U.S. patent application number 09/802370 was filed with the patent office on 2002-07-25 for methods of suppressing hepatitis virus infection using immunomodulatory polynucleotide sequences.
Invention is credited to Eiden, Joseph J. JR., Van Nest, Gary.
Application Number | 20020098199 09/802370 |
Document ID | / |
Family ID | 26883932 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098199 |
Kind Code |
A1 |
Van Nest, Gary ; et
al. |
July 25, 2002 |
Methods of suppressing hepatitis virus infection using
immunomodulatory polynucleotide sequences
Abstract
Methods are provided for the treatment of hepatitis B virus
(HBV) and hepatitis C virus (HCV) infections. A polynucleotide
comprising an immunostimulatory sequence is administered to a
individual who has been exposed to or infected by HBV and/or HCV.
The polynucleotide is not administered with a HCV or HBV antigen.
Administration of the polynucleotide results in amelioration of
symptoms of HBV and/or HCV infection.
Inventors: |
Van Nest, Gary; (Martinez,
CA) ; Eiden, Joseph J. JR.; (Danville, CA) |
Correspondence
Address: |
Karen R. Zachow, Ph.D.
Morrison & Foerster LLP
755 Page Mill Road
Palo Alto
CA
94304-1018
US
|
Family ID: |
26883932 |
Appl. No.: |
09/802370 |
Filed: |
March 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60188301 |
Mar 10, 2000 |
|
|
|
Current U.S.
Class: |
424/189.1 ;
514/44R |
Current CPC
Class: |
A61P 31/14 20180101;
A61K 31/7088 20130101; A61P 1/16 20180101; A61P 43/00 20180101;
A61K 31/713 20130101; A61P 37/04 20180101 |
Class at
Publication: |
424/189.1 ;
514/44 |
International
Class: |
A61K 048/00; A61K
039/29 |
Claims
What is claimed is:
1. A method of reducing viremia in an individual infected with
hepatitis B virus (HBV), comprising administering a composition
comprising a polynucleotide comprising an immunostimulatory
sequence (ISS) to said individual, wherein the ISS comprises the
sequence 5'-C, G-3', wherein an HBV antigen is not administered in
conjunction with administration of said composition, and wherein
said composition is administered in an amount sufficient to reduce
HBV viremia.
2. The method of claim 1, wherein the ISS comprises the sequence
5'-T, C, G-3'.
3. The method of claim 1, wherein the ISS comprises the sequence
5'-purine, purine, C, G, pyrimidine, pyrimidine, C, G-3' or
5'-purine, purine, C, G, pyrimidine, pyrimidine, C, C-3'.
4. The method of claim 3, wherein the ISS comprises a sequence
selected from the group consisting of 5'-AACGTTCC-3',
5'-AACGTTCG-3', 5'-GACGTTCC-3' and 5'-GACGTTCG-3'.
5. The method of claim 1, wherein the ISS comprises the sequence
5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1).
6. The method of claim 1, wherein the individual is a mammal.
7. The method of claim 1, wherein administration is intravenous or
subcutaneous.
8. A method of reducing blood levels of a hepatitis virus antigen
in an individual infected with hepatitis B virus (HBV), comprising
administering a composition comprising a polynucleotide comprising
an immunostimulatory sequence (ISS) to said individual, wherein the
ISS comprises the sequence 5'-C, G-3', wherein an HBV antigen is
not administered in conjunction with administration of said
composition, and wherein said composition is administered in an
amount sufficient to reduce blood levels of a hepatitis virus
antigen.
9. The method of claim 8, wherein the ISS comprises the sequence
5'-T, C, G-3'.
10. The method of claim 8, wherein the ISS comprises the sequence
5'-purine, purine, C, G, pyrimidine, pyrimidine, C, G-3' or
5'-purine, purine, C, G, pyrimidine, pyrimidine, C, C-3'.
11. The method of claim 10, wherein the lSS comprises a sequence
selected from the group consisting of 5'-AACGTTCC-3',
5'-AACGTTCG-3', 5'-GACGTTCC-3' and 5'-GACGTTCG-3'.
12. The method of claim 8, wherein the ISS comprises the sequence
5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1).
13. The method of claim 8, wherein the individual is a mammal.
14. The method of claim 8, wherein administration is intravenous or
subcutaneous.
15. The method of claim 8, wherein the hepatitis virus antigen is
HBsAg.
16. A kit for reducing viremia in an individual infected with or
exposed to HBV, comprising a composition comprising a
polynucleotide comprising an immunostimulatory sequence (ISS),
wherein the ISS comprises the sequence 5'-C, G-3', wherein said kit
does not comprise an HBV antigen; and wherein the kit comprises
instructions for administration of said composition to an
individual infected with or exposed to HBV to reduce viremia.
17. The kit of claim 16, wherein the ISS comprises the sequence
5'-T, C, G-3'.
18. The kit of claim 16, wherein the ISS comprises the sequence
5'-purine, purine, C, G, pyrimidine, pyrimidine, C, G-3' or
5'-purine, purine, C, G, pyrimidine, pyrimidine, C, C-3'.
19. The kit of claim 18, wherein the ISS comprises the sequence
5'-AACGTTCG-3'.
20. The kit of claim 16, wherein the ISS comprises the sequence
5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Application No. 60/188,301, filed Mar. 10, 2000, which
is hereby incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] This invention is in the field of immunomodulatory
polynucleotides, more particularly their use in ameliorating or
preventing hepatitis viral infection and/or symptoms of hepatitis
virus infection.
BACKGROUND ART
[0003] Hepatitis is a generic term for disease involving
inflammation of the liver. A variety of agents can cause hepatitis,
including viruses, drugs, toxins, and autoimmune disorders.
Additionally, hepatitis can arise secondary to non liver-related
disorders. Viral infection is the most common cause of
hepatitis.
[0004] At least 8 different hepatitis viruses are believed to
exist, and include the A, B, C, D, E, F, G and cryptogenic
hepatitis viruses. The hepatitis viruses are spread through a
number of different virus families. Of these viruses, hepatitis B
virus (HBV, a hepadnavirus) and hepatitis C virus (HCV, a
flavivirus) pose the greatest public health problem in
industrialized countries. Both hepatitis B and C are bloodborne
diseases, although both viruses may also be transmitted perinatally
and via sexual contact.
[0005] Hepatitis B and C can each give rise to acute and chronic
infections. A relatively low level of mortality is due to acute B
and C hepatitis (primarily due to fulminant hepatitis). However,
the chronic forms of each disease pose significant medical issues.
HBV is the most prevalent chronic infectious disease in the world,
and poses a substantially larger perinatal transmission risk than
HCV, with 90% of children born to HBV-infected mothers becoming
lifelong carriers. HCV is currently the leading cause of liver
transplants in the United States.
[0006] Only about half of HBV infections, and even fewer HCV
infections, are symptomatic in the acute phase, which typically
presents with symptoms such as jaundice, fatigue, abdominal pain
and/or loss of appetite. Subclinical infections can be detected
using diagnostic testing for viral antigens and/or DNA.
[0007] The vast majority (95-98%) of adults infected with HBV
resolve their disease and experience no further ill effects,
although newborns are at substantial risk of developing a chronic
infection, with approximately 80-90% of perinatally infected
individuals developing chronic disease. Chronic HBV infection is
typically asymptomatic, although some symptoms of acute hepatitis B
may be present. The long term sequelae of chronic HBV infection
include liver fibrosis/cirrhosis, liver cancer, liver failure and
death.
[0008] Chronic HBV infection is a substantial public health issue
in Asia, where comparatively large percentages of the population
are chronically infected with HBV. Mirroring these high rates of
chronic infection are rates of hepatocellular carcinoma (HCC), a
liver cancer associated with chronic HBV infection.
[0009] In the U.S., acute HCV infections are substantially less
common than acute HBV infections, by a factor of approximately ten.
However, due to the substantially greater risk of progression to
chronic infection (.gtoreq.85%), the prevalence of chronic HCV
infection is two to three times greater than that for chronic HBV
infection. Additionally, HCV infection carries a much greater risk
of the development of chronic liver disease and liver failure.
[0010] Although HBV and HCV are very different viruses, treatments
for chronic infections with the two viruses are virtually
identical. Currently available treatments for chronic hepatitis B
and C infection are limited to interferons. Interferon .alpha.-2a,
interferon .alpha.-2b and interferon alfacon-1 (a recombinant,
non-naturally occurring interferon 1 variant) are currently used
for the treatment of chronic hepatitis virus infection, although
"combination" therapy with ribavirin (an anti-viral drug) and
interferon .alpha.-2b has also been approved for the treatment of
chronic hepatitis C. However, these drugs require frequent
administration and are associated with a large number of side
effects, including "flu-like" symptoms (e.g., fatigue, fever,
myalgia), leukopenia, thrombocytopenia, nausea, vomiting, and
arthralgia. One rare complication of interferon administration is
hepatotoxicity, which can be fatal. Unfortunately, only about 40%
of patients show any improvement with interferon treatment, and may
relapse after treatment is completed.
[0011] Currently, a number of new drugs are being developed for
treatment of chronic HBV infection, including interferon .beta.,
interferon .gamma., interleukin 2, thymosin, acyclovir, lamivudine
(3TC), and granulocyte colony factor. These drugs typically require
long courses of administration, and most are accompanied by
significant side effects.
[0012] Administration of certain DNA sequences, generally known as
immunostimulatory sequences or "ISS," induces an immune response
with a Th1-type bias as indicated by secretion of Th1-associated
cytokines. The Th1 subset of helper cells is responsible for
classical cell-mediated functions such as delayed-type
hypersensitivity and activation of cytotoxic T lymphocytes (CTLs),
whereas the Th2 subset functions more effectively as a helper for
B-cell activation. The type of immune response to an antigen is
generally influenced by the cytokines produced by the cells
responding to the antigen. Differences in the cytokines secreted by
Th1 and Th2 cells are believed to reflect different biological
functions of these two subsets. See, for example, Romagnani (2000)
Ann. Allergy Asthma Immunol. 85:9-18.
[0013] Administration of an immunostimulatory polynucleotide with
an antigen results in a Th1-type immune response to the
administered antigen. Roman et al. (1997) Nature Med. 3:849-854.
For example, mice injected intradermally with Escherichia coli (E.
coli) .beta.-galactosidase (.beta.-Gal) in saline or in the
adjuvant alum responded by producing specific IgGI and IgE
antibodies, and CD4.sup.+ cells that secreted IL-4 and IL-5 , but
not IFN-.gamma., demonstrating that the T cells were predominantly
of the Th2 subset. However, mice injected intradernally (or with a
tyne skin scratch applicator) with plasmid DNA (in saline) encoding
.beta.-Gal and containing an ISS responded by producing IgG2a
antibodies and CD4.sup.+ cells that secreted IFN-.gamma., but not
IL-4 and IL-5, demonstrating that the T cells were predominantly of
the Th1 subset. Moreover, specific IgE production by the plasmid
DNA-injected mice was reduced 66-75%. Raz et al. (1996) Proc. Natl.
Acad. Sci. USA 93:5141-5145. In general, the response to naked DNA
immunization is characterized by production of IL-2, TNF.alpha. and
IFN-.gamma. by antigen-stimulated CD4.sup.+ T cells, which is
indicative of a Th1-type response. This is particularly important
in treatment of allergy and asthma as shown by the decreased IgE
production. The ability of immunostimulatory polynucleotides to
stimulate a Th1-type immune response has been demonstrated with
bacterial antigens, viral antigens and with allergens (see, for
example, WO 98/55495).
[0014] Other references describing ISS include: Krieg et al. (1989)
J. Immunol. 143:2448-2451; Tokunaga et al. (1992) Microbiol.
Immunol. 36:55-66; Kataoka et al. (1992) Jpn. J. Cancer Res.
83:244-247; Yamamoto et al. (1992) J. Immunol. 148:4072-4076;
Mojcik et al. (1993) Clin. Immuno. and Immunopathol. 67:130-136;
Branda et al. (1993) Biochem. Pharmacol. 45:2037-2043; Pisetsky et
al. (1994) Life Sci. 54(2):101-107; Yamamoto et al. (1994a)
Antisense Research and Development. 4:119-122; Yamamoto et al.
(1994b) Jpn. J. Cancer Res. 85:775-779; Raz et al. (1994) Proc.
Natl. Acad. Sci. USA 91:9519-9523; Kimura et al. (1994) J. Biochem.
(Tokyo) 116:991-994; Krieg et al. (1995) Nature 374:546-549;
Pisetsky et al. (1995) Ann. N.Y Acad. Sci. 772:152-163; Pisetsky
(1996a) J. Immunol. 156:421-423; Pisetsky (1996b) Immunity
5:303-310; Zhao et al. (1996) Biochem. Pharmacol 51:173-182; Yi et
al. (1996) J. Immunol. 156:558-564; Krieg (1996) Trends Microbiol.
4(2):73-76; Krieg et al. (1996) Antisense Nucleic Acid Drug Dev.
6:133-139; Klinman et al. (1996) Proc. Natl. Acad. Sci. USA.
93:2879-2883; Raz et al. (1996); Sato et al. (1996) Science
273:352-354; Stacey et al. (1996) J. Immunol. 157:2116-2122; Ballas
et al. (1996) J. Immunol. 157:1840-1845; Branda et al. (1996) J.
Lab. Clin. Med. 128:329-338; Sonehara et al. (1996) J. Interferon
and Cytokine Res. 16:799-803; Klinman et al. (1997) J. Immunol.
158:3635-3639; Sparwasser et al. (1997) Eur. J. Immunol.
27:1671-1679; Roman et al. (1997); Carson et al. (1997) J. Exp.
Med. 186:1621-1622; Chace et al. (1997) Clin. ImmunoL and
Immunopathol. 84:185-193; Chu et al. (1997) J. Exp. Med.
186:1623-1631; Lipford et al. (1997a) Eur. J. Immunol.
27:2340-2344; Lipford et al. (1997b) Eur. J. Immunol. 27:3420-3426;
Weiner et al. (1997) Proc. Natl. Acad. Sci. USA 94:10833-10837;
Macfarlane et al. (1997) Immunology 91:586-593; Schwartz et al.
(1997) J. Clin. Invest. 100:68-73; Stein et al. (1997) Antisense
Technology, Ch. 11 pp. 241-264, C. Lichtenstein and W. Nellen,
Eds., IRL Press; Wooldridge et al. (1997) Blood 89:2994-2998;
Leclerc et al. (1997) Cell. Immunol. 179:97-106; Kline et al.
(1997) J. Invest. Med. 45(3):282A; Yi et al. (1998a) J. Immunol
160:1240-1245; Yi et al. (1998b) J. Immunol. 160:4755-4761; Yi et
al. (1998c) J. Immunol. 160:5898-5906; Yi et al. (1998d) J. Immunol
161:4493-4497; Krieg (1998) Applied Antisense Oligonucleotide
Technology Ch. 24, pp. 431-448, C. A. Stein and A. M. Krieg, Eds.,
Wiley-Liss, Inc.; Krieg et al. (1998a) Trends Microbiol. 6:23-27;
Krieg et al. (1998b) J. Immunol. 161:2428-2434; Krieg et al.
(1998c) Proc. Natl. Acad. Sci. USA 95:12631-12636; Spiegelberg et
al. (1998) Allergy 53(45S):93-97; Homer et al. (1998) Cell Immunol.
190:77-82; Jakob et al. (1998) J. Immunol. 161:3042-3049; Redford
et al. (1998) J. Immunol. 161:3930-3935; Weeratna et al. (1998)
Antisense & Nucleic Acid Drug Development 8:351-356; McCluskie
et al. (1998) J. Immunol. 161(9):4463-4466; Gramzinski et al.
(1998) Mol. Med. 4:109-118; Liu et al. (1998) Blood 92:3730-3736;
Moldoveanu et al. (1998) Vaccine 16: 1216-1224; Brazolot Milan et
al. (1998) Proc. Natl. Acad. Sci. USA 95:15553-15558; Broide et al.
(1998) J. Immunol. 161:7054-7062; Broide et al. (1999) Int. Arch.
Allergy Immunol. 118:453-456; Kovarik et al. (1999) J. Immunol.
162:1611-1617; Spiegelberg et al. (1999) Pediatr. Pulmonol. Suppl.
18:118-121; Martin-Orozco et al. (1999) Int. Immunol. 11:1111-1118;
EP 468,520; WO 96/02555; WO 97/28259; WO 98/16247; WO 98/18810; WO
98/37919; WO 98/40100; WO 98/52581; WO 98/55495; WO 98/55609 and WO
99/11275. See also Elkins et al. (1999) J. Immunol 162:2291-2298,
WO 98/52962, WO 99/33488, WO 99/33868, WO 99/51259 and WO 99/62923.
See also Zimmermann et al. (1998) J. Immunol. 160:3627-3630; Krieg
(1999) Trends Microbiol. 7:64-65; U.S. Pat. Nos. 5,663,153,
5,723,335, 5,849,719 and 6,174,872. See also WO 99/56755, WO
00/06588, WO 00/16804; WO 00/21556; WO 00/67023 and WO
01/12223.
[0015] There exists a need in the art for effective treatments of
acute and chronic hepatitis B and C.
[0016] All publications and patent applications cited herein are
hereby incorporated by reference in their entirety.
DISCLOSURE OF THE INVENTION
[0017] The invention provides methods of suppressing and/or
ameliorating hepatitis infection in an individual using
immunostimulatory polynucleotide sequences. Accordingly, the
invention provides methods for preventing, palliating,
ameliorating, reducing, and/or eliminating one or more symptoms of
HBV or HCV infection without administering HBV or HCV antigens. A
polynucleotide comprising an immunostimulatory sequence (an ISS) is
administered to an individual who has been exposed to HBV and/or
HCV or is infected with HBV and/or HCV. The ISS-containing
polynucleotide is administered without any HBV or HCV antigens
(i.e., HBV or HCV antigen is not co-administered). Administration
of the ISS results in reduced incidence and/or severity of one or
more symptoms of HBV and/or HCV infection.
[0018] In one embodiment, the invention provides methods for
preventing a symptom of acute hepatitis B virus (HBV) or hepatitis
C virus (HCV) infection in an individual which entail administering
an effective amount of a composition comprising a polynucleotide
comprising an immunostimulatory sequence (ISS), (i.e., an amount of
the composition sufficient to prevent a symptom of acute HBV or HCV
infection) to the individual, wherein the ISS comprises the
sequence 5'-C, G-3' and wherein an HBV or HCV antigen is not
administered in conjunction with administration of the composition
(i.e., antigen is not administered with the ISS-containing
polynucleotide), thereby preventing a symptom of acute HBV or HCV
infection. The individual may have been exposed to and/or infected
by HBV or HCV.
[0019] Another embodiment of the invention provides methods of
reducing severity of a symptom of acute HBV and/or HCV infection in
an individual which entail administering an effective amount of a
composition comprising a polynucleotide comprising an ISS to the
individual, wherein the ISS comprises the sequence 5'-C, G-3' and
wherein an HBV or HCV antigen is not administered in conjunction
with administration of the composition, thereby reducing severity
of a symptom of acute HBV or HCV infection. The individual may have
been exposed to and/or infected by HBV or HCV.
[0020] Another embodiment of the invention provides methods of
delaying development of a symptom of acute HBV or HCV infection in
an individual which entail administering an effective amount of a
composition comprising a polynucleotide comprising an ISS to the
individual, wherein the ISS comprises the sequence 5'-C, G-3' and
wherein an HBV or HCV antigen is not administered in conjunction
with administration of the composition, thereby delaying
development of a symptom of acute HBV or HCV infection. The
individual may have been exposed to and/or infected by HBV and/or
HCV.
[0021] Another embodiment of the invention provides methods of
reducing duration of a symptom of acute HBV or HCV infection in an
individual which entail administering an effective amount of a
composition comprising a polynucleotide comprising an ISS to the
individual, wherein the ISS comprises the sequence 5'-C, G-3' and
wherein an HBV or HCV antigen is not administered in conjunction
with administration of the composition, thereby reducing duration
of a symptom of acute HBV or HCV infection The individual may have
been exposed to and/or infected by HBV and/or HCV.
[0022] Another embodiment of the invention provides methods for
preventing a symptom of chronic HBV or HCV infection in an
individual which entail administering an effective amount of a
composition comprising a polynucleotide comprising an ISS to the
individual, wherein the ISS comprises the sequence 5'-C, G-3' and
wherein an HBV or HCV antigen is not administered in conjunction
with administration of the composition, thereby preventing a
symptom of chronic HBV or HCV infection. The individual may have
been exposed to and/or infected by HBV and/or HCV.
[0023] Another embodiment of the invention provides methods of
reducing severity of a symptom of chronic HBV or HCV infection in
an individual which entail administering an effective amount of a
composition comprising a polynucleotide comprising an ISS to the
individual, wherein the ISS comprises the sequence 5'-C, G-3' and
wherein an HBV or HCV antigen is not administered in conjunction
with administration of the composition, thereby reducing severity
of a symptom of chronic HBV or HCV infection. The individual may
have been exposed to and/or infected by HBV and/or HCV.
[0024] Another embodiment of the invention provides methods of
delaying development of a symptom of chronic HBV or HCV infection
in an individual which entail administering an effective amount of
a composition comprising a polynucleotide comprising an ISS to the
individual, wherein the ISS comprises the sequence 5'-C, G-3' and
wherein an HBV or HCV antigen is not administered in conjunction
with administration of the composition, thereby delaying
development of a symptom of chronic HBV or HCV infection. The
individual may have been exposed to and/or infected by HBV and/or
HCV.
[0025] Another embodiment of the invention provides methods of
reducing duration of a symptom of chronic HBV or HCV infection in
an individual which entail administering an effective amount of a
composition comprising a polynucleotide comprising an ISS to the
individual, wherein the ISS comprises the sequence 5'-C, G-3' and
wherein an HBV or HCV antigen is not administered in conjunction
with administration of the composition, thereby reducing duration
of a symptom of chronic HBV or HCV infection. The individual may
have been exposed to and/or infected by HBVand/or HCV.
[0026] Another embodiment of the invention provides methods of
suppressing an HBV or HCV infection in an individual infected with
or at risk of being infected with HBV or HCV which entail
administering an effective amount of a composition comprising a
polynucleotide comprising an ISS to the individual, wherein the ISS
comprises the sequence 5'-C, G-3' and wherein an HBV or HCV antigen
is not administered in conjunction with administration of the
composition, thereby suppressing an HBV or HCV infection.
[0027] In further aspect, the invention provides methods for
reducing viremia in an individual exposed to and/or infected with
HBV and/or HCV which entail administering an effective amount of a
composition comprising a polynucleotide comprising an ISS to the
individual, wherein the ISS comprises the sequence 5'-C, G-3' and
wherein an HBV or HCV antigen is not administered in conjunction
with administration of the composition, thereby reducing HBV or HCV
viremia.
[0028] In a further aspect, the invention provides methods for
reducing blood levels of hepatitis virus antigens, preferably HBV
or HCV antigens, in an individual which entail administering an
effective amount of a composition comprising a polynucleotide
comprising an ISS to the individual, wherein the ISS comprises the
sequence 5'-C, G-3' and wherein an HBV or HCV antigen is not
administered in conjunction with administration of the composition,
thereby reducing blood levels of hepatitis virus antigens. The
individual may have been exposed to and/or infected by HBV or
HCV.
[0029] In another aspect, the invention provides kits for use in
ameliorating a symptom of acute or chronic HBV or HCV infection in
an individual exposed to and/or infected with HBV or HCV. The kits
comprise a composition comprising a polynucleotide comprising an
ISS, wherein the ISS comprises the sequence 5'-C, G-3', wherein the
kit does not comprise an HBV or HCV antigen, and wherein the kits
comprise instructions for administration of the composition to an
individual infected with or exposed to HBV or HCV.
[0030] In some embodiments of the methods and kits of the
invention, the ISS comprises the sequence 5'-purine, purine, C, G,
pyrimidine, pyrimidine, C, G-3' or 5'-purine, purine, C, G,
pyrimidine, pyrimidine, C, C-3'. In further embodiments of the
methods and kits, the ISS comprises a sequence selected from the
group consisting of AACGTTCC, AACGTTCG, GACGTTCC, and GACGTTCG.
[0031] In some embodiments of the methods and kits of the
invention, the ISS comprises the sequence 5'-T, C, G-3'. In some
embodiments of the methods and kits of the invention, the ISS
comprises the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID
NO:1).
[0032] In some embodiments of the methods and kits of the
invention, the individual is a mammal. In further embodiments, the
mammal is human.
[0033] In some embodiments of the methods and kits of the
invention, the virus is HBV.
[0034] In some embodiments of the methods and kits of the
invention, the virus is HCV.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1(A)-(D) are graphs depicting effects of administration
of ISS and control reagents to STC mice on viral titer. Results
shown are blood viral DNA titer (in copies per milliliter) over
time (in days). FIG. 1(A) depicts results for STC mice injected
with ISS at day 0, 7, and 14 (week 0, 1 and 2); FIG. 1(B) depicts
results for STC mice injected with ISS at day 14 (week 2) only;
FIG. 1(C) depicts results for STC mice injected with 100 ng of
murine IL-12 on days 12, 13 and 14; and FIG. 1(D) depicts results
for STC mice injected with phosphate buffered saline (PBS) on days
0, 7 and 14. Error bars indicate.+-.one standard deviation
(SD).
[0036] FIG. 2 is a graph depicting effects of administration of ISS
and control reagents to STC mice on hepatitis B surface antigen
(HBsAg) levels. Results are shown as percent of value at day -1
over time (in days). Open squares indicate results for STC mice
injected with ISS at day 0, 7, and 14 (week 0, 1 and 2); closed
diamonds indicate results for STC mice injected with ISS at day 14
(week 2) only; closed square indicate results for STC mice injected
with 100 ng of murine IL-12 on days 12, 13 and 14; and open
diamonds indicate results for STC mice injected with phosphate
buffered saline on days 0, 7 and 14.
MODES FOR CARRYING OUT THE INVENTION
[0037] We have discovered methods for the treatment of hepatitis B
and C which are applicable to acute and/or chronic phases of
infection. A polynucleotide comprising an immunostimulatory
sequence (an "ISS") is administered to an individual exposed to
and/or infected with hepatitis B virus (HBV) or hepatitis C virus
(HCV). Administration of the ISS without co-administration of viral
antigen, preferably without hepatitis antigen, results in reduced
titer of hepatitis B as well as reduced HBV serum antigens in an
animal model of chronic hepatitis B infection. We reasonably expect
that such reduction would translate to reduction of severity of
infection, including amelioration or even prevention of one or more
symptoms associated with acute and/or chronic infection.
[0038] The invention also relates to kits for treatment and/or
prevention of hepatitis B and/or hepatitis C infection in exposed
individuals. The kits, which do not contain a hepatitis viral
antigen, comprise a polynucleotide comprising an ISS and
instructions describing the administration of an ISS to an
individual for the intended treatment. Kits intended for use on
individuals exposed to or infected with hepatitis B do not include
hepatitis B viral antigens. Kits intended for use on individuals
exposed to or infected with hepatitis C do not include hepatitis C
viral antigens. Kits intended for use on individuals infected with
both hepatitis B and hepatitis C contain neither hepatitis B nor
hepatitis C viral antigens.
[0039] General Techniques
[0040] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook et
al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984);
Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in
Enzymology (Academic Press, Inc.); Handbook of Experimental
Immunology (D. M. Weir & C. C. Blackwell, eds.); Gene Transfer
Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds.,
1987); Current Protocols in Molecular Biology (F. M. Ausubel et
al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et
al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et
al., eds., 1991); The Immunoassay Handbook (David Wild, ed.,
Stockton Press N.Y., 1994); and Methods ofImmunological Analysis
(R. Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH
Verlags gesellschaft mbH, 1993).
[0041] Definitions
[0042] The term "hepatitis B virus" or "HBV" is a term
well-understood in the art and refers to a virus which is a member
of the family hepadnaviridae, which family consists of the genus
Orthohepadnavirus (hepadnaviruses which infect mammals) and
Avihepadnaviridae (hepadnaviruses which infect birds). HBV is a
Orthohepadnavirus which infects humans. Hepatitis B virus is a
lipid-enveloped virus having a diameter of approximately 42 nm and
comprises a circular, double stranded DNA genome. The genome is
contained in a capsid which is enclosed by a lipid envelope studded
with the surface, or "s", antigen (HBsAg). The 20 nm diameter
particles are non-infectious. The main structural component of the
25-27 nm diameter capsid is the core or "c" protein (HBcAg). Also
included in the capsid is a polymerase ("P protein"). The HBV
genome encodes a number of additional products, including the "e"
protein or "HBeAg", which is secreted by infected cells
independently from the virion and other virion-related particles.
Such antigens and/or antibodies against such antigens are generally
diagnostic for HBV.
[0043] The term "hepatitis C virus" or "HCV" is a term
well-understood in the art and refers to a virus which is the sole
member of an unnamed genus of the family flaviviridae. Unlike most
flaviviridae, HCV does not utilize a vector such as an insect, and
humans are the only known host (although chimpanzees may be
infected experimentally). Hepatitis C virus is a lipid-enveloped
virus having a diameter of approximately 30-80 nm in diameter.
Unlike HBV, HCV has a positive strand RNA genome. However, HCV does
not integrate like a retrovirus. Serum titers of HCV tend to be
relatively low, so most diagnostic assays rely on detecting patient
antibodies to a viral protein such as the nucleocapsid protein or
the NS3, NS4 and/or NS5 proteins. Additionally, diagnostic assays
are available which detect genomic viral RNA.
[0044] The term "acute hepatitis infection", as used herein, refers
to acute hepatitis B and/or acute hepatitis C infection, although
not all individuals infected with HBV and/or HCV will exhibit
clinical symptoms of acute hepatitis. Clinical symptoms of acute
hepatitis include elevated bilirubin levels (up to an including
frank jaundice), nausea, fatigue, elevated blood levels of liver
enzymes (e.g., alanine aminotransferase or ALT and/or aspartate
aminotransferase or AST), nausea, and joint and/or abdominal pain.
Acute hepatitis may be the result of an initial infection by HBV or
HCV, or may be the result of a "flare up" or relapse in a
chronically infected patient. Acute hepatitis as a result of an
initial infection by HBV or HCV may be distinguished from recurrent
acute hepatitis by examination of anti-hepatitis virus
immunoglobin. High levels of anti-virus IgM (and low levels of
anti-virus IgG) are found in acute hepatitis due to an initial
infection of HBV or HCV, while the reverse is found in relapsing or
recurring acute hepatitis.
[0045] The term "chronic hepatitis", as used herein, refers to a
disorder in which liver inflammation due to chronic HBV or HCV
infection is present for at least six continuous months. Chronic
hepatitis patients may suffer from fatigue, general malaise and/or
abdominal pain. Chronic hepatitis due to HBV or HCV may be
diagnosed by the use of diagnostic testing for the presence of HBV
or HCV. Chronic hepatitis may be divided into two types (either or
both of which are included in the invention), chronic active
hepatitis and chronic persistent hepatitis. Chronic active
hepatitis is hepatitis which is causing active damage to the liver,
such as ongoing hepatocellular necrosis. Chronic persistent
hepatitis is a hepatitis infection which is not currently causing
damage, although pre-existing liver damage may be present. While
the prognosis for patients with chronic persistent hepatitis is
better than that for those with chronic active hepatitis, chronic
persistent hepatitis may develop into chronic active hepatitis. The
sequelae of chronic hepatitis include portal hypertension,
cirrhosis, and hepatocellular carcinoma (HCC).
[0046] "Exposure" to a virus denotes encounter with HBV or HCV
which allows infection, such as, for example, upon transfer of
blood or a blood product from an infected individual such as by
transfusion of contaminated blood, or a "needle stick" accident or
incident involving a needle used on an HBV or HCV positive
individual.
[0047] An individual is "seronegative" for a virus if antibodies
specific to the virus cannot be detected in blood or serum samples
from the individual using methods standard in the art, such as
ELISA. Conversely, an individual is "seropositive" for a virus if
antibodies specific for the virus can be detected in blood or serum
samples from the individual using methods standard in the art, such
as ELISA. An individual is said to "seroconvert" for a virus when
antibodies to the virus can be detected in blood or serum from an
individual who was previously seronegative.
[0048] A "symptom of HBV or HCV" refers to a symptom HBV and/or HCV
infection. Such symptoms are well known in the art and include
symptoms of acute and chronic hepatitis B and C. Symptoms of HBV or
HCV include physical symptoms such as jaundice, abdominal pain,
fatigue, malaise, nausea, and vomiting, as well as
clinical/laboratory findings associated with hepatitis, such as
elevated liver enzyme levels (e.g., alanine aminotransferase, ALT,
aspartate aminotransferase, AST, and/or lactate dehydrogenase,
LDH), elevated bilirubin, HBV and/or HCV viremia or antigen levels,
portal hypertension, cirrhosis, anorexia, and other symptoms
recognized in the art.
[0049] "Suppressing" hepatitis virus infection refers to any aspect
of hepatitis B or C virus infection, such as a physical symptom
(e.g., jaundice, fatigue, abdominal pain), a hepatitis-associated
laboratory finding (e.g., liver enzyme levels in blood or
cirrhosis), viral replication, or amount (titer) of virus, which is
curtailed, inhibited, or reduced (in terms of severity and/or
duration) in an individual or a population of individuals treated
with an ISS-containing polynucleotide in accordance with the
invention as compared to an aspect of viral infection in an
individual or a population of individuals not treated in accordance
with the invention. Reduction in viral titer includes, but is not
limited to, elimination of the virus from an infected site or
individual. Viral infection can be assessed by any means known in
the art, including, but not limited to, detection of symptoms,
measurement of liver function by laboratory testing, liver biopsy,
direct or indirect measurement of liver portal vein pressure, and
measurement of virus particles, viral nucleic acid or viral antigen
titer and detection and/or measurement of anti-virus antibodies.
Anti-virus antibodies are widely used to detect and monitor viral
infection and generally are commercially available.
[0050] "Palliating" a disease or one or more symptoms of a disease
or infection means lessening the extent and/or time course of
undesirable clinical manifestations of a disease state or infection
in an individual or population of individuals treated with an ISS
in accordance with the invention.
[0051] As used herein, "delaying" development of viral infection or
a symptom of hepatitis means to defer, hinder, slow, retard,
stabilize, and/or postpone development of the disease or symptom
when compared to not using the method(s) of the invention. This
delay can be of varying lengths of time, depending on the history
of the disease and/or individual being treated. As is evident to
one skilled in the art, a sufficient or significant delay can, in
effect, encompass prevention, in that the individual does not
develop the disease.
[0052] "Reducing severity of a symptom" or "ameliorating a symptom"
of viral infection means a lessening or improvement of one or more
symptoms of hepatitis as compared to not administering an
ISS-containing polynucleotide. "Reducing severity" also includes
shortening or reduction in duration of a symptom. For hepatitis B
and hepatitis C, these symptoms are well known in the art and
include, but are not limited to, jaundice, abdominal pain, fatigue,
malaise, nausea, and vomiting, as well as clinical/laboratory
findings associated with hepatitis, such as elevated liver enzyme
levels (e.g., ALT, AST, and/or LDH), elevated bilirubin, HBV and/or
HCV viremia or antigen levels, portal hypertension, cirrhosis,
anorexia, and other symptoms recognized in the art.
[0053] "Reducing duration of viral infection" means the length of
time of viral infection (usually indicated by symptoms) is reduced,
or shortened, as compared to not administering an ISS-containing
polynucleotide.
[0054] "Preventing a symptom of infection" by a hepatitis virus
means that the symptom does not appear after exposure to the
virus.
[0055] The term "infected individual", as used herein, refers to an
individual who has been infected by HBV and/or HCV. Symptoms of HBV
infection include seropositivity for anti-HBsAg, HBeAg, or HBcAg,
presence of HBsAg, HBeAg, or HBcAg in samples from the individual,
or presence of HBV DNA in samples from the individual, as well as
other symptoms known in the art. Symptoms of HCV infection include
seropositivity for antibodies to nucleocapsid protein or the NS3,
NS4 and/or NS5 proteins, presence of nucleocapsid protein or the
NS3, NS4 and/or NS5 proteins in samples from the individual, or
presence of HCV RNA or DNA in samples from the individual, as well
as other symptoms known in the art.
[0056] A "biological sample" encompasses a variety of sample types
obtained from an individual and can be used in a diagnostic or
monitoring assay. The definition encompasses blood and other liquid
samples of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures or cells derived therefrom, and the
progeny thereof. The definition also includes samples that have
been manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components, such as proteins or polynucleotides. The term
"biological sample" encompasses a clinical sample, and also
includes cells in culture, cell supernatants, cell lysates, serum,
plasma, biological fluid, and tissue samples.
[0057] "Viral titer" is a term well-known in the art and indicates
the amount of virus in a given biological sample. "Viremia" is a
term well-known in the art as the presence of virus in the
bloodstream and/or viral titer in a blood or serum sample. Amount
of virus are indicated by various measurements, including, but not
limited to, amount of viral nucleic acid; presence of viral
particles (such as HBsAg or hepatitis B surface antigen particles);
replicating units (RU); plaque forming units (PFU). Generally, for
fluid samples such as blood and urine, amount of virus is
determined per unit fluid, such as milliliters. For solid samples
such as tissue samples, amount of virus is determined per weight
unit, such as grams. Methods for determining amount of virus are
known in the art and described herein.
[0058] An "individual" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to,
humans, farm animals, sport animals, rodents, primates and certain
pets. Vertebrates also include, but are not limited to, birds
(i.e., avian individuals) and reptiles (i.e., reptilian
individuals).
[0059] The term "ISS" as used herein refers to polynucleotide
sequences that effect a measurable immune response as measured in
vitro, in vivo and/or ex vivo. Examples of measurable immune
responses include, but are not limited to, antigen-specific
antibody production, secretion of cytokines, activation or
expansion of lymphocyte populations such as NK cells, CD4.sup.+ T
lymphocytes, CD8.sup.+ T lymphocytes, B lymphocytes, and the like.
Preferably, the ISS sequences preferentially activate a Th1-type
response. A polynucleotide for use in methods of the invention
contains at least one ISS.
[0060] As used interchangeably herein, the terms "polynucleotide"
and "oligonucleotide" include single-stranded DNA (ssDNA),
double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) and
double-stranded RNA (dsRNA), modified oligonucleotides and
oligonucleosides or combinations thereof. The oligonucleotide can
be linearly or circularly configured, or the oligonucleotide can
contain both linear and circular segments.
[0061] "Adjuvant" refers to a substance which, when added to an
immunogenic agent such as antigen, nonspecifically enhances or
potentiates an immune response to the agent in the recipient host
upon exposure to the mixture.
[0062] An "effective amount" or a "sufficient amount" of a
substance is an amount sufficient to effect beneficial or desired
results, including clinical results. An effective amount can be
administered in one or more administrations. A "therapeutically
effective amount" is an amount to effect beneficial clinical
results, including, but not limited to, alleviation of one or more
symptoms associated with viral infection as well as prevention of
disease (e.g., prevention of one or more symptoms of
infection).
[0063] A microcarrier is considered "biodegradable" if it is
degradable or erodable under normal mammalian physiological
conditions. Generally, a microcarrier is considered biodegradable
if it is degraded (i.e., loses at least 5% of its mass and/or
average polymer length) after a 72 hour incubation at 37.degree. C.
in normal human serum. Conversely, a microcarrier is considered
"nonbiodegradable" if it is not degraded or eroded under normal
mammalian physiological conditions. Generally, a microcarrier is
considered nonbiodegradable if it not degraded (i.e., loses less
than 5% of its mass and/or average polymer length) after at 72 hour
incubation at 37.degree. C. in normal human serum.
[0064] The term "immunostimulatory sequence-microcarrier complex"
or "ISS-MC complex" refers to a complex of an ISS-containing
polynucleotide and a microcarrier. The components of the complex
may be covalently or non-covalently linked. Non-covalent linkages
may be mediated by any non-covalent bonding force, including by
hydrophobic interaction, ionic (electrostatic) bonding, hydrogen
bonds and/or van der Waals attractions . In the case of hydrophobic
linkages, the linkage is generally via a hydrophobic moiety (e.g.,
cholesterol) covalently linked to the ISS.
[0065] As used herein, the term "comprising" and its cognates are
used in their inclusive sense; that is, equivalent to the term
"including" and its corresponding cognates.
[0066] As used herein, the singular form "a", "an", and "the"
includes plural references unless indicated otherwise. For example,
"a" symptom of viral infection includes one or more additional
symptoms.
[0067] Methods of the Invention
[0068] The invention provides methods of ameliorating (i.e.,
reducing severity) and/or preventing one or more symptoms of acute
and/or chronic HBV and HCV virus infection which may include
reducing incidence of or delaying of appearance of sequelae of HBV
and/or HCV infection (i.e., cirrhosis or fulminant liver failure)
by administering an ISS-containing polynucleotide (used
interchangeably herein with "ISS") to an individual without
administering a HBV or HCV antigen. The invention also provides
methods of reducing viremia as well as methods of reducing levels
of hepatitis viral antigen(s) in blood. Controlling and/or reducing
viral load in an individual has several beneficial aspects, since
hepatitis viruses not only cause acute disease but can also lead to
chronic infection and other disease states. In addition,
transmission of hepatitis B and C can occur through blood and blood
products, perinatally and via sexual contact. The hepatitis virus
may be HBV or HCV, although concurrent infections with both HBV and
HCV may also be treated. It should be noted that hepatitis D virus
infection may be present in individuals infected with HBV.
[0069] An ISS-containing composition which includes neither a HBV
nor HCV antigen is administered to an individual exposed to,
infected with, and/or exhibiting one or more symptoms of infection
by HBV and/or HCV. Individuals receiving ISS are preferably
mammals, more preferably humans. In accordance with the invention,
neither HBV or HCV antigen is administered to the individual in
conjunction with administration of an ISS (i.e., is not
administered in a separate administration at or about the time of
administration of the ISS).
[0070] In some embodiments, the individual has been exposed to HBV
and/or HCV. An exposed individual can be easily identified by a
skilled clinician or epidemiologist. Generally, an exposed
individual is an individual that has been exposed to HBV and/or HCV
by a route through which HBV and/or HCV can be transmitted. For
example, an exposed individual may be a person who has been
percutaneously exposed to blood or a blood product derived from an
individual infected with HBV and/or HCV (e.g., by transfusion or by
a "needle stick" accident). Alternately, the exposed individual may
be a child born to an individual infected with HBV or HCV or the
sexual partner of an HBV or HCV infected individual not practicing
barrier methods of contraception.
[0071] In other embodiments, the individual is infected with HBV
and/or HCV. Infection by HBV or HCV may be detected by diagnostic
testing, or by clinical assessment of the infected individual.
Because not all infected individuals exhibit overt symptoms of
hepatitis, diagnostic assays which detect viral antigen(s), viral
DNA or RNA, or host antibodies against viral antigen(s) are
considered more reliable indicators of infection. Generally, HBsAg
is a diagnostic antigen for HBV and the nucleocapsid, NS3, NS4
and/or NS5 proteins are diagnostic antigens for HCV. Infection is
usually engendered by percutaneous exposure to blood or blood
products from an infected individual, such as through transfusion,
sharing of needles during intravenous drug use, or a "needle stick"
incident, although sexual transmission and "vertical" transmission
from mother to child during childbirth or the perinatal period are
also routes for infection.
[0072] In some embodiments, the individual may have chronic or
acute hepatitis B and/or hepatitis C. Acute hepatitis may be easily
recognized by one of skill in the art, and is characterized by
jaundice, fatigue, malaise, elevated blood levels of liver enzymes
such as AST and/or ALT, dark urine and other symptoms known to
those of skill in the art. However, as most types of hepatitis are
characterized by these symptoms, positive diagnostic test for HBV
or HCV is required to identify hepatitis B or hepatitis C,
respectively. Chronic hepatitis B and hepatitis C are generally not
characterized by any specific overt symptoms, although the sequelae
of these disorders, such as hepatomegaly, disrupted clotting (due
to reduced levels of clotting factors produced by the liver),
ascites formation, cirrhosis, portal hypertension, and the like,
are easily recognized by the clinician. However, altered liver
fimction (as demonstrated by increased blood levels of liver
enzymes) can be detected by laboratory testing. Additionally,
chronic hepatitis B and hepatitis C sufferers may be subject to
occasional "flare ups", in which the symptoms of acute hepatitis
return.
[0073] ISS
[0074] The methods of this invention entail administering a
polynucleotide comprising an ISS (or a composition comprising such
a polynucleotide). In accordance with the present invention, the
immunomodulatory polynucleotide contains at least one ISS, and can
contain multiple ISSs. The ISSs can be adjacent within the
polynucleotide, or they can be separated by additional nucleotide
bases within the polynucleotide. Alternately, multiple ISSs may be
delivered as individual polynucleotides.
[0075] ISS have been described in the art and may be readily
identified using standard assays which indicate various aspects of
the immune response, such as cytokine secretion, antibody
production, NK cell activation and T cell proliferation. See, e.g.,
WO 97/28259; WO 98/16247; WO 99/11275; Krieg et al. (1995);
Yamamoto et al. (1992); Ballas et al. (1996); Klinman et al.
(1997); Sato et al. (1996); Pisetsky (1996a); Shimada et al. (1986)
Jpn. J. Cancer Res. 77:808-816; Cowdery et al. (1996) J. Immunol.
156:4570-4575; Roman et al. (1997); and Lipford et al. (1997a).
[0076] The ISS can be of any length greater than 6 bases or base
pairs and generally comprises the sequence 5'-cytosine, guaineg-3',
preferably greater than 15 bases or base pairs, more preferably
greater than 20 bases or base pairs in length. As is well-knowm in
the art, the cytosine of the 5'-cytosine, guanine-3' sequence is
unmethylated. An ISS may also comprise the sequence 5'-purine,
purine, C, G. pyrimidine, pyrimidine, C, G-3'. An ISS may also
comprise the sequence 5'-purine, purine, C, G, pyrimidine,
pyrimidine, C, C-3'. As indicated in polynucleotide sequences
below, an ISS may comprise (i.e., contain one or more of) the
sequence 5'-T, C, G-3'. In some embodiments, an ISS may comprise
the sequence 5'-C, G, pyrimidine, pyrimidine, C, G-3' (such as
5'-CGTTCG-3'). In some embodiments, an ISS may comprise the
sequence 5'-C, G, pyrimidine, pyrimidine, C, G, purine, purine-3'.
In some embodiments, an ISS comprises the sequence 5'-purine,
purine, C, G, pyrimidine, pyrimidine-3' (such as 5'-AACGTT-3').
[0077] In some embodiments, an ISS may comprise the sequence
5'-purine, T, C, G, pyrimidine, pyrimidine-3'.
[0078] In some embodiments, an ISS-containing polynucleotide is
less than about any of the following lengths (in bases or base
pairs): 10,000; 5,000; 2500; 2000; 1500; 1250; 1000; 750; 500; 300;
250; 200; 175; 150; 125; 100; 75; 50; 25; 10. In some embodiments,
an ISS-containing polynucleotide is greater than about any of the
following lengths (in bases or base pairs): 8; 10; 15; 20; 25; 30;
40; 50; 60; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 500;
750; 1000; 2000; 5000; 7500; 10000; 20000; 50000. Alternately, the
ISS can be any of a range of sizes having an upper limit of 10,000;
5,000; 2500; 2000; 1500; 1250; 1000; 750; 500; 300; 250; 200; 175;
150; 125; 100; 75; 50; 25; or 10 and an independently selected
lower limit of 8; 10; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125;
150; 175; 200; 250; 300; 350; 400; 500; 750; 1000; 2000; 5000;
7500, wherein the lower limit is less than the upper limit.
[0079] In some embodiments, the ISS comprises any of the following
sequences:
1 In some embodiments, the ISS comprises any of the following
sequences: GACGCTCC; GACGTCCC; GACGTTCC; GACGCCCC; AGCGTTCC;
AGCGCTCC; AGCGTCCC; AGCGCCCC; AACGTCCC; AACGCCCC; AACGTTCC;
AACGCTCC; GGCGTTCC; GGCGCTCC; GGCGTCCC; GGCGCCCC; GACGCTCG;
GACGTCCG; GACGCCCG; GACGTTCG; AGCGCTCG; AGCGTTCG; AGCGTCCG;
AGCGCCCG; AACGTCCG; AACGCCCG; AACGTTCG; AACGCTCG; GGCGTTCG;
GGCGCTCG; GGCGTCCG; GGCGCCCG. In some embodiments, the
immunomodulatory polynucleotide comprises the sequence
5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1). In some embodiments,
the ISS comprises any of the following sequences: GACGCU; GACGUC;
GACGUU; GACGUT; GACGTU; AGCGUU; AGCGCU; AGCGUC; AGCGUT; AGCGTU;
AACGUC; AACGUU; AACGCU; AACGUT; AACGTU; GGCGUU; GGCGCU; GGCGUC;
GGCGUT; GGCGTU. In some embodiments, the ISS comprises any of the
following sequences: GABGCTCC; GABGTCCC; GABGTTCC; GABGCCCC;
AGBGTTCC; AGBGCTCC; AGBGTCCC; AGBGCCCC; AABGTCCC; AABGCCCC;
AABGTTCC; AABGCTCC; GGBGTTCC; GGBGCTCC; GGBGTCCC; GGBGCCCC;
GABGCTCC; GABGTCCC; GABGCCCC; GABGTTCC; AGBGCTCG; AGBGTTCG;
AGBGTCCG; AGBGCCCG; AABGTCCG; AABGCCCG; AABGTTCG; AABGCTCG;
GGBGTTCG; GGBGCTCG; GGBGTCCG; GGBGCCCG; GABGCTBG; GABGTCBG;
GABGCCBG; GABGTTBG; AGBGCTBG; AGBGTTBG; AGBGTCBG; AGBGCCBG;
AABGTCBG; AABGCCBG; AABGTTBG; AABGCTBG; GGBGTTBG; GGBGCTBG;
GGBGTCBG; GGBGCCBG, where B is 5-bromocytosine. In some
embodiments, the ISS comprises any of the following sequences:
GABGCUCC; GABGUCCC; GABGUTCC; GABGTUCC; GABGUUCC; AGBGUUCC;
AGBGTUCC; AGBGUTCC; AGBGCUCC; AGBGUCCC; AABGUCCC; AABGUUCC;
AABGUTCC; AABGTUCC; AABGCUCC; GGBGUUCC; GGBGUTCC; GGBGTUCC;
GGBGCUCC; GGBGUCCC; GABGCUCG; GABGUCCG; GABGUUCG; GABGUTCG;
GABGTUCG; AGBGCUCG; AGBGUUCG; AGBGUTCG; AGBGTUCG; AGBGUCCG;
AABGUCCG; AABGUUCG; AABGUTCG; AABGTUCG; AABGCUCG; GGBGUUCG;
GGBGUTCG; GGBGTUCG; GGBGCUCG; GGBGUCCG; GABGCUBG; GABGUCBG;
GABGUUBG; GABGUTBG; GABGTUBG; AGBGCUBG; AGBGUUBG; AGBGUCBG;
AGBGUTBG; AGBGTUBG; AABGUCBG; AABGUUBG; AABGUTBG; AABGTUBG;
AABGCUBG; GGBGUUBG; GGBGUTBG; GGBGTUBG; GGBGCUBG; GGBGUCBG, where B
is 5-bromocytosine. In other embodiments, the ISS comprises any of
the sequences: 5'-TGACCGTGAACGTTCGAGATGA- -3' (SEQ ID NO:2);
5'-TCATCTCGAACGTTCCACAGTCA-3' (SEQ ID NO:3);
5'-TGACTGTGAACGTTCCAGATGA-3' (SEQ ID NO:4);
5'-TCCATAACGTTCGCCTAACGTTCGTC-3' (SEQ ID NO:5);
5'-TGACTGTGAABGTTCCAGATGA-3' (SEQ ID NO:6), where B is
5-bromocytosine; 5'-TGACTGTGAABGTTCGAGATGA-3' (SEQ ID NO:7), where
B is 5-bromocytosine and 5'-TGACTGTGAABGTTBGAGATGA-3' (SEQ ID
NO:8), where B is 5-bromocytosine.
[0080] An ISS and/or ISS-containing polynucleotide may contain
modifications. Modifications of ISS include any known in the art,
but are not limited to, modifications of the 3'-OH or 5'-OH group,
modifications of the nucleotide base, modifications of the sugar
component, and modifications of the phosphate group. Various such
modifications are described below.
[0081] An ISS may be single stranded or double stranded DNA, as
well as single or double-stranded RNA or other modified
polynucleotides. An ISS may or may not include one or more
palindromic regions, which may be present in the motifs described
above or may extend beyond the motif. An ISS may comprise
additional flanking sequences, some of which are described herein.
An ISS may contain naturally-occurring or modified, non-naturally
occurring bases, and may contain modified sugar, phosphate, and/or
termini. For example, phosphate modifications include, but are not
limited to, methyl phosphonate, phosphorothioate, phosphoramidate
(bridging or non-bridging), phosphotriester and phosphorodithioate
and may be used in any combination. Other non-phosphate linkages
may also be used. Preferably, oligonucleotides of the present
invention comprise phosphorothioate backbones. Sugar modifications
known in the field, such as 2'-alkoxy-RNA analogs, 2'-amino-RNA
analogs and 2'-alkoxy- or amino-RNA/DNA chimeras and others
described herein, may also be made and combined with any phosphate
modification. Examples of base modifications include, but are not
limited to, addition of an electron-withdrawing moiety to C-5
and/or C-6 of a cytosine of the ISS (e.g., 5-bromocytosine,
5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine).
[0082] The ISS can be synthesized using techniques and nucleic acid
synthesis equipment which are well known in the art including, but
not limited to, enzymatic methods, chemical methods, and the
degradation of larger oligonucleotide sequences. See, for example,
Ausubel et al. (1987); and Sambrook et al. (1989). When assembled
enzymatically, the individual units can be ligated, for example,
with a ligase such as T4 DNA or RNA ligase. U.S. Pat. No.
5,124,246. Oligonucleotide degradation can be accomplished through
the exposure of an oligonucleotide to a nuclease, as exemplified in
U.S. Pat. No. 4,650,675.
[0083] The ISS can also be isolated using conventional
polynucleotide isolation procedures. Such procedures include, but
are not limited to, hybridization of probes to genomic or cDNA
libraries and synthesis of particular native sequences by the
polymerase chain reaction.
[0084] Circular ISS can be isolated, synthesized through
recombinant methods, or chemically synthesized. Where the circular
ISS is obtained through isolation or through recombinant methods,
the ISS will preferably be a plasmid. The chemical synthesis of
smaller circular oligonucleotides can be performed using any method
described in the literature. See, for instance, Gao et al. (1995)
Nucleic Acids Res. 23:2025-2029; and Wang et al. (1994) Nucleic
Acids Res. 22:2326-2333.
[0085] The techniques for making oligonucleotides and modified
oligonucleotides are known in the art. Naturally occurring DNA or
RNA, containing phosphodiester linkages, is generally synthesized
by sequentially coupling the appropriate nucleoside phosphoramidite
to the 5'-hydroxy group of the growing oligonucleotide attached to
a solid support at the 3'-end, followed by oxidation of the
intermediate phosphite triester to a phosphate triester. Once the
desired oligonucleotide sequence has been synthesized, the
oligonucleotide is removed from the support, the phosphate triester
groups are deprotected to phosphate diesters and the nucleoside
bases are deprotected using aqueous ammonia or other bases. See,
for example, Beaucage (1993) "Oligodeoxyribonucleotide Synthesis"
in Protocols for Oligonucleotides and Analogs, Synthesis and
Properties (Agrawal, ed.) Humana Press, Totowa, N.J.; Warner et al.
(1984) DNA 3:401 and U.S. Pat. No. 4,458,066.
[0086] The ISS can also contain phosphate-modified
oligonucleotides. Synthesis of polynucleotides containing modified
phosphate linkages or non-phosphate linkages is also know in the
art. For a review, see Matteucci (1997) "Oligonucleotide Analogs:
an Overview" in Oligonucleotides as Therapeutic Agents, (D. J.
Chadwick and G. Cardew, ed.) John Wiley and Sons, New York, N.Y.
The phosphorous derivative (or modified phosphate group) which can
be attached to the sugar or sugar analog moiety in the
oligonucleotides of the present invention can be a monophosphate,
diphosphate, triphosphate, alkylphosphonate, phosphorothioate,
phosphorodithioate or the like. The preparation of the above-noted
phosphate analogs, and their incorporation into nucleotides,
modified nucleotides and oligonucleotides, per se, is also known
and need not be described here in detail Peyrottes et al. (1996)
Nucleic Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucleic
Acids Res. 24:2318-2323; and Schultz et al (1996) Nucleic Acids
Res. 24:2966-2973. For example, synthesis of phosphorothioate
oligonucleotides is similar to that described above for naturally
occurring oligonucleotides except that the oxidation step is
replaced by a sulfurization step (Zon (1993) "Oligonucleoside
Phosphorothioates" in Protocols for Oligonucleotides and Analogs,
Synthesis and Properties (Agrawal, ed.) Humana Press, pp. 165-190).
Similarly the synthesis of other phosphate analogs, such as
phosphotriester (Miller et al. (1971) JACS 93:6657-6665),
non-bridging phosphoramidates (Jager et al. (1988) Biochem.
27:7247-7246), N3' to P5' phosphoramidates (Nelson et al. (1997)
JOC 62:7278-7287) and phosphorodithioates (U.S. Pat. No. 5,453,496)
has also been described. Other non-phosphorous based modified
oligonucleotides can also be used (Stirchak et al. (1989) Nucleic
Acids Res. 17:6129-6141). Oligonucleotides with phosphorothioate
backbones can be more immunogenic than those with phosphodiester
backbones and appear to be more resistant to degradation after
injection into the host. Braun et al. (1988) J. Immunol.
141:2084-2089; and Latimer et al. (1995) Mol. Immunol.
32:1057-1064.
[0087] ISS-containing polynucleotides used in the invention can
comprise ribonucleotides (containing ribose as the only or
principal sugar component), deoxyribonucleotides (containing
deoxyribose as the principal sugar component), or, as is known in
the art, modified sugars or sugar analogs can be incorporated in
the ISS. Thus, in addition to ribose and deoxyribose, the sugar
moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose,
arabinose, xylose, lyxose, and a sugar "analog" cyclopentyl group.
The sugar can be in pyranosyl or in a furanosyl form. In the ISS,
the sugar moiety is preferably the furanoside of ribose,
deoxyribose, arabinose or 2'-0-alkylribose, and the sugar can be
attached to the respective heterocyclic bases either in .alpha. or
.beta. anomeric configuration. Sugar modifications include, but are
not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs and
2'-alkoxy- or amino-RNA/DNA chimeras. The preparation of these
sugars or sugar analogs and the respective "nucleosides" wherein
such sugars or analogs are attached to a heterocyclic base (nucleic
acid base) per se is known, and need not be described here, except
to the extent such preparation can pertain to any specific example.
Sugar modifications may also be made and combined with any
phosphate modification in the preparation of an ISS.
[0088] The heterocyclic bases, or nucleic acid bases, which are
incorporated in the ISS can be the naturally-occurring principal
purine and pyrimidine bases, (namely uracil or thymine, cytosine,
adenine and guanine, as mentioned above), as well as
naturally-occurring and synthetic modifications of said principal
bases.
[0089] Those skilled in the art will recognize that a large number
of "synthetic" non-natural nucleosides comprising various
heterocyclic bases and various sugar moieties (and sugar analogs)
are available in the art, and that as long as other criteria of the
present invention are satisfied, the ISS can include one or several
heterocyclic bases other than the principal five base components of
naturally-occurring nucleic acids. Preferably, however, the
heterocyclic base in the ISS includes, but is not limited to,
uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl,
guanin-8-yl, 4-aminopyrrolo [2.3-d] pyrimidin-5-yl,
2-amino-4-oxopyrolo [2,3-d] pyrimidin-5-yl, 2-amino-4-oxopyrrolo
[2.3-d] pyrimidin-3-yl groups, where the purines are attached to
the sugar moiety of the ISS via the 9-position, the pyrimidines via
the 1-position, the pyrrolopyrimidines via the 7-position and the
pyrazolopyrimidines via the 1-position.
[0090] The ISS may comprise at least one modified base as
described, for example, in the commonly owned international
application WO 99/62923. As used herein, the term "modified base"
is synonymous with "base analog", for example, "modified cytosine"
is synonymous with "cytosine analog." Similarly, "modified"
nucleosides or nucleotides are herein defined as being synonymous
with nucleoside or nucleotide "analogs." Examples of base
modifications include, but are not limited to, addition of an
electron-withdrawing moiety to C-5 and/or C-6 of a cytosine of the
ISS. Preferably, the electron-withdrawing moiety is a halogen. Such
modified cytosines can include, but are not limited to,
azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine,
chlorinated cytosine, cyclocytosine, cytosine arabinoside,
5-fluorocytosine, fluoropyrimidine, fluorouracil,
5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil,
5-nitrocytosine, uracil, and any other pyrimidine analog or
modified pyrimidine.
[0091] The preparation of base-modified nucleosides, and the
synthesis of modified oligonucleotides using said base-modified
nucleosides as precursors, has been described, for example, in U.S.
Pat. Nos. 4,910,300, 4,948,882, and 5,093,232. These base-modified
nucleosides have been designed so that they can be incorporated by
chemical synthesis into either terminal or internal positions of an
oligonucleotide. Such base-modified nucleosides, present at either
terminal or internal positions of an oligonucleotide, can serve as
sites for attachment of a peptide or other antigen. Nucleosides
modified in their sugar moiety have also been described (including,
but not limited to, e.g., U.S. Pat. Nos. 4,849,513, 5,015,733,
5,118,800, 5,118,802) and can be used similarly.
[0092] The ISS used in the methods of the invention may be produced
as ISS microcarrier complexes. ISS-microcarrier complexes comprise
an ISS-containing polynucleotide bound to a microcarrier (MC).
ISS-MC complexes comprise an ISS bound to the surface of a
microcarrier (i.e., the ISS is not encapsulated in the MC),
adsorbed within a microcarrier (e.g., adsorbed to PLGA beads), or
encapsulated within a MC (e.g., incorporated within liposomes).
[0093] ISS-containing oligonucleotides bound to microparticles
(SEPHAROSE.RTM. beads) have previously been shown to have
immunostimulatory activity in vitro (Liang et al., (1996), J. Clin.
Invest. 98:1119-1129). However, recent results show that
ISS-containing oligonucleotides bound to gold, latex and magnetic
particles are not active in stimulating proliferation of 7TD1
cells, which proliferate in response to ISS-containing
oligonucleotides (Manzel et al., (1999), Antisense Nucl. Acid Drug
Dev. 9:459-464).
[0094] Microcarriers are not soluble in pure water, and are less
than about 50-60 .mu.m in size, preferably less than about 10 .mu.m
in size, more preferably from about 10 nm to about 10 .mu.m, 25 nm
to about 5 .mu.m, 50 nm to about 4.5 .mu.m or 1.0 .mu.m to about
2.0 .mu.m in size. Microcarrers may be any shape, such as
spherical, ellipsoidal, rod-shaped, and the like, although
spherical microcarriers are normally preferred. Preferred
microcarriers have sizes of or about 50 nm, 200 nm, 1 .mu.m, 1.2
.mu.m, 1.4 .mu.m, 1.5 .mu.m, 1.6 .mu.m, 1.8 .mu.m, 2.0 .mu.m, 2.5
.mu.m or 4.5 .mu.m. The "size" of a microcarier is generally the
"design size" or intended size of the particles stated by the
manufacturer. Size may be a directly measured dimension, such as
average or maximum diameter, or may be determined by an indirect
assay such as a filtration screening assay. Direct measurement of
microcarrier size is typically carried out by microscopy, generally
light microscopy or scanning electron microscopy (SEM), in
comparison with particles of known size or by reference to a
micrometer. As minor variations in size arise during the
manufacturing process, microcarriers are considered to be of a
stated size if measurements show the microcarriers are.+-.about
5-10% of the stated measurement. Size characteristics may also be
determined by dynamic light scattering. Alternately, microcarrier
size may be determined by filtration screening assays. A
microcarrier is less than a stated size if at least 97% of the
particles pass through a "screen-type" filter (i.e., a filter in
which retained particles are on the surface of the filter, such as
polycarbonate or polyethersulfone filters, as opposed to a "depth
filter" in which retained particles lodge within the filter) of the
stated size. A microcarrier is larger than a stated size if at
least about 97% of the microcarrier particles are retained by a
screen-type filter of the stated size. Thus, at least about 97%
microcarriers of about 10 .mu.m to about 10 nm in size pass through
a 10 .mu.m pore screen filter and are retained by a 10 nm screen
filter.
[0095] As above discussion indicates, reference to a size or size
range for a microcarrier implicitly includes approximate variations
and approximations of the stated size and/or size range. This is
reflected by use of the term "about" when referring to a size
and/or size range, and reference to a size or size range without
reference to "about" does not mean that the size and/or size range
is exact.
[0096] Microcarriers may be solid phase (e.g., polystyrene beads)
or liquid phase (e.g., liposomes, micelles, or oil droplets in an
oil and water emulsion). Liquid phase microcarriers include
liposomes, micelles, oil droplets and other lipid or oil-based
particles. One preferred liquid phase microcarrier is oil droplets
within an oil-in-water emulsion. Preferably, oil-in-water emulsions
used as microcarriers comprise biocompatible substituents such as
squalene. Liquid phase microcarriers are normally considered
nonbiodegradable, but may be biodegradable liquid phase
microcarriers may be produced by incorporation of one or more
biodegradable polymers in the liquid microcarrier formulation. In
one preferred embodiment, the microcarrier is oil droplets in an
oil-in-water emulsion prepared by emulsification of squalene,
sorbitan trioleate, TWEEN 80.RTM. in an aqueous pH buffer.
[0097] Solid phase microcarriers for use in ISS-microcarrier
complexes may be made from biodegradable materials or
nonbiodegradable materials, and may include or exclude agarose or
modified agarose microcarriers. Useful solid phase biodegradable
microcarriers include, but are not limited to: biodegradable
polyesters, such as poly(lactic acid), poly(glycolic acid), and
copolymers (including block copolymers) thereof, as well as block
copolymers of poly(lactic acid) and poly(ethylene glycol);
polyorthoesters such as polymers based
on3,9-diethylidene-2,4,8,10-tetrao- xaspiro[5.5]undecane (DETOSU);
polyanhydrides such as poly(anhydride) polymers based on sebacic
acid, p-(carboxyphenoxy)propane, or p-(carboxyphenoxy)hexane;
polyanhydride imides, such as polyanhydride polymers based on
sebacic acid-derived monomers incorporating amino acids (i.e.,
linked to sebacic acid by imide bonds through the amino-terminal
nitrogen) such as glycine or alanine; polyanhydride esters;
polyphosphazenes, especially poly(phosphazenes) which contain
hydrolysis-sensitive ester groups which can catalyze degradation of
the polymer backbone through generation of carboxylic acid groups
(Schacht et al. (1996) Biotechnol. Bioeng. 1996:102); and
polyamides such as poly(lactic acid-co-lysine). A wide variety of
nonbiodegradable materials suitable for manufacturing microcarriers
are also known, including, but not limited to polystyrene,
polyethylene, latex, gold, and ferromagnetic or paramagnetic
materials. Solid phase microcarriers may be covalently modified to
incorporate one or more moieties for use in linking the ISS, for
example by addition of amine groups for covalent linking using
amine-reactive crosslinkers.
[0098] The ISS-microcarrier complexes may be covalently or
non-covalently linked. Covalently linked ISS-MC complexes may be
directly linked or be linked by a crosslinking moiety of one or
more atoms (typically the residue of a crosslinking agent). The ISS
may be modified to allow or augment binding to the MC (e.g., by
incorporation of a free sulfhydryl for covalent crosslinking or
addition of a hydrophobic moieties such as lipids, steroids,
sterols such as cholesterol, and terpenes, for hydrophobic
bonding), although unmodified ISS may be used for formation of
non-covalent ISS-MC complex formation by electrostatic interaction
or by base pairing (e.g., by base pairing at least one portion of
the ISS with a complementary oligonucleotide bound to the
microcarrier). ISS-containing polynucleotides may be linked to
solid phase microcarriers or other chemical moieties to facilitate
ISS-MC complex formation using conventional technology known in the
art, such as use of available heterobifunctional crosslinkers
(e.g., succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
or its sulfo-derivatives for covalently linking an
amine-derivatized microcarrier and an ISS modified to contain a
free sulfhydryl) or by addition of compounds such as cholesterol
(e.g., by the method of Godard et al. (1995) Eur. J. Biochem.
232:404-410) to facilitate binding to hydrophobic microcarriers
such as oil droplets in oil-in-water emulsions. Alternatively,
modified nucleosides or nucleotides, such as are known in the art,
can be incorporated at either terminus, or at internal positions in
the ISS. These can contain blocked functional groups which, when
deblocked, are reactive with a variety of functional groups which
can be present on, or attached to, the microcarrier or a moiety
which would facilitate binding to a microcarrier. Certain
embodiments of noncovalently linked ISS-MC complexes utilize a
binding pair (e.g., an antibody and its cognate antigen or biotin
and streptavidin or avidin), where one member of the binding pair
is bound to the ISS and the microcarrier is derivatized with the
other member of the binding pair (e.g., a biotinylated ISS and a
streptavidin-derivatized microcarrier may be combined to form a
noncovalently linked ISS-MC complex).
[0099] Non-covalent ISS-MC complexes bound by electrostatic binding
typically exploit the highly negative charge of the polynucleotide
backbone. Accordingly, microcarriers for use in non-covalently
bound ISS-MC complexes are generally positively charged at
physiological pH (e.g., about pH 6.8-7.4). The microcarrier may
intrinsically possess a positive charge, but microcarriers made
from compounds not normally possessing a positive charge may be
derivatized or otherwise modified to become positively charged. For
example, the polymer used to make the microcarrier may be
derivatized to add positively charged groups, such as primary
amines. Alternately, positively charged compounds may be
incorporated in the formulation of the microcarrier during
manufacture (e.g., positively charged surfactants may be used
during the manufacture of poly(lactic acid)/poly(glycolic acid)
copolymers to confer a positive charge on the resulting
microcarrier particles.
[0100] Solid phase microspheres are prepared using techniques known
in the art. For example, they can be prepared by emulsion-solvent
extraction/evaporation technique. Generally, in this technique,
biodegradable polymers such as polyanhydrates,
poly(alkyl-.alpha.-cyanoac- rylates) and poly(.alpha.-hydroxy
esters), for example, poly(lactic acid), poly(glycolic acid),
poly(D,L-lactic-co-glycolic acid) and poly(caprolactone), are
dissolved in a suitable organic solvent, such as methylene
chloride, to constitute the dispersed phase (DP) of emulsion. DP is
emulsified by high-speed homogenization into excess volume of
aqueous continuous phase (CP) that contains a dissolved surfactant,
for example, polyvinylalcohol (PVA) or polyvinylpirrolidone (PVP).
Surfactant in CP is to ensure the formation of discrete and
suitably-sized emulsion droplet. The organic solvent is then
extracted into the CP and subsequently evaporated by raising the
system temperature. The solid microparticles are then separated by
centrifugation or filtration, and dried, for example, by
lyophilization or application of vaccum, before storing at
4.degree. C.
[0101] Generally, to prepare cationic microspheres, cationic lipids
or polymers, for example, 1,2-dioleoyl-
1,2,3-trimethylammoniopropane (DOTAP), cetyltrimethylammonium
bromide (CTAB) or polylysine, are added either to DP or CP, as per
their solubility in these phases.
[0102] Physico-chemical characteristics such as mean size, size
distribution and surface charge of dried microspheres may be
determined. Size characteristics are determined, for example, by
dynamic light scattering technique and the surface charge was
determined by measuring the zeta potential.
[0103] Generally, ISS-containing polynucleotides can be adsorbed
onto the cationic microspheres by overnight aqueous incubation of
ISS and the particles at 4.degree. C. Microspheres are
characterized for size and surface charge before and after ISS
association. Selected batches may then evaluated for activity as
described herein.
[0104] Administration
[0105] An ISS-containing polynucleotide may be administered after
exposure to HBV and/or HCV and/or after infection by HBV and/or
HCV. In certain instances, the ISS-containing polynucleotide may be
administered to an infected individual in the absence of physical
symptoms of viral infection (e.g., jaundice, fatigue, etc.).
Accordingly, administration of ISS-containing polynucleotide may be
at various times with respect to exposure to, infection by and/or
onset of symptoms of infection by HBV and/or HCV. Additionally,
treatments employing an ISS-containing polynucleotide may also be
employed in conjunction with other treatments or as `second line`
treatments employed after failure of a `first line` treatment
(e.g., ISS-containing polynucleotide therapy may be employed after
failure of interferon therapy). Further, an ISS-containing
polynucleotide may be administered in a single dose or in multiple
doses. If the ISS-containing polynucleotide is administered on
multiple occasions, the ISS may be administered on any schedule
selected by the clinician, such as daily, every other day, every
three days, every four days, every five days, every six days,
weekly, biweekly, monthly or at ever longer intervals (which may or
may not remain the same during the course of treatment) Where
multiple administrations are given, the ISS-containing
polynucleotide may be given in 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
separate administrations.
[0106] In some embodiments, when IS S-containing polynucleotide is
administered to an individual who has been exposed to HBV and/or
HCV, ISS-containing polynucleotide may be administered prior to the
appearance of physical symptom(s) of HBV and/or HCV. ISS-containing
polynucleotide is preferably administered to an individual exposed
to HBV and/or HCV less than about 28, 21, or 14 days after exposure
to HBV and/or HCV, preferably less than about 10 days after
exposure to HBV and/ or HCV, more preferably less than about 7 days
after exposure to HBV and/or HCV, even more preferably less than
about 5 days after exposure to HBV and/or HCV. In some embodiments,
ISS-containing polynucleotide is administered about 3 days after
exposure to HBV and/or HCV. In other embodiments, the
ISS-containing polynucleotide is administered as soon as possible
following a known exposure (e.g., after a needle stick or other
percutaneous exposure to a bodily fluid or other material known or
thought to be contaminated with HBV and/or HCV). In such
embodiments, the ISS-containing polynucleotide is preferably
administered within 48, 36, 24, or 12 hours after exposure.
[0107] In another embodiment, the ISS-containing polynucleotide is
administered upon or after appearance of at least one symptom of
HBV or HCV infection. Preferably, ISS-containing polynucleotide is
administered within about 28, 21, 14, 7, 5 or 3 days following
appearance of a symptom of HBV and/or HCV infection. However, some
infected individuals exhibiting symptoms will already have
undertaken one or more courses of treatment with another therapy
(e.g., interferon-based therapy). In such individuals, or in
individuals who failed to appreciate the import of their symptoms,
the ISS-containing polynucleotide may be administered at any point
following infection.
[0108] Some individuals infected with HBV and/or HCV are
asymptomatic, and identified through routine screening (e.g., when
donating blood). Accordingly, for individuals presenting without
appreciable or noticeable physical symptoms, the ISS-containing
polynucleotide may be administered at any point following
infection.
[0109] ISS polynucleotides may be formulated in any form known in
the art, such as dry powder, semi-solid or liquid formulations. For
parenteral administration ISS polynucleotides preferably
administered in a liquid formulation, although solid or semi-solid
formulations may also be acceptable, particularly where the ISS
polynucleotide is formulated in a slow release depot form.
[0110] ISS polynucleotide formulations may contain additional
components such as salts, buffers, bulking agents, osmolytes,
antioxidants, detergents, surfactants and other
pharmaceutically-acceptable excipients as are known in the art.
Generally, liquid ISS polynucleotide formulations made in USP water
for injection and are sterile, isotonic and pH buffered to a
physiologically-acceptable pH, such as about pH 6.8 to 7.5.
[0111] ISS-containing polynucleotides may be formulated in delivery
vehicles such as liposomes, oil/water emulsion or slow release
depot formulations. Methods of formulating polynucleotides in such
forms are well known in the art.
[0112] ISS-containing polynucleotide formulations may also include
or exclude immunomodulatory agents such as adjuvants and
immunostimulatory cytokines, which are well known in the art.
[0113] A suitable dosage range or effective amount is one that
provides the desired reduction of symptom(s) and/or suppression of
viral infection and depends on a number of factors, including the
particular hepatitis virus, ISS sequence of the polynucleotide,
molecular weight of the polynucleotide and route of administration.
Dosages are generally selected by the physician or other health
care professional in accordance with a variety of parameters known
in the art, such as severity of symptoms, history of the patient
and the like. Generally, for an ISS-containing polynucleotide of
about 20 bases, a dosage range may be selected from, for example,
an independently selected lower limit such as about 0.1, 0.25, 0.5,
1, 2, 5, 10, 20, 30 40, 50 60, 80, 100, 200, 300, 400 or 500
.mu.g/kg up to an independently selected upper limit, greater than
the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750,
1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or
10,000 .mu.g/kg. For example, a dose may be about any of the
following: 0.1 to 100 .mu.g/kg, 0.1 to 50 .mu.g/kg, 0.1 to 25
.mu.g/kg, 0.1 to 10 .mu.g/kg, 1 to 500 .mu.g/kg, 100 to 400
.mu.g/kg, 200 to 300 .mu.g/kg, 1 to 100 .mu.g/kg, 100 to 200
.mu.g/kg, 300 to 400 .mu.g/kg, 400 to 500 .mu.g/kg, 500 to 1000
.mu.g/kg, 500 to 5000 .mu.g/kg, or 500 to 10,000 .mu.g/kg.
Generally, parenteral routes of administration require higher doses
of ISS compared to more direct application to infected tissue, as
do ISS-containing polynucleotides of increasing length.
[0114] Polynucleotides comprising an ISS may be administered by
systemic (e.g., parenteral) or local/regional administration,
although systemic administration is preferred, due to the relative
inaccessability of the site of infection. For local/regional
administration, polynucleotides comprising an ISS may be
administered into the portal vein, but this route of administration
is not preferred because of the invasiveness of the procedure.
[0115] In other embodiments, the ISS-containing polynucleotide is
administered parenterally. Parenteral routes of administration
include, but are not limited to, transdermal, transmucosal,
nasopharyngeal, pulmonary and direct injection. Parenteral
administration by injection may be by any parenteral injection
route, including, but not limited to, intravenous (IV),
intraperitoneal (IP), intramuscular (IM), subcutaneous (SC) and
intradermal (ID) routes. Transdemnal and transmucosal
administration may be accomplished by, for example, inclusion of a
carrier (e.g., dimethylsulfoxide, DMSO), by application of
electrical impulses (e.g., iontophoresis) or a combination thereof.
A variety of devices are available for transdermal administration
which may be used in accordance with the invention.
[0116] Nasopharyngeal and pulmonary routes of administration
include, but are not limited to, intranasal, inhalation,
transbronchial and transalveolar routes. The ISS-containing
polynucleotide may thus be administered by inhalation of aerosols,
atomized liquids or powders. Devices suitable for administration by
inhalation of ISS-containing compositions include, but are not
limited to, nebulizers, atomizers, vaporizers, and metered-dose
inhalers. Nebulizers, atomizers, vaporizers and metered-dose
inhalers filled with or employing reservoirs containing
formulations comprising the ISS-containing polynucleotide(s) are
among a variety of devices suitable for use in inhalation delivery
of the ISS-containing polynucleotide(s). Other methods of
delivering to respiratory mucosa include delivery of liquid
formulations, such as by nose drops.
[0117] IV, IP, IM and ID administration may be by bolus or infusion
administration. For SC administration, administration may be by
bolus, infusion or by implantable device, such as an implantable
minipump (e.g., osmotic or mechanical minipump) or slow release
implant. The ISS polynucleotide(s) may also be delivered in a slow
release formulation adapted for IV, IP, IM, ID or SC
administration. Administration by inhalation is preferably
accomplished in discrete doses (e.g., via a metered dose inhaler),
although delivery similar to an infusion may be accomplished
through use of a nebulizer. Administration via the transdermal and
transmucosal routes may be continuous or pulsatile.
[0118] Assessment
[0119] In some embodiments, administration of an ISS-containing
polynucleotide results in prevention, palliation, and/or
improvement in one or more symptoms of HBV or HCV. The exact form
of prevention, palliation or improvement will depend on the
particular hepatitis virus, the symptoms experienced by the
patient, and the stage of the hepatitis, but includes reduction or
improvement in one or more physical symptoms such as jaundice,
fatigue, abdominal pain and the like and/or clinical/laboratory
findings associated with hepatitis such as viremia, blood levels of
liver enzymes, portal hypertension, cirrhosis, and the like.
[0120] Symptoms of infection may be assessed before and/or after
administration of ISS-containing polynucleotide. As will be
apparent to one of skill in the art, the symptoms measured and the
method of their measurement will vary depending on the particular
hepatitis virus and the stage of infection. Physical symptoms of
acute HBV and/or HCV infection include jaundice, fatigue, abdominal
pain, dark urine, and other symptoms known in the art. Subjective
physical symptoms such as abdominal pain and fatigue may be
measured on a qualitative (e.g., presence/absence) basis or may be
quantitated using a visual scale system. Jaundice may also be
measured on a qualitative basis or may be quantitated by
measurement of blood or serum levels of bilirubin.
[0121] Clinical/laboratory findings associated with hepatitis are
normally measured through clinical assessment, diagnostic assays,
and histologic testing. For example, blood/serum levels of liver
enzymes may be quantitated by running a standard clinical
laboratory liver function panel of tests which include quantitation
of AST and ALT levels in the individual's blood or serum. Viremia
(i.e., viral titer in a blood or serum sample) may be measured by
any method known in the art, such as quantitation of viral
particles (for example, by isolation and visualization or by assay
of DNase resistant particles), detection of viral antigens in blood
or serum samples, detection of antivirus antibodies in blood or
serum samples and/or detection of viral nucleic acid (e.g., by PCR
amplification using HBV or HCV specific primers or by in situ
hybridization with virus-specific probes). Viral titer may also be
measured in liver tissue biopsies, generally by quantitation of
viral nucleic acid, although viral antigens may also be used for
calculation of viral titer. Viral titer from tissue samples is
calculated in virus particles per unit weight of tissue.
[0122] Kits of the Invention
[0123] The invention provides kits for carrying out the methods of
the invention (i.e., treatment and/or prevention of HBV and/or HCV
infection). Accordingly, a variety of kits are provided. The kits
may be used for any one or more of the following (and, accordingly,
may contain instructions for any one or more of the following
uses): reducing levels of a hepatitis B and/or hepatitis C antigen
in blood in an individual who has been infected with hepatitis B
and/or hepatitis C; reducing viremia in an individual infected with
or exposed to hepatitis B and/or hepatitis C; preventing one or
more symptoms of hepatitis B and/or hepatitis C infection in an
individual exposed to hepatitis B, hepatitis C, or both hepatitis B
and C; reducing severity of one or more symptoms of hepatitis B
and/or hepatitis C infection in an individual who has been infected
with hepatitis B, hepatitis C, or both hepatitis B and C; delaying
development of one or more symptoms of hepatitis B and/or hepatitis
C infection in an individual who has been infected with hepatitis
B, hepatitis C, or both hepatitis B and C; reducing duration of one
or more symptoms of hepatitis B and/or hepatitis C infection in an
individual who has been infected with hepatitis B, hepatitis C, or
both hepatitis B and C; reducing severity of one or more symptoms
of chronic hepatitis B and/or hepatitis C infection in an
individual infected with hepatitis B, hepatitis C, or both
hepatitis B and C; preventing of one or more symptoms of chronic
hepatitis B and/or hepatitis C infection in an individual who has
been infected with hepatitis B, hepatitis C, or both hepatitis B
and C; delaying development of one or more symptoms of chronic
hepatitis B and/or hepatitis C infection in an individual who has
been infected with hepatitis B, hepatitis C, or both hepatitis B
and C; reducing duration of one or more symptoms of chronic
hepatitis B and/or hepatitis C infection in an individual who has
been infected with hepatitis B, hepatitis C, or both hepatitis B
and C. As is understood in the art, any one or more of these uses
would be included in instructions directed to treating or
preventing hepatitis B and/or hepatitis C infection.
[0124] The kits of the invention comprise one or more containers
comprising an ISS-containing polynucleotide and a set of
instructions, generally written instructions although electronic
storage media (e.g., magnetic diskette or optical disk) containing
instructions are also acceptable, relating to the use and dosage of
the ISS-containing polynucleotide for the intended treatment (e.g.,
reducing levels of a hepatitis B and/or hepatitis C antigen in
blood in an individual who has been infected with hepatitis B
and/or hepatitis C; reducing viremia in an individual infected with
or exposed to hepatitis B and/or hepatitis C; preventing one or
more symptoms of hepatitis B and/or hepatitis C infection in an
individual exposed to hepatitis B, hepatitis C, or both hepatitis B
and C; reducing severity of one or more symptoms of hepatitis B
and/or hepatitis C infection in an individual who has been infected
with hepatitis B, hepatitis C, or both hepatitis B and C; delaying
development of one or more symptoms of hepatitis B and/or hepatitis
C infection in an individual who has been infected with hepatitis
B, hepatitis C, or both hepatitis B and C; reducing duration of one
or more symptoms of hepatitis B and/or hepatitis C infection in an
individual who has been infected with hepatitis B, hepatitis C, or
both hepatitis B and C; reducing severity of one or more symptoms
of chronic hepatitis B and/or hepatitis C infection in an
individual infected with hepatitis B, hepatitis C, or both
hepatitis B and C; preventing one or more symptoms of chronic
hepatitis B and/or hepatitis C infection in an individual infected
with hepatitis B, hepatitis C, or both hepatitis B and C; delaying
development of one or more symptoms of chronic hepatitis B and/or
hepatitis C infection in an individual infected with hepatitis B,
hepatitis C, or both hepatitis B and C and/or reducing duration of
one or more symptoms of chronic hepatitis B and/or hepatitis C
infection in an individual infected with hepatitis B, hepatitis C,
or both hepatitis B and C). The instructions included with the kit
generally include information as to dosage, dosing schedule, and
route of administration for the intended treatment. The containers
of ISS may be unit doses, bulk packages (e.g., multi-dose packages)
or sub-unit doses.
[0125] The kits of the invention do not include any packages or
containers which contain viral antigens from the hepatitis
virus(es) the kit is intended to be used to treat. Accordingly,
neither the container comprising the ISS-containing polynucleotide
nor any other containers in the kit contain hepatitis B viral
antigens in kits intended for use on individuals exposed to or
infected with hepatitis B, neither the container comprising the
ISS-containing polynucleotide nor any other containers in the kit
contain hepatitis C viral antigens in kits intended for use on
individuals exposed to or infected with hepatitis C, and neither
the container comprising the ISS-containing polynucleotide nor any
other containers in the kit contain hepatitis B or C viral antigens
in kits intended for use on individuals infected with both
hepatitis B and hepatitis C.
[0126] The ISS component of the kit may be packaged in any
convenient, appropriate packaging. For example, if the ISS is a
freeze-dried formulation, an ampoule with a resilient stopper is
normally used, so that the drug may be easily reconstituted by
injecting fluid through the resilient stopper. Ampoules with
non-resilient, removable closures (e.g., sealed glass) or resilient
stoppers are most conveniently used for injectable forms of ISS.
Also, prefilled syringes may be used when the kit is supplied with
a liquid formulation of the ISS-containing polynucleotide. Also
contemplated are packages for use in combination with a specific
device, such as an inhaler, nasal administration device (e.g., an
atomizer) or an infusion device such as a minipump.
[0127] As stated above, any ISS-containing polynucleotide described
herein may be used, such as, for example, any polynucleotide
comprising any of the following ISS: the sequence 5'-cytosine,
guanine-3', the sequence 5'-T, C, G-3', the sequence 5'-C, G,
pyrimidine, pyrimidine, C, G-3', the sequence 5'-purine, purine, C,
G, pyrimidine, pyrimidine, C, G-3', the sequence 5'-purine, purine,
C, G, pyrimidine, pyrimidine, C, C-3'; the sequence SEQ ID NO:
1018; the sequence 5'-purine, purine, B, G, pyrimidine,
pyrimidine-3' wherein B is 5-bromocytosine or the sequence
5'-purine, purine, B, G, pyrimidine, pyrimidine, C, G-3' wherein B
is 5-bromocytosine.
[0128] The following Examples are provided to illustrate, but not
limit, the invention.
EXAMPLES
Example 1
[0129] Administration of an ISS in an Animal Model of Chronic HBV
Infection
[0130] ISS activity was tested in an animal model of chronic
hepatitis. An ISS-containing phosphorothioate oligonucleotide
(5'-TGACTGTGAACGTTCGAGATG- A-3') (SEQ ID NO:1), was delivered to
STC strain transgenic mice, followed by measurement of HBV DNA and
HBsAg production.
[0131] STC line mice were developed at Stanford University by
Patricia Marion. The majority of these mice secrete HBV of the Ayw
genotype (Galibert et al. (1979) Nature 281:646) to titers of
10.sup.6-8 viral genome equivalents per ml of serum STC mice were
derived from the FVB strain, and were constructed by microinjection
of HBV genomic DNA. STC mice have been shown to be responsive to
drugs which inhibit HBV replication, and so are considered a good
model of chronic HBV.
[0132] Approximately one month old mice were bled and tested for
serum levels of HBsAg, which is predictive of viral DNA titer. A
pool of 40 STC mice with approximately equal levels of HBsAg were
selected and randomly assigned to four treatment groups of 10
animals each. The groups were treated as follows:
[0133] 1. 100 .mu.g of ISS injected subcutaneously, once per week
for 3 weeks (days 0, 7, 14)
[0134] 2. 100 .mu.g of ISS injected subcutaneously, one injection
at day 14
[0135] 3. 100 ng of murine 1L-12 injected intraperitoneally on days
12, 13, and 14.
[0136] 4. PBS injected subcutaneously (days 0, 7, 14)
[0137] Blood samples were taken at day 0, 7, 14, 15 (22 hr after
last IL-12 injection), 18, 28 and 35. Serum prepared from the blood
samples was tested for HBV DNA by quantitative PCR (testing
performed under contract by Hepadnavirus Testing, kic.), and HBsAg
using a commercially available EIA kit for HBsAg from Abbott
Laboratories. Animals were sacrificed at day 35 and livers were
collected for histologic analysis.
[0138] The results of the quantitative PCR assays for serum HBV DNA
levels in HBV-producing mice treated with ISS, murine IL-12 or PBS,
are summarized in FIG. 1. The results are plotted as means of the
HBV DNA levels of each of the 4 groups in each of the serial
samples. Samples were blinded to the person conducting the assays.
Both ISS and murine IL-12 were effective in reducing viral titer in
STC mice. The most dramatic titer drop was seen in Group 2 (single
subcutaneous injection of ISS at day 14), where the mean viral DNA
titer was reduced by 90 fold three days after injection.
[0139] The results of the assays for serum HBsAg levels in
HBV-producing mice treated with ISS, murine IL-12 or PBS are
summarized in FIG. 2. The results are plotted as averages of the
antigen levels of each of the 4 groups in each of the serial
sample. The data showed a trend towards decreased average HBsAg
values of animals treated with ISS compared to control animals
treated with PBS.
[0140] It should be noted that, as with all lineages of
HBV-producing mice, some animals sharply dropped titer during the
observation period, even before treatments, or with treatment with
the control. Despite the randomizing at -7 days, more of these mice
were found in groups 3 and 4 (IL-12 and control, respectively),
possibly obscuring a more dramatic effect by the ISS.
[0141] The present invention has been detailed both by direct
description and by example. Equivalents and modifications of the
present invention will be apparent to those skilled in the art, and
are encompassed within the scope of the invention
* * * * *