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.
Gene Silencing using Polypurine Reverse Hoogsteen Hairpins
1. 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
2.
• 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
3. 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
4. • 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
5. 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
6. • Intracellular
protein
of
16.5-‐
kDa
• Belongs
to
IAP
family
(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
cells,
undetectable
in
normal
9ssue
7. 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.
8. 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).
9. 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.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
CONTROL
30
100
300
100
100
Hps-‐SC
Hps-‐WC
Survivin
mRNA
levels
(relaDve
to
CONTROL)
PPRHs
(nM)
HpsPr-‐B
HpsPr-‐C
0
20
40
60
80
100
Survivin
protein
levels
(relaDve
to
CONTROL)
PPRHs
(100nM)
HpsPr-‐B
HpsPr-‐C
Protein
levels.
WB
of
survivin
levels
of
PC3
when
transfected
with
100nM
of
HpsPr-‐B
and
HpsPr-‐C.
10. 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
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
CONTROL
DOTAP
HpsPr-‐B
HpsPr-‐C
HpsE3-‐C
HpsI1-‐C
Hps-‐WC
Hps-‐Sc
%
apoptosis
(relaDve
to
CONTROL)
PPRHs
(100nM)
Caspase-‐3
acDvaDon
in
PC3
when
transfected
with
PPRHs
against
survivin
gene
0
10
20
30
40
50
60
70
CONTROL
DOTAP
HpsPr-‐B
HpsPr-‐C
HpsE3-‐C
HpsI1-‐C
HpsPr-‐Sc
HpsPr-‐WC
%
apoptoDc
cells
PPRHs
(100
nM)
Apoptosis
when
transfected
with
PPRHs
against
survivin
HCT116
MiaPaCa
2
PC3
11. 1. CODING
VERSUS
TEMPLATE
NON-‐TUMORAL
CELLS
Survivin
mRNA
levels
in
HUVEC
(rela9ve
to
PC3
levels)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
PC3
HUVEC
Survivin
mRNA
levels
(relaDve
to
PC3)
Cell
line
Survivin
19
KDa
AcDn
42
Kda
PC3
HUVEC
0"
20"
40"
60"
80"
100"
120"
140"
DOTAP" HpsPr1B" HpsPr1C" Hps1Sc"
%"viability"
"(rela-ve"to"DOTAP)"
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
12. 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
13. 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
14. 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)
15. 0.0
0.5
1.0
1.5
2.0
CNT
PPRH
siRNA
Protein
levels
(relaDve
to
control)
NF-‐kB
protein
levels
a)
b)
0
2
4
6
8
10
12
14
16
18
CNT
PPRH
sIRNA
Protein
levels
(relaDve
to
control)
IRF3
protein
levels
IRF3
Tubulin
NF-‐kB
3.
PROPERTIES:
IMMUNOGENICITY
siRNA
vs
PPRH
siRNA
induces
an
increase
in
NF-‐κβ
and
IRF3
17. siRNA
induces
Caspase-‐1
cleavage
and
IL-‐1β
acDvaDon
Caspase-‐1
proteolyDc
acDvity.
Determina9on
by
luciferase
assay.
3.
PROPERTIES:
IMMUNOGENICITY
siRNA
vs
PPRH
0
1
2
3
4
5
6
CNT
DTP
PPRH
MET
(1,5)
siRNA
(1,5)
LPS/ATP
F12
Caspase-‐1
proteolyDc
acDvity
(relaDve
to
control)
Supernatant
18. 3.
STABILITY:
siRNA
vs
PPRH
y
=
100e-‐6E-‐04x
y
=
100e-‐0.004x
10
100
0
100
200
300
400
%
of
INPUT
IncubaDon
Dme
(min)
F-‐PPRH
vs
F-‐siRNA
stability
in
mouse
serum
y
=
100e-‐4E-‐04x
y
=
100e-‐0.003x
10
100
0
100
200
300
400
%
of
INPUT
IncubaDon
Dme
(min)
F-‐PPRH
vs
F-‐siRNA
stability
in
human
serum
y
=
100e-‐0.001x
y
=
100e-‐0.011x
1
10
100
0
100
200
300
400
%
of
INPUT
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
Fluorescence
intensity
(%
relaDve
to
t
=
24h)
Decay
Dme
(h)
F-‐PPRH
vs
F-‐siRNA
stability
in
PC3
cells
19. 3.
STABILITY:
siRNA
vs
PPRH
PPRHs
are
more
stable
than
siRNAs
in
fetal,
mouse,
human
serum
and
in
PC3
cells
20. 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