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Cancer Patent Abstract
The present invention provides a pharmaceutical composition, comprising
a non-infectious, non-integrating polynucleotide construct comprising
a polynucleotide encoding an interferon .omega. and one or more
cationic compounds. The present invention also provides methods
of treating cancer in a mammal, comprising administering into a
muscle of the mammal a non-infectious, non-integrating DNA polynucleotide
construct comprising a polynucleotide encoding a cytokine. In addition,
the present invention also relates to the methodology for selective
transfection of malignant cells with polynucleotides expressing
therapeutic or prophylactic molecules in intra-cavity tumor bearing
mammals. More specifically, the present invention provides a methodology
for the suppression of an intra-cavity dissemination of malignant
cells, such as intraperitoneal dissemination. Furthermore, the invention
relates to compositions and methods to deliver polynucleotides encoding
polypeptides to vertebrate cells in vivo, where the composition
comprises an aqueous solution of sodium phosphate.
Cancer Patent Claims
What is claimed is:
1. A method of treating cancer or metastasis thereof in a mammal,
comprising: administering into a muscle of a mammal with cancer
or metastasis thereof, a DNA plasmid comprising a polynucleotide
which encodes interferon-alpha or an active fragment thereof, operably
associated with a promoter; wherein said muscle has not been treated
with an agent that destroys muscle tissue; wherein said DNA plasmid
is administered free from ex vivo cells; wherein said interferon
alpha is expressed in vivo, and is present in the blood stream of
said mammal in an amount effective to treat said cancer, or metastasis
thereof.
2. The method of claim 1, wherein said plasmid further comprises
a polyadenylation signal and transcription termination signal operably
associated with said polynucleotide.
3. The method of claim 1, wherein said cancer is selected from
the group consisting of renal cell carcinoma, colorectal carcinoma,
lymphoma, Kaposi's sarcoma, melanoma, prostate cancer, ovarian cancer,
lung cancer, liver cancer, head and neck cancer, bladder cancer,
uterine cancer, bone cancer, leukemia, breast cancer, non-melanoma
skin cancer, glioma, solid cutaneous tumor, epidermoid carcinoma,
metastases of any of thereof, and combinations of any of thereof.
4. The method of claim 3, wherein said cancer is a lung metastasis
of any of said cancers.
5. The method of claim 3, wherein said cancer is a liver metastasis
of any of said cancers.
6. The method of claim 1, wherein said muscle tissue is skeletal
muscle.
7. The method of claim 1, wherein said interferon alpha is a polypeptide
comprising amino acids 1 to 166 of SEQ ID NO:10.
8. The method of claim 7, wherein said interferon alpha is a polypeptide
comprising amino acids -23 to 166 of SEQ ID NO:10.
9. The method of claim 1, wherein said DNA plasmid is VR4112 (SEQ
ID NO:2).
10. The method of claim 1, wherein said cancer is melanoma or metastasis
thereof.
11. The method of claim 10, wherein said cancer is metastasis of
melanoma.
12. The method of claim 11, wherein the metastasis of melanoma
is lung metastasis.
13. The method of claim 1, wherein said cancer is glioma.
14. The method of claim 1, wherein said cancer is epidermoid carcinoma.
15. The method of claim 1, wherein said DNA plasmid is dissolved
in an aqueous solution.
16. The method of claim 1, wherein said DNA plasmid is administered
free from association with transfection-facilitating proteins, viral
particles, liposomes, cationic lipids, and calcium phosphate precipitating
agents.
17. The method of claim 1, wherein said DNA plasmid is administered
as a complex of said DNA plasmid and one or more cationic compounds
selected from the group consisting of cationic lipids, cationic
peptides, cationic proteins, cationic polymers other than lipids
or peptides, and mixtures thereof.
18. The method of claim 17, wherein said one or more cationic compounds
are one or more cationic lipids.
19. The method of claim 18, wherein said compounds further comprise
one or more neutral lipids.
20. The method of claim 1, wherein said DNA plasmid further comprises
a region regulating expression operably associated with said polynucleotide.
21. A method of treating cancer, or metastasis thereof, in a mammal,
comprising: the method of claim 1 in combination with one or more
additional cancer treatment methods selected from the group consisting
of surgery, radiation therapy, chemotherapy, immunotherapy, and
gene therapy.
22. The method of claim 21, wherein said DNA plasmid is administered
prior to the commencement of said one or more additional cancer
treatment methods.
23. The method of claim 21, wherein said DNA plasmid is administered
during the practice of said one or more additional cancer treatment
methods.
24. The method of claim 21, wherein said DNA plasmid is administered
after the end of said one or more additional cancer treatment methods.
25. The method of claim 1, wherein said mammal is human.
26. A method of treating cancer in a mammal with cancer or metastasis
thereof, comprising: administering into the peritoneal cavity of
said mammal, a DNA plasmid comprising a polynucleotide which encodes
interferon alpha or an active fragment thereof, operably associated
with a promoter, wherein said DNA plasmid is administered free from
ex vivo cells or ex vivo cellular material; and wherein said interferon
alpha is delivered to a tumor, or metastases thereof, in a therapeutically
effective amount.
27. The method of 26, wherein said tumor disseminates in said peritoneal
cavity.
28. The method of claim 26, wherein said DNA plasmid is free from
association with transfection-facilitating proteins, viral particles,
and calcium phosphate precipitating agents.
29. The method of claim 26, wherein said DNA plasmid is administered
as a complex with one or more cationic lipids.
30. The method of claim 29, wherein said complex further comprises
one or more neutral lipids.
31. The method of claim 30, wherein said DNA plasmid is complexed
with (.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanimi-
nium bromide and 1,2-dioleoyl-glycero-3-phosphoethanolamine.
32. The method of claim 26, wherein said mammal is a human.
33. A method of transfecting malignant cells in a mammal with cancer
or metastasis thereof, comprising: administering into the peritoneal
cavity of said mammal, a DNA plasmid comprising a polynucleotide
encoding interferon alpha, or an active fragment thereof, operably
associated with a promoter, wherein said DNA plasmid is administered
free from ex vivo cells or ex vivo cellular material; and wherein
said plasmid is delivered to and expressed in malignant cells within
said peritoneal cavity.
34. The method of claim 33, wherein said DNA plasmid is free from
association with transfection-facilitating proteins, viral particles,
and calcium phosphate precipitating agents.
35. The method of claim 33, wherein said DNA plasmid is administered
as a complex with one or more cationic lipids.
36. The method of claim 33, wherein said complex further comprises
one or more neutral lipids.
37. The method of claim 36, wherein said DNA plasmid is complexed
with (.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanimi-
nium bromide and 1,2-dioleoyl-glycero-3-phosphoethanolamine.
Cancer Patent Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to treatment of cancer in mammals.
Generally, the present invention provides methods of treating cancer
in a mammal by administering a polynucleotide construct comprising
a polynucleotide encoding a cytokine. In addition, the present invention
relates to the methodology for selective transfection of malignant
cells with polynucleotides expressing therapeutic or prophylactic
molecules in intra-cavity tumor bearing mammals. More specifically,
the present invention provides a methodology for the suppression
of an intra-cavity dissemination of malignant cells, such as intraperitoneal
dissemination.
The present invention further relates generally to compositions
and methods useful for in vivo polynucleotide-based polypeptide
delivery into cells of vertebrates. More particularly, the present
invention provides the use of sodium phosphate solutions in compositions
and methods useful for direct polynucleotide-based polypeptide delivery
into the cells of vertebrates.
2. Related Art
Cytokines have been demonstrated both in pre-clinical animal models
as well as in humans to have potent anti-tumor effects. In particular
IFN's have been tried for the treatment of a number of human concerns.
The interferons (IFNs) are a family of cytokines with potent anti-viral,
antiproliferative, and immunomodulatory activities and play important
roles in the body's defensive response to viruses, bacteria, and
tumors (Baron, S. et al., JAMA 266:1375 (1991)). On the basis of
antigenicity, biochemical properties, and producer cell, the interferon's
have been divided into two classes, type I interferon and type II
interferon. IFN.alpha., IFN.beta., IFN.omega., and IFN.tau. are
type I interferons, and bind to the same .alpha./.beta. receptor.
IFN.gamma. is a type II interferon, and binds to the .gamma. receptor
(Pestka, S., Ann. Rev. Biochem. 56:727 (1987)). IFN.alpha. and IFN.beta.
are naturally expressed in many cells upon viral infection. IFN.gamma.
is produced by activated T lymphocytes and natural killer (NK) cells.
IFN.tau. is believed to possess hormone activity, and plays an important
role in pregnancy in cattle, sheep, and related ruminants (Imakawa,
K. et al., Nature 330:377 (1987); Stewart, H. J. et al., J. Endocrinology
115:R13 (1987)). Due to the pleiotropic activities of IFNs, these
cytokines have been studied for their therapeutic efficacy in a
number of diseases, particularly cancers and viral infectious diseases.
IFN.omega. was discovered independently by three different groups
in 1985 (Capon, D. J., et al., Molec. Cell. Biol. 5: 768-779 (1985),
Feinstein, S. et al., Molec. Cell. Biol 5:510 (1985); and Hauptmann
and Swetly, Nucl. Acids Res. 13: 4739-4749 (1985)). Unlike IFN.alpha.,
for which at least 14 different functional nonallelic genes have
been identified in man, IFN.omega. is encoded by a single functional
gene. IFN.omega. genes are believed to be present in most mammals,
but have not been found in dogs, rats or mice. The mature IFN.omega.
polypeptide is 172 amino acids and shares 60% sequence homology
with the human IFN.alpha.'s. Due to the sequence similarity with
IFN.alpha., IFN.omega. was originally considered to be a member
or a subfamily of IFN.alpha., and was originally termed IFN.alpha.-.sub.II.
IFN.omega. is a significant component (.apprxeq.10%) of human leukocyte-derived
interferon, the natural mixture of interferon produced after viral
infection (Adolf, G. et al., Virology 175:410 (1990)). IFN.omega.
has been demonstrated to bind to the same .alpha./.beta. receptor
as IFN.alpha. (Flores, I. et al., J. Biol. Chem. 266: 19875-19877
(1991)), and to share similar biological activities with IFN.alpha.,
including anti-proliferative activity against tumor cells in vitro
(Kubes, M. et al., J. Interferon Research 14:57 (1994) and immunomodulatory
activity (Nieroda et al., Molec. Cell. Differentiation 4: 335-351
(1996)).
Recombinant IFN.alpha. polypeptide has been approved for use in
humans for hairy cell leukemia, AIDS-related Kaposi's sarcoma, malignant
melanoma, chronic hepatitis B and C, chronic myleogenous leukemia,
and condylomata acuminata (Baron, S. et al., JAMA266:1375 (1991)).
However, for each of these indications, IFN.alpha. polypeptide must
be administered repeatedly, often on a daily basis, for extended
periods of time to maintain effective serum levels due to the short
half-life (hours) of the polypeptide in the serum (Friedman, Interferons:
A Primer, Academic Press, New York, pp. 104-107 (1981); Galvani
and Cawley, Cytokine Therapy, Cambridge University Press, Cambridge,
pp. 114-115 (1992)). Thus, in spite of producing clinical benefit
for many disease conditions, the use of IFN.alpha. polypeptide is
associated with acute and chronic side effects in most patients
(Jones, Cancer 57: 1709-1715 (1986); and Quesda et al., Blood 68:
493-497 (1986)). The severity of the adverse reaction correlates
with peak serum interferon levels.
Viral or plasmid vectors containing IFN.alpha. genes have been
used in ex vivo therapy to treat mouse tumors. For example, tumor
cells were transfected in vitro with viral or plasmid vectors containing
an IFN.alpha. gene, and the transfected tumor cells were injected
into mice (Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216
(1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993);
Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994); Kaido,
T. et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H. et al.,
Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., Human
Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy
4:1246-1255 (1997)). In another ex vivo study, cervical carcinoma
and leukemia cells were transfected with a viral vector containing
the interferon-consensus gene, and the transfected cells were injected
into mice (Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)).
In all of these ex vivo studies, varying levels of anti-tumor efficacy,
such as tumor regression and/or prolonged survival, have been observed.
Viral or plasmid vectors containing interferon genes have also
been used in in vivo therapy for tumor-bearing mice. For example,
a viral vector containing the interferon-consensus gene was injected
into mice bearing transplanted MDA-MB-435 breast cancer, hamster
melanoma, or K562 leukemia, and tumor regression was reported (Zhang,
J.-F. et al., Proc. Natl. Acad. Sci. USA 93: 4513-4518 (1996)).
In a similar study, a plasmid vector containing human IFN.beta.
gene complexed with cationic lipid was injected intracranially into
mice bearing a human glioma, and tumor regression was reported (Yagi,
K. et al., Biochemistry and Molecular Biology International 32:
167-171 (1994)). In a murine model of renal cell carcinoma the direct
intratumoral injection of an IL-2 plasmid DNA:lipid complex has
been shown to result in complete tumor regression and a significant
induction of a tumor specific CTL response increase in survival
(Saffran et al., Cancer Gene Therapy 5: 321-330 (1998)).
Plasmid vectors containing cytokine genes have also been reported
to result in systemic levels of the encoded cytokine and in some
cases, biological effects characteristic of each cytokine in mice.
For example, the intramuscular injection of plasmid DNA encoding
either TGF.beta., IL-2, IL-4, IL-5, or IFN.alpha. resulted in physiologically
significant amounts in the systemic circulation of the corresponding
cytokine polypeptide (Raz, E. et al., Proc. Natl. Acad. Sci. USA
90: 4523-4527 (1993); Raz, E. et al., Lupus 4: 266-292 (1995); Tokui,
M. et al., Biochem. Biophys. Res. Comm. 233: 527-531 (1997); Lawson,
C. et al., J. Interferon Cytokine Res. 17: 255-261 (1997); Yeow,
W.-S. et al., J. Immunol. 160: 2932-2939 (1998)).
U.S. Pat. No. 5,676,954 reports on the injection of genetic material,
complexed with cationic liposomes carriers, into mice. U.S. Pat.
Nos. 4,897,355; 4,946,787; 5,049,386; 5,459,127; 5,589,466; 5,693,622;
5.580,859; 5,703,055; and International Patent Application No. PCT/US94/06069
(publication no. WO 94/29469) provide cationic lipids for use in
transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466,
5.693,622, 5,580,859, 5,703,055, and international patent application
no. PCT/US94/06069 (publication no. WO 04/9469) provide methods
for delivering DNA-cationic lipid complexes to mammals.
Even though some viral vectors used in ex vivo and in vivo cancer
therapy in murine models showed anti-tumor efficacy, the use of
viral vectors to deliver interferon-expressing genes in vivo could
induce anti-viral immune responses or result in viral integration
into host chromosomes, causing disruption of essential host genes
or activation of oncogenes (Ross et al. Human Gene Therapy 7: 1781-1790
(1996)).
For treatment of multiple metastatic carcinomas of a body cavity
are treated using laparoscopy (Childers et al, Gynecol. Oncol. 59:
25-33. (1995)), catheterization (Naumann et al, Gynecol. Oncol.
50: 291-3, (1993)) or other access devices (Almadrones et al, Semin.
Oncol. Nurs. 11: 194-202, (1995)). Treatment is usually by surgical
removal of primary and large metastatic tumors and postoperative
chemotherapy (Kigwawa et al, Am. J. Clin. Oncol. 17: 230-3, (1994);
Markman et al, J. Clin. Oncol. 10: 1485-91. (1992)) or radiotherapy
(Fjeld et al, Acta. Obstet. Gynecol. Scand. Suppl. 155: 105-11.
(1992)). Tumor recurrence is monitored by magnetic resonance imaging
(Forstner et al, Radiology 196: 715-20, (1995)), ascites cytology
(Clement, Am. J. Clin. Pathol. 103: 673-6, (1995); Forstner et al,
Radiology 196: 715-20, 1995) and blood analyses (Forstner et al,
Radiology 196: 715-20. (1995)). Many intraperitoneal (i.p.) cancers,
such as ovarian cancer, eventually metastasize via the lymphatic
system to the lungs or other vital organs, and the prognosis for
the patient is very poor (Kataoka et al, Nippon Sanka Fujinka Gakkai
Zasshi 46: 337-44, 1994; Hamilton, Curr. Probl. Cancer 16: 1-57.
(1992)).
Human ovarian cancer is often diagnosed at an advanced stage when
the effectiveness of surgery and chemotherapy are limited. The lack
of effective treatment options for late-stage patients warrants
the development of new treatment modalities for this disease. There
have been several attempts to develop an effective immunotherapy
for the treatment of ovarian cancer.
The early work in this area involved mouse studies in which bacteria-derived
immunostimulants, such as Bacillus Calmette-Guerin (BCG) and Corynebacterium
parvum, were injected i.p. as non-specific activators of the immune
system. (Knapp and Berkowitz, Am. J. Obstet. Gynecol., 128: 782-786,
(1977); Bast et al., J. Immunol., 123: 1945-1951, (1979); Vanhaelen,
et al., Cancer Research, 41: 980-983, (1981); and Berek, et al.,
Cancer Research, 44, 1871-1875, (1984)). These studies generally
resulted in a non-specific immune response that often did not prevent
the growth of later tumors. In addition, if the bacterial antigens
were injected more than 24 hours after tumor cell inoculation, there
was minimal antitumor response, suggesting that treatment of late-stage
ovarian cancer patients with this type of therapy would not be effective.
More recent studies in both mice and humans have involved the i.p.
or intravenous (i.v.) administration of cytokine proteins as more
specific activators of the immune response (Adachi, et al, Cancer
Immunol. Immunother. 37: 1-6, (1993); Lissoni, et al, Tumori. 78:
118-20, (1992)). Treating murine ovarian tumors with a combination
of recombinant IL-2 and GM-CSF proteins had some beneficial effect
in inhibiting ascites production; however, IL-2 was only effective
if it was combined with GM-CSF (Kikuchi, et al., Cancer Immunol.
Immunother., 43: 257-261, (1996)). Similarly, a combination of IL-2
and lymphokine-activated killer (LAK) cells was able to reduce i.p.
sarcomas in mice, while IL-2 protein alone was not as effective
(Ottow, et al., Cellular Immunology, 104: 366-376, (1987)). Human
clinical trials evaluating IL-2 protein therapy of ovarian cancer
patients indicated some antitumor effects (Chapman et al., Investigational
New Drugs, 6:179-188. (1988); West et al. N. Engl. J. Med. 316:898-905,
1987; Lotze et al., Arch. Surg. 121:1373-1379, 1986; Benedetti Panici
et al., Cancer Treatment Review, 16A:123-127, 1989; Beller et al.,
Gynecol. Oncol., 34:407-412, 1989; Urba et al., J. Natl. Cancer
Inst., 81:602-611, 1989; Stewart et al., Cancer Res., 50:6302-6310,
1990; Steis et al., J. Clin. Oncol., 8:1618-1629, 1990; Lissoni
et al., Tumori, 78:118-120, 1992; Sparano et al., J. of Immunotherapy,
16:216-223, 1994; Freedman et al., J. of Immunotherapy, 16:198-210,
1994; Edwards et al., J. Clin. Oncol., 15:3399-3407, 1997).
Recent studies in mice have involved the injection of DNA constructs
encoding "suicide" genes followed by treatment with prodrugs.
This approach has successfully caused regression of some small tumors
but has been less successful on larger tumor masses. (Szala, et
al. Gene Therapy 3: 1025-1031, 1996; Sugaya, et al. Hum Gene Ther
7: 223-230 (1996)). In another study, liposome-mediated E1A gene
therapy for mice bearing ovarian cancers that overexpress HER-2/neu
resulted in reduced mortality among these tumor bearing mice. (Yu,
et al. Oncogene, 11: 1383-1388 (1995)). Similarly, the successful
treatment of murine ovarian carcinoma (MOT) has been demonstrated
using cisplatin-induced gene transfer of DNA constructs encoding
IFN.gamma. via i.p. injection. (Son, Cancer Gene Therapy 4: 391-396
(1997)). However, this study demonstrated that tumors were poorly
responsive to either the IFN.gamma. gene or cisplatin alone, suggesting
that the effectiveness of the cisplatin-based gene therapy protocol
was mainly due to enhanced sensitization of cisplatin-exposed tumor
cells to transfection by the IFN.gamma. gene. (Son, Cancer Gene
Therapy 4: 391-396, 1997).
Clearly, there is a need for superior therapeutic compositions
and methods for treating mammalian cancer. Further, there is a need
for an in vivo delivery system for IFN.omega.. The present invention
provides a simple and safe yet effective compositions and methods
for treatment of mammalian cancer.
The present invention also solves the problems inherent in prior
attempts to treat body cavity malignancies. The inventors show herein
that the malignant cell dissemination into body cavities, such as
into the peritoneal cavity during late stage ovarian cancer, can
be suppressed simply by administering as few as two to six doses
of a polynucleotide formulation directly into the body cavity. This
treatment results in selective transfection of malignant cells,
and subsequent long-term local production of an effective amount
of therapeutic molecules.
The in vivo delivery of a polynucleotide (e.g., plasmid DNA) into
vertebrate tissues has been shown to result in the cellular uptake
and expression of the polynucleotide into a desired polypeptide
(Wolff, J. A. et al., Science 247:1465-1468 (1990); Wheeler, C.
J. et al., Proc. Natl. Acad. Sci. USA 93:11454-11459 (1996)). Potential
human therapeutic uses of such polynucleotide-based polypeptide
delivery include immune response induction and modulation, therapeutic
polypeptide delivery, and amelioration of genetic defects. For example,
a polynucleotide may encode an antigen that induces an immune response
against an infectious pathogen or against tumor cells (Restifo,
N. P. et al., Folia Biol. 40:74-88 (1994); Ulmer, J. B. et al.,
Ann. NY Acad. Sci. 772:117-125 (1995); Horton, H. M. et al., Proc.
Natl. Acad. Sci. USA 96:1553-1558 (1999); Yagi, K. et al., Hum.
Gene Ther. 10: 1975-1982 (1999)). The polynucleotide may encode
an immunomodulatory polypeptide, e.g., a cytokine, that diminishes
an immune response against self antigens or modifies the immune
response to foreign antigens, allergens, or transplanted tissues
(Qin, L. et al., Ann. Surg. 220:508-518 (1994); Dalesandro, J. et
al., J. Thorac. Cardiovasc. Surg. 111: 416-421 (1996); Moffatt,
M. and Cookson, W., Nat. Med. 2:515-516 (1996); Ragno, S. et al.,
Arth. and Rheum. 40:277-283 (1997); Dow, S. W. et al., Hum. Gene
Ther. 10:1905-1914 (1999); Piccirillo, C. A. et al., J. Immunol.
161:3950-3956 (1998); Piccirillo, C. A. and Prud'homme, G. J., Hum.
Gene Ther. 10: 915-1922 (1999)). For therapeutic polypeptide delivery,
the polynucleotide may encode, for example, an angiogenic protein,
hormone, growth factor, or enzyme (Levy, M. Y. et al., Gene Ther.
3:201-211 (1996); Tripathy, S. K. et al., Proc. Natl. Acad. Sci.
USA 93:10876-10880 (1996); Tsurumi, Y. et al., Circulation 94:3281-3290
(1996); Novo, F. J. et al., Gene Ther. 4:488-492 (1997); Baumgartner,
I. et al., Circulation 97:1114-1123 (1998); Mir, L. M. et al., Proc.
Natl. Acad. Sci. USA 96:4262-4267 (1999)). For amelioration of genetic
defects, the polynucleotide may encode normal copies of defective
proteins such as dystrophin or cystic fibrosis transmembrane conductance
regulator (Danko, I. et al., Hum. Mol. Genet. 2:2055-2061 (1993);
Cheng, S. H. and Scheule, R. K., Adv. Drug Deliv. Rev. 30:173-184
(1998)).
However, the efficiency of a polynucleotide uptake and expression,
especially when the polynucleotide is not associated with infectious
agents, is relatively low. For example, Doh, S. G. et al., Gene
Ther. 4:648-663 (1997) reports that the administration of plasmid
DNA into mouse muscle results in the detectable transduction of
an average of only 6%, e.g., about 234 out of approximately 4000,
of the myofibers in the injected muscle. Wheeler, C. G. et al.,
ibid., showed that administration of plasmid DNA complexed with
cationic lipid into a mouse lung results in the transduction of
less than 1% of the lung cells.
Attempts have been made to increase the efficiency of in vivo polynucleotide
administration into vertebrates using chemical agents or physical
manipulations. Such chemical agents include cellular toxins such
as bupivacaine or barium chloride (Wells, D. J., FEBS Letters 332:179-182
(1993); Vitadello, M. et al., Hum. Gene. Ther. 5:11-18 (1994); Danko,
I. et al., Hum. Mol. Genet. 2:2055-2061 (1993)) which act to cause
muscle damage followed by muscle regeneration by cell division which
makes the cells more receptive to DNA entry (Thomason, D. B. and
Booth, F. W., Am. J. Physiol. 258:C578-81 (1990)); polymers such
as polyvinyl pyrollidine that coat the DNA and protect it from DNases
(Mumper, R. J., et al., Pharm. Res. 13:701-709 (1996); Mumper R.
J. et al., J. Cont. Rel. 52:191-203 (1998); Anwer, K. et al., Pharm.
Res. 16:889-95 (1999)); bulking agents such as sucrose that are
injected before DNA injection to help expand the spaces between
muscle cells and therefore allow better distribution of the subsequently
injected DNA (Davis, H. L. et al., Hum. Gene Ther. 4:151-159 (1993));
DNA binding agents such as histones or intercalaters that protect
the DNA from DNases (Manthorpe, M. et al., Hum. Gene Ther. 4:419-431
(1993); Wolff. J. A., Neuromuscul. Disord. 7:314-318 (1997); WO
99/31262). Physical manipulations include removal of nerves that
control muscle contraction (Wolff, J. A. et al., BioTechniques 11:575-485
(1991)); electroporation that electrically opens muscle cell pores
allowing more DNA entry (Aihara, H. and Miyazaki, J., Nature Biotechnol.
16:867-870 (1998); Mir, L. M. et al. CR Acad. Sci. III 321:893-899
(1998), Mir, L. M. et al., Proc. Natl. Acad. Sci. USA 96:4262-4267
(1999); Mathiesen, I., Gene Ther. 6:508-514 (1999); Rizzuto, G.
et al., Proc. Natl. Acad. Sci. USA 96:6417-6422 (1999)); use of
intravascular pressure (Budker, V. et al., Gene Ther. 5:272-276
(1998)); use of sutures coated with plasmid DNA (Labhasetwar, V.
et al., J. Pharm. Sci. 87:1347-1350 (1998); Qin, Y. et al., Life
Sci. 65:2193-2203 (1999)); use of sponges soaked with DNA as intramuscular
depots to prolong DNA delivery (Wolff, J. A. et al. (1991), ibid.);
use of special needle-based injection methods (Levy, M. Y. et al.,
Gene Ther. 3:201-211 (1996); Doh, S. G. et al. (1997), ibid.); and
of needleless-injectors that propel the DNA into cells (Gramzinski,
R. A. et al., Molec. Med. 4:109-118 (1998); Smith, B. F. et al.,
Gene Ther. 5:865-868 (1998); Anwer, K. et al. (1999) ibid.). In
addition, Wolff, J. A. et al. (1991) ibid. and Manthorpe, M. et
al. (1993) ibid. refers to conditions affecting direct gene transfer
into rodent muscle in vivo.
WO99/64615 identifies the use of products and methods useful for
delivering formulated nucleic acid molecules using electrical pulse
voltage delivery. Examples include the formulation of plasmid DNA
in a saline solution containing agents that promote better delivery
of the plasmid DNA into cells in vivo when the formulation is delivered
with an electrical pulse. Electrical pulse delivery often comprises
electroporation where an electrical pulse is delivered to a tissue
that is previously injected with a drug. Electroporation of a tissue
causes transient interruption of cell membranes allowing more drug
to enter the cell through the interruptions or "pores".
The agents in the saline DNA solution that promote delivery of the
DNA into electroporated tissues include propylene glycols, polyethylene
glycols, poloxamers (block copolymers of propylene oxide and ethylene
oxide), or cationic lipids. They claim that the way that these agents
enhance deliver % of the DNA into cells is by either protecting
the DNA from degradation by DNases or by condensing the DNA into
a smaller form, or both.
Many of these attempts to enhance tissue transduction have used
agents that destroy muscle (bupivacaine, barium chloride) and actually
lower expression (Norman, J. et al., Methods in Molec. Med. 29:185-196
(1999)); have to be pre-injected before the DNA (sucrose); are expensive
organic polymers (polyvinyl pyrollidine), mutagens (intercalaters),
antigenic proteins (histones) or devices that destroy muscle tissue
(needless or needle-free injectors); or need to be inserted surgically
(sutures, sponges, intravascular pressure). Furthermore, most of
these methods may be expensive and not suitable or practical for
human use.
On the other hand, little attention has been given to the use of
alternative salt solutions and/or auxiliary agents in the pharmaceutical
formulation as a way of enhancing the efficiency of a polynucleotide-based
polypeptide delivery. Investigators in this field routinely use
normal saline or phosphate buffered saline (PBS 0.9% (i.e., about
154 mM) NaCl and 10 mM Na-phosphate) solutions for polynucleotide
delivery, e.g., by intramuscular injection, because they are physiologically
isotonic, isoosmotic, stable, non-toxic, and also because they have
been traditionally used for human intramuscular injections of other
drugs. However, sodium phosphate, in the absence of saline, has
been used in humans for delivery of non-polynucleotide-based drugs
(e.g., small molecules) administered via the intramuscular or intravenous
routes (See generally, Physician's Desk Reference. Medical Economics
Co, Monyvale, N.J. (1998)).
Sodium or potassium phosphate have been reported to enhance Lipofectin.TM.-mediated
transfection of human osteosarcoma cells in vitro (Kariko, K., et
al., Biochim Biophys Acta 1369:320-334 (1998)), and the use of RPMI
cell culture medium buffered with NaHCO.sub.3/Na.sub.2HPO.sub.4
were reported to be the best medium for forming DNA/cationic lipid
complexes in vitro. (Kichler, A., et al., Gene Ther. 5:855-860 (1998)).
There remains a need in the art for a convenient and safe way of
improving the effectiveness of in vivo polypeptide delivery via
direct administration of a polynucleotide. Aqueous solutions of
certain salts including sodium phosphate have been used in humans
(i.e., intramuscular injection of various small molecule drugs),
and detergents or surfactants as auxiliary agents are common additives
in drugs administered into human tissues. However, the use of certain
salts or auxiliary agents, or a combination thereof to improve the
transduction, i.e., the entry into cells, and/or expression-enhancing
efficiency of polynucleotides delivered in vivo is new.
SUMMARY OF THE INVENTION
The present invention is broadly directed to treatment of cancer
by administering in vivo, into a tissue of a mammal suffering from
cancer, a polynucleotide construct comprising a polynucleotide encoding
a cytokine. The polynucleotide construct is incorporated into the
cells of the mammal in vivo, and a therapeutically effective amount
of a cytokine is produced in vivo, and delivered to tumor cells.
Combinations of cytokine-encoding polynucleotides can be administered.
The present invention provides a pharmaceutical composition comprising
about 1 ng to 20 mg of a non-infectious, non-integrating polynucleotide
construct comprising a polynucleotide selected from the group consisting
of (a) a polynucleotide that hybridizes under stringent conditions
to the nucleotide sequence of SEQ ID No. 7 or the complement thereof,
wherein the polynucleotide sequence encodes a polypeptide that has
antiproliferative activity when added to NIH-OVCAR3 cells in vitro;
(b) a polynucleotide that encodes a polypeptide comprising an amino
acid sequence which, except for at least one but not more than 20
amino acid substitutions, deletions, or insertions, is identical
to amino acids -23 to 172 or 1 to 172 in SEQ ID No. 8, wherein the
polypeptide has antiproliferative activity when added to NIH-OVCAR3
cells in vitro; and (c) a polynucleotide that encodes a polypeptide
comprising amino acids 86-172 in SEQ ID No. 8, wherein the polypeptide
has antiproliferative activity when added to NIH-OVCAR3 cells in
vitro; and any of the above group complexed with one or more cationic
compounds selected from the group consisting of cationic lipids,
cationic peptides, cationic proteins, cationic polymers, and mixtures
thereof.
The present invention also provides a pharmaceutical composition
obtained by complexing a polynucleotide selected from the group
consisting of (a) a polynucleotide that hybridizes under stringent
conditions to the nucleotide sequence of SEQ ID No. 7 or the complement
thereof, wherein the polynucleotide sequence encodes a polypeptide
that has antiproliferative activity when added to NIH-OVCAR3 cells
in vitro; (b) a polynucleotide that encodes a polypeptide comprising
an amino acid sequence which, except for at least one but not more
than 20 amino acid substitutions, deletions, or insertions, is identical
to amino acids -23 to 172 or 1 to 172 in SEQ ID No. 8, wherein the
polypeptide has antiproliferative activity when added to NIH-OVCAR3
cells in vitro; (c) a polynucleotide that encodes a polypeptide
comprising amino acids 86-172 in SEQ ID No. 8, wherein the polypeptide
has antiproliferative activity when added to NIH-OVCAR3 cells in
vitro, with one or more cationic compounds selected from the group
consisting of cationic lipids, cationic peptides, cationic proteins,
cationic polymers, and mixtures thereof.
The present invention also provides a method of treating cancer
in a mammal, comprising administering into a tissue of the mammal
a non-infectious, non-integrating polynucleotide construct comprising
a polynucleotide encoding a cytokine, or an active fragment thereof,
such that the polynucleotide is expressed in vivo, and such that
the cytokine, or active fragment thereof, is delivered systemically
to a tumor tissue in an amount effective to treat the cancer.
The present invention also provides a method of treating cancer
in a mammal, comprising administering into a tissue of the mammal
a non-infectious, non-integrating polynucleotide construct comprising
a polynucleotide encoding a cytokine selected from the group consisting
of interferon-.omega., interferon-.alpha., and a combination thereof,
such that the polynucleotide or an active fragment thereof is expressed,
and such that the cytokine is delivered locally to a tumor tissue
in an amount effective to treat the cancer. Preferably, the polynucleotide
construct is complexed with a cationic vehicle, more preferably,
the cationic vehicle may be a cationic lipid, and most preferably,
the cationic lipid may be mixed with a neutral lipid.
Another object of the invention is to provide a method of selectively
transfecting malignant cells in a body cavity of a tumor-bearing
mammal, comprising administering into the body cavity at least one
non-infectious, non-integrating polynucleotide complexed with a
cationic vehicle, such that the polynucleotide is expressed substantially
in the malignant cells of the body cavity. Preferably, the cationic
vehicle comprises one or more cationic lipids, and more preferably,
the cationic vehicle comprises a cationic and neutral lipid mixture.
In a preferred embodiment, the present invention is used to suppress
peritoneal dissemination of malignant cells in a tumor-bearing mammal.
In particular, the mammal may have ovarian cancer, or metastasis
of ovarian cancer. Preferred polynucleotides may encode cytokines,
or active fragments thereof. Most preferably, the polynucleotide
may encode IL-2, or an active fragment thereof.
Compared to injection of recombinant cytokine polypeptides, the
methods described herein have several important advantages. The
present invention shows that in vivo transfection of cells with
encoding polynucleotide, such as an IL-2 or IFN.omega., results
in serum levels of the corresponding cytokine that have therapeutic
effects, and yet are lower than the maximal serum levels typically
required when cytokine polypeptides are injected. Further, injecting
frequent high doses of cytokine polypeptides can produce debilitating
side effects. The methods of the present invention provide cytokine
therapy requiring less frequent injections of cytokine-encoding
nucleic acids. The injection of polynucleotide constructs encoding
cytokines produces sustained, low levels of biologically active
cytokines that have beneficial effects, while minimizing adverse
side effects.
Compared to the delivery of cytokine genes via a viral gene delivery
vectors, the present method also has important advantages. Injection
of non-viral vectors of the present method does not induce significant
toxicity or pathological immune responses, as described, for example,
in mice, pigs or monkeys (Parker, et al., Human Gene Therapy 6:
575-590 (1995); and San, et al., Human Gene Therapy 4: 781-788 (1993)).
Thus, a non-viral vector is safer and can be repeatedly injected.
The present invention is further directed to compositions, and
methods for using such compositions, for improving the effectiveness
of polypeptide delivery into a vertebrate by administering in vivo,
a polypeptide-encoding polynucleotide in an aqueous solution sodium
phosphate. The polynucleotide is incorporated into the cells of
the vertebrate in vivo, and encodes a detectable amount or a prophylactically
or therapeutically effective amount, of a desired polypeptide.
The present invention further provides a composition selected from
the group consisting of (a) a composition comprising about 1 ng
to about 30 mg of a polynucleotide in aqueous solution, where the
polynucleotide operably encodes a polypeptide within vertebrate
cells in vivo; and sodium phosphate dissolved in the aqueous solution
at a molar concentration from about 20 mM to about 300 mM, and reaction,
association, or dissociation products thereof; and (b) a composition
comprising: about 1 ng to about 30 mg of a polynucleotide in aqueous
solution, where the polynucleotide operably encodes a polypeptide
within vertebrate cells in vivo; sodium phosphate dissolved in the
aqueous solution at a molar concentration from about 0.1 mM to about
150 mM, and reaction, association, or dissociation products thereof;
and a cationic lipid suspended in said aqueous solution; where the
aqueous solution is substantially free of chloride anion.
Another aspect of the present invention is a method for delivering
a polypeptide into a vertebrate, comprising administering to the
vertebrate a composition selected from the group consisting of (a)
a composition comprising about 1 ng to about 30 mg of a polynucleotide
in aqueous solution, where the polynucleotide operably encodes a
polypeptide within vertebrate cells in vivo; and sodium phosphate
dissolved in the aqueous solution at a molar concentration from
about 20 mM to about 300 mM, and reaction, association, or dissociation
products thereof; and (b) a composition comprising: about 1 ng to
about 30 mg of a polynucleotide in aqueous solution, where the polynucleotide
operably encodes a polypeptide within vertebrate cells in vivo;
sodium phosphate dissolved in the aqueous solution at a molar concentration
from about 0.1 mM to about 150 mM, and reaction, association, or
dissociation products thereof; and a cationic lipid suspended in
said aqueous solution; where the aqueous solution is substantially
free of chloride anion; such that the polypeptide encoded by the
delivered polynucleotide is expressed in the vertebrate, in an amount
sufficient to be detectable.
Another aspect of the present invention is a method for delivering
a therapeutic polypeptide into a vertebrate, comprising administering
to a vertebrate in need of such a therapeutic polypeptide a composition
selected from the group consisting of (a) a composition comprising
about 1 ng to about 30 mg of a polynucleotide in aqueous solution,
where the polynucleotide operably encodes a polypeptide within vertebrate
cells in vivo; and sodium phosphate dissolved in the aqueous solution
at a molar concentration from about 20 mM to about 300 mM, and reaction,
association, or dissociation products thereof; and (b) a composition
comprising: about 1 ng to about 30 mg of a polynucleotide in aqueous
solution, where the polynucleotide operably encodes a polypeptide
within vertebrate cells in vivo; sodium phosphate dissolved in the
aqueous solution at a molar concentration from about 0.1 mM to about
150 mM, and reaction, association, or dissociation products thereof;
and a cationic lipid suspended in said aqueous solution; where the
aqueous solution is substantially free of chloride anion; such that
a therapeutic polypeptide encoded by the delivered polynucleotide
is expressed in the vertebrate, in a therapeutically effective amount.
The present invention also provides a method of producing antibodies
to a polypeptide in a vertebrate, comprising administering to the
vertebrate a composition selected from the group consisting of (a)
a composition comprising about 1 ng to about 30 mg of a polynucleotide
in aqueous solution, where the polynucleotide operably encodes a
polypeptide within vertebrate cells in vivo; and sodium phosphate
dissolved in the aqueous solution at a molar concentration from
about 20 mM to about 300 mM, and reaction, association, or dissociation
products thereof; and (b) a composition comprising: about 1 ng to
about 30 mg of a polynucleotide in aqueous solution, where the polynucleotide
operably encodes a polypeptide within vertebrate cells in vivo;
sodium phosphate dissolved in the aqueous solution at a molar concentration
from about 0.1 mM to about 150 mM, and reaction, association, or
dissociation products thereof; and a cationic lipid suspended in
said aqueous solution; where the aqueous solution is substantially
free of chloride anion; such that a polypeptide encoded by the delivered
polynucleotide is expressed in the vertebrate, in a sufficient amount
to generate antibody to the encoded polypeptide in the vertebrate.
The present invention also provides a method of enhancing or modulating
an immune response in a vertebrate in need of such an enhanced or
modulated immune response, comprising administering to the vertebrate
a composition selected from the group consisting of (a) a composition
comprising about 1 ng to about 30 mg of a polynucleotide in aqueous
solution, where the polynucleotide operably encodes a polypeptide
within vertebrate cells in vivo; and sodium phosphate dissolved
in the aqueous solution at a molar concentration from about 20 mM
to about 300 mM, and reaction, association, or dissociation products
thereof; and (b) a composition comprising: about 1 ng to about 30
mg of a polynucleotide in aqueous solution, where the polynucleotide
operably encodes a polypeptide within vertebrate cells in vivo;
sodium phosphate dissolved in the aqueous solution at a molar concentration
from about 0.1 mM to about 150 mM, and reaction, association, or
dissociation products thereof; and a cationic lipid suspended in
said aqueous solution; where the aqueous solution is substantially
free of chloride anion; such that an immunogenic and/or immunomodulatory
polypeptide encoded by the delivered polynucleotide is expressed
in the vertebrate, in a sufficient amount to induce a desired immune
response in the vertebrate to prevent disease or treat disease,
i.e., cure disease, reduce the severity of disease symptoms, or
prolong the life of the vertebrate.
The invention further provides a method of delivering a physiologically
or metabolically necessary polypeptide to a vertebrate incapable
of making a functional form of the polypeptide, comprising administering
to the vertebrate a composition selected from the group consisting
of (a) a composition comprising about 1 ng to about 30 mg of a polynucleotide
in aqueous solution, where the polynucleotide operably encodes a
polypeptide within vertebrate cells in vivo; and sodium phosphate
dissolved in the aqueous solution at a molar concentration from
about 20 mM to about 300 mM, and reaction, association, or dissociation
products thereof; and (b) a composition comprising: about 1 ng to
about 30 mg of a polynucleotide in aqueous solution, where the polynucleotide
operably encodes a polypeptide within vertebrate cells in vivo;
sodium phosphate dissolved in the aqueous solution at a molar concentration
from about 0.1 mM to about 150 mM, and reaction, association, or
dissociation products thereof; and a cationic lipid suspended in
said aqueous solution; where the aqueous solution is substantially
free of chloride anion; such that a functional self polypeptide,
i.e., a physiologically or metabolically necessary polypeptide encoded
by the delivered polynucleotide is expressed in the vertebrate,
in a sufficient amount to supply the vertebrate's requirements for
the polypeptides.
The present invention also provides a pharmaceutical kit selected
from the group consisting of: (a) a pharmaceutical kit comprising:
a container or containers holding about 1 ng to about 30 mg of a
polynucleotide which operably encodes a polypeptide within vertebrate
cells in vivo; an amount of sodium phosphate which, when dissolved
in a prescribed volume of distilled water, results in an aqueous
solution with a molar concentration of sodium phosphate from about
20 mM to about 300 mM, and reaction, association, or dissociation
products thereof; and optionally, an administration means and/or
an instruction sheet; whereby the polynucleotide is provided in
a prophylactically or therapeutically effective amount to treat
a vertebrate; and (b) a pharmaceutical kit comprising: a container
or containers holding about 1 ng to about 30 mg of a polynucleotide
which operably encodes a polypeptide within vertebrate cells in
vivo; an amount of sodium phosphate which, when dissolved in a prescribed
volume of distilled water, results in an aqueous solution with a
molar concentration of said salt from about 0.1 mM to about 150
mM, and reaction, association, or dissociation products thereof,
and where the aqueous solution formed thereby is essentially free
of chloride anion; a cationic lipid; and optionally, an administration
means and/or an instruction sheet; whereby the polynucleotide is
provided in a prophylactically or therapeutically effective amount.
Any of components of the pharmaceutical kit can be provided in a
single container, or in multiple containers packaged together.
The inventors have discovered that delivery of the compositions
provided herein to a vertebrate results in much improved in vivo
polypeptide expression over the delivery of existing nucleic acid-based
compositions, e.g., compositions comprising polynucleotides which
encode a polypeptide and an aqueous solution consisting of sterile
water, normal saline (i.e., 154 mM sodium chloride), or phosphate
buffered saline (i.e., 154 mM sodium chloride plus 10 mM sodium
phosphate).
BRIEF DESCRIPTION OF THE FIGURES
A more complete appreciation of the invention and many of the attendant
advantages thereof will be readily obtained as the same become better
understood by reference to the following detailed description when
considered in connection with the accompanying figures.
FIG. 1 shows the plasmid map of VR4151 (SEQ ID No. 4). The cytomegalovirus
immediate-early gene promoter enhancer and 5' untranslated sequences
(5'UTR+intron A) drive the expression of the human interferon .omega.
coding sequence. The transcriptional terminator region includes
polyadenylation and termination signals derived from the rabbit
.beta.-globin gene.
FIGS. 2A and 2B show the pharmacokinetics of hIFN.omega. in the
serum of C57BL/6 mice (FIG. 2A) and nude mice (FIG. 2B) after a
single intramuscular (i.m.) injection of hIFN.omega. plasmid DNA
(VR4151). Mice were injected i.m. with 100 .mu.g of VR4151. Following
the intramuscular injection, mice were bled daily, and serum was
collected and assayed for hIFN.omega. polypeptide using an ELISA.
Each point represents an average of four mice. In C57BL/6 mice,
the single i.m. injection resulted in peak serum levels of 254 pg/ml
on day 6 after injection, and serum levels were still detectable
14 days after injection (50 pg/ml) (FIG. 2A). In nude mice, the
single i.m. injection resulted in peak serum levels of 648 pg/ml
on day 7, and serum levels were still detectable 14 days after injection
(134 pg/ml) (FIG. 2B).
FIGS. 3A-3F show that systemic mIFN.alpha. treatment reduces tumor
volume (FIGS. 3A, 3C, and 3E) and increases survival (FIGS. 3B,
3D, and 3F) in three murine tumor models. C57BL/6 mice bearing subcutaneous
B16F10 melanoma (FIGS. 3A and 3B), subcutaneous glioma 261 (FIGS.
3C and 3D), or DBA/2 mice bearing subcutaneous Cloudman melanoma
(FIGS. 3E and 3F) were injected with 100 .mu.g either of VR4111
(mIFN.alpha. plasmid) or VR1055 (control plasmid), twice per week
for three weeks, beginning on day 4 after tumor cell injection (n=8-10
mice per group).
FIGS. 4A and 4B show that systemic mIFN.alpha., mIL-2 or mIL-12
plasmid DNA treatment reduces tumor volume (FIG. 4A) and mIFN.alpha.
or mIL-12 plasmid DNA treatment increases survival (FIG. 4B) in
the subcutaneous B16F10 melanoma model. C57BL/6 mice bearing subcutaneous
B16F10 melanoma were injected with 100 .mu.g of VR4111 (mIFN.alpha.),
VR4001 (mIL-12), VR1110 (mIL-2), or VR1012 (control plasmid) (n=15-16
mice per group) twice per week for three weeks.
FIGS. 5A and 5B show that i.m. administration of hIFN.omega. pDNA
reduces tumor volume (FIG. 5A) and increases survival (FIG. 5B)
in nude mice bearing human A431 epidermoid carcinoma tumors. Mice
bearing human A431 tumors between 30-80 mm.sup.3 were injected i.m.
with 200 .mu.g of either VR4151 (hIFN.omega. plasmid) or VR1055
(control plasmid) twice per week for three weeks (n=15)
FIG. 6 shows that i.m. administration of mIFN.alpha. pDNA reduces
B16F10 melanoma lung metastases in C57BL/6 mice. Mice bearing lung
metastases of B16F10 melanoma were injected i.m. with 100 .mu.g
of either VR4111 or VR1055 twice per week for three weeks, beginning
on day 4 after tumor cell injection (n=10 mice per group). "TNTC"
means too numerous to count as seen in the control group.
FIGS. 7A and 7B show that i.m. administration of mIFN.alpha. pDNA
reduces intradermal M5076 primary tumor growth (FIG. 7A) as well
as liver metastases (FIG. 7B) in C57BL/6 mice bearing murine M5076
reticulum cell sarcoma cells. Mice bearing M5076 tumors were injected
i.m. with 100 .mu.g of either VR4111 or VR1055 twice per week for
three weeks, beginning on day 4 after tumor cell inoculation (n=10-13
mice per group).
FIGS. 8A-8D show a comparison of different dosages and frequencies
of mIFN.alpha. pDNA administration in the subcutaneous B16F10 melanoma
model. C57BL/6 mice bearing subcutaneous B16F10 melanoma were injected
i.m. with 50 .mu.g or 100 .mu.g of either VR4111 or VR1055 twice
a week for 3 weeks beginning 4 days after tumor cell inoculation
(n=10 mice per group). All groups treated with 100 .mu.g of VR4111
showed significant reduction in tumor growth by day 21 (p=0.002)
and significant enhancement in survival (p<0.008) with all treatments
tested (FIGS. 8A and 8B). In mice treated with 50 .mu.g VR4111,
tumor growth was significantly reduced by day 21 (p=0.005), and
survival was significantly increased (p<0.003) in the groups
of mice that were injected twice per week or once per week. The
group injected every other week with 50 .mu.g VR4111 was not significantly
different from the mice that received the control plasmid (FIGS.
8C and 8D).
FIGS. 9A-9D shows the results of experiments performed to determine
the role of NK and T cells in the antitumor response induced by
mIFN.alpha. plasmid DNA. Nude mice (T cell deficient) (FIGS. 9A
and 9B), and beige-nude mice (NK and T cell deficient) (FIGS. 9C
and 9D) bearing subcutaneous B16F10 melanoma tumors were injected
i.m. with 100 .mu.g of either VR4111 or VR1055 twice per week for
three weeks, beginning on day 4 after tumor cell injection (n=15
mice per group). No significant reduction in tumor volume or increase
in survival was found for nude or nude-beige mice treated with VR4111,
suggesting that T cells are involved in the mIFN.alpha. antitumor
response.
FIGS. 10A and 10B show the results of experiments performed to
evaluate the role of CD4.sup.+ and CD8.sup.+ T cells in the mIFN.alpha.
DNA antitumor response. For depletion of CD4.sup.+ and CD8.sup.+
T cells, C57BL/6 mice bearing subcutaneous B16F1 melanoma tumors
were injected i.p. with 500 .mu.g of either the anti-CD4 mAb (clone
GK1.5, rat IgG) (ATCC, Rockville, Md.) or anti-CD8 mAb (clone 2.43,
rat IgG) (ATCC, Rockville, Md.) one day after each i.m. injection
of 100 .mu.g of either VR4111 or VR1055 twice per week for three
weeks (n=10 mice per group). The mIFN.alpha. plasmid DNA therapy
significantly reduced tumor growth (p.ltoreq.0.002) and enhanced
survival (p.ltoreq.0.008) of both normal mice and mice depleted
of CD4.sup.+ T cells, suggesting that CD4.sup.+ T cells were not
required for the response. In contrast, mice depleted of CD8.sup.+
T cells and injected with VR4111 had tumor volumes and survival
that were not significantly different from mice treated with the
control plasmid DNA, indicating a requirement for CD8.sup.+ T cells
in the antitumor response.
FIGS. 11A and 11B show that intratumoral hIFN.omega. (VR4151) and
hIFN.alpha. (VR4112) treatment reduces tumor volume in the human
A375 melanoma model (FIG. 11A) and human NIH-OVCAR3 (FIG. 11B) in
nude mice. Mice bearing subcutaneous tumor received direct intratumoral
injections of a complex of DNA:DMRIE/DOPE (1:1 DNA:lipid mass ratio,
100 .mu.g of plasmid DNA) for 6 consecutive days followed by an
additional 5 treatments every other day for a total of 11 injections
(A375 melanoma model), or for every other day for a total of 11
injections (NIH-OVCAR3 ovarian cancer model).
FIGS. 12A and 12B show that intratumoral mIFN.alpha. (VR4101) plasmid
DNA treatment reduces tumor volume (FIG. 12A) and increases survival
(FIG. 12B) in the subcutaneous B16F10 melanoma model in C57BL/6
mice. Mice received a subcutaneous implantation of 10.sup.4 B16F10
cells into the flank. Beginning at day 12 post tumor implant, mice
received six consecutive intratumoral injections of a complex of
pDNA:DMRIE/DOPE (1:1 DNA:DMRIE mass ratio 100 .mu.g of plasmid DNA).
FIGS. 13A and 13B show luciferase activity in peritoneal tissues
and MOT ascites in mice after i.p. injection of luciferase DNA:lipid
complex. The results show high levels of reporter gene expression
in ascites but low levels in peritoneal tissue. MOT tumor-bearing
C3H/HeN mice received i.p. injections of a complex of pDNA:DMRIE/DOPE
(1:1 DNA:DMRIE mass ratio, 100 .mu.g of plasmid DNA) on days 5 and
6 after tumor cell implant. Tissues were collected 1 day (FIG. 13A)
or 3 days (FIG. 13B) following the DNA:lipid injection.
FIGS. 14A and 14B show serum levels of IL-2 after i.p. injection
of either IL-2 pDNA or protein in MOT tumor bearing mice. The serum
levels of IL-2 were much lower than levels in ascites. Ascites and
serum were collected at 4 hours and days 1, 2, 3, 6 and 10 post
DNA or protein injection (5 mice for each time point), and analyzed
for mIL-2 polypeptide using an ELISA.
FIGS. 15A-15F show a significant reduction in MOT tumor growth
(p=0.01) (FIG. 15A) and increased survival (p=0.04) (FIG. 15B) of
mice treated with i.p. injection of IL-2 pDNA:lipid on days 5-10
after tumor cell injection. The DNA was complexed at either a 1:1
(15A and 15B) or 5:1 (FIGS. 15C and 15D) DNA:DMRIE mass ratio (100
.mu.g pDNA). Plasmid DNA without lipid was not effective (FIGS.
15E and 15F)
FIGS. 16A and 16B show, that i.p. mIL-2 plasmid DNA (VR1110):lipid
treatment inhibits tumor growth (FIG. 16A) and enhances survival
(FIG. 16B) in the MOT tumor model in C3H/HeN mice. MOT tumor-bearing
mice received three alternative-day i.p. injections of a complex
of pDNA:DMRIE/DOPE (1:1 DNA:DMRIE mass ratio, 100 .mu.g of plasmid
DNA).
FIGS. 17A and 17B show a significant reduction in MOT tumor growth
and increased survival of mice treated with i.p. injection of IL-2
DNA:lipid followed by debulking of tumor ascites. MOT tumor-bearing
mice received six consecutive intraperitoneal injections of a complex
of pDNA:DMRIE/DOPE (1:1 DNA:DMRIE mass ratio, 100 .mu.g of pDNA)
and debulked of 5 ml of tumor ascites 4 days after the last DNA:lipid
injection (n=10).
FIGS. 18A and 18B show dose-response of mIL-2 pDNA (VR110):lipid
treatment in the MOT tumor model. C3H/HeN mice bearing MOT tumor
were injected with 25, 50 or 100 .mu.g of VR1110:DMRIE/DOPE on days
5, 8 and 11 after MOT tumor cell injection. In mice treated with
50 or 100 .mu.g of VR1110, tumor growth was significantly reduced
(p=0.002) and survival significantly enhanced (p=0.01) by day 15
post tumor cell inoculation compared to the control. Tumor-bearing
mice treated with 25 .mu.g of VR1110:lipid were not significantly
different from the control mice for either tumor volume or survival
(n=15).
FIGS. 19A-19D show the cytokine profile of ovarian tumor ascites
in C3H/HeN mice MOT tumor model following mIL-2 pDNA (VR1110):lipid
treatment. Mice received i.p. injections of a complex of pDNA/DMRIE/DOPE
(1:1 DNA/DMRIE mass ratio, 100 .mu.g of plasmid DNA) on days 5,
8 and 11 after tumor cell implant. Two days after each injection,
mice were sacrificed (5 mice for each time point), and the ascites
were collected and analyzed for cytokine concentration. The level
of IL-2 (days 7, 10 and 13) as well as IFN.gamma. and GM-CSF (days
10 and 13) were markedly elevated suggesting that IL-2 upregulates
IFN.gamma. and GM-CSF production.
FIGS. 20A and 20B show that i.p. mIFN.alpha. pDNA (VR4111):lipid
treatment enhances survival (FIG. 20B) in the MOT tumor model in
C3H/HeN mice. MOT tumor-bearing mice received three alternative-day
i.p. injections of a complex of pDNA:DMRIE/DOPE (1:1 DNA:DMRIE mass
ratio, 100 .mu.g of plasmid DNA).
FIG. 21 shows the schematic contents of plasmid DNAs used in the
examples that follow. All vectors contain a pUC19 origin of replication,
human cytomegalovirus intron A, and the bacterial kanamycin resistance
gene. "Lux" denotes the coding region encoding luciferase,
from the firefly, Photinus pyralis; "CMV" denotes the
human cytomegalovirus immediate early region--promoter and enhancer;
"BGH" denotes the bovine growth hormone transcriptional
terminator; "LacZ" denotes the coding region encoding
the .beta.-galactosidase protein of Escherichia coli; "RSV"
denotes the Rous sarcoma virus promoter and enhancer; "EPO"
denotes the coding region encoding murine erythropoietin; "SEAP"
denotes the coding region for secreted human placental alkaline
phosphatase; "Rat preproinsulin" denotes the coding region
for rat preproinsulin containing a point mutation to change histidine
B10 (codon CAC), to aspartic acid (codon GAC), Abai, A. M., et al.
Human Gene Therapy 10:2637-2649 (1999); "IFN-omega" denotes
the coding region encoding human interferon-.omega.; "mRGB"
denotes the modified rabbit .beta.-globin transcriptional terminator;
and "NP" denotes the coding region encoding the nucleoprotein
of influenza virus A/PR/8/34. Intermediate and parental plasmids
*VR1012, **VR1255 and ***VIJ were prepared as described by Manthorpe,
M. et al., Hum. Gene Ther. 4:419-431 (1993), Hartikka, J. et al.,
Hum. Gen. Ther. 7:1205-1217 (1996), and Montgomery, D. L. et al.,
DNA Cell Biol. 12:777-783 (1993), respectively. VR1043 was derived
from VR1012 by replacing the SacI-NdeI CMV promoter enhancer fragment
with the RSV promoter enhancer.
FIG. 22A is a bar graph demonstrating the effectiveness of sodium
phosphate concentration on luciferase expression in mouse muscle.
Fifty .mu.g of plasmid VR1223 DNA per 50 .mu.l sodium phosphate
solution at the indicated molar concentrations was injected into
mouse quadriceps and the muscles were extracted and assayed for
enzyme activity 7 days later. Bars represent Standard Error of the
Mean (n=50, 5 experiments each with n=10 per concentration). Peak
expression occurred with DNA dissolved in 150 mM sodium phosphate,
and yielded 386 ng luciferase per muscle which is 4.3-fold higher
than the saline average (dashed line at 89 ng luciferase per muscle).
The 80, 100, 150 and 200 mM sodium phosphate values were significantly
higher than saline by Mann-Whitney rank sum test (p<0.05).
FIG. 22B is a bar graph demonstrating the effect of pH of the sodium
phosphate and potassium phosphate solutions on luciferase expression
in mouse muscle. Fifty .mu.g of plasmid VR1223 DNA per 50 .mu.l
sodium phosphate and potassium phosphate solution at the indicated
pH was injected into mouse quadriceps and the muscles were extracted
and assayed for enzyme activity 7 days later. Bars represent Standard
Error of the Mean (n=20 muscles per group).
FIG. 22C is a graph plotting the effect of pH of the various salt
solutions listed in Table 11-A on luciferase expression in mouse
muscle.
FIG. 22D is a graph plotting the effect of osmolarity of the various
salt solutions listed in Table 11-B on luciferase expression in
mouse muscle.
FIG. 23 is a bar graph demonstrating the reproducibility of the
enhancement of luciferase expression in muscle upon delivery in
150 mM sodium phosphate. In each of nine experiments, ten quadriceps
muscles in 5 mice per group were injected with 50 .mu.g of plasmid
VR1223 DNA dissolved in 50 .mu.l saline or in 150 mM sodium phosphate
(NaP). Bars represent the average ng luciferase per muscle for each
experiment numbered 1 through 9. Error bars represent Standard Error
of the Mean.
FIG. 24 shows the comparison of the effect of a 150 mM sodium phosphate
solution on the expression of three reporter genes. Fifty .mu.g
of plasmid VR1223 (luciferase), 10 .mu.g of plasmid VR1418 (.beta.-galactosidase,
or LacZ) or 50 .mu.g of plasmid VR4151 (human IFN.omega.) dissolved
in 50 .mu.l saline or in 150 mM sodium phosphate solution were injected
into the quadriceps muscles of BALB/c mice. For luciferase and LacZ
DNAs, the muscles were extracted and assayed 7 days later for enzyme
activity. For IFN-.omega. DNA, serum was collected at 7 days after
the injection and assayed for IFN-.omega. protein. Values are expressed
as average ng of gene product per muscle or per ml serum. Bars represent
Standard Error of the Mean. For luciferase, n.sub.Saline=413, n.sub.NaP=120;
for .beta.-galactosidase, n.sub.Saline=119, n.sub.NaP=180; for IFN-.omega.,
n.sub.Saline=10, n.sub.NaP=9. The average expression in NaP was
significantly higher than saline by Mann-Whitney rank sum test for
luciferase (p=0.001), .beta.-galactosidase (p=0.001) and IFN.omega.
(p=0.02).
FIG. 25 shows long-term effects of a 150 mM sodium phosphate solution
on the expression of secreted reporter gene products. Compositions
comprising plasmids VR3301 encoding human placental alkaline phosphatase
(SEAP), VR3502 encoding rat preproinsulin, and VR2901 encoding mouse
erythropoietin, dissolved in saline or in 150 mM sodium phosphate,
were injected bilaterally into mice as described in Example 1. At
the indicated times after injections, serum was collected and assayed
for SEAP or proinsulin expression, or hematocrits were measured
as an indication of erythropoietin expression. Control mice injected
with plasmid DNA encoding canine clotting Factor IX (open triangles
in the lower graph) in 150 mM sodium phosphate exhibited an average
hematocrit of 46. Bars represent Standard Error of the Mean (n=10).
By the Mann-Whitney rank sum test, the sodium phosphate values were
significantly different (p values all <0.007) from the saline
values for each time point and for all three reporters.
FIG. 26 shows the effect of a 150 mM sodium phosphate solution
on DNA degradation in mouse muscle extract or serum. VR1255 plasmid
DNA dissolved in each of 4 aqueous solutions was spiked with 10%
(v/v) unbuffered mouse Muscle Extract or Serum and the spiked solutions
were incubated for 2 hours at 37.degree. C. The reactions were neutralized
with SDS+EDTA and analyzed by agarose gel electrophoresis. The top
row of four lanes are from the solutions spiked with muscle extract
and the bottom row of lanes are from the solutions spiked with serum.
The DNA samples from left to right are in: Control solution (pre-neutralized
sample in water), Water, Saline, or NaP (150 mM sodium phosphate).
On the right side of the right lane are indicated the position of
the bands corresponding to nicked, linear, closed circular, and
degraded plasmid DNA. The numbers at the bottom of the lower lanes
are 7 day luciferase expression values taken from Tables 1 and 2
where DNA in the indicated vehicle was injected into muscle.
FIGS. 27A and 27B show the effects of a 150 mM sodium phosphate
solution on DNA vaccination. Mice were vaccinated bilaterally in
the quadriceps muscle with 5 .mu.g of plasmid VR4700, encoding the
influenza virus nucleoprotein, which was dissolved in 50 .mu.l of
saline or in 50 .mu.l of 150 mM sodium phosphate on days 0 and 21.
(A) Serum was collected at day 42 and assayed for anti-NP antibody
titer by ELISA. Three separate experiments were performed with n=10
mice each, labeled 1-3. The average (Avg.) of all three experiments
is indicated in the black bar. Values are expressed as anti-NP specific
titer (n=10, 2 experiments with n=5). Error bars represent Standard
Error of the Mean. Average anti-NP titers from NaP groups 1-3 were
significantly different from the saline averages by Mann-Whitney
rank sum test (p<0.04) as was the average titers from all 3 groups
(p<0.001). (B) At day 60 the spleens were collected, dissociated
and assayed for the presence of NP-specific cytolytic T lymphocyte
activity. Splenocytes from unvaccinated mice served as controls
("Naive"). Average % NP specific lysis from the saline
and NaP groups were not significantly different by Mann Whitney
rank sum test.
FIG. 28 shows the effects of sodium phosphate solutions on luciferase
expression in lung following delivery of compositions comprising
plasmid DNA encoding luciferase. Mouse lungs were intranasally instilled
with compositions comprising 132 .mu.g of plasmid VR1223 encoding
luciferase, complexed with GAP-DLRIE/DOPE (1:1) cationic liposomes
at a molar ratio of 4:1 DNA to lipid in water or in various aqueous
solutions of sodium phosphate. The lungs were extracted 3 days later
and assayed for luciferase activity. Values are expressed in ng
luciferase per lung +/- Standard Error of the Mean (n.sub.water
and n.sub.25mMNaP=35; n.sub.10mMNaP, and n.sub.150mMNap=15 with
n=5 per each individual experiment). The 2.5 mM NaP solution averages
were significantly different by Mann-Whitney rank sum test from
all the other groups (p=<0.001).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is broadly directed to treatment of cancer
by administering in vivo, into a tissue of a mammal suffering from
cancer, at least one polynucleotide construct comprising at least
one polynucleotide encoding at least one cytokine, or at least one
active fragment thereof. The polynucleotide construct is incorporated
into the cells of the mammal in vivo, and a therapeutically effective
amount of a cytokine is produced in vivo, and delivered to tumor
cells. Combinations of cytokine-encoding polynucleotides can be
administered.
The present invention provides a pharmaceutical composition comprising
about 1 ng to 20 mg of a non-infectious, non-integrating polynucleotide
construct comprising a polynucleotide selected from the group consisting
of (a) a polynucleotide that hybridizes under stringent conditions
to the nucleotide sequence of SEQ ID No. 7 or the complement thereof,
wherein the polynucleotide sequence encodes a polypeptide that has
antiproliferative activity when added to NIH-OVCAR3 cells in vitro;
(b) a polynucleotide that encodes a polypeptide comprising an amino
acid sequence which, except for at least one but not more than 20
amino acid substitutions, deletions, or insertions, is identical
to amino acids -23 to 172 or 1 to 172 in SEQ ID No. 8, and wherein
the polypeptide has antiproliferative activity when added to NIH-OVCAR3
cells in vitro; and (c) a polynucleotide that encodes a polypeptide
comprising amino acids 86-172 in SEQ ID No. 8, wherein the polypeptide
has antiproliferative activity when added to NIH-OVCAR3 cells in
vitro; and one or more cationic compounds selected from the group
consisting of cationic lipids, cationic peptides, cationic proteins,
cationic polymers, and mixtures thereof. The pharmaceutical composition
can be used to practice all of the methods of the present invention.
The present invention also provides a pharmaceutical composition
obtained by complexing a polynucleotide selected from the group
consisting of (a) a polynucleotide that hybridizes under stringent
conditions to the nucleotide sequence of SEQ ID No. 7 or the complement
thereof, wherein the polynucleotide sequence encodes a polypeptide
that has antiproliferative activity when added to NIH-OVCAR3 cells
in vitro; (b) a polynucleotide that encodes a polypeptide comprising
an amino acid sequence which, except for at least one but not more
than 20 amino acid substitutions, deletions, or insertions, is identical
to amino acids -23 to 172 or 1 to 172 in SEQ ID No. 8, wherein the
polypeptide has antiproliferative activity when added to NIH-OVCAR3
cells in vitro; and (c) a polynucleotide that encodes a polypeptide
comprising amino acids 86-172 in SEQ ID No. 8, wherein the polypeptide
has antiproliferative activity when added to NIH-OVCAR3 cells in
vitro, with one or more cationic compounds selected from the group
consisting of cationic lipids, cationic peptides, cationic proteins,
cationic polymers, and mixtures thereof.
The pharmaceutical composition of the present invention can be
a polynucleotide construct comprising a polynucleotide that hybridizes
under stringent conditions to the nucleotide sequence of SEQ ID
No. 7 or the complement thereof, wherein the polynucleotide sequence
encodes a polypeptide that has antiproliferative activity when added
to NIH-OVCAR3 cells in vitro, and one or more cationic compounds
selected from the group consisting of cationic lipids, cationic
peptides, cationic proteins, cationic polymers, and mixtures thereof.
Alternatively, the pharmaceutical composition of the present invention
can be a polynucleotide construct comprising a polynucleotide that
encodes a polypeptide comprising an amino acid sequence which, except
for at least one but not more than 20 amino acid substitutions,
deletions, or insertions, is identical to amino acids -23 to 172
or 1 to 172 in SEQ ID No. 8, wherein the polypeptide has antiproliferative
activity when added to NIH-OVCAR3 cells in vitro, and one or more
cationic compounds selected from the group consisting of cationic
lipids, cationic peptides, cationic proteins, cationic polymers,
and mixtures thereof. Alternatively, the pharmaceutical composition
of the present invention can be a polynucleotide construct comprising
a polynucleotide that encodes a polypeptide comprising amino acids
86-172 in SEQ ID No. 8, wherein the polypeptide has antiproliferative
activity when added to NIH-OVCAR3 cells in vitro; and one or more
cationic compounds selected from the group consisting of cationic
lipids, cationic peptides, cationic proteins, cationic polymers,
and mixtures thereof.
The pharmaceutical composition of the present invention comprises
at least one polynucleotide construct comprising at least one polynucleotide
encoding an IFN.omega., or an active fragment thereof. Preferably,
the polynucleotide construct contains a polynucleotide encoding
a human IFN.omega.. More preferably, IFN.omega. is encoded by nucleotides
1 to 585 in SEQ ID No. 7 (corresponding to amino acids -23 to 172
in SEQ ID No. 8), or by nucleotides 70 to 585 in SEQ ID No. 7 (corresponding
to amino acids 1 to 172 in SEQ ID No. 8). Most preferably, the polynucleotide
construct is VR4151 in SEQ ID No. 4. The polynucleotide construct
may be complexed with one or more cationic compounds selected from
the group consisting of cationic lipids, cationic peptides, cationic
proteins, cationic polymers, and mixtures thereof. Preferably, the
polynucleotide construct is complexed with one or more cationic
lipids. More preferably, the polynucleotide construct is complexed
with one or more cationic lipids and one or more neutral lipids.
Still more preferably, the cationic lipid is (.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanimi-
nium bromide (DMRIE) and the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine
(DOPE) such that the mass ratio of polynucleotide construct to lipid
is from about 10:1 and about 0.5:1. More preferably, the mass ratio
of polynucleotide construct to lipid is from about 5:1 and about
1:1. Still more preferably, the mass ratio of polynucleotide construct
to lipid is about 5:1.
Cytokine-encoding plasmids discussed herein include VR4102 (hIFN.alpha.
in the VR1012 vector) (SEQ ID No. 1), VR4112 (hIFN.alpha. in the
VR1055 vector) (SEQ ID No. 2), VR4150 (hIFN.omega. in the VR1012
vector) (SEQ ID No. 3), VR4151 (hIFN.omega. in the VR1055 vector)
(SEQ ID No. 4), VR4101 (mIFN.alpha. in the VR1012 vector) (SEQ ID
No. 5), VR4111 (mIFN.alpha. in the VR1055 vector) (SEQ ID No. 6),
and VR1110 (mIL-2 in the VR1012 vector), VR1103 (hIL-2 in the VR1012
vector) (SEQ ID No: 25), VR4001 (mIL-12 in the VR1033 vector), and
VR1700 (mGM-CSF in the VR1012 vector).
Cytokine-encoding cDNAs discussed herein include the cDNA for hIFN.omega.
(SEQ ID No. 7), the cDNA for hIFN.alpha. (SEQ ID No. 9), the cDNA
for mIFN.alpha. (SEQ ID No. 11), the cDNA for hIL-2 (SEQ ID No.
13 and the coding portion of SEQ ID No. 25), the cDNA for mIL-2
(for example, as disclosed in Kashima et al., Nature 313:402-404
(1985), which is hereby incorporated by reference) the cDNA for
mIL-12 (for example, as disclosed in Tone et al., Eur. J. Immunol.
26:1222-1227(1996), which is hereby incorporated by reference),
and the cDNA for mGM-CSF (for example, as disclosed in Gough et
al., EMBO J. 4:645-653 (1985), which is hereby incorporated by reference).
Cytokine polypeptides discussed herein include hIFN.omega. (SEQ
ID No. 8), hIFN.alpha. (SEQ ID No. 10), mIFN.alpha. (SEQ ID No.
12), and hIL-2 (SEQ ID No. 14 and SEQ ID No. 26).
By "stringent conditions" is intended a hybridization
by overnight incubation at 42.degree. C. in a solution comprising:
50% formamide, 5.times.SSC (750 mM NaCl, 15 mM trisodium citrate),
50 mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA, followed by repeatedly washing the filters (at least three
times) in 0.1.times.SSC and 0.1% sodium dodecyl sulfate (w/v) for
20 minutes at about 65.degree. C.
By "active fragment" is intended a fragment of a cytokine
that displays the antiproliferative activity of the mature or full
length cytokine. For example, a full length hIFN.omega. is set forth
in amino acids -23 to 172 of SEQ ID No. 8. The corresponding mature
hIFN.omega. is set forth in amino acids 1 to 172 of SEQ ID No. 8.
Active fragments of hIFN.omega. include, but are not limited to
a polypeptide comprising amino acids 86-172 in SEQ ID No. 8, a polypeptide
comprising amino acids 61-172 in SEQ ID No. 8, a polypeptide comprising
amino acids 41-172 in SEQ ID No. 8, and a polypeptide comprising
amino acids 21-172 in SEQ ID No. 8. A full length hIFN.alpha. is
set forth in amino acids -23 to 166 of SEQ ID No. 10. The corresponding
mature hIFN.alpha. is set forth in amino acids 1 to 166 of SEQ ID
No. 10. Active fragments of hIFN.alpha. include, but are not limited
to a polypeptide comprising amino acids 83-166 in SEQ ID No. 10,
a polypeptide comprising amino acids 61-166 in SEQ ID No. 10, a
polypeptide comprising amino acids 41-166 in SEQ ID No. 10, and
a polypeptide comprising amino acids 21-166 in SEQ ID No. 10. Full
length hIL-2 is set forth in amino acids -20 to 133 of SEQ ID No.
14. The corresponding mature hIL-2 is set forth in amino acids 1
to 133 of SEQ ID No. 14. Active fragments of hIL-2 include, but
are not limited to a polypeptide comprising amino acids 58 to 105
in SEQ ID No. 14, and a polypeptide comprising amino acids 20 to
126 in SEQ ID No. 14.
Assays of antiproliferative activity in vitro are well known to
those of ordinary skill in the art. For example, one antiproliferation
assay that can be used is to treat cultured cells, such as human
ovarian NIH-OVCAR3 cells (ATCC, Rockville, Md.), with supernatants
from human melanoma UM449 cells transfected with the polynucleotide
construct containing a polynucleotide encoding an IFN.omega. or
an active fragment thereof. In this antiproliferation assay, NIH-OVCAR3
cells are cultured and plated in 96 well-tissue culture plates.
The plates are incubated for 24 hours at 37.degree. C. in a humidified
5% CO.sub.2 atmosphere. Twenty .mu.l of tissue culture supernatants
from transfected UM449 cells are added to duplicate wells. An interferon
reference standard (e.g., human leukocyte interferon, Sigma Chemical
Co., St. Louis, Mo.) is included in each assay. The cells are incubated
with the test samples or the interferon standard for an additional
72 hours at 37.degree. C. To quantitate the effects on cell proliferation,
50 .mu.l of XTT/ECR substrate (Cell Proliferation Kit, Boehringer
Mannheim, Indianapolis, Ind.) is added to each well and the plates
are incubated for an additional 24 hours at 37.degree. C. prior
to measurement of the OD.sub.490. Other cell lines can be used in
the antiproliferation assay. For example, any of the cells listed
on Table 1 can be used. Another antiproliferation assay that can
be used is provided in Nieroda, et al (Mol. Cell. Differentiation
4: 335-351 (1996)).
For treatment of cancer, a polynucleotide construct comprising
a polynucleotide encoding a cytokine can be delivered locally, systemically
or intra-cavity. In the "systemic delivery" embodiment
of the invention, one or more polynucleotide construct comprising
one or more polynucleotide encoding one or more cytokine is administered
into a tissue such that the polynucleotide is expressed as the cytokine
in vivo and the cytokine is released into the circulation, and such
that a therapeutically effective amount of the cytokine is systemically
delivered to the tumor. In this embodiment, the polynucleotide construct
can be administered within ex vivo cells or associated with ex vivo
cellular material. Preferably, the cytokine is an IFN.omega., IFN.alpha.,
IFN.tau., IFN.gamma., IFN.beta., IL-1, IL-2, IL-4, IL-7, IL-12,
IL-15, IL-18, GM-CSF, or any combination of these, or any combination
of one or more of these and one or more additional cytokines. More
preferably, the cytokine is an IFN.alpha., IFN.omega., IL-2, or
IL-12. Most preferably, the cytokine is an IFN.alpha. or IFN.omega..
Examples of the combination are a polynucleotide encoding an IFN.omega.
and an IFN.alpha.; a polynucleotide encoding an IFN.omega. and an
IL-2; a polynucleotide encoding an IFN.alpha. and an IL-2; and a
polynucleotide encoding an IFN.alpha., an IFN.alpha., and an IL-2.
More preferably, the polynucleotide construct contains a polynucleotide
encoding an IFN.omega. and/or an IFN.alpha.. Even more preferably,
the polynucleotide construct contains a polynucleotide encoding
a human IFN.omega. and/or a human IFN.alpha.. Even more preferably,
the polynucleotide encodes a human IFN.omega.. Preferably, the polynucleotide
construct is administered free from ex vivo cells and free from
ex vivo cellular material.
In this embodiment, administration can be into tissue including
but not limited to muscle, skin, brain, lung, liver, spleen, bone
marrow, thymus, heart, lymph nodes, blood, bone, cartilage, pancreas,
kidney, gall bladder, stomach, intestine, testis, ovary, uterus,
rectum, nervous system, eye, gland, or connective tissue. Preferably,
the administration is into muscle tissue. i.e., skeletal muscle,
smooth muscle, or myocardium, and the polynucleotide construct is
naked. Most preferably, the muscle is skeletal muscle. For polynucleotide
constructs in which the polynucleotide encoding a cytokine is DNA,
the DNA can be operably linked to a cell-specific promoter that
directs substantial transcription of the DNA only in predetermined
cells.
By "naked" is meant that the polynucleotide construct
is free from association with any delivery vehicle known in the
art that can act to facilitate entry into cells, for example, from
transfection-facilitating proteins, viral particles, liposomes,
cationic lipids, and calcium phosphate precipitating agents.
As used herein, "ex vivo" cells are cells into which
the polynucleotide construct is introduced, for example, by transfection,
lipofection, electroporation, bombardment, or microinjection. The
cells containing the polynucleotide construct are then administered
in vivo into mammalian tissue. Such ex vivo polynucleotide constructs
are well-known to those of ordinary skill in the art. For example,
see Belldegrun, A., e al., J. Natl. Cancer Inst. 85: 207-216 (1993);
Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini,
M. et al., J. Immunology 153: 4604-4615 (1994); Kaido. T., et al.,
Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research
50: 5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy
7:1-10 (1996); Santodonato. L., et al., Gene Therapy 4:1246-1255
(1997); and Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996).
The polynucleotide construct is administered in a "cell-free"
fashion when it is administered independently, i.e., free of ex
vivo cells or ex vivo cellular material.
In the "local cytokine delivery" embodiment of the present
invention, a polynucleotide construct comprising a polynucleotide
encoding IFN.omega. and/or IFN.alpha. is administered in vivo into
or near a tumor of a mammal, such that the polynucleotide is incorporated
into the cells of the tumor. Tumor cells subsequently express the
interferon polypeptide in an amount effective to treat cancer.
In this embodiment, a polynucleotide construct comprising a polynucleotide
encoding an IFN.omega. and/or an IFN.alpha. can be administered
into the tumor. Alternatively, the polynucleotide construct can
be administered into non-tumor cells surrounding a tumor, near a
tumor, or adjacent to a tumor, such that a therapeutically effective
amount of an IFN.omega. and/or an IFN.alpha. is produced in vivo
near or within the tumor and is delivered to the malignant cells
of the tumor. One way to provide local delivery of the polynucleotide
construct is by administering intravenously a polynucleotide construct
comprising a tumor-targeted promoter, wherein the polynucleotide
is incorporated into the cells of the tumor and the cytokine is
expressed in the tumor in an amount effective to treat cancer. Preferably,
the polynucleotide construct is administered into the tumor.
In the "intra-cavity delivery" embodiment, the present
invention provides a method of selectively transfecting malignant
cells in a tumor-bearing body cavity of a mammal by introducing
a polynucleotide construct into the body cavity, wherein the polynucleotide
is incorporated into tumor cells and the tumor cells subsequently
express the protein encoded by the polynucleotide in an amount effective
to treat cancer. The polynucleotide construct is administered free
from ex vivo cells and free from ex vivo cellular material.
A cavity is a space within the body that can confine a fluid volume
for some period of time. The cavity can either be present in a normal
animal, or it can be produced as a result of disease, surgery or
trauma. Cavities in the normal animal include the peritoneum, the
cerebrospinal fluid space, the ventricles of the brain, the plural
space around lung, the bronchiolar airways, the nasal sinus, the
bladder, the vagina, the ear, the synovium of various joints (knee,
hip etc.), the internal network of salivary gland tissue, and the
gastrointestinal tract including stomach. Surgical removal of tumor
tissue can also produce a space which fits the definition of a cavity.
An open wound produced by trauma or surgery and closed by suture
can be defined as a cavity, and the area under a blister produced
by an infection, abrasion or a burn also fits the definition.
There are special bioavailability considerations when a gene delivery
system is administered into a cavity. First the fluid volume in
the cavity can be substantially comprised of the vehicle in which
the delivery system is suspended. Second, the delivery system can
have particular access to cells that are either suspended in the
cavity, or that are lining the surface of the cavity. Third, in
some cases normally differentiated cells that are lining the cavity
may be embedded in an extracellular matrix and, may not be accessible
to the delivery system. Thus, the delivery system may preferentially
transfect cells that are growing outside the normal extracellular
matrix and avoid the cells that are growing within the extracellular
matrix, conferring a kind of cell selectivity to the delivery system.
With respect to the first point, body fluids such as serum, have
been shown to inhibit gene delivery systems. For example, the transfection
activities of Lipofectin and LipofectAMINE are inhibited by serum.
It is thought that serum factors bind to cationic lipid/DNA complexes
and block their uptake into cells. In cavity models the endogenous
fluid volume can be removed, the cavity can be washed, and the delivery
system can be administered into the cavity in a vehicle that is
compatible with optimal gene delivery efficacy. Thus the cavity
model allows the investigator to create a fluid environment which
allows for optimal gene delivery potency.
With respect to the second point, cells that are either floating
in the cavity or are lining the surface of the cavity have preferential
access to the delivery system and can be preferentially transfected
relative to other cells in the body. Since the delivery system is
confined within the cavity, peripheral cells in the body outside
of the cavity will not be transfected. Thus, there is tissue targeting
to the cells within the cavity. For example, gene delivery systems
administered into the peritoneal cavity will have access to metastatic
tumor cells derived from colon or ovarian cancers that are floating,
in the peritoneum or are attached to the surfaces of the peritoneum.
Delivery systems administered into the plural space should transfect
cancer cells in the plural effusion. Delivery systems administered
into the cerebral spinal fluid should have access to metastatic
cancer cells present there.
With respect to the third point, differentiated cells that are
present in normal tissues are often embedded into an extracellular
matrix. This matrix can be difficult to penetrate with large particulate
delivery systems. Some cells, such as poorly differentiated tumor
cells, that are present in cavities can grow outside of the normal
extracellular matrix and are therefore more accessible to gene delivery
systems. In this way the delivery system can preferentially transfect
those cells that are growing outside of the extracellular matrix
and not transfect those cells that are growing within the extracellular
matrix. This is another form of in vivo, cell type specific targeting.
Examples of normal cells that are not embedded in an extracellular
matrix and are therefore more accessible to gene delivery systems
are, bronchial airway cells, lung cells in the plural space, and
ependimal cells lining the surface of the ventricles of the brain.
Normal bladder cells that line the surface of the bladder are embedded
in a tight extracellular matrix and are therefore not readily accessible
to a gene delivery system delivered into the bladder, but tumor
cells which grow up and out of the extracellular matrix into the
bladder vesicle are accessible to gene delivery systems administered
into the bladder vesicle. Thus normal bladder tissue would be expected
to resist transfection whereas, bladder tumor would be expected
to be transfectable.
A preferred application of the intra-cavity delivery embodiment
is in the treatment of peritoneally disseminated cancers. More specifically,
a mammal bearing peritoneal tumor may be injected i.p. with an effective
amount of a polynucleotide complexed with a lipid in a physiologically
acceptable diluent in a total volume sufficient to access the entire
body cavity. The mammal may have tumor ascites in the peritoneal
cavity as in an ovarian cancer. In the most preferred application,
this methodology may be used in treating ovarian cancer of a human.
Debulking of tumor ascites is commonly performed on human ovarian
cancer patients. Debulking involves removal of tumor ascites from
the peritoneal cavity. In humans bearing ovarian tumor ascites,
the ascites fluid would be debulked by insertion of a catheter i.p.
followed by periodic draining of ascites fluid. It is contemplated
that the tumor ascites would be debulked before and/or after the
i.p. administration of the polynucleotide formulation of the present
invention.
Transfection efficacy of the intra-cavity delivery embodiment may
be determined by collecting the tumor ascites and serum at various
times after the injection and performing diagnostic assays appropriate
for the encoded molecule(s). Naturally, other means of determining
tumor mass, growth, and viability may also be used to assess the
effectiveness of the present invention.
Preferred polynucleotides for the intra-cavity delivery embodiment
may encode not only immunogenic molecules such as cytokines (e.g.,
interleukins 1-18 and .alpha./.beta./.gamma./.omega.-interferons,
colony stimulating factors, e.g., G-CSF, GM-CSF, M-CSF, and tumor
necrosis factors), but also chemokines (e.g., C-X-C and C-C), Class
I and II histocompatibility antigens, costimulatory molecules (e.g.,
B7-1, B7-2, CAMs, and flt3 ligand), growth factors (e.g., epidermal
growth factors, fibroblast growth factors, transforming growth factors
and growth hormone), and the like. The polynucleotide may also encode
bacterial antigens, viral glycoproteins, enzymes (e.g., lysozymes),
recombinant antibodies, molecules that interfere with cellular adhesion,
adhesion molecules, proliferation and vascular inhibitory factors,
ribozymes, and antisense RNAs targeted toward key oncogenic or tumor
growth proteins. Moreover, selective delivery of toxic peptides
(e.g., ricin, diphtheria toxin, or cobra venom factor) or proteins
capable of synthesizing toxic compounds (e.g. thymidine kinase and
cytosine deaminase) to the malignant cells may have therapeutic
benefits. The polynucleotide may also comprise a tumor suppressor
gene (e.g., p53). Preferred polynucleotides encode cytokines. Preferred
cytokines are IL-2, IFN.omega., and IFN.alpha.. IL-2 is most preferred.
For treatment of cancer by any of the above disclosed embodiments,
any polynucleotide encoding an IFN.omega., or an active fragment
thereof, can be used. For example, the polynucleotide construct
can be a construct comprising a polynucleotide that hybridizes under
stringent conditions to the nucleotide sequence of SEQ ID No. 7
or the complement thereof, wherein the polynucleotide sequence encodes
a polypeptide that has antiproliferative activity when added to
NIH-OVCAR3 cells in vitro, and one or more cationic compounds selected
from the group consisting of cationic lipids, cationic peptides,
cationic proteins, cationic polymers, and mixtures thereof. Alternatively,
the construct can be a polynucleotide construct comprising a polynucleotide
that encodes a polypeptide comprising an amino acid sequence which,
except for at least one but not more than 20 amino acid substitutions,
deletions, or insertions, is identical to amino acids -23 to 172
or 1 to 172 in SEQ ID No. 8, wherein the polypeptide has antiproliferative
activity when added to NIH-OVCAR3 cells in vitro, and one or more
cationic compounds selected from the group consisting of cationic
lipids, cationic peptides, cationic proteins, cationic polymers,
and mixtures thereof. Alternatively, the construct can be a polynucleotide
construct comprising a polynucleotide that encodes a polypeptide
comprising amino acids 86-172 in SEQ ID No. 8, wherein the polypeptide
has antiproliferative activity when added to NIH-OVCAR3 cells in
vitro; and one or more cationic compounds selected from the group
consisting of cationic lipids, cationic peptides, cationic proteins,
cationic polymers, and mixtures thereof. Preferably, IFN.alpha.
is encoded by nucleotides 1 to 585 in SEQ ID No. 7 (corresponding
to amino acids -23 to 172 in SEQ ID No. 8), or by nucleotides 70
to 585 in SEQ ID No. 7 (corresponding to amino acids 1 to 172 in
SEQ ID No. 8). More preferably, the polynucleotide construct is
VR4151.
For treatment of cancer, any polynucleotide encoding IFN.alpha.,
or active fragment thereof, can also be used. For example, the polynucleotide
construct can be a construct comprising a polynucleotide that hybridizes
under stringent conditions to the nucleotide sequence of SEQ ID
No. 9 or the complement thereof, wherein the polynucleotide sequence
encodes a polypeptide that has antiproliferative activity when added
to NIH-OVCAR3 cells in vitro, and one or more cationic compounds
selected from the group consisting of cationic lipids, cationic
peptides, cationic proteins, cationic polymers, and mixtures thereof.
Alternatively, the construct can be a polynucleotide construct comprising
a polynucleotide that encodes a polypeptide comprising an amino
acid sequence which, except for at least one but not more than 20
amino acid substitutions, deletions, or insertions, is identical
to amino acids -23 to 166 or 1 to 166 in SEQ ID No. 10, wherein
the polypeptide has antiproliferative activity when added to NIH-OVCAR3
cells in vitro, and one or more cationic compounds selected from
the group consisting of cationic lipids, cationic peptides, cationic
proteins, cationic polymers, and mixtures thereof. Alternatively,
the construct can be a polynucleotide construct comprising a polynucleotide
that encodes a polypeptide comprising amino acids 83-166 in SEQ
ID No. 10, wherein the polypeptide has antiproliferative activity
when added to NIH-OVCAR3 cells in vitro; and one or more cationic
compounds selected from the group consisting of cationic lipids,
cationic peptides, cationic proteins, cationic polymers, and mixtures
thereof. Preferably, IFN.alpha. is encoded by nucleotides 1 to 567
in SEQ ID No. 9 (corresponding to amino acids -23 to 166 in SEQ
ID No. 10), or by nucleotides 1 to 567 in SEQ ID No. 9 (corresponding
to amino acids 1 to 166 in SEQ ID No. 10). Preferably, the polynucleotide
construct is VR4112.
For polynucleotide constructs that do not contain a polynucleotide
encoding IFN.omega., the polynucleotide construct is preferably
a cell-free construct. For polynucleotide constructs that contain
a polynucleotide encoding IFN.omega., the polynucleotide construct
can be administered either within ex vivo cells or free of ex vivo
cells or ex vivo cellular material. Preferably, the polynucleotide
construct is administered free of ex vivo cells or ex vivo cellular
material.
In the "local delivery" and "intra-cavity delivery"
embodiments, the polynucleotide construct is preferably complexed
with one or more cationic compounds. More preferably, the polynucleotide
construct is complexed with one or more cationic lipids by ionic
interaction. Generally, the complex then contacts the cell membrane
and is transfected into the cell. This transfection mechanism is
referred to as "lipofection," and is a highly efficient
transfection procedure (Felgner, et al., Proc. Natl. Acad. Sci.
USA 84:7413-7417, 1987); and Felgner, et al., Nature 337:387-388,
1989). Still more preferably, the polynucleotide construct is complexed
with one or more cationic lipids and one or more neutral lipids.
For purposes of the present invention, lipid refers to a synthetic
or naturally occurring compound that possesses both a lipophilic
region and a polar region, commonly referred to as a head group.
Preferred cationic compounds are cationic lipids. Cationic lipids
are described in U.S. Pat. Nos. 4,897,355; 4,946,787; 5,049,386;
5,264,618; 5,279,833; 5,334,761; 5,429,127; 5,459,127; 5,589,466;
5,676,954; 5,693,622; 5,580,859; 5,703,055; and 5,578,475; and international
publications WO 04/9469, WO 95/14381, 95/14651, 95/17373, 96/18372,
96/26179, 96/40962, 96/40963, 96/41873, and 97/00241, and documents
cited therein. As illustrated in the above-cited patents and patent
applications, cationic lipids comprise structural features that
may be present in a variety of core molecular classes.
Examples of cationic lipids are 5-carboxyspermylglycine dioctadecylamide
(DOGS) and dipalmitoyl-phophatidylethanolamine-5-carboxyspermylamide
(DPPES). Cationic cholesterol derivatives are also useful, including
{3.beta.-[N--N',N'-dimethylamino)ethane]-carbomoyl}-cholesterol
(DC-Chol). Dimethyldioctdecyl-ammonium bromide (DDAB), N-(3-aminopropyl)-N,N-(bis-(2-tetradecyloxyethyl))-N-methyl-ammonium
bromide (PA-DEMO), N-(3-aminopropyl)-N,N-(bis-(2-dodecyloxyethyl))-N-methyl-ammonium
bromide (PA-DELO), N,N,N-tris-(2-dodecyloxy)ethyl-N-(3-amino)propyl-ammonium
bromide (PA-TELO), and N'-(3-aminopropyl)((2-dodecyloxy)ethyl)-N.sup.2-(2-dodecyloxy)ethyl-1-pip-
erazinaminium bromide (GA-LOE-BP) can also be employed in the present
invention.
Non-diether cationic lipids, such as DL-1,2-dioleoyl-3'-dimethylaminopropyl-p-hydroxyethylammonium
(DORI diester), 1-O-oleyl-2-oleoyl-3-dimethylaminopropyl-.beta.-hydroxyethylamm-
onium (DORI ester/ether), and their salts promote in vivo gene delivery.
Preferred cationic lipids comprise groups attached via a heteroatom
attached to the quaternary ammonium moiety in the head group. A
glycyl spacer can connect the linker to the hydroxyl group.
Preferred cationic lipids are 3,5-(N,N-dilysyl)-diaminobenzoyl-3-(DL-1,2-dioleoyl-dimethylaminopropyl-p-
-hydroxyethylamine) (DLYS-DABA-DORI diester), 3,5-(N,N-di-lysyl)diamino-benzoylglycyl-3-(DL-1,2-dioleoyl-dimethylaminop-
ropyl-p-hydroxyethylamine) (DLYS-DABA-GLY-DORI diester), and (.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanimi-
nium bromide (DMRIE).
Also preferred are (O)-N,N-dimethyl-N-[2-(sperminecarboxamido)ethyl]-2,3-bis(dioleyloxy)-1-p-
ropaniminium pentahydrochloride (DOSPA), (.+-.)-N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanimini-
um bromide (.beta.-aminoethyl-DMRIE or PAE-DMRIE) (Wheeler, et al.,
Biochim. Biophys. Acta 1280:1-11 (1996)), and (.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminium
bromide (GAP-DLRIE) (Wheeler, et al., Proc. Natl. Acad. Sci. USA
93:11454-11459 (1996)), which have been developed from DMRIE.
Other examples of DMRIE-derived cationic lipids that are useful
for the present invention are (.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-decyloxy)-1-propanaminium
bromide (GAP-DDRIE), (.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-tetradecyloxy)-1-propanami-
nium bromide (GAP-DMRIE), (.+-.)-N-((N''-methyl)-N'-ureyl)propyl-N,N-dimethyl-2,3-bis(tetradecyloxy-
)-1-propanaminium bromide (GMU-DMRIE), (.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminiu-
m bromide (DLRIE), and (.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis-([Z]-9-octadecenyloxy)prop-
yl-1-propaniminium bromide (HP-DORIE).
The lipids of the lipid-containing formulation can comprise a cationic
lipid alone, or further comprise a neutral lipid such as cardiolipin,
phosphatidylcholine, phosphatidylethanolamine, dioleoylphosphatidylcholine,
dioleoylphosphatidyl-ethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine
(DOPE), sphingomyelin, and mono-, di- or tri-acylglycerol. Other
additives, such as cholesterol, fatty acid, ganglioside, glycolipid,
neobee, niosome, prostaglandin, sphingolipid, and any other natural
or synthetic amphiphiles, can also be used. A preferred molar ratio
of cationic lipid to neutral lipid in these lipid-containing formulations
is from about 9:1 to about 1:9; an equimolar ratio is particularly
preferred. The lipid-containing formulation can further comprise
a lyso lipid (e.g., lysophosphatidylcholine, lysophosphatidyl-ethanolamine,
or a lyso form of a cationic lipid).
More preferably, the cationic lipid is (.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanimi-
nium bromide (DMRIE) and the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine
(DOPE) such that the mass ratio of polynucleotide construct to lipid
is from about 10:1 and about 0.5:1. Still more preferably, the mass
ratio of polynucleotide construct to lipid is from about 5:1 and
about 1:1. Still more preferably, the mass ratio of polynucleotide
construct to lipid is about 5:1.
Lipid-containing pharmaceutical composition for use in a complex
with the polynucleotide construct of the present invention can also
comprise cationic lipid together with an effective amount of a lysophosphatide.
The lysophosphatide can have a neutral or a negative head group.
Lysophosphatidylcholine and lysophosphatidyl-ethanolamine are preferred,
and 1-oleoyl lysophosphatidylcholine is particularly preferred.
Lysophosphat |