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Summary
Lancet 2010; 375: 1896905
See Comment page 1850
*Contributed equally to the
report
National Emerging Infectious
Diseases Laboratories Institute
(Prof T W Geisbert PhD,
J B Geisbert), Department of
Microbiology
(Prof T W Geisbert), and
Department of Medicine
(Prof T W Geisbert), Boston
University School of Medicine,
Boston, MA, USA; Virology
Division, US Army Medical
Research Institute of Infectious
Diseases, Fort Detrick, MD, USA
(A N Honko PhD, J C Johnson BSc,
L E Hensley PhD); and Tekmira
Pharmaceuticals, Burnaby, BC,
Canada (A C H Lee MSc,
M Robbins PhD, V Sood MSc,
S de Jong PhD, I Tavakoli BSc,
A Judge PhD, I MacLachlan PhD)
Correspondence to:
Prof Thomas W Geisbert, Boston
University School of Medicine,
National Emerging Infectious
Diseases Laboratories Institute,
620 Albany Street, Room 401B,
Boston, MA 02118, USA
geisbert@bu.edu
1896
Background We previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV)
RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs
when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful
for screening prospective countermeasures, they are frequently not useful for prediction of ecacy in the more
stringent non-human primate models. We therefore assessed the ecacy of modied non-immunostimulatory
siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever.
Methods A combination of modied siRNAs targeting the ZEBOV L polymerase (EK-1 mod), viral protein (VP)
24 (VP24-1160 mod), and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given
these pooled anti-ZEBOV siRNAs (2 mg/kg per dose, bolus intravenous infusion) after 30 min, and on days 1, 3, and
5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after
30 min, and on days 1, 2, 3, 4, 5, and 6 after challenge with ZEBOV.
Findings Two (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs
were protected from lethal ZEBOV infection, whereas all macaques given seven postexposure treatments were
protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that
might have been related to viral infection.
Interpretation This complete postexposure protection against ZEBOV in non-human primates provides a model for
the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an
eective postexposure treatment strategy for people infected with Ebola virus, and suggest that this strategy might
also be useful for treatment of other emerging viral infections.
Funding Defense Threat Reduction Agency.
Introduction
For more than 30 years, Ebola virus (EBOV) has been
associated with periodic episodes of haemorrhagic fever
in Central Africa that produce severe disease in infected
patients. Mortality rates during outbreaks have ranged
from 50% with the Sudan species of EBOV (SEBOV) to
up to 90% with the Zaire species (ZEBOV).1 An outbreak
towards the end of 2007, caused by an apparently new
species of EBOV in Uganda, seemed to be less
pathogenic than SEBOV or ZEBOV with a case fatality
rate of about 25%.2
EBOV particles contain a non-infectious RNA genome
of roughly 19 kilobases that encodes seven structural
proteins and one non-structural protein. The gene order
is 3leader, nucleoprotein, virion protein (VP) 35 (VP35),
VP40, glycoprotein, VP30, VP24, polymerase L protein,
and 5 trailer.3 Four of these proteinsnucleoprotein,
VP30, VP35, and the polymerase L proteinare associated
with the viral genomic RNA in the ribonucleoprotein
complex. The L protein and VP35 make up the polymerase
complex, which transcribes and replicates the EBOV
genome. The L protein provides the RNA-dependent RNA
Articles
Quantitative RT-PCR
Methods
Design and in-vitro screening of siRNAs
We designed siRNAs to target regions of the ZEBOV
(Mayinga strain) polymerase L, VP24, and VP35 genes
using a conventional siRNA design algorithm. We
excluded siRNAs with sequence homology to any human
reference mRNAs of 16 or more contiguous bases within
the core duplex using the Basic Local Alignment Search
Tool (version 2.2.13). The siRNA duplexes were
synthesised by Dharmacon (Chicago, IL, USA) or
Integrated DNA Technologies BVBA (Leuven, Belgium).
EK-1 was previously described.11 siRNAs for targeting the
VP24 and VP35 genes were identied by screening in
vitro for reduction in expression of the ZEBOV VP24 or
ZEBOV VP35 viral transgene inserted in the psiCHECK2
dual-luciferase plasmid system (Promega, Madison, WI,
USA) under the control of the SV40 promoter in HepG2
cells. 10 L of Lipofectamine 2000 (Invitrogen, Life
Technologies, Carlsbad, CA, USA) complexes containing
075 g of plasmid and 10 L of siRNA (VP24-775, VP24978, VP24-1160, or a non-specic negative control siRNA
at 13 nmol/L, 25 nmol/L, 50 nmol/L, 100 nmol/L,
200 nmol/L, and 400 nmol/L; or VP35-219, VP35-349,
VP35-687, VP35-855, or a non-specic negative control
www.thelancet.com Vol 375 May 29, 2010
5 RACE assays
RNA extraction and 5 RACE were done as described11
except we used gene-specic primers (GSP) for cDNA
production: EK-1 GSP (5-TTTGTGATTCGTCCTTTT
1897
Articles
EK-1 mod
ZEBOV target
Sequence
L polymerase
VP24-775
VP24
VP24-978
VP24
VP24-1160
VP24
VP24-1160 mod
VP24
VP35-219
VP35
VP35-349
VP35
VP35-687
VP35
VP35-855
VP35
VP35-855 mod
VP35
Luc
NA
Luc mod
NA
Sequences used in the non-human primate studies contain an m in front of the base that designates a 2-O-methyl modication (unmodied versions do not have any
2-O-methyl modied bases). mod=modied. Luc mod=modied luciferase. NA=not applicable.
Table 1: Targets and target sequences in Zaire Ebola virus (ZEBOV) of small interfering RNAs
Mouse studies
Mouse studies were completed in accordance with the
Canadian Council on Animal Care guidelines following
approval by the local animal care and use committee at
Tekmira Pharmaceuticals. Animal research was done in
compliance with the animal welfare act, and other federal
statutes and regulations relating to animals and
experiments involving animals, and adheres to the
principles stated in the Guide for the Care and Use of
Laboratory Animals, National Research Council, 1996.
The facilities used are fully accredited by the Association
for Assessment and Accreditation of Laboratory Animal
Care International.
68-week-old female CD1 ICR mice (n=16; immunostimulation studies [ four mice per group]), and 68-weekold male (n=20) and female B6C3F1 mice (n=20; tolerability
www.thelancet.com Vol 375 May 29, 2010
Articles
A
120
100
80
60
40
20
0
1 3
25
13
25
1 3
B
120
100
80
60
40
20
0
08
40 200
VP35-219
08 40 200
VP35-349
08
40 200
VP35-687
08
40 200
VP35-855
08 40 200
Negative control
siRNA
0
Plasmid
only
siRNA (nmol/L)
Figure 1: Eect of small interfering RNAs (siRNAs) targeting virion protein genes VP24 (A) and VP35 (B) of Zaire Ebola virus (ZEBOV) expressed in a non-viral
plasmid-based system in HepG2 cells
Error bars represent SD of triplicate tissue culture wells.
Articles
1200
4000
C
50249
1000
3500
Inteferon (pg/mL)
Interleukin 6 (pg/mL)
3000
800
600
400
2500
2000
1500
1000
200
086
053
043
500
0
PBS
Luc
Luc
mod
ZEBOV
cocktail
PBS
Luc
Luc
mod
ZEBOV
cocktail
PBS
Luc
Luc
mod
ZEBOV
cocktail
D
500
450
400
Interferon (pg/mL)
350
300
250
200
150
100
50
0
PBS
100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400
VP24-1160
VP24-1160 mod
VP35-855
VP35-855 mod
Luc mod
EK-1
EK-1 mod
Luc
siRNA (nmol/L)
Figure 2: Eect of small interfering RNAs (siRNAs) on synthesis of interleukin 6 (A) and interferon (B), induction of interferon-induced protein with
tetratricopeptide repeats (IFIT1) mRNA in mice (C), and induction of interferon in human peripheral blood mononuclear cell cultures (D)
Data are mean (SD). Error bars represent SD. Luc=luciferase. PBS=phosphate-buered saline. Luc mod=modied luciferase. GAPDH=glyceraldehyde 3-phosphate
dehydrogenase.
Results
VP24-1160 was the most ecacious siRNA at silencing
ZEBOV VP24 gene in HepG2 cells, and VP35-855 was
most ecacious at silencing ZEBOV VP35 gene (gure 1).
These sequences, and EK-1,11 were selected as the siRNAs
of the ZEBOV SNALP cocktail. Since non-selective
chemical modication might attenuate the potency of the
siRNA, we selectively modied lead siRNAs by substituting
2-O-methyl guanosine or uridines in both strands to
eliminate the immune stimulatory capacity of the siRNA
duplex,1315 and conrmed the gene silencing activity of the
modied versions using the in-vitro plasmid-based
ZEBOV gene expression system (data not shown).
SNALPs containing modied ZEBOV siRNAs did not
induce interferon or interleukin 6 in the plasma of
mice, whereas SNALPs containing chemically unmodied
Luc siRNA induced high concentrations of both plasma
cytokines (gure 2A and B). Analysis of liver IFIT1
mRNA showed no dierences between SNALPs
containing PBS (negative control) and those containing
the 2-O-methyl-modied Luc or ZEBOV siRNA cocktail,
whereas the SNALPs containing unmodied Luc induced
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A
810
610
410
210
PBS
Luc mod
siRNA in SNALP (50 nmol/L)
B
Lane 1
10
11
12
ZEBOV
13 14 15 16
17
400 bp
300 bp
282 bp
200 bp
205 bp
100 bp
EK-1 RACE
VP24-1160 RACE
VP35-855 RACE
Figure 3: Rapid amplication of cDNA ends (RACE)-PCR of small interfering RNA (siRNA)-mediated cleavage
of Zaire Ebola virus (ZEBOV) L polymerase, virion protein (VP) 24 (VP24), and VP35 mRNAs in ZEBOVinfected Vero E6 cells
Vero E6 cells were treated with stable nucleic acid-lipid particles (SNALPs) containing ZEBOV siRNA cocktail, modied
luciferase (Luc mod), or phosphate-buered saline (PBS). (B) Vero E6 cells were treated with SNALPs containing
EK-1-mod, VP24-1160-mod, VP35-855-mod, ZEBOV siRNA cocktail, Luc mod, or PBS. The order of samples for each
RACE PCR shown in the gel is SNALPs containing PBS, single gene-specic siRNAs (EK-1-mod, VP24-1160-mod, or
VP35-855-mod), ZEBOV cocktail, and Luc mod. Lanes 1 and 17 are the 100 base pair (bp) ladder, lanes 25 are EK-1
RACE PCR, lanes 710 are VP24-1160 RACE, and lanes 1215 are VP35-855 RACE. Lanes 6, 11, and 16 are empty.
Articles
53%
39%
34%
0
CD34+
CD11b+
CD14+
CD11c+
Cell marker
Figure 4: Proportion of dierentiated cells with uptake of stable nucleic acid-lipid particles (SNALPs)
containing uorescein-isothiocyanate-labelled modied luciferase small interfering RNAs
Articles
Alanine aminotransferase
Aspartate aminotransferase
Sorbitol dehydrogenase
70
19
37 30
PBS
37
21
35 41 27
26
siRNAs
20 mg/kg
siRNAs
285 mg/kg
28
43
28
23
siRNAs
43 mg/kg
40
PBS
51
24
28
45
24
20
siRNAs
20 mg/kg
Male
43
22
37
siRNAs
285 mg/kg
siRNAs
43 mg/kg
siRNAs
285 mg/kg
siRNAs
43 mg/kg
Female
SNALPs
B
6
4
3
2
1
0
PBS
siRNAs
20 mg/kg
siRNAs
285 mg/kg
siRNAs
43 mg/kg
PBS
Male
siRNAs
20 mg/kg
Female
SNALPs
Figure 5: Eect of daily administration of stable nucleic acid-lipid particles (SNALPs) containing Zaire Ebola virus (ZEBOV) small interfering RNAs on activities
(siRNAs) of alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase (A), and blood cell counts (B) in mice
PBS=phosphate-buered saline.
Articles
Status
Animal 1
Anorexia (days 79), lymphopenia (day 6), >5-fold increase in AST (day 10)
Survived
Animal 2
Fever (day 6), mild rash (days 8,11, and 12), moderate rash (days 9 and 10), depression (days 711), anorexia (days 7-11), diarrhoea (day 12),
lymphopenia (days 6 and 14), thrombocytopenia (day 6), 23-fold increase in ALP (day 10), 23-fold increase in ALT (day 10), 23-fold increase in AST
(day 6), >5-fold increase in AST (day 10), >5-fold increase in GGT (day 10)
Survived
Animal 3
Mild rash (days 610), depression (days 6-10), anorexia (days 610), bleeding at venepuncture site (day 10), recumbency (day 10), thrombocytopenia Died on day 10
(day 6), 23-fold increase in ALP (day 6), >5-fold increase in ALP (day 10), 45-fold increase in ALT (day 10), 23-fold increase in AST (day 3), >5-fold
increase in AST (days 6 and 10), >5-fold increase in BUN (day 10), >5-fold increase in creatinine (day 10), >5-fold increase in GGT (day 10)
Control 1*
Study 1
Died day 6
Study 2
Animal 4
Survived
Animal 5
>5-fold increase in AST (day 6), 23-fold increase in AST (day 10)
Survived
Animal 6
Fever (day 10), lymphopenia (day 6), thrombocytopenia (days 6, 10, and 14), 23-fold increase in AST (day 10)
Survived
Animal 7
Fever (day 10), lymphopenia (day 6), thrombocytopenia (day 6), >5-fold increase in AST (day 10)
Survived
Control 2
Fever (day 6), moderate rash (day 10), recumbency (day 10), thrombocytopenia (day 6) 23-fold increase in ALT (day 6), >5-fold increase in ALT
(day 10), >5-fold increase in AST (days 6 and 10), >5-fold increase in BUN (day 10), 23-fold increase in creatinine (day 10)
Died on day 10
Fever was dened as a temperature that was 25F greater than baseline temperature or at least 15F greater than baseline temperature, and at least 1035F. Mild rash was dened as focal petechiae covering less
than 10% of skin. Moderate rash was dened as petechiae covering between 10% and 40% of skin. Severe rash was dened as petechiae or ecchymoses covering more than 40% of skin. Lymphopenia was dened
as at least a 35% reduction in numbers of lymphocytes; and thrombocytopenia was dened as at least a 35% reduction in numbers of platelets. AST=aspartate aminotransferase. ALP=alkaline phosphatase.
ALT=alanine aminotransferase. GGT= glutamyltransferase. BUN=blood urea nitrogen. *Not given any siRNAs. Given seven treatments with non-specic siRNAs.
Table 2: Macaques given four (study 1) or seven treatments (study 2) with stable nucleic acid-lipid particles containing Zaire Ebola virus (ZEBOV) small interfering RNAs (siRNAs) after
ZEBOV challenge
Discussion
Seven treatments with anti-ZEBOV siRNAs inhibited the
replication of the virus and completely protected rhesus
monkeys against death from haemorrhagic fever. In
previous studies, ZEBOV-infected rhesus monkeys
succumbed when viraemia on days 610 after exposure
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100
Day 3
Survival (%)
80
60
40
Study 1
Study 2
Control
0
0
10
15
20
Days after challenge
25
30
1904
Day 10
Day 14
Study 1
20
Day 6
Animal 1
Animal 2
22
Animal 3
37
68
NA
Control 1*
NA
NA
NA
0
Study 2
Animal 4
20
Animal 5
24
Animal 6
20
21
Animal 7
21
Control 2
41
67
NA
NA=not applicable. *Not given any siRNAs. Given seven treatments with nonspecic siRNAs.
Table 3: Plasma viral load in rhesus monkeys infected with Zaire Ebola
virus (ZEBOV) and given four (study 1) and seven (study 2)
postexposure treatments with anti-ZEBOV small interfering RNAs
(siRNAs)
Articles
Contributors
TWG, ACHL, MR, AJ, LEH, and IM conceived and designed the
experiments. TWG wrote the research programme plan and the animal
protocol for the studies. LEH wrote modications for the animal
protocol. TWG and LEH selected the ZEBOV L polymerase as a target.
TWG, ACHL, VS, MR, AJ, LEH, and IM selected ZEBOV VP24 and
VP35 as targets. TWG co-designed the L polymerase siRNA, and ACHL
and VS co-designed the VP24 and VP35 siRNAs. ACHL, VS, and AJ
designed the modications to the siRNAs. VS and ANH did the in vitro
siRNA assays. JBG and JCJ did the Ebola challenge experiments in nonhuman primates and the clinical pathology assays. JBG did the Ebola
virus infectivity assays. SDJ did the immunology and uptake assays. MR
and IT did the 5RACE assays. TWG, ACHL, MR, JBG, ANH, JCJ, AJ,
LEH, and IM analysed the data. TWG and MR co-wrote the report. LEH
and IM edited the report. All authors have seen and approved the nal
version of the report.
Conicts of interest
TWG, ACHL, MR, VS, AJ, LEH, and IM claim intellectual property
regarding RNA interference for the treatment of loviral infections. ACHL,
MR, VS, SDJ, IT, AJ, and IM are employees of Tekmira Pharmaceuticals.
JCJ, ANH, and JBG declare that they have no conicts of interest.
Acknowledgments
Work on loviruses at US Army Medical Research Institute of Infectious
Diseases was funded by the Defense Threat Reduction Agency (project
number 04-4-7J-012). Opinions, interpretations, conclusions, and
recommendations are those of the authors and are not necessarily
endorsed by Boston University or the US Army.
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