Preclinical study of a combinatorial RNAi/vaccination therapy
as a potential functional cure for chronic hepatitis B
Thomas Michler1,2, Anna Kosinska1, Till Bunse1,2, Mathias Heikenwalder1,3, Dirk Grimm3, Stuart Milstein4, Laura Sepp-Lorenzino4, and Ulrike Protzer1,2
1 Institute
of Virology, Technical University of Munich /Helmholtz Zentrum München, Munich, Germany
2 German Center for Infection Research (DZIF), Munich partner site
3 Department of Infectious Diseases/Virology, Heidelberg University, Heidelberg, Germany
4 Alnylam Pharmaceuticals, 300 Third Street, Cambridge MA 02142
B Effect of pretreatment on HBV parameters A METHODS
transcripts
HBV genome (3.2kb)
The heterologous prime-­‐boost vaccina%on induced B-­‐cell immunity and an%-­‐HBs-­‐seroconversion in all animals but there was no effect of HBV an%gen suppression on B cell responses. No an%-­‐HBe an%bodies could be detected (not shown). Monthly subcutaneous injec%ons of 3 mg/kg GalNAc-­‐
siRNAs as well as i.v. injected 1x1011 par%cles AAV-­‐
shHBV efficiently suppressed viral transcripts in liver (A) and HBV an%gens and DNA in serum (B). HBsAg and HBV DNA were reduced by 2 log10 and HBeAg by 1 log10. ETV strongly reduced HBV DNA by 4 log10 but an%gen levels remained unchanged. siRNA and subsequent therapeu%c vaccina%on showed an addi%ve effect on HBsAg and HBV-­‐DNA levels cumula%ng in >4 log10 reduc%ons compared to pretreatment levels. D Effect of pretreatment on T cell responses A HBV-­‐specific CD8 T cell responses were only seen in animals with lower an%gen %ters aKer siRNA or shRNA pretreatment. MVA-­‐specific CD8 T cell responses were not influenced by HBV an%gen levels. siRNA/shRNA target region
Conjuga%on of siRNAs to N-­‐Acetylgalactosamine (GalNAc) allows hepatocyte-­‐specific delivery (B) 5′-sense
Asialoglycoprotein Receptor (ASGPR)
Highly expressed in hepatocytes High rate of uptake Recycling %me ~15 minutes Conserved across species Clathrin-coated pit
Target protein
Recycling
ASGPR
mRNA
cleavage
Adapted from Essentials of Glycobiology (2009)
RISC loading
Endosome
HBeAg (PEI U/ml)
C HBsAg (IU/ml)
10000
400
1000
300
100
20010
HBVtg mice 100 1
(HBVxfs) 0.1
0
0.01
Immunisation
Vaccina%on P P M Limit of detection
00 22 44 6
1012
1214141616
17 17
6 88 10
Week HBsAg (IU/ml)
High viremic HBV-­‐transgenic mice were pretreated with nucleoside analogue Entecavir, an shRNA-­‐expressing Adeno-­‐Associated Virus vector (AAV-­‐shHBV7/
TuDHBV7) (Ref. 2) or GalNAc-­‐conjugated siRNAs before 10000
applying a HBV core (HBc) and surface (HBs) protein 1000
100
prime vaccina%on and a Modified Vaccinia Ankara virus Immunisation
10
(MVA)-­‐boost immuniza%on (Ref. 3) (C). 500
1
DURATION OF ANTIGEN SUPPRESSION
B Experimental setup Effect on T cell responses We now compared different dura%ons of an%gen suppression to determine the op%mal %me point of vaccina%on. siRNA therapy was started 3, 6, or 8 weeks prior to start of therapeu%c vaccina%on (A). The dura%on of siRNA pretreatment posi%vely correlated with the intrahepa%c HBV-­‐specific CD8 T cell responses aKer therapeu%c vaccina%on (B). A Immunisation
1
0.1
10
1
0.1
0.01
100
10
1
We developed a combinatorial RNAi/vaccina%on therapy for hepa%%s B that, aKer finite treatment, allows recons%tu%on of HBV-­‐specific T cell responses and suppression of HBsAg to undetectable levels in a preclinical mouse model of chronic hepa%%s B, sugges%ng substan%al poten%al for clinical transla%on. Effect on HBV parameters Vaccina%on P P M Control siRNA 8 weeks
HBV siRNA-1 3 weeks
HBV siRNA-1 6 weeks
HBV siRNA-1 8 weeks
HBVtg mice Week before/aKer start of vaccina%on P = HBsAg+HBcAg+PCEP+CPG Limit of detection
0.1 (HBVxfs) M = MVA-­‐HBsAg+MVA-­‐HBcAg MOCK
P = HBsAg+HBcAg+c-­‐di-­‐AMP Entecavir 1 µg/ml drinking water 0.01
Untreated
-8
-6
-2 0 2 4 6
M -4
= MVA-­‐HBsAg+MVA-­‐HBcAg AAV-shHBV7/TuDHBV7
1x10e11 i.v.
Entecavir
1 µg/ml drinking water
Weeks before/after start of vaccination
siTTR AD-57727.93 3mg/kg
AAV-shHBV7/TuDHBV7
1x1011 i.v.
siRNA dosing: 3 µg/kg s.c. Control
3mg/kg s.c.
siHBVsiRNA
AD-66816.7
3mg/kg
HBV
siRNA-1
3mg/kg s.c.3mg/kg
siHBV
AD-66810.16
HBV siRNA-2 3mg/kg s.c.
10
100
MVA(B8R)-specific response in liver
CONCLUSIONS
REFERENCES C Conform with stronger HBV-­‐
specific CD8 T cell responses, there was an increased suppression of HBsAg with longer siRNA-­‐pretreatment before vaccina%on. The best treatment scheme resulted in a >5 log10 reduc%on of HBsAg to undetectable levels in all 10000
treated animals. 1000
100
1. Michler&Kosinska et al. EASL 2016 (Abstract#ILC2016-­‐RS-­‐1449) 2. Michler et. al. EMBO Molecular Medicine. 2016 3. Backes&Jäger et al. Vaccine. 2016 Immunisation
Immunisation
10000
1000
100
10
sAg (IU/ml)
ASGPR
IU/ml)
B 100
HBc(C93)-specific response in liver
En
AA tec
V- avi
r1
sh
H
BV µg/
7/ ml Un
t
d
T
C uD rin rea
on
te
ki
H
n
d
tro BV
g
H l si 7 1 wa
BV R
te
N x1
A
0 11 r
si
R
3
H
BV NA mg i.v
/k .
si -1
g
R
3
s.
m
N
Ag/ c.
kg
2
3m
s.
g/ c.
kg
s.
En
c.
AA tec
V- avi
r1
sh
H
BV µg/
7/ ml Un
tre
dr
Tu
C
i
on DH nki ate
d
tro BV ng
H l si 7 1 wa
BV R
te
N x1
A
0 11 r
si
R
3
H
BV NA mg i.v
/k .
si -1
g
R
3
s.
m
N
Ag/ c.
kg
2
3m
s.
g/ c.
kg
s.
c.
En
AA tec
V- avi
r1
sh
H
BV µg/
7/ ml Un
t
d
T
C uD rin rea
on
te
ki
H
n
d
tro BV
g
H l si 7 1 wa
BV R
te
N x1
A
0 11 r
si
R
3
H
BV NA mg i.v
/k .
si -1
g
R
3
s.
m
N
Ag/ c.
kg
2
3m
s.
g/ c.
kg
s.
c.
The HBV genome is highly condensed and all transcripts have an iden%cal 3´end which allows simultaneous suppression of all HBV transcripts and proteins with one siRNA/shRNA (A). HBs(S208)-specific response in liver
IFNy+ / CD8+ (%)
Hepa%%s B cure is highly desired, but rarely achieved with current therapies. Hepa%%s B Virus (HBV) persistence was found to correlate with a failure to develop an efficient virus-­‐specific T cell response due to high HBV an%gen load (Ref. 1). RNAi is a promising approach to control HBV replica%on and lower an%gen load. We evaluated the capacity of stabilized, liver-­‐targeted siRNAs to (i) suppress HBV gene expression, (ii) allow recovery of HBV-­‐specific B-­‐ and T cell responses. C IFNy+ / CD8+ (%)
RESULTS
IFNy+ / CD8+ (%)
BACKGROUND and AIM
Effect of pretreatment on B cell responses Control siRNA 8 weeks
HBV siRNA-1 3 weeks
HBV siRNA-1 6 weeks
Control siRNA 8 weeks
HBV siRNA-1 3 weeks
HBV siRNA-1 6 weeks
HBV siRNA-1 8 weeks
CONTACT INFORMATION
[email protected]