Shooting the Messenger: Harnessing RNA Interference for Therapy Judy Lieberman, MD, PhD Center for B

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Shooting the MessengerHarnessing RNA
Interference for TherapyJudy Lieberman, MD,
PhDCenter for Blood Research Harvard Medical
School
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DNA makes RNA and RNA makes protein.
DNA
mRNA
Protein
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RNA interference (RNAi) is an ancient mechanism
for degrading RNA
DNA
mRNA
Protein
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• RNA interference (RNAi) may have evolved as a
  defense mechanism to protect against genetic
  elements that can insert into the genome, such as
  viruses and transposons.
• Almost two years ago, Tom Tuschl and colleagues
  showed that RNAi operates not only in plants,
  flies and worms, but also in mammalian cells.
• In the past year, many labs have begun to
  investigate whether RNAi can be harnessed as a
  potent therapeutic tool to treat disease,
  including viruses such as HIV and hepatitis C.

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Small interfering RNA (siRNA)-mediated gene
silencing
dsRNA
DICER (RNase III enzyme)
19-nt duplex
siRNA (21-23 nt)
siRNA/protein complex
2-nt
2-nt
RNA Induced Silencing Complex (RISC)
Target mRNA
Cleaved target mRNA
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Possible advantages of RNAi for therapy

• Exquisite specificity one base pair difference
  in target blocks effect no silencing of
  unrelated genes
• Potent gene silencing
• Bioavailability and stability of double stranded
  RNAs
• Cheap and simple to make
• A broad spectrum of target cells and diseases can
  be targeted

Main therapeutic challenge How to deliver siRNAs
to different cells and tissues in vivo
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Developing RNAi against HIV
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Both HIV genes and host receptor or
HIV-interacting genes are fair targets of siRNA
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CD4 is the cellular HIV receptor
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CD4 silencing in Magi-CCR5 cells transfected with
CD4 siRNA
Experimental Timetable
Day 1 Transfect with CD4 siRNA
Day 3 Flow cytometry and Northern blot analysis
of CD4 expression
Infect with HIV
Day 5 p24 ELISA, b-gal infection assay, syncytia
assay
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siRNA-directed gene silencing is more effective
than antisense RNA
Mock CD4-siRNA
CD4-antisense RNA
HPRT-siRNA
Northern blot of mRNA
HeLa ------- HeLa-CD4 --------
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Decreased syncytia formation in CD4-silenced Magi
cells
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CD4 silencing inhibits replication of production
of 2 types of HIV viruses
R5
X4
p24 antigen
(ng/ml)
(pg/ml)
AS CD4
Control siRNA
CD4 siRNA
AS CD4
Control siRNA
CD4 siRNA
X4
R5
b-gal cells
CD4-AS Ctrl-si CD4-si
CD4-AS Ctrl-si CD4-si
AS CD4
Control siRNA
CD4 siRNA
AS CD4
Control siRNA
CD4 siRNA
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CD4 silencing decreases viral entry
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siRNA can target HIV gene expression at multiple
sites
?
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Organization of the HIV genome
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(No Transcript)
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p24 siRNA silences viral gene expression
p24 siRNA
no siRNA
uninfected
control siRNA
100
1
4
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R
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Experimental Timetable
Day 1 Transfect with p24 siRNA
Day 2 Infect with HIV
Day 4 FACS for p24 expression
Northern blot
Day 5 p24 ELISA
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p24 siRNA silences multiple viral transcripts
Stable infection
No infection
No siRNA
Control siRNA
p24 siRNA
p24 probe
9.2 kB
9.2 kB
nef probe
6.5 kB
1.6 kB
b-actin probe
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p24 silencing inhibits cell free virus production
uninfected
no siRNA
p24 siRNA
control siRNA
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All regions of HIV genomic RNA are potential
targets of siRNAs
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Goals of recent HIV work

• Are primary macrophages (an important reservoir
  for HIV-1 infection that is relatively
  drug-insensitive) and primary T cells good
  targets for RNAi?
• Can exogenously delivered siRNAs provide
  long-lasting protection?
• Is there synergy using siRNAs that target
  different parts of the viral life cycle?
• Can lentiviral vectors that express stem-loop
  siRNAs induce a stable HIV-resistant state in
  primary cells?

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p24-siRNA inhibits HIV infection in primary T
cells
uninfected no siRNA
p24-siRNA GFP-siRNA
p24 RD1
Experiment Day 1 Transfect CD4 T cell
blasts Day 2 Infect with HIV Day 4 Measure
HIV production by intracellular p24
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Approach
Generate monocyte derived macrophages (MDM) from
PBMCs
Transfect with Cy5-labeled or unlabeled siRNA
1-2 days
Infect with macrophage tropic HIVBAL
Examine HIV inhibition by
p24 ELISA
In situ hybridization for HIV RNA
Intracellular p24 expression
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Efficient siRNA transduction of primary
macrophages
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Silencing CD4 or CCR5 decreases viral entry
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Reduced HIV RNA 7 days after macrophage infection
f-Actin
HIV RNA
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Synergistic suppression of HIV replication by
targeting CCR5 and p24 in primary macrophages
GFP-siRNA
mock
p24-siRNA
CCR5-siRNA
CCR5- and p24-siRNA
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Inhibition of macrophage HIV infection for 15 days
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CCR5 silencing is sustained for 3 weeks in
macrophages
---- mock ----- - CCR5-siRNA -
1 4 7 15 1 4 7 15 days
p-transfection
Ctrl
RT-PCR
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Just as for antiviral drug therapy, inhibiting
HIV at multiple sites in its life cycle is
synergistic for blocking infection.
?
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p24-siRNA inhibits HIV production in previously
infected macrophages
MF infected for 16 d (90 of cells HIV) were
transfected with siRNA and assayed after
indicated times for intracellular HIV p24.
Although p24-siRNA persists for less than 1 week
in uninfected cells, silencing is prolonged in
infected cells. This suggests that target RNA
may enable siRNA to persist.
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Expression of stem-loop siRNA from a lentiviral
vector
Lentivirus (pLL3.7) Lois et al. Science (2002)
Predicted CCR5 stem loop


CTCTGCTTCGGTGTCGAAA

GAGACGAAGCCACAGCUUU

C
G
UU
A
A
A
AG
UU
G
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HIV suppressed in HeLa CD4 T cells infected with
rev stem-loop-expressing lentivirus
Day 1 Infect with stem-loop lentivirus
Day 6 Infect sorted for GFP cells with HIV-1
Day 8 Analyze for intracellular p24
Day 6
Day 8
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Stem-loop lentivirus efficiently silences CCR5
expression in primary macrophages
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Summary

• Combination of siRNAs targeting cellular and
  viral genes can completely abrogate HIV-1
  infection in macrophages.
• A single application of siRNA provides long
  lasting viral suppression in non-dividing cells
  such as macrophages.
• Suppression is also effective for cells that are
  already infected with HIV.
• Stable sustenance of exogenously delivered siRNA
  may require the presence of the target mRNA.
• Lentiviruses containing siRNAs stem-loops can be
  used for stable endogenous expression of siRNA in
  primary cells.

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Can siRNA be delivered for in vivo therapy?
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Hepatocytes efficiently take up intravenously
injected duplex siRNAs. (McCaffrey et al
Nature 2002)Most forms of fulminant hepatitis
of diverse etiology are fas-mediated.PLAN Test
effectiveness of siRNA injection in 2 models of
autoimmune hepatitis - conA injection - Fas
agonistic antibody
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Hepatocytes efficiently take up siRNA after
hydrodynamic injection
50 mg siRNA in 1 ml x 3
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Sustained specific silencing of fas mRNA in
hepatocytes after hydrodynamic injection of
fas-siRNA
GFP-siRNA
Fas-siRNA
Saline
No rx
1 5 10 days post injection
FAS
RNase protection assay
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Sustained specific suppression of fas protein in
hepatocytes after hydrodynamic injection of
fas-siRNA
GFP-siRNA
Fas-siRNA
Saline
No rx
-
1 5 10 days post injection
Fas
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Hepatocytes from Fas-siRNA treated mice are
resistant to fas-mediated apoptosis in vitro
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Hepatocytes from Fas-siRNA treated mice resist
apoptosis induced by infiltrating liver
mononuclear cells from conA-treated mice
Control
Fas-siRNA
Plt0.0.001
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Mouse challenge with concanavalin A
1 day
conA i.v.
Control or FAS-siRNA
20 hr
Liver histology, serum transaminases
Untreated mice develop acute hepatitis.
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Liver protection in conA-induced hepatitis
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Reduced hepatocyte death in conA-induced hepatitis
Plt0.001
Liver cell death monitored by serum levels of
liver enzymes.
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FAS-siRNA treatment of chronic hepatitis induced
by conA
Week 7 liver histology, markers of fibrosis
conA i.v. weekly X 6
FAS-siRNA weeks 2, 4
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Fas-siRNA protects against liver fibrosis
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Liver fibrosis reduced by fas-siRNA treatment
Plt0.05
Plt0.01
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Mouse challenge with fas antibody
1 day
Fas antibody i.p.
Control or FAS-siRNA
Observe x10 d, autopsy day 10
Untreated mice challenged with fas antibody die
of fulminant hepatitis within a few days.
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Three fas-siRNAs silenced liver cell expression
in vivo by gt80
FAS
RNase protection assay
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Fas-siRNA treated mice survive injection of
anti-Fas antibody
Fas-siRNAs that silence
Survival ()
Control siRNAs and mock treatment
Control siRNAs and Fas-siRNAs that dont silence
Days after anti-fas treatment
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Conclusions

• Hepatocytes efficiently take up intravenously
  injected siRNAs. Silencing is effective and
  specific.
• siRNA-mediated silencing is long-lived ( 10
  days) in hepatocytes in vivo.
• Silencing fas can protect mice from fulminant
  hepatitis in 2 models of autoimmune hepatitis.
• Treatment can be initiated even after the
  destructive process has started.
• Extension to other types of hepatitis, to other
  diseases, to other cellular targets, to
  primates.?

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Acknowledgments

• Center for Blood Research
• Premlata Shankar
• Paul Beresford
• Erwei Song
• Sang-Kyung Lee
• Nedim Ince
• Manju Swamy
• Jan Cerny
• Vanessa Francois-Bongarcon

• Center for Cancer Research, MIT
• Carl Novina
• Derek Dykxhoorn
• Michael Murray
• Phil Sharp
• Luk Van Parijs

• U Penn School of Medicine
• Jonathan Reiss
• Ron Collman

• Sun Yat Sen Memorial Hospital, Guangzhou
• Jie Wang
• Jun Min
• Jisheng Chen

• Guangzhou Medical Institute
• Nengtai Ouyang