Note: Descriptions are shown in the official language in which they were submitted.
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MODULATING IMMUNE RESPONSES
GOVERNMENT RIGHTS
[0001] The
invention described herein was made with U.S. government support under
grant number R01A1079336 awarded by the National Institutes of Health. The
U.S.
government has certain rights in the invention.
FIELD OF THE INVENTION
[0002]
Embodiments of the invention relate to compositions and methods for modulating
innate and adaptive immunity in a subject and/or for the treatment of an
immune-related
disorder, cancer, autoimmunity, treating and preventing infections.
BACKGROUND
[0003] Cellular
host defense responses to pathogen invasion principally involves the
detection of pathogen associated molecular patterns (PAMPs) such as viral
nucleic acid or
bacterial cell wall components including lipopolysaccharide or flagellar
proteins that results
in the induction of anti-pathogen genes. For example, viral RNA can be
detected by
membrane bound Toll-like receptors (TLR's) present in the endoplasmic
reticulum (ER)
and/or endosomes (e.g. TLR 3 and 7/8) or by TLR-independent intracellular
DExD/H box
RNA helicases referred to as retinoic acid inducible gene 1 (RIG-I) or
melanoma
differentiation associated antigen 5 (MDA5, also referred to as IFIH1 and
helicard). These
events culminate in the activation of downstream signaling events, much of
which remains
unknown, leading to the transcription of NF-KB and IRF3/7- dependent genes,
including type
I IFN.
SUMMARY
[0004] STING
(Stimulator of Interferon Genes), a molecule that plays a key role in the
innate immune response, includes 5 putative transmembrane (TM) regions,
predominantly
resides in the endoplasmic reticulum (ER), and is able to activate both NF-KB
and IRF3
transcription pathways to induce type I IFN and to exert a potent anti-viral
state following
expression. See U.S. patent application serial no. 13/057,662 and
PCT/U52009/052767.
Loss of STING reduced the ability of polyIC to activate type I IFN and
rendered murine
embryonic fibroblasts lacking STING (-/- MEFs) generated by targeted
homologous
recombination, susceptible to vesicular stomatitis virus (VSV) infection. In
the absence of
STING, DNA-mediated type I IFN responses were inhibited, indicating that STING
may play
an important role in recognizing DNA from viruses, bacteria, and other
pathogens which can
infect cells. Yeast-two hybrid and co-immunoprecipitation studies indicated
that STING
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interacts with RIG-I and with Ssr2/TRAP3, a member of the translocon-
associated protein
(TRAP) complex required for protein translocation across the ER membrane
following
translation. RNAi ablation of TRAPP inhibited STING function and impeded the
production
of type I IFN in response to polyIC.
[0005] Further
experiments showed that STING itself binds nucleic acids including
single- and double-stranded DNA such as from pathogens and apoptotic DNA, and
plays a
central role in regulating proinflammatory gene expression in inflammatory
conditions such
as DNA-mediated arthritis and cancer. Various new methods of, and compositions
for,
upregulating STING expression or function are described herein along with
further
characterization of other cellular molecule which interact with STING. These
discoveries
allow for the design of new adjuvants, vaccines and therapies to regulate the
immune system
and other systems.
[0006]
Described herein are methods for modulating an immune response in a subject
having a disease or disorder associated with aberrant STING function. These
methods can
include the step of administering to the subject an amount of a pharmaceutical
composition
including an agent which modulates STING function and a pharmaceutically
acceptable
carrier, wherein amount the pharmaceutical composition is effective to
ameliorate the
aberrant STING function in the subject. The agent can be a small molecule that
increases or
decreases STING function, or a nucleic acid molecule that binds to STING under
intracellular
conditions. The STING-binding nucleic acid molecule can be a single-stranded
DNA
between 40 and 150 base pairs in length or a double-stranded DNA between 40
and 150, 60
and 120, 80 and 100, or 85 and 95 base pairs in length or longer. The STING-
binding nucleic
acid molecule can be nuclease-resistant, e.g., made up of nuclease-resistant
nucleotides. It
can also be associated with a molecule that facilitates transmembrane
transport. In these
methods, the disease or disorder can be a DNA-dependent inflammatory disease.
[0007] Also
described herein are methods of treating cancer in a subject having a
cancerous tumor infiltrated with inflammatory immune cells. These methods can
include the
step of administering to the subject an amount of a pharmaceutical composition
including an
agent which downregulates STING function or expression and a pharmaceutically
acceptable
carrier, wherein amount the pharmaceutical composition is effective to reduce
the number of
inflammatory immune cells infiltrating the cancerous tumor by at least 50%
(e.g., at least 50,
60, 70, 80, or 90%, or until reduction of inflammatory cell infilitration is
detectably reduced
by histology or scanning).
DESCRIPTION OF THE DRAWINGS
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[0008] Figs. 1A-
G show STING dependent innate immune signaling. Fig. 1A: Human
Telomerase Fibroblasts (hTERT-BJ1) were transfected with various nucleotides
(3 ug/m1) for
16h. Endogenous IFN[3 levels were measured. hTERT-BJ1 cells were transfected
with FITC
conjugated dsDNA90 was examined by fluorescent microscopy to ensure efficient
transfection. Fig. 1B: hTERT-BJ1 cells were transfected with mock, random or
two
independent human STING siRNAs (siRNA 3 or 4) for 3 days followed by dsDNA90
transfection (3 ug/m1) for 16 hours. Endogenous IFN[3 levels were measured.
Silencing of
hSTING protein was demonstrated by immunoblotting, with [3-actin serving as a
control. Fig.
36C: Primary STING, STING-/-, STAT1 / or STAT1-/- MEFs were transfected with
or
without dsDNA90 (3 ug/m1) for 3 hours. Total RNA was purified and examined for
gene
expression by Illumina Sentrix BeadChip Array (Mouse WG6 version 2). Fig. 1D:
hTERT-
BJ1 cells were treated NS or STING siRNA. After 3days, cells were treated with
dsDNA90,
ssDNA90 or ssDNA45 (3 ug/m1). IFN[3 mRNA levels were measured by real time RT-
PCR
after 16 hours. Fig. 1E: hTERT-BJ1 cells were treated NS or STING siRNA. At
3days, cells
were treated with dsDNA90, ssDNA90 or ssDNA45 (3 ug/m1). Endogenous IFN[3
levels
were measured after 16 hours. Fig. 1F: Primary STING or or STING-/- MEFs were
transfected with or without dsDNA90 (3 ug/m1). After 3h, the same as Fig. 1C.
Fig. 1G:
hTERT-BJ1 cells were treated with or without dsDNA90 (3 ug/m1) for 3 hours and
stained
with anti-HA antibody and calreticulin as an ER marker.
[0009] Figs. 2A-
J show that STING binds to DNA. Fig. 2A: 293T cells were transfected
with indicated plasmids. Cell lysates were precipitated with biotin-dsDNA90
agarose and
analyzed by immunoblotting using anti-HA antibody. Fig. 2B: Schematic of STING
mutants.
Fig. 2C: Same as Fig. 2A. Fig. 2D: Same as Fig. 2A. STING variants unable to
bind DNA
are labeled in red. Fig. 2E: hTERT-BJ1 cells were transfected biotin
conjugated dsDNA90
(B-d5DNA90; 3 ug/m1) for 6h and treated with DSS. Lysates were precipitated
using
streptavidin agarose and analyzed by immunoblotting using anti-HA antibody.
Fig. 2F:
STING was expressed in 293T cells and after 36 hours lysates were incubated
with dsDNA90
agarose in the presence of competitor dsDNA90, Poly(I:C), B-DNA or ssDNA90 and
analyzed by immunoblotting using anti-HA antibody. Fig. 2G: 293T cells were
transfected
with HA-tagged STING, GFP or TREX1. Cells were lysed and biotin labeled ssDNA
or
dsDNA added with streptavidin agarose beads.
Precipitates were analyzed by
immunoblotting using anti-HA antibody. Fig. 2H: 293T cells were transfected
with IFN[3-
Luciferase and STING variants and luciferase activity measured. Fig. 21: hTERT-
BJ1 cells
were transfected with dsDNA90 and crosslinked with formaldehyde. STING was
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precipitated and bound DNA detected by PCR using dsDNA90 specific primers. NC:
negative control. PC: positive control, dsDNA90. Fig. 2J: STING or or STING4-
MEFs were
transfected with dsDNA90 and then same as Fig. 21. Error bars indicates s.d.
Data are
representative of at least two independent experiments.
[0010] Figs. 3A-
3H show that TREX1 is negative regulator of STING signaling. Fig.
3A: Immunoblot confirming STING and/or TREX1 knockdown in siRNA treated hTERT-
BJ1 cells. Fig. 3B: siRNA treated hTERT-BJ1 cells were transfected with
dsDNA90 (3
Out). Endogenous IFNP levels were measured after 16 hours. *P<0.05. Fig. 3C:
siRNA
treated hTERT-BJ1 cells were infected with HSV-1 (m.o.i=1) and virus titers
measured.
*P<0.05. Fig. 38D: siRNA treated hTERT-BJ1 cells were infected with 734.5
deleted-HSV
and virus titers measured. *P<0.05. Fig. 3E: Immunoblot of NS or STING siRNA
treated
TREX1' or TREX1' - MEFs, confirming STING knockdown. Fig. 3F: siRNA treated
TREX1' or TREX14- MEFs were treated with dsDNA90 and IFNP levels were
measured
after 16 hours. *P<0.05. Fig. 3G: siRNA treated TREX1' or TREX14- MEFs were
infected
HSV-1 (m.o.i=1) and virus titers measured. *P<0.05. Fig. 3H:
Immunofluorescence analysis
using anti-TREX1 or anti-STING antibody of hTERT-BJ1 cells transfected with or
without
dsDNA90. *P<0.05, Student's t-test. Error bars indicated s.d. Data are
representative of at
least two independent experiments.
[0011] Figs. 4A-
J: show TREX1 associayes with oligosaccharyltransferase complex. Fig.
4A shows a schematic of TREX1. Red indicates RPN1 binding site. Fig. 4B shows
a
schematic of STING. Red indicates DAD1 binding site. Fig. 4C: RPN1 interacts
with
TREX1 in yeast two hybrid analysis (1.pGBKT7, 2.pGBKT7-NFAR M9, 3.pGBKT7-
TREX1, 4.pGBKT7-STING full length, 5.pGBKT7-STING C-terminal). Fig. 4D: 293T
cells
were co-transfected with TREX1-tGFP and RPN1-Myc. Lysates were
immunoprecipitated
using anti-Myc antibody and analyzed by immunoblotting. Fig. 4H: hTERT-BJ1
cells were
treated with or without dsDNA90 (3 it' g/ml). At 6h after transfection, cells
were examined by
immunofluorescence using anti-STING or anti-DAD1 antibody. Fig. 41: Immunoblot
analysis of microsome fractions after sucrose gradient centrifugation using
indicated
antibodies. I: input. Fig. 4J: hTERT-BJ1 cells were treated with TREX1,
Sec61A1, TRAPP,
NS or STING siRNA. After 72h, cells were treated with dsDNA90 (3 it' g/ml) for
16h and
then endogenous IFNP levels were measured. *P<0.05, Student's t-test. Error
bars indicated
s.d. Data are representative of at least two independent experiments.
[0012] Figs. 5A-
G show that cytoplasmic DNA induces STING-dependent genes in
hTERT-BJ1 cells. Fig. 5A: hTERT-BJ1 cells were treated with STING siRNA. After
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days, cells were treated with or without dsDNA90 for 3h. Total RNA was
purified and
examined for gene expression using Human HT-12_V4_Bead Chip. Figs. 5B-G: hTERT-
BJ
1 cells were treated as in Fig. 5A. Total RNAs were examined by real time PCR
for IFN[3
(Fig. 5B), PMAIP1 (Fig. 5C), IFIT1 (Fig. 5D), IFIT2 (Fig. 5E), IFIT3 (Fig. 5F)
and PTGER4
(Fig. 5G). Real time PCR was performed using TaqMan gene Expression Assay
(Applied
Biosystem). *P<0.05, Student s t-test. Error Bars indicated s.d. Data are
representative of at
least two independent experiments.
[0013] Figs. 6A-
H show that STING-dependent genes are induced by cytoplasmic DNA
in MEFs. Primary STAT1 / or STAT14- MEFs were treated dsDNA90, IFNa or
dsDNA90
with IFNa. Total RNAs were purified and examined by real time PCR for IFN[3
(Fig. 6A),
IFIT1 (Fig. 6B), IFIT2 (Fig. 6C), IFIT3 (Fig. 6D), CXCL 10 (Fig. 6E), GBP1
(Fig. 6F),
RSAD2 (Fig. 6G) and CCL5 (Fig. 6H). *P<0.05, Student' s t-test. Error Bars
indicated s.d.
Data are representative of at least two independent experiments.
[0014] Figs. 7A-
H show that cytoplasmic DNAs induce STING-dependent genes in
MEFs. In Figs. 7A-H, STING or or STING-/- MEFs were treated with or without
dsDNA45,
dsDNA90, ssDNA45 or ssDNA90 for 3h. Total RNAs were purified and examined by
real
time PCR for IFN[3 (Fig. 7A), IFIT1 (Fig. 7B), IFIT2 (Fig. 7C), IFIT3 (Fig.
7D), CCL5 (Fig.
7E), CXCL 10 (Fig. 7F), RSAD2 (Fig. 7G) or GBP1 (Fig. 7H). *P<0.05, Student' s
t-test.
Error Bars indicated s.d. Data are representative of at least two independent
experiments.
[0015] Figs. 8A-
D show STING localization and dimerization. Fig. 8A: MEFs stably
expressing STING-HA were treated with ssDNA45, dsDNA45, ssDNA90 or dsDNA90.
After 3h, cells were stained using anti-HA or anti-calreticulin antibody. Fig.
8B: 293T cells
were transfected with STING-HA and Myc-STING. Lysates were precipitated by
anti-Myc
antibody and analyzed by immunoblotting using anti-HA antibody. Fig. 8C. hTERT-
BJ 1
cells were treated with or without the cross linker DSS. Cell lysates were
subjected to
immunoblot using anti-STING antibody. Fig. 8D: 293T cells were transfected
with indicated
plasmids and treated with DSS. Cell lysates were analyzed by immunoblotting
using anti-HA
antibody.
[0016] Figs. 9A-
I show that DNA virus induces STING-dependent genes in MEFs. Fig.
9A: MEFs were infected with 734.5 deleted-HSV (m.o.i.= 1) for 3h. Total RNA
was purified
and examined for gene expression using Illumina Sentric Bead Chip array (Mouse
WGS
yersion2). Figs. 9B-I: STING, STING-/- or STAT-i7STING / MEFs were treated
with or
without dsDNA90, HSV or 734.5 deleted-HSV for 3h. Total RNAs were purified and
examined by real time PCR for IFN[3 (Fig. 9B), IFIT1 (Fig. 9C), IFIT2 (Fig.
9D), IFIT3 (Fig.
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9E), CCL5 (Fig. 9F), CXCL 10 (Fig. 9G), RSAD2 (Fig. 9H) or GBP1 (Fig. 91).
Error Bars
indicate s.d.
[0017] Figs.
10A-F show that STING interacts with IRF3/7 and NFic-B. Fig. 10A: IRF7
binding sites in the promoter regions of STING dependent dsDNA90 stimulatory
genes.
Fig.s 10B-D: Nudear extract was isolated from mock treated or dsDNA90 treated
STING /
or STING-/- MEFs and were examined for IRF3 (Fig. 10B), IRF7 (Fig. 10C) and NF-
KB (Fig.
10D) activation following the manufacturer's instruction. Nuclear extract kit,
Trans AM
IRF3, TransAM IRF7 and Tran5AMNFO3 family Elisa kits were from Active Motif.
*P<0.05, Student s t-test. Error Bars indicated s.d. Data are representative
of at least two
independent experiments. Fig. 10E: STING or or
STING-/- MEFs were treated with
poly(I:C). dsDNA90 or HSV1 and cells were stained by anti-IRF3 antibody. Fig.
10F.
STING or or STING-/- MEFs were treated dsDNA90 or HSV1 and cells were stained
by anti-
p65 antibody. Loss of STING did not affect poly(I:C) mediated innate immune
signaling.
[0018] Figs.
11A-F show that STING binds DNA in vitro. Fig. 11A: In vitro translation
products were precipitated with biotin conjugated dsDNA90 and immunoblotted by
anti-HA
antibody. Fig. 11B: schematic of STING variants. Figs. 11C, 11D: Same as Fig.
11A.
STING variants lacking aa 242-341 (red) failed to bind DNA. Figs. 11E-F: In
vitro
translation products were precipitated with biotin conjugated ssDNA90 and
immunoblotted
by anti-HA antibody.
[0019] Figs.
12A-G show that STING binds DNA in vivo and in vitro. hTERT BJ1 cells
were transfected with biotin-dsDNA90 and crosslinked by UV. Cells were lysed
and
precipitated by streptavidin agarose and analyzed by immunoblotting. Figs. 12B-
C hTERT-
BJ1 cells were treated with IF116 (Fig. 12B) or STING (Fig. 12C) siRNA and
then same as in
Fig. 12A. Fig. 12D: STING or or STING4- MEFs were treated as in Fig. 12A. Fig.
12E:
STING-Flag expressing 293T cells were treated with or without biotin-dsDNA90
and
crosslinked by DSS. Lysates were precipitated and analyzed by immunoblotting.
Fig. 12F:
293T cells were transfected with dsDNA90 or ssDNA90 and crosslinked by UV or
DSS and
then precipitated and analyzed by immunoblotting. Fig. 12G: STING-Flag
expression 293T
cells were lysed and incubated with dsONA90 or Poly(I:C) and biotin-dsDNA90
agarose and
then same as Fig. 12E.
[0020] Figs.
13A-C show that STING binds viral DNA. Fig. 13A: Oligonucleotide
sequences of HSV, cytomegalovirus (CMV) or adenovirus (ADV). Figs. 13B-C: 293T
cells
were transfected with indicated plasmids. Cell-lysates were precipitated with
biotin-
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dsDNA90, biotin-HSV DNA 120 mer, biotin-ADV DNA 120 mer or biotin-CMV DNA 120
mer agarose and analyzed by immunoblotting using anti-HA antibody.
[0021] Figs.
14A-C show that STING binds DNA. Fig. 14A: Schematic of STING
ELISA. Fig. 14B: Process of an embodiment of a STING ELISA. Fig. 14C: Binding
capacity of dsDNA90 was measured by ELISA. *P<0.05, Student s t-test. Error
Bars
indicated s.d. Data are representative of at least two independent
experiments.
[0022] Figs.
15A-D show that TREX1 is a negative regulator of STING signalling. Fig.
15A: hTERT-BJ1 cells were treated with TREX1, STING or STING and TREX1 siRNA.
After 3 days, cells were infected with HSV-Luc ( m.o.i =1) for 48h. Lysates
were measured
luciferase activity. Fig. 15B: Primary TREX1' or TREX-/- MEFs were
transfected with NS
or STING siRNA. After 3 days, cells were infected with HSV-Luc (m.o.i. = 1)
for 48h.
Lysates were measured. Fig. 15C: Primary TREX1' or TREX14-MEFs were
transfected
with wild type TREX1. After 48h, cells were infected with HSV and measured HSV
replication. TREX1 expression was checked by immunoblotting. *P<0.05, Student'
s t-test.
Error Bars indicated s.d. Data are representative of at least two independent
experiments.
Fig. 15D: STING or or STING4- MEFs were treated with or without dsDNA90 for
3h. Total
RNAs were purified and examined for gene expression by Illumina Sentrix Bead
Chip array
(Mouse WG6 version2).
[0023] Fig. 16
shows that STING regulates ssDNA90-mediated IFN[3 production in
TREX-/- MEFs. siRNA treated TREX1 or TREX14- MEFs were treated with ssDNA45 or
ssDNA90 and IFN[3 levels were measured after 16 hours. *P<0.05, Student' s t-
test. Error
Bars indicated s.d. Data are representative of atleast two independent
experiments.
[0024] Figs.
17A-H show that TREX1 is not a negative regulator of STING-dependent
genes. Fig. 17A: TREX1' or TREX14- MEFs were treated with HSV1, IFNa,
dsDNA90,
triphosphate RNA (TPRNA) and VSV. TPRNA and VSV weakly activated IFN induced
genes. Total RNAs were purified and examined for gene expression by Illumina
Sentrix
Bead Chip array (Mouse WG6 version2). Figs. 17B-H: Total RNAs were examined by
RT-
PCR for IFN[3 (Fig. 17B), IFIT1 (Fig. 17C), IFIT2 (Fig. 17D), IFIT3 (Fig.
17E), CCL5 (Fig.
17F), CXCL 10 (Fig. 17G), RSAD2 (Fig. 17H). No significant difference in IFN
induced
genes was observed in TREX1 lacking cells. *P<0.05, Student' s t-test. Error
bars indicated
s.d. Data are representative of at least two independent experiments.
[0025] Figs.
18A-D show that TREX1 assoicates with oligosaccharyltransferase complex.
An IFN-treated hTERT cDNA library was used to develop a yeast two hybrid
library
(AH109). Full length TREX 1 was used as bait to screen the library.
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million cDNA expressing yeast were screened (Clontech). 44 clones were
isolated from 3
independent yeast mating procedures. RPN1 was isolated 8 times in total (three
times in
screen 1, twice in screen 2 and three times in screen 3). The majority of the
clones, aside
from RPN1, failed to interact with IREX1 after re-testing. Of these eight RPN1
isolated
clones, four clones encoded aa 258-397, two clones aa 220-390 and two clones
aa 240-367.
TREX1 variants were generated and the interation between TREX1 (aa.241-369)
and RPN 1
(aa 256-397) was mapped. To isolate DAD1, the C-terminal region of STING (aa
173-379)
was used to screen the same library. 24 isolated clones, full length DAD I was
found twice.
The majority of the clones, aside from DAD1, failed to interact with STING
after re-
comfirmation studies. Full length DAD 1 was seen to associate with region 242-
310 of
STING. Fig. 18A: Schematic of TREX1 mutants. Fig. 18B: GAL4 binding domain
fused to
TREX1 or TREX1-4 interact with RPN1 fused to the GAL4 activation domain in
yeast two
hybrid screening. Fig. 18C: Schematic of STING mutants. Fig. 18D. GAL4 binding
domain
fused to STING-C-terminal or STING-C2 interact with DAD1 fused to the GAL4
activation
domain in yeast two hybrid screening.
[0026] Figs. 19A-C show that TREX1 and STING associate with
oligosaccharyltransferase complex. Fig. 19A: 293T cells were co-transfected
with TREX1-
tGFP and RPN1-Myc. Lysates were immunoprecipitated with anti-tGFP antibody or
IgG
control and analyzed by immunoblotting using indicated antibodies. Fig. 19B:
293T cells
were co-transfected with Myc-STING or GFP-DAD1 and cells were lysed. Lysates
were
immunoprecipitated with anti-Myc antibody or IgG control and immunoblotted
using anti-
GFP antibody. Fig. 19C: 293T cells were co-transfected with TREX1-tGFP and
RPN1-Myc,
GFP-DAD1 or STING-HA. Lysates were immunoprecipitated by anti-tGFP antibody or
IgG
control and analyzed by immunoblotting using anti-tGFP, anti-Myc, anti-GFP or
anti=HA
antibodies.
[0027] Fig. 20
shows that TREX1 localizes in the endoplasmic reticulum. hTERT-BJ1
cells were transfected with RPN1-Myc. After
48h, cells were examined by
immunofluorescence using anti-TREX1 antibody (red), anti-Myc antibody (green)
or anti-
calreticulin antibody (blue) as an endoplasmic reticulum marker.
[0028] Figs.
21A-H show that exogenously expressed STING in 293T cells reconstitutes
dsDNA90 response. Fig. 21A: 293T cells were infected with control lentivirus
or hSTING
lentivirus. 1 day afer infection, cells were treated with dsDNA90. After 6h,
cells were
stained using anti-STING or anti-calreticulin antibody. Fig. 21B: Cell lysates
(from Fig.
56A) were subjected to immunoblot using anti-STING antibody. Figs. 21C-D:
Lentivirus
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infected 293T cells were stimulated with dsDNA90 for 6h. Total RNAs were
purified and
examined by real time PCR for IFNP (Fig. 21C) or IFIT2 (Fig. 21D). Fig. 21E:
293T cells
stably transduced with control or hSTING lentivirus were subjected to
Brefeldin A (BFA)
experiment as shown in the flow chart. Fig. 21F: Cell lysates (from Fig. 21E)
were subjected
to immunoblot using anti-STING antibody. Fig. 21G: Cell lysates (from Fig.
21E) were
measured for IFNP-luciferase activity. Fig. 21H: Primary STING-/-.MEFs were
stably
transduced with control or mSTING. Cells were treated with dsDNA90 and
endogenous
IFNP levels were measured by ELISA. *P<0.05, Student s t-test. Error bars
indicated s.d.
Data are representative of at least two independent experiments.
[0029] Fig. 22 shows that translocon members regulate HSV1 replication.
hTERT-BJ1
cells were treated withTREX1, Sec61A1, TRAPP, NS or STING siRNA.
[0030] Figs. 23A-C show that IF116 is not required for IFN-production by
dsDNA90 in
hTER-BJ1 cells. Fig. 23A: hTERT-B.It cells were treated with NS, IF116, STING
or TREX1
siRNA. After 3 days, cells were lysed and checked expression levels by
immunoblotting.
Fig. 23B: siRNA treated hTERT-BJ1 cells were stimulated with dsDNA90 and IFNP
production was measured by ELISA. Fig. 23C: siRNA treated hTERT-BJ1 cells were
infected with HSV-luciferase (m.o.i=0.1). At 2 days after infection, cells
were lysed and
luciferase activity was measured. *P<0.05, Student' s t-test. Error bars
indicated s.d. Data
are representative of at least two independent experiments.
[0031] Fig. 24 shows an embodiment of a STING cell based assay.
[0032] Fig. 25 shows that Drug "A" induces STING trafficking.
[0033] Fig. 26 shows that drug "X" inhibits IFNP mRNA production.
[0034] Fig. 27 is a schematic showing that STING is phosphorylated in
response to
cytoplamic DNA. hTERT-BJ1 cells were transfected with 4 pg/m1 of ISD for 6 h.
The cell
lysates were prepared in TNE buffer and then subjected to immunoprecipitation
with anti-
STING antibody followed by SDS-PAGE. The gel was stained with CBB and then
bands
including STING were analyzed by mLC/MS/MS at the Harvard Mass Spectrometry
and
Proteomics Resource Laboratory. Alignment of STING amino acid sequence from
different
species and the phosphorylation sites identified by mass spectrometry. Serine
345, 358, 366,
and 379 were identified by mass spectrometry. Serine 358 and S366 are
important for
STING function.
[0035] Fig. 28 shows that Serine 366 (S366) of STING is important for IFNP
production
in cytoplasmic DNA pathway. 293T cells were transfected with plasmid encoding
mutant
STING and reporter plasmid. After 36 hr, luciferase activity was measured.
STING-/- MEF
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cells were reconstituted with mutant STING and then the amount of IFN[3 in
culture media
was measured by ELISA. S366 is important for IFN production by STING and S358
is also
likely to play an important role.
[0036] Figs.
29A-D show that STING deficient mice are resistant to DMBA induced
inflammation and skin oncogenesis: STING +1+ and STING-/- mice were either
mocked treated
with acetone or treated with 10 ug of DMBA on the shaved dorsal weekly for 20
weeks. Fig.
29A: STING deficient animals are resistant to DNA-damaging agants that cause
skin cancer.
Percentages of skin tumor-free mice were shown in the Kaplan¨Meier curve. Fig.
29B:
Pictures of representative mice of each treatment groups were shown. Fig. 29C:
Histopathological examinations were performed by H&E staining on mock or DMBA
treated
skin/skin tumor biopsies. Images were taken at 20X magnification. Fig. 29D:
Cytokine
upregulation in STING expressing mice exposed to carcinogens. RNAs extracted
from mock
or DMBA treated skin/skin tumor biopsies were analyzed by Illumina Sentrix
BeadChip
Array (Mouse WG6 version 2) in duplicate. Total gene expression was analyzed.
Most
variable genes were selected. Rows represent individual genes; columns
represent individual
samples. Pseudo-colors indicate transcript levels below, equal to, or above
the mean (green,
black, and red, respectively). Gene expression; fold change 10g10 scale ranges
between -5 to
5. No cytokines were observed in the skin of STING-deficient animals.
DETAILED DESCRIPTION
[0037]
Described herein are methods and compositions for modulating an immune
response in a subject having a disease or disorder associated with aberrant
STING function.
The below described preferred embodiments illustrate adaptation of these
compositions and
methods. Nonetheless, from the description of these embodiments, other aspects
of the
invention can be made and/or practiced based on the description provided
below.
[0038] Methods
and compositions for modulating an immune response in a subject (e.g.,
a human being, dog, cat, horse, cow, goat, pig, etc.) having a disease or
disorder associated
with aberrant STING function involve a pharmaceutical composition including an
agent
which modulates STING function and a pharmaceutically acceptable carrier,
wherein amount
the pharmaceutical composition is effective to ameliorate the aberrant STING
function in the
subject.
[0039] Diseases
or disorders associated with aberrant STING function can be any where
cells having defective STING function or expression cause or exacerbate the
physical
symtoms of the disease or disorder. Commonly, such diseases or disorders are
mediated by
immune system cells, e.g., an inflammatory condition, an autoimmune condition,
cancer (e.g.,
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breast, colorectal, prostate, ovarian, leukemia, lung, endometrial, or liver
cancer),
atherosclerosis, arthritis (e.g., osteoarthritis or rheumatoid arthritis), an
inflammatory bowel
disease (e.g., ulcerative colitis or Crohn's disease), a peripheral vascular
disease, a cerebral
vascular accident (stroke), one where chronic inflammation is present, one
characterized by
lesions having inflammatory cell infiltration, one where amyloid plaques are
present in the
brain (e.g., Alzheimer's disease), Aicardi-Goutieres syndrome, juvenile
arthritis,
osteoporosis, amyotrophic lateral sclerosis, or multiple sclerosis.
[0040] The
agent can be a small molecule (i.e., an organic or inorganic molecule having a
molecular weight less than 500, 1000, or 2000 daltons) that increases or
decreases STING
function or expression or a nucleic acid molecule that binds to STING under
intracellular
conditions (i.e., under conditions inside a cell where STING is normally
located). The agent
can also be a STING-binding nucleic acid molecule which can be a single-
stranded (ss) or
double-stranded (ds) RNA or DNA. Preferably the nucleic acid is between 40 and
150, 60
and 120, 80 and 100, or 85 and 95 base pairs in length or longer. The STING-
binding nucleic
acid molecule can be nuclease-resistant, e.g., made up of nuclease-resistant
nucleotides or in
cyclic dinucleotide form. It can also be associated with a molecule that
facilitates
transmembrane transport.
[0041] Methods
and compositions for treating cancer in a subject having a cancerous
tumor infiltrated with inflammatory immune cells involve a pharmaceutical
composition
including an agent which downregulates STING function or expression and a
pharmaceutically acceptable carrier, wherein amount the pharmaceutical
composition is
effective to reduce the number of inflammatory immune cells infiltrating the
cancerous tumor
by at least 50% (e.g., at least 50, 60, 70, 80, or 90%, or until reduction of
inflammatory cell
infilitration is detectably reduced by histology or scanning).
[0042] The
compositions described herein might be included along with one or more
pharmaceutically acceptable carriers or excipients to make pharmaceutical
compositions
which can be administered by a variety of routes including oral, rectal,
vaginal, topical,
transdermal, subcutaneous, intravenous, intramuscular, insufflation,
intrathecal, and
intranasal administration. Suitable formulations for use in the present
invention are found in
Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia,
Pa., 17th ed.
(1985).
[0043] The
active ingredient(s) can be mixed with an excipient, diluted by an excipient,
and/or enclosed within a carrier which can be in the form of a capsule,
sachet, paper or other
container. When the excipient serves as a diluent, it can be a solid, semi-
solid, or liquid
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material, which acts as a vehicle, carrier or medium for the active
ingredient. The
compositions can be in the form of tablets, pills, powders, lozenges, sachets,
cachets, elixirs,
suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid
medium),
ointments, soft and hard gelatin capsules, suppositories, sterile injectable
solutions, sterile
liquids for intranasal administration (e.g., a spraying device), or sterile
packaged powders.
The formulations can additionally include: lubricating agents such as talc,
magnesium
stearate, and mineral oil; wetting agents; emulsifying and suspending agents;
preserving
agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and
flavoring
agents. The compositions of the invention can be formulated so as to provide
quick, sustained
or delayed release of the active ingredient after administration to the
patient by employing
procedures known in the art.
[0044] For
preparing solid formulations such as tablets, the composition can be mixed
with a pharmaceutical excipient to form a solid preformulation composition
containing a
homogeneous mixture of a compound. Tablets or pills may be coated or otherwise
compounded to provide a dosage form affording the advantage of prolonged
action. For
example, the tablet or pill can comprise an inner dosage and an outer dosage
component, the
latter being in the form of an envelope over the former. The two components
can be separated
by an enteric layer which serves to resist disintegration in the stomach and
permit the inner
component to pass intact into the duodenum or to be delayed in release. A
variety of
materials can be used for such enteric layers or coatings, such materials
including a number
of polymeric acids and mixtures of polymeric acids with such materials as
shellac, cetyl
alcohol, and cellulose acetate.
[0045] Liquid
forms of the formulations include suspensions and emulsions. To enhance
serum half-life, the formulations may be encapsulated, introduced into the
lumen of
liposomes, prepared as a colloid, or incorporated in the layers of liposomes.
A variety of
methods are available for preparing liposomes, as described in, e.g., Szoka,
et al., U.S. Pat.
Nos. 4,235,871, 4,501,728 and 4,837,028 each of which is incorporated herein
by reference.
[0046] The
compositions are preferably formulated in a unit dosage form of the active
ingredient(s). The amount administered to the patient will vary depending upon
what is being
administered, the purpose of the administration, such as prophylaxis or
therapy, the state of
the patient, the manner of administration, and the like all of which are
within the skill of
qualified physicians and pharmacists. In therapeutic applications,
compositions are
administered to a patient already suffering from a disease in an amount
sufficient to cure or at
least partially arrest the symptoms of the disease and its complications.
Amounts effective for
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this use will depend on the disease condition being treated as well as by the
judgment of the
attending clinician depending upon factors such as the severity of the
symptoms, the age,
weight and general condition of the patient, and the like.
[0047] All
documents mentioned herein are incorporated herein by reference. All
publications and patent documents cited in this application are incorporated
by reference for
all purposes to the same extent as if each individual publication or patent
document were so
individually denoted. By their citation of various references in this
document, Applicants do
not admit any particular reference is "prior art" to their invention.
Embodiments of inventive
compositions and methods are illustrated in the following examples.
EXAMPLES
Example 1: Translocon-Associated STING Complexes with Cytoplasmic DNA to
Regulate
Innate Immunity
[0048]
Previously the isolation of a new transmembrane component of the endoplasmic
reticulum (ER), referred to as STING (Stimulator of Interferon Genes), which
was
demonstrated as essential for the production of type I IFN in fibroblasts,
macrophages and
dendritic cells (DC's) in response to cytoplasmic dsDNA as well as DNA viruses
and
intracellular bacteria was described (see U.S. patent application serial no.
13/057,662 and
PCT/U52009/052767). The minimum size of dsDNA required to activate STING-
dependent
type I IFN signaling in murine cells was noted to be approximately 45 base
pairs in murine
cells. In normal human cells (hTERT), however, it was observed that dsDNA of
approximately 90 base pairs (referred to herein as interferon stimulatory
d5DNA90) were
required to fully activate type I IFN. Using RNAi knockdown procedures, it was
additionally
confirmed that STING is indeed essential for the production of type I IFN in
hTERTs (Fig.
1B). Further analysis using microarray procedures to measure mRNA expression
indicated
that cytoplasmic dsDNA can induce a wide array of innate immune genes, in
addition to type
I IFN, in hTERT cells (Figs. 5A-G). The induction of these innate molecules
which included
members of the IFIT family appeared STING-dependent since RNAi knockdown of
STING
in hTERTs greatly eliminated their stimulation by cytoplasmic dsDNA (Figs. 5A-
G). That
cytoplasmic dsDNA induced a variety of innate immune genes in a STING-
dependent
manner was confirmed using STING +4 or -/- murine embryonic fibroblasts (MEFs)
(Fig. 1C).
To confirm that the induction of these mRNAs were STING-dependent genes (SDG)
and not
stimulated through type I IFN dependent autocrine or paracrine signaling, type
I IFN-
signaling defective STAT1 -/- MEF's were similarly treated with dsDNA and
verified that the
production of the SDG's remained unaffected (Fig. 1C). Reverse-transcriptase
(RT) PCR
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analysis confirmed the array results (Figs. 6A-H and 7A-H). It was noted that
ssDNA of 45
nucleotides (55DNA45) weakly induced innate immune gene production in hTERTS
and less
so in MEFs. However, transfected ssDNA comprising 90 nucleotides (55DNA90) was
observed to more robustly activate an array of genes, including type I IFN in
hTERT cells
(Figs. 1D, 1E). That cytoplasmic ssDNA90 induced the production of innate
immune genes
in a STING-dependent manner was similarly confirmed using STING +1+ or -/-
murine
embryonic fibroblasts (MEFs) (Figs. 1F and 7A-H). It was observed that STING
likely
resides as a homodimer in the ER of both human and murine cells, and migrates
from the ER
to perinuclear regions in the presence of cytoplasmic ssDNA or dsDNA ligands
to activate
type I IFN-dependent transcription factors (Figs. 1G and 8A-E). HSV1 was
similarly
observed to activate innate immune gene production in a STING dependent manner
(Figs.
9A-I). It was confirmed that many of the SDG's contained IRF7 binding sites in
their
promoter region (Figs. 10A-F). Thus, cytoplasmic ssDNA or dsDNA, which
includes
transfected plasmid DNA, can potently induce the transcription of a wide array
of innate
immune related genes that is dependent on STING.
[0049] To
further evaluate the possibility that STING itself could associate with DNA
species, 293T cells were transfected with STING and after cell lysis observed
that the C-
terminal region of STING (aa 181-349) could be precipitated using biotin-
labeled dsDNA90
(Fig. 2A). The N-terminal region of STING (aa 1-195) and three similarly HA-
tagged
controls (GFP, NFAR1 and IPS1) did not associate with dsDNA90. The DNA binding
exonuclease TREX1 served as a positive control. A further series of extensive
studies
indicated that amino acid region 242-341 of STING was likely responsible for
binding
dsDNA since STING variants lacking this region failed to associate with
nucleic acid (Figs.
2B-D). In vitro expressed STING also bound to dsDNA under high salt and high
detergent
conditions (except for those variants similarly lacking region 242-341) (Figs.
11A-F).
Further evidence that STING could complex with dsDNA, likely as a dimer, was
achieved by
transfecting biotin-labeled dsDNA90 into hTERT's and treating such cells with
a reversible
cross-linking reagent (DSS) or UV light. In both treatment cases STING was
observed to
retain its association with DNA after cell lysis (Fig. 2E and Figs. 12A-G).
RNAi knockdown
of STING in hTERT cells eliminated the observed binding and STING-DNA
complexes were
also only observed in wild type MEFs (+4) but not MEFs lacking STING (4)
(Figs. 12A-G).
It was similarly confirmed that HSV1, cytomegalovirus (CMV) as well as
adenovirus (ADV)
related dsDNA Competition experiments suggested that STING also could bind to
ssDNA
(55DNA90) as well as dsDNA, but not dsRNA (Fig. 2F). This was confirmed by
expressing
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STING in vitro and observing association with ssDNA90 (Fig. 2G). All STING
variants
analyzed lacked the ability to activate the type IFN promoter in 293T cells
(Fig. 2H). dsDNA
was also transfected into hTERT or MEFs cells and treated with formaldehyde to
cross-link
cellular proteins to the nucleic acid. Subsequent CHIP analysis following
STING pull down,
further confirmed that transfected DNA can directly associate with STING as
determined
using dsDNA90 specific primers (Figs. 21 and 2J). It was observed that STING
could bind to
biotin-labeled DNA in ELISA assays (Figs. 14A-C). The data indicated that
ssDNA and
dsDNA-mediated innate signaling events were dependent on STING and evidence
that
STING itself was able to complex to these nucleic acid structures to help
trigger these events.
[0050] TREX1, a
3'->5' DNA exonuclease is also an ER associated molecule, and
important for degrading checkpoint activated ssDNA species that could
otherwise activate the
immune system. RNAi used to silence TREX1 in hTERT cells significantly
increased
STING-dependent, production of type I IFN by dsDNA90 (Figs. 3A and 3B).
Concomitantly, the replication of the dsDNA virus HSV1 was greatly reduced in
hTERT
cells lacking TREX1, likely due to the elevated production of type I IFN and
antiviral IFN
stimulated genes (ISG's) (Fig 3C and D). Luciferase expression from a
recombinant HSV-
expressing the luciferase gene was also significantly lower in hTERT infected
cells treated
with RNAi to silence TREX1 (Figs. 15A-D). These observations were extended by
using
TREX1 deficient MEFs, which similarly indicated that cytoplasmic dsDNA-
dependent gene
induction was greatly elevated in the absence of TREX1 and that HSV1
replication was
significantly reduced (Figs. 3E-G). To determine if STING was responsible for
the elevated
production of type I IFN observed in the absence of TREX1, STING was silenced
in TREX1
lacking hTERTs or TREX1 -/- MEFs and treated these cells with cytoplasmic
dsDNA or
HSV1. The results indicated greatly reduced type I IFN production in TREX1
deficient cells
(both hTERTs and MEFs) lacking STING indicating that the elevated levels of
type I IFN
observed in the absence of TREX1 are STING-dependent (Figs. 3A-F). Similarly,
it was
noted that RNAi knockdown of STING in TREX -/- MEFs also eliminated ssDNA90-
mediated type I IFN production and innate gene stimulation (Fig. 16). Confocal
analysis
confirmed that TREX1 and STING colocalized in the ER (Fig. 3H). However,
cytoplasmic
dsDNA did not potently induce the trafficking of TREX1 from the ER to
perinuclear regions
similar to STING (Fig. 3H). Thus, it was not observed that STING and TREX1
interacted
robustly, as determined by coimmunpreciptation analysis. Neither was a
dramatic difference
noticed in the expression of STING-dependent genes in TREX1 +/+ or -/- MEFs
under non-
stimulated conditions (Figs. 17A-H). However, TREX1 is a dsDNA-induced gene,
which
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was confirmed and can be upregulated in a STING-dependent manner (Figs. 17A-
H). Thus,
it is plausible that dsDNA species complex with STING and accessory molecules
to mediate
trafficking and downstream signaling events that activate the transcription
factors IRF3/7 and
NF-KB, responsible for the induction of primary innate immune genes including
TREX1.
The evidence indicates that STING-activated TREX1 resides in the ER region to
degrade
activator dsDNA and repress cytoplasmic dsDNA signaling in a negative-feedback
manner.
Thus, TREX1 is a negative regulator of STING.
[0051] The data
herein demonstrated that STING resides in the ER as part of the
translocon complex, associating with translocon associated protein 13 (TRAPP).
The
translocon complex includes Sec61 a p and 7 coupled with TRAP a p, 7 and 6,
which can
attach to ribosomes. Secretory and membrane proteins are translocated into the
ER for
proper folding and glycosylation prior to being exported. To identify TREX1
binding
partners full length TREX1 was used as bait in a two hybrid yeast screen. The
results
indicated that TREX1 recurrently interacted with a protein referred to as
Ribophorin I
(RPN1), a 68 kDa type I transmembrane protein and member of the
oligosaccharyltransferase
(OST) complex (Figs. 4A-E; Figs. 18A-D). The OST complex catalyses the
transfer of
mannose oligosaccharides onto asparagines residues of nascent polypeptides as
they enter the
ER through the translocon. At least seven proteins include the OST complex
including
RPN1, RPN2, 05T48, 05T4, STT3A/B, TUSC3 and DAD1. Significantly, a similar
screen
using STING as bait determined that STING could associate also with DAD1
(defender
against apoptotic cell death), a 16 kDa transmembrane protein (Figs. 14F-H).
Further
analysis of these associations using yeast-two hybrid approaches indicated
that the C-terminal
region of TREX1 comprising its transmembrane region (amino acids 241-369) was
responsible for binding to amino acids 258-397 of RPN1 (Figs. 18A-D). Further,
amino
acids 242-310 of STING were accountable for association with full length DAD1
(Figs. 18A-
D). Coimmunoprecipitation studies confirmed the interaction of these molecules
(Figs. 4D
and 4G and Figs. 29A-C). Further co-immunoprecipitation experiments indicated
the
association of TREX1 with DAD1 (Figs. 19A-C). Confocal analysis confirmed that
TREX1
and RPN1 co-localized in the cell though did not traffic in response to
cytoplasmic dsDNA
(Fig. 4E and Fig. 20). Similarly, STING and DAD1 colocalized in the ER of the
cell
although the latter molecule did not accompany STING to endosomal compartments
in the
presence of cytoplasmic dsDNA (Fig. 4H). Cellular microsome compartments
comprising
the ER were isolated by fractionation and examined by sucrose gradient
analysis. This study
indicated that TREX1 and STING cofractionated with the ER markers RPN1 and
RPN2,
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DAD1 and calreticulin, but not nuclear histone H3, confirming that their
subcellular
localization is indistinguishable from components of the translocon/OST
complex (Fig. 41).
Thus, TREX1 is targeted to the OST/translocon complex of the ER, that includes
STING, and
this association occurs through association with RPN1, although the TM region
of TREX1
was found to be involved in TREX1's localization to the ER. To identify
whether members
of the OST, TRAP or SRP (signal recognition peptide) complex influenced dsDNA-
dependent signaling, an RNAi screen was carried out to silence the expression
of these
components. However, aside from repressing STING (essential for DNA-mediated
type I
IFN production; Figs. 21A-H) and TREX1, which greatly elevated type I IFN
production,
only Sec61 a and TRAPP silencing significantly affected signaling and HSV1
replication,
evidencing a role for these translocon members in controlling this pathway
(Fig. 4J and Fig.
22). Silencing of IF116, also implicated in cytoplasmic DNA sensing, was
observed to not
robustly suppress dsDNA-dependent signaling, at least in hTERT cells (Figs.
23A-C).
Neither did loss of IF116 rescue augmented IFN-production by dsDNA in the
absence of
TREX1, similar to loss of STING (Figs. 23A-C). However, reduced IF116 enabled
more
proficient HSV1 gene expression confirming an important role for this molecule
in
influencing viral replication. Silencing of RPN1 or 2 also lead to an increase
in HSV1 gene
expression but did not significantly affect type I IFN production either,
evidencing that these
components of the OST may be predominantly involved in N-glycosylation.
[0052] The data
evidences that STING can complex with cytoplasmic intracellular
ssDNA and dsDNA, which can include plasmid-based DNA and gene therapy vectors,
can
regulate the induction of a wide array of innate immune genes such as type I
IFN, the IFIT
family, and a variety of chemokines important for antiviral activity and for
initiating adaptive
immune responses. STING activation facilitates the escort of TBK1 to clathrin
covered
endosomal compartments plausibly to activate IRF3/7 by mechanisms that remain
to be fully
clarified. TREX1 appears present in low levels in the cell and is itself
inducible by STING.
After translation, TREX1 localizes to the OST complex in close proximity to
unactivated
STING (which also resides in the OST/translocon complex) where presumably it
degrades
DNA species that can otherwise provoke STING action. Components of the
translocon/OST
complex, which now involve STING and TREX1, regulate cytoplasmic ssDNA and
dsDNA-
mediated innate immune signaling. Since loss of TREX1 manifests autoimmune
disorders
through elevated type I IFN production, it is possible that these diseases are
induced through
STING activity.
Example 2: STING Modulators
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[0053] Drug
libraries were screened to identify agents that modulate STING expression,
function, activity, etc. Fig. 24 shows the steps of a STING cell based assay.
[0054] The
libraries included, BioMol ICCB known Bioactives Library, 500 targets;
LOPAC1280TM Library of Pharmacologically-Active Compounds; Enzo Life Sciences,
ScreenWellTM Phosphatase Inhibitor Library, 33 known phosphatase inhibitors;
[0055]
MicroSource Spectrum Collection 2000 components, 50% drug components,
30% natural products, 20% other bioactive components; EMD: InhibitorSelectTM
96-Well
Protein Kinase Inhibitor Library I, InhibitorSelectTM 96-Well Protein Kinase
Inhibitor Library
II, InhibitorSelectTM 96-Well Protein Kinase Inhibitor Library Ina; Kinase
Library B Kinase
TrueClone collection; Kinase Deficient TrueClone collection.
[0056] The
results showed that one drug (termed 'Drug A") induced STING trafficking
(Fig. 60). Another drug (termed "drug X") inhibited IFN[3 mRNA production
(Fig. 61).
[0057] Table 2:The following were identified as STING inhibitors:
Name Description
Chemical Name
Diclofenac sodium 2-[(2,6- Cyclooxygenase
inhibitor;
Dichlorophenyl)amino]benzeneacetic anti-inflammatory
acid sodium
R(-)-2,10,11- R(-)-2-Hydroxyapomorphine D2 dopamine receptor
Trihydroxyaporphine hydrobromide agonist
hybrobromide
Dipropyldopamine Dopamine receptor agonist
hydrobromide
2,2'-Bipyridyl alpha,alpha'-Bipyridyl Metalloprotease inhibitor
( ) trans-U-
50488 trans-( )-3,4-Dichloro-N-methyl-N-[2- Selective kappa opioid
methanesulfonate (1-pyrrolidiny1)-cyclohexyl]- receptor agonist.
benzeneacetamide
methanesulfonate
SP600125 Anthrapyrazolone; 1,9-
Selective c-Jun N-terminal
Pyrazoloanthrone kinase (c-JNK) inhibitor.
Doxazosin mesylate 1-(4-amino-6,7-dimethoxy-2- alpha1 adrenoceptor
blocker
quinazoliny1)-4-[4-(1,4-benzodioxan-
2-yl)carpiperazin-1-y1)]-6,7-
dimethoxyquinazoline mesylate
Mitoxantrone 1,4-Dihydroxy-5,8-bis([2-([2- DNA synthesis inhibitor
hydroxyethyl]amino)ethyl]amino)-
9,10-anthracenedione
MRS 2159 P2X1 receptor antagonist
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Nemadipine-A 1,4-Dihydro-2,6-dimethy1-4- An L-type calcium channel
(pentafluorophenyI)-3,5- alpha1 -subunit antagonist.
pyridined icarboxylic acid diethyl
ester
( )-PPHT hydrochloride ( )-2-(N-Phenylethyl)-N- Potent D2
dopamine
propyl)amino-5-hydroxytetralin receptor agonist
hydrochloride
SMER28 6-Bromo-N-2-propeny1-4- Small molecule modulator of
quinazolinamine mammalian autophagy.
Quinine sulfate K+ channel blocker;
antimalarial, anticholinergic,
antihypertensive and
hypoglycemic agent; alkaloid
isolated from the bark of the
Cinchona family of South
American trees
(+)-Quisqualic acid L(+)-alpha-Amino-3,5-dioxo-1,2,4- Active
enantiomer of
oxadiazolidine-2-propanoic acid quisqualic acid; excitatory
amino acid at glutamate
receptors;
anthelmentic
agent
[0058] Activators of STING included dihydroouabain and BNTX maleate salt
hydrate.
Example 3: STING Manifests Self DNA-Dependent Inflammatory Disease
[0059] Bone marrow derived macrophages (BMDM) were obtained from Sting+/+
and Sting
mice and transfected them with 90 base pair dsDNA (dsDNA90) to activate the
STING pathway, or
with apoptotic DNA (aDNA) derived from dexamethasone (Dex)-treated thymocytes.
It was
observed that both types of DNA potently induced the production of IFNf3 in
BMDM and
conventional dendritic cells (BMDC's) in a STING dependent manner. DNA
microarray experiments
confirmed that aDNA triggered STING-dependent production of a wide array of
innate immune and
inflammatory related cytokines in BMDM such as IFNf3 as well as TNFa (Table
3). These data were
confirmed by measuring cytokine production in Sting+/+ or Sting' BMDM treated
with aDNA. Thus,
STING can facilitate apoptotic DNA-mediated pro-inflammatory gene production
in BMDM's as well
as BMDC' s.
[0060] Table 3
shows the gene expression of higher expressed genes in BMDM treated
with apoptic DNA (aDNA).
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[0061] To
determine if STING played a role in DNase II related inflammatory disease,
STING and/or DNase II was knocked down in THP1 cells or BMDM using RNAi and it
was
noticed that loss of DNase II facilitated the upregulation of cytokines,
including type I IFN,
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in response to aDNA in a STING-dependent manner. Since DNase IT/- mice usually
die
before birth, DNase IT/ - , Stine , or Stine DNase III- DKO 17 day embryos
(E17 days) were
analyzed. Genotyping analysis, including RT-PCR and immunoblot confirmed that
the
embryos lacked Sting, DNase II or both functional genes. It was observed that
DNase IT'
embryos exhibited anemia, as described above, which was in significant
contrast to Stine
DNase IT/- DKO embryos or controls which noticeably lacked this phenotype.
Lethal anemia
has been reported to be due to type I IFN inhibition of erythropoiesis during
development. It
was subsequently observed by hematoxylin and eosin staining that the livers of
DNase IT'
embryoscontained numerous infiltrating macrophages full of engulfed apoptotic
cells
responsible for producing high levels of cytokines. In contrast to control
mice, the livers of
Stine DNase IT/- embryos exhibited a similar phenotype. Analysis of fetal
livers by TUNEL
(terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling)
confirmed
that the Stine DNase IT/- embryos and DNase 11-deficient but not wild-type
fetal livers
contained numerous large inappropriately digested dying cells. In vitro
analysis has indicated
that macrophages from the embryos of of wild-type or DNase IT' - mice engulf
apoptotic cells
adequately. However, while the DNA of the engulfed apoptotic cells is
efficiently degraded
in the lysosomes of wild-type macrophages, DNase II-/- macrophages accumulate
engulfed
nuclei and cannot digest DNA. This event leads to the stimulation of innate
immune
signaling pathways and production of autoimmune related cytokines. Given this,
the ability
of embryonic liver derived macrophages that lacked both DNase II and STING
were
evaluated as to whether they to engulf apoptotic cells and digest DNA. It was
noted that
Stine- DNase II-/- macrophages, similar to DNase II-/- macrophages, were not
able to digest
the engulfed nuclei from dexamethasone treated apoptotic thymocytes compared
to control
macrophages taken from wild type or Stine- mice. Thus, macrophages harvested
from the
livers of Stine- DNase II-/- embryonic mice similarly exhibit an inability to
digest engulfed
apoptotic cells, analogous to DNase II-/- macrophages.
[0062] The
above analysis was complemented with analyzing mRNA expression levels in
the livers of the embryonic mice. This study indicated very little
inflammatory gene
production in the livers of wild type or Stine- embryos. However, it was
observed that the
livers of DNase le- embryos contained abnormally high levels of cytokine
related mRNA.
-/-
Significantly, the livers of Stine- DNase II mice had dramatically reduced
levels of innate
immune gene expression activity compared to DNase le- mice. These results were
confirmed by analyzing the mRNA expression levels of select innate immune
genes in
embryonic livers by RT-PCR. For example, the production of IFN[3 was reduced
several fold
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in Stine- DNase le- mice compared to DNase le- mice. The production of key
interferon-
stimulated genes (ISG's) such as the 2'-5' oligoadenylate synthetases (OAS),
interferon-
induced proteins with tetratricopeptide repeats (IFITs) interferon-inducible
protein 27 (IF127)
and ubiquitin-like modifier (ISG15) were also dramatically reduced. Pro-
inflammatory
cytokines such as TNFa and ILlp were also decreased in the embryonic livers of
Stine- and
-I- -I- -I-
Sting DNase II compared to DNase II mice. While the production of innate
immune
genes was dramatically suppressed in the absence of STING, the presence of
some genes
remained slightly elevated in Stine- DNase II-/- mice, albeit in low levels as
determined by
anay analysis, which may be due to variation in mRNA expression between the
animals
analyzed, or perhaps due to the stimulation of other pathways. Many of these
genes are
regulated by NF-KB and interferon regulatory factor (IRF) pathways. The
function of these
transcription factors was thus evaluated in Stine- DNase II-/- or control
murine embryonic
fibroblasts (MEFs), developed from 14 day embryos (E14 days). Principally, a
defect was
observed in NF-KB activity (p65 phosphorylation) in Stine- DNase IC- MEFs when
exposed
to cytoplasmic DNA. The same defect was obtained in Stine DNase IT/- BMDM when
exposed to apoptotic DNA as well as cytoplasmic DNA. This was confirmed by
noting that
NF-03 as well as IRF3 also failed to translocate in Stine DNase III- MEF's but
not in
control MEFs following exposure to dsDNA. Thus, STING is likely responsible
for
controlling self DNA-induced inflammatory cytokine production that is
responsible for
causing lethal embryonic erythropoiesis.
[0063] To
extend of the importance of STING in mediating self-DNA-facilitated lethal
erythropoiesis, it was evaluated whether DNase IT/- mice could be born in the
absence of
STING. Significantly, it was observed that DNase IT/- mice were born, with
apparent
Mendelian frequency, when crossed onto a Stine- background. PCR genotyping,
Northern
blot, RT-PCR and immunoblot analysis confirmed DNase II and STING deficiency
in the
progeny mice. Stine DNase III- double knockout mice (DKO) appeared to grow
normally
and exhibited similar size and weight compared to control mice although it was
noted that
Sting micemice were somewhat larger for reasons that remain unclear.
Preliminary
immunological evaluation also indicated that the Stine DNase IT/- DKO animals
shared a
similar CD4 /CD8+ profile similar to Sting andand wild type mice, although the
DKO's were
noted to develop splenomegaly as they aged. Splenomegaly was also noted in
surviving
DNase II deficient mice that lacked type I IFN signaling (DNase IT/- Ifnar14-
mice) and has
been reported to be due to enlargement of the red pulp. However, analysis of
serum from
--
Sting DNase IT/- mice indicated no detectable abnormal cytokine production
compared to
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control mice at 8 weeks of age, due to the general low immeasurable levels of
cytokines
produced. Through these studies it was noted in vitro that Stine DNase IT/-
macrophages,
similar to DNase IT/- macrophages, were not able to digest the engulfed nuclei
from apoptotic
thymocytes (Dex+) compared to control macrophages taken from wild type or
Stine- mice.
The accumulation of undigested DNA in DNase le- Stine- macrophages was less
pronounced when WT thymocytes were used as targets (Dex-). Thus, BMDM derived
from
Stine- DNase III- mice are also incapable of digesting DNA from apoptotic
cells, although in
contrast to DNase IT/- BMDM do not produce inflammatory cytokine responses.
[0064] While
DNase II mediated embryonic lethality can be avoided by crossing DNase
ii+/- mice with type I IFN defective Ifnarl-/- mice, the resultant progeny
suffer from severe
polyarthritis approximately 8 weeks after birth (arthritis score of 2) since
undigested DNA
activates innate immune signaling pathways and triggers the production of
inflammatory
cytokines such as TNFa. Significantly, it was noted that Stine- DNase le- mice
did not
manifest any signs of polyarthritis following birth.
Arthritis scores remained at
approximately zero (no score) in the Stine DNase IT/-, up to 12 months of age
in contrast to
reported DNase IT/- Ifnarl-/- mice which exhibited an arthritis score of up to
7 after a similar
period. While H&E and TUNEL staining of spleen and thymus tissues of DNase 11-
/-Sting-/-
mice illustrated the presence of infiltrating macrophages that also contained
apoptotic DNA,
histology of joints from 6 month old Stine- DNase 111- mice exhibited normal
bone (B)
synovial joint (S) and cartilage (C) structure with no evidence of pannus
infiltration in the
joint structure. Levels of TNFa, ILlp and IL6 from sera of Stine- DNase IT/-
mice remained
at low levels as predicted from our array analysis of BMDM that lacked STING
(Table 3).
Neither was there evidence of CD4, CD68 or TRAP positive cells infiltration
within the
joints of Stine- DNase 11-1- mice. Analysis of the serum of Stine- DNase IT/-
mice also
indicated no elevated levels of Rheumatoid Factor (RF), anti-dsDNA antibody or
MMP3.
Thus, loss of STING eliminates pro-inflammatory cytokine production
responsible for self
DNA-mediated polyarthritis.
[0065] STING is
responsible for inflammatory disease such as for example, Aicardi-
Goutieres syndrome (AGS). AGS is genetically determined encephalopathy and is
characterized by calcification of basal ganglia and white matter,
demyelination. High levels
of lymphocytes and type I IFN in cerebrospinal fluid. The features mimic
chronic infection.
Serum levels of type I IFN are also raised in autoimmune syndrome systemic
lupus
erythromatosis (SLE). AGS is caused by mutations in 3'-5' DNA exonuclease
TREX1. Loss
of TREX1 function- DNA species accumulates in the ER of cells and activates
cytoplasmic
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DNA sensors (STING). TREX1 digests this DNA source (housekeeping function) to
prevent
innate immune gene activation.
[0066] Given that STING seems responsible for inflammatory disease in mice
defective
in apoptosis, it was next evaluated whether other types of self-DNA triggered
disease
occurred through activation of the STING pathway. For example, patients
defective in the 3'
repair exonuclease 1 (Trexl) suffer from Aicardi-Goutieres Syndrome (AGS)
which
instigates lethal encephalitis characterized by high levels of type I IFN
production being
present in the cerebrospinal fluid. Trexl-deficient mice exhibit a median life
span of
approximately 10 weeks since as yet uncharacterized self-DNA, presumably
normally
digested by Trexl, activates intracellular DNA sensors which triggers cytokine
production
and causes lethal inflammatory aggravated myocarditis. Recent data indicates
that loss of
STING can extend the lifespan of Trexl-/- mice although the causes are
unknown. These
studies were extended and it was noted that there were slightly elevated
levels of type I IFN
production in Trexl deficient BMDC (Trexl-/- BMDC) exposed to dsDNA90.
Significantly,
loss of STING (Stine Trex11- BMDC) eliminated the ability of DNA to augment
type I IFN
production in BMDM's deficient in Trexl. Interestingly, a size reduction of
the hearts of
-- -/-F -z --
Stine Trexl , Sting Trexl was observed when compared to Trexl mice. Evidence
of
myocarditis was also note to be dramatically reduced in Stine Trexl-/-
compared to Trexl-/-
alone. In addition, anti-nuclear autoantibody (ANA) observed to be highly
prevalent in the
sera of Trexii- mice, was almost completely absent in the sera of Stine Trexl-
/- mice.
Microarray analysis demonstrated dramatically, reduced levels of pro-
inflammatory genes in
the hearts of Stine- Trex14-, Sting4+ Trexl-/- compared to Trex 14- mice.
Collectively, these
data indicates that STING is responsible for pro-inflammatory gene induction
in Trexl
deficient mice and plausibly AGS.
Example 4: Screening of kinases for S366 of STING
[0067] The activity of 217 protein kinase targets were evaluated against 2
peptides (A366
and S366) as substrate. Protein kinases were mixed with each peptide and 33P-
ATP and then
activity (CPM) was measured. The below kinases were identified as
phosphorylating S366 in
STING. Identification of kinases which target this serine opens up avenues for
drug
discovery. Drugs that target this association may inhibit STING activity and
be used for
therapeutic purposes to inhibit STING activity. STING over activity may lead
to
inflammatory diseases which can exacerbate cancer.
[0068] Table 4: Screening of kinases for S366 of STING
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Activity (cpm)
Protein Kinase . Fold induction
A366 peptide 5366 peptide
CK16 15569 25185 1.62
GRK7 7263 13124 1.81
IKKet 2432 6889 2.83
IRAK4 7435 50353 6.77
MEKK1 11316 24866 2.20
NEK2 8702 162331 18.65
NEK6 5512 35556 6.45
NEK7 3619 19112 5.28
NEK9 6977 26284 3.77
PIM2 1414 3715 2.63
PKCL 6812 12820 1.88
RIPK5 1327 15966 12.03
TBK1 6603 16651 2.52
ULK1 12221 209014 17.10
ULK2 7377 155880 21.13
Example 5: STING is responsible for inflammation-associated cancer
[0069]=
STING WT and STING animals were treated with DNA damaging agents and
mice lacking STING were resistant to tumor formation. This is because
infiltrating immune
cells such as dendritic cells, macrophages etc eat the damaged cells that have
undergone
necrosis or apoptosis and the DNA or other ligands from such cells activate
STING and the
production of cytokines that promote tumor formation. STING may be involved in
facilitating tumor progression in a wide variety of other cancers.
[0070] Figs.
29A-D show that STING deficient mice are resistant to DMBA induced
inflammation and skin oncogenesis: STING +4 and STING-/- mice were either
mocked treated
with acetone or treated with 10 ug of DMBA on the shaved dorsal weekly for 20
weeks. Fig.
29A: STING deficient animals are resistant to DNA-damaging agants that cause
skin cancer.
Percentages of skin tumor-free mice were shown in the Kaplan¨Meier curve. Fig.
29B:
Pictures of representative mice of each treatment groups were shown. Fig. 29C:
Histopathological examinations were performed by H&E staining on mock or DMBA
treated
skin/skin tumor biopsies. Images were taken at 20X magnification. Fig. 29D:
Cytokine
upregulation in STING expressing mice exposed to carcinogens. RNAs extracted
from mock
or DMBA treated skin/skin tumor biopsies were analyzed by Illumina Sentrix
BeadChip
Anay (Mouse WG6 version 2) in duplicate. Total gene expression was analyzed.
Most
variable genes were selected. Rows represent individual genes; columns
represent individual
samples. Pseudo-colors indicate transcript levels below, equal to, or above
the mean. Gene
expression; fold change 10g10 scale ranges between -5 to 5. No cytokines were
observed in
the skin of STING-deficient animals.
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