Biology,
School of Pharmacy, University of Barcelona
• Double-‐stranded DNA molecule:
– Reverse Hoogsteen bonds between an9parallel purine strands – Linked by 5-‐T loop – Watson-‐Crick with genomic DNA – pH-‐independent, Salts required
INTRODUCTION PPRHs
PPRHs = PolyPurine Reverse-‐Hoogsteen Hairpins
r-‐H r-‐H WC
WC
INTRODUCTION PPRHS
Binding of PPRH causes strand displacement
Watson-‐Crick bond Reverse-‐Hoogsteen bond Coma et al. OLIGONUCLEOTIDES (2005)
WC
WC
Decrease in gene expression
• Types: Template-‐PPRH Coding-‐PPRH
INTRODUCTION PPRHS
Watson-‐Crick bond Reverse-‐Hoogsteen bond
5’
3’
3’
5’
3’ 5’
5’ 3’ mRNA
Ribosoma
Protein
3’
5’
3’
5’
5’
3’
3’
5’
De Almagro et al. THE JOURNAL OF BIOLOGICAL CHEMISTRY (2009) De Almagro et al. HUMAN GENE THERAPY (2011)
Splicing alteraDon
InhibiDon of transcripDon
New gene silencing tool
1. Comparison Coding-‐ and Template-‐PPRHs in different cell lines in terms of decrease in viability, mRNA levels and apoptosis:
– MiaPaCa 2 à Pancrea9c cancer – PC3 à Prostate cancer – HCT116 à Colon cancer – HUVEC à normal cells
2. In vivo administra9on of PPRHs: Proof of principle
3. PPRH’s Proper9es: – Immunogenicity – Stability
GOALS
• Intracellular protein of 16.5-‐kDa
• Be long s t o I AP f am i l y (inhibitor of apoptosis)
• Involved in: – Cellular division – Apoptosis supression – Angiogenesis – Chemoresistance
INTRODUCCIÓN 1. CODING VERSUS TEMPLATE SURVIVIN
Altieri D.C. NATURE REVIEWS CANCER (2003; 2007)
GOOD TARGET
Human survivin structure (1XOX)
Apopto9c pathways
• Overexpressed in cancer ce l l s , undetectable in normal 9ssue
DISEÑO PPRHs
Survivin. Survivin gene structure and localiza9on of designed PPRHs (arrows).
INTRODUCCIÓN 1. CODING VERSUS TEMPLATE PPRHs DESIGN
NegaDve controls. Hps-‐WC has intramolecular Watson-‐Crick bonds instead of reverse-‐Hoogsteen bonds. Hps-‐Sc has a randon polypurine sequence without target in the human genome.
INTRODUCCIÓN 1. CODING VERSUS TEMPLATE VIABILITY
Most effecDve concentraDon 100 nM
≈ range siRNA
HpsPr-‐B and HpsPr-‐C efficient in all lines
Viability assays. Comparison between coding-‐ and template-‐PPRHs designed against survivin gene in three different cell lines : PC3 (prostate cancer), MiaPaCa 2 (pancrea9c cancer) and HCT116 (colon cancer).
1. CODING VERSUS TEMPLATE mRNA and protein LEVELS
Both Template and Coding-‐PPRHs against the promoter sequence of the survivin gene decrease mRNA and protein levels of the targeted gene
mRNA levels. qRT-‐PCR of survivin levels of PC3 when transfected with increasing doses of HpsPr-‐B and HpsPr-‐C.
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CONTR
OL 30
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Hps-‐SC Hps-‐WC
Survivin m
RNA levels
(relaD
ve to
CONTR
OL)
PPRHs (nM)
HpsPr-‐B
HpsPr-‐C
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Survivin protein levels
(relaD
ve to
CONTR
OL)
PPRHs (100nM)
HpsPr-‐B
HpsPr-‐C
Protein levels. WB of survivin levels of PC3 when transfected with 100nM of HpsPr-‐B and HpsPr-‐C.
INTRODUCCIÓN 1. CODING VERSUS TEMPLATE APOPTOSIS
ApoptoDc assays. Flow cytometry by Rhodamine method or Caspase-‐3/7 Assay. Comparison between coding-‐ and template-‐PPRHs against survivin in 3 different cell lines : PC3 (prostate cancer), MiaPaCa 2 (pancrea9c cancer) and HCT116 (colon cancer).
Coding-‐PPRHs cause more apoptosis than Template-‐PPRHs at 24h
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CONTR
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HpsPr-‐B
HpsPr-‐C
HpsE3-‐C
HpsI1-‐C
Hps-‐WC
Hps-‐Sc
% apo
ptosis
(relaD
ve to
CONTR
OL)
PPRHs (100nM)
Caspase-‐3 acDvaDon in PC3 when transfected with PPRHs against survivin gene
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HpsPr-‐B
HpsPr-‐C
HpsE3-‐C
HpsI1-‐C
HpsPr-‐Sc
HpsPr-‐WC
% apo
ptoD
c cells
PPRHs (100 nM)
Apoptosis when transfected with PPRHs against survivin
HCT116 MiaPaCa 2 PC3
1. CODING VERSUS TEMPLATE NON-‐TUMORAL CELLS
Survivin mRNA levels in HUVEC (rela9ve to PC3 levels)
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PC3 HUVEC
Survivin m
RNA levels
(relaD
ve to
PC3
)
Cell line
Survivin 19 KDa
AcDn 42 Kda
PC3 HUVEC 0"
20"
40"
60"
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100"
120"
140"
DOTAP" HpsPr1B" HpsPr1C" Hps1Sc"
%"viability"
"(rela-
ve"to
"DOTA
P)"
PPRHs"(100nM)"
Survivin protein levels in HUVEC (rela9ve to PC3 levels)
Viability assays. Comparison between HpsPr-‐B and HpsPr-‐C in HUVEC
The most cytotoxic PPRHs (HpsPr-‐B and HpsPr-‐C) DO NOT cause decrease in viability in HUVEC, which DO NOT express survivin
2. IN VIVO ASSAYS Intratumoral versus Intravenous administraDon
Efficacy Assay. Administra9on of HpsPr-‐C to animals with a xenograced tumor of prostate cancer (PC3). Tumor volume is represented.
A. Intratumoral administra9on (10µg/animal twice a week)
B. Intravenous administra9on (50µg/animal twice a week)
Intratumoral or intravenous of the Coding-‐PPRH against survivin administered induces a significant anD-‐tumor effect without effect in
animal body weight loss
3. PROPERTIES: IMMUNOGENICITY siRNA vs PPRH Transcriptional induction of pro-inflammatory genes Inflammasome-dependent caspase-1 activation
dsRNA
ssRNA
CpG DNA
Cytoplasm
Adapted from Atianand MK, Fitzgerald KA. J Immunol. 2013
RNA
TLR-‐3/7/8 RIG1, PKR
DNA
TLR-‐9 DAI, IFI16, AIM2
3. PROPERTIES: IMMUNOGENICITY siRNA vs PPRH
IFN-‐α, TNF-‐α, IL-‐6 IFN-‐β, IL-‐6, IL-‐8
IFN and proinflammatory
cytokines
Inflammasome
ê
Caspase-‐1
ê
IL-‐1β, IL-‐18
IFN-‐α, IFN-‐β, TNF-‐α and IL-‐6
IFN-‐β
Robbins et al. OLIGONUCLEOTIDES (2009) Barker B.R. et al. CURRENT OPINION IN IMMUNOLOGY (2011)
Choubey D. CLINICAL IMMUNOLOGY (2012)
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CNT PPRH siRNA
Protein levels
(relaD
ve to
con
trol)
NF-‐kB protein levels a) b)
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CNT PPRH sIRNA
Protein levels
(relaD
ve to
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trol)
IRF3 protein levels
IRF3
Tubulin
NF-‐kB
3. PROPERTIES: IMMUNOGENICITY siRNA vs PPRH
siRNA induces an increase in NF-‐κβ and IRF3
0 5 10 15 20 25 30 35 40 45 50
100 nM
100 nM
CNT DTP PPRH MTF siRNA LPS
mRN
A levels
(relaD
ve to
con
trol)
IFN-‐ß mRNA levels in THP-‐1 cells
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0.5
1
1.5
2
2.5
100 nM
100 nM
CNT DTP PPRH MTF siRNA LPS
mRN
A levels
(relaD
ve to
con
trol)
IFN-‐α mRNA levels in THP-‐1 cells
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100 nM
CNT DTP PPRH MTF siRNA LPS
mRN
A levels
(relaD
ve to
con
trol)
IL-‐6 mRNA levels in THP-‐1 cells
3. PROPERTIES: IMMUNOGENICITY siRNA vs PPRH
siRNA induces an increase in IL-‐6, TNF-‐α and IFN-‐β
levels
0
5
10
15
20
25
100 nM
100 nM
CNT DTP PPRH MTF siRNA LPS
mRN
A levels
(relaD
ve to
con
trol)
TNF-‐α mRNA levels in THP-‐1 cells
siRNA induces Caspase-‐1 cleavage and IL-‐1β acDvaDon
Caspase-‐1 proteolyDc acDvity. Determina9on by luciferase assay.
3. PROPERTIES: IMMUNOGENICITY siRNA vs PPRH
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CNT DTP PPRH MET (1,5) siRNA (1,5)
LPS/ATP F12
Caspase-‐1 proteo
lyDc acDvity
(relaD
ve to
con
trol)
Supernatant
3. STABILITY: siRNA vs PPRH
y = 100e-‐6E-‐04x
y = 100e-‐0.004x
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100
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% of INPU
T
IncubaDon Dme (min)
F-‐PPRH vs F-‐siRNA stability in mouse serum
y = 100e-‐4E-‐04x
y = 100e-‐0.003x
10
100
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% of INPU
T
IncubaDon Dme (min)
F-‐PPRH vs F-‐siRNA stability in human serum
y = 100e-‐0.001x
y = 100e-‐0.011x 1
10
100
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% of INPU
T
IncubaDon Dme (min)
F-‐PPRH vs F-‐siRNA stability in FCS 100%
y = 100e-‐0.01x
y = 100e-‐0.023x 10
100
0 20 40 60 80
Fluo
rescen
ce intensity
(%
relaDv
e to t = 24h)
Decay Dme (h)
F-‐PPRH vs F-‐siRNA stability in PC3 cells
3. STABILITY: siRNA vs PPRH
PPRHs are more stable than siRNAs in fetal, mouse, human serum and in PC3 cells
CONCLUSIONS
1. Coding-‐PPRHs against an9-‐apopto9c genes decrease viability, at least, as efficiently as Template-‐PPRHs.
2. Coding-‐PPRHs cause a higher apopto9c effect than Template-‐PPRHs at 24h
3. Administra9on of PPRHs in xenograced tumors is effec9ve.
4. PPRHs are less immunogenic than siRNAs in THP-‐1 cells.
5. PPRHs are much more stable than siRNAs in FCS, mouse and human serum and inside the cells.
ü Effec9ve in different cell lines
ü Effec9ve in xenograced tumors
ü Low immunogenicity
ü High stability
AGRADECIMIENTOS
ACKNOWLEDGEMENTS