U.S. patent application number 11/449152 was filed with the patent office on 2007-01-11 for oligonucleotide compositions and their use to induce differentiation of cells.
This patent application is currently assigned to Bioniche Life Sciences, Inc.. Invention is credited to Mario C. Filion, Nigel C. Phillips.
Application Number | 20070010472 11/449152 |
Document ID | / |
Family ID | 26825829 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070010472 |
Kind Code |
A1 |
Filion; Mario C. ; et
al. |
January 11, 2007 |
Oligonucleotide compositions and their use to induce
differentiation of cells
Abstract
The present invention provides compositions comprising a 3'-OH,
5'-OH, chemically unmodified, synthetic phosphodiester nucleotide
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and a pharmaceutically
acceptable carrier, wherein the compositions are useful to induce
differentiation of cells or to stimulate differentiation or
production of pluripotent cells. The present invention provides
methods of using these compositions to induce differentiation of
pluripotent cells, including bone marrow derived cells, and to
treat disease associated with insufficient differentiation of cells
in animals and humans, including but not limited to leukemia,
lymphoma, non-malignant blood disorders such as hemoglobinopathies,
sickle cell disease, myelodysplastic syndrome, pancytopenia,
anemia, thrombocytopenia and leukopenia.
Inventors: |
Filion; Mario C.; (Laval,
CA) ; Phillips; Nigel C.; (Pointe-Claire,
CA) |
Correspondence
Address: |
JOHN S. PRATT, ESQ;KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
ATLANTA
GA
30309
US
|
Assignee: |
Bioniche Life Sciences,
Inc.
|
Family ID: |
26825829 |
Appl. No.: |
11/449152 |
Filed: |
June 8, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10127645 |
Apr 22, 2002 |
7087586 |
|
|
11449152 |
Jun 8, 2006 |
|
|
|
60286158 |
Apr 24, 2001 |
|
|
|
Current U.S.
Class: |
514/44R ;
536/23.2 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/7105 20130101; A61P 7/00 20180101; A61P 7/06 20180101; A61K
38/00 20130101; A61P 35/02 20180101; C12N 15/117 20130101; C12N
2310/18 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/044 ;
536/023.2 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/04 20060101 C07H021/04 |
Claims
1. A composition comprising a 3'-OH, 5'-OH, chemically unmodified,
synthetic phosphodiester nucleotide sequence selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and
SEQ ID NO: 4, and a pharmaceutically acceptable carrier, wherein
the composition is effective to induce differentiation of cells, to
increase cells derived from pluripotent cells, or to treat disease
associated with insufficient differentiation of cells when
administered to the cells in vivo or in vitro.
2. The composition of claim 1, wherein the induction of
differentiation of cells is induction of differentiation of
leukemia cells.
3. The composition of claim 1, wherein the induction of
differentiation of cells is induction of differentiation of
pluripotent stem cells, myeloid stem cells, lymphoid stem cells,
progenitor cells, immune cell precursors, or cells derived from the
pluripotent stem cells, the myeloid stem cells, the lymphoid stem
cells, the progenitor cells, or the immune cell precursors.
4. The composition of claim 1, wherein the disease is leukemia,
lymphoma, a non-malignant blood disorder, hemoglobinopathy, sickle
cell disease, myelodysplastic syndrome, pancytopenia, anemia,
thrombocytopenia or leukopenia.
5. A method comprising administration of an amount of a composition
comprising a 3'-OH, 5'-OH, chemically unmodified, synthetic
phosphodiester nucleotide sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID
NO: 4, and a pharmaceutically acceptable carrier, to an animal or a
human wherein the amount is effective to induce differentiation of
cells in the animal or the human.
6. The method of claim 5, wherein the animal or the human has a
disease associated with insufficient differentiation of cells.
7. The method of claim 6, wherein the disease is leukemia,
lymphoma, a non-malignant blood disorder, hemoglobinopathy, sickle
cell disease, myelodysplastic syndrome, pancytopenia, anemia,
thrombocytopenia or leukopenia.
8. The method of claim 6, wherein the disease is leukemia.
9. The method of claim 5, wherein induction of differentiation of
cells is induction of erythrocyte-like phenotype, monocyte-like
phenotype, megakaryocyte-like phenotype, inhibition of
proliferation or induction of hemoglobin synthesis in cells.
10. A method comprising administration of an amount of a
composition comprising a 3'-OH, 5'-OH, chemically unmodified,
synthetic phosphodiester nucleotide sequence selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and
SEQ ID NO: 4, and a pharmaceutically acceptable carrier, to an
animal or a human, wherein the amount is effective to increase
differentiation of pluripotent cells in the animal or the
human.
11. The method of claim 10, wherein the animal or the human has
received chemotherapy or radiotherapy.
12. The method of claim 4, further comprising administration of a
therapeutic agent.
13. The method of claim 12, wherein the therapeutic agent is a
chemotherapeutic drug, an immunosuppressive agent, a
differentiating agent, an immunotherapeutic agent, an antimicrobial
agent, an antiviral agent, radiotherapy, or a combination
thereof.
14. The method of claim 10, wherein the pluripotent cells are
derived from bone marrow, liver, spleen, lymph nodes, thymus or
cord blood.
15. The method of claim 10, wherein the pluripotent cells are
derived from bone marrow.
16. A method comprising administration of an amount of a
composition comprising a 3'-OH, 5'-OH, chemically unmodified,
synthetic phosphodiester nucleotide sequence selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and
SEQ ID NO: 4, and a pharmaceutically acceptable carrier, to an
animal or a human having a disease associated with insufficient
differentiation of cells, wherein the amount is effective to induce
differentiation of cells in the animal or the human.
17. The method of claim 16, wherein the disease is leukemia and the
amount is effective to treat the leukemia.
Description
PRIOR RELATED APPLICATIONS
[0001] The present application claims priority to U.S. provisional
patent application Ser. No. 60/286,158 filed Apr. 24, 2001. This
application is a divisional of U.S. patent application Ser. No.
10/127,645 filed Apr. 22, 2002, now allowed, the contents of which
are incorporated herein by reference.
SEQUENCE LISTING
[0002] The content of the sequence listing information is identical
to the paper sequence listing provided in U.S. patent application
Ser. No. 10/127,645 filed Apr. 22, 2002, and includes no new
matter.
FIELD OF THE INVENTION
[0003] The present invention provides compositions comprising
specific oligonucleotides combined with a pharmaceutically
acceptable carrier, wherein the compositions are useful to induce
differentiation of cells, including pluripotent cells, leukemic
cells, lymphoma cells and bone marrow-derived cells, and to treat
diseases such as leukemia, lymphoma and disorders associated with
insufficient differentiation of cells.
BACKGROUND OF THE INVENTION
[0004] Numerous diseases and conditions in animals and humans are
associated with insufficient differentiation of cells or with an
insufficiency of cells. Many of these cells are derived from bone
marrow. Such diseases and conditions include but are not limited to
leukemia, lymphoma, and non-malignant blood disorders such as
hemoglobinopathies, sickle cell disease, myelodysplastic syndrome
and insufficient production of bone marrow derived cells following
therapies such as radiation and chemotherapy.
[0005] Differentiation therapy of leukemia cells in diseases such
as acute promyelocytic leukemia (APL), acute myeloid leukemia
(AML), chronic promyelocytic leukemia (CPL) and chronic myeloid
leukemia (CML), has provided an alternative strategy for the
treatment of leukemia. In differentiation therapy, immature
leukemia cells are induced by different chemical compounds to
attain a mature phenotype resulting in arrest of their growth.
[0006] A number of differentiation compounds and also radiation
have been reported to induce the differentiation of leukemia cells.
Hemin, butyric acid, 5-azacytidine, cytosine arabinoside,
hydroxyurea, guanosine, guanine, retinoic acid, trimidox,
gamma-irradiation, mithramycin and chromomycin have been reported
to induce differentiation of leukemia cells (Rutherford et al.,
Nature 280:164, 1979; Gambari et al., Biochem. Biophys. Acta,
886:203, 1986; Bianchi et al., Cancer Res. 46:6327, 1986; Adunyah
et al., Biochem. Biophys. Acta, 1263:123, 1995; Osti et al.,
Haematologica 82:395, 1997; Cortesi et al., Eur. J. Haematol.
61:295, 1998; Iyamu et al., Biochem. Biophys. Res. Com. 247:759,
1998; Schwenke et al., Leuk. Res. 19:955, 1995; and Bianchi et al.,
Br. J. Haematol. 104:258, 1999).
[0007] Synthetic oligonucleotides are polyanionic sequences that
are internalized in cells (Vlassov et al. Biochim. Biophys. Acta
1197:95, 1994). Synthetic oligonucleotides are reported to bind
selectively to nucleic acids (Wagner, R. Nature: 372:333, 1994), to
specific cellular proteins (Bates et al. J. Biol. Chem. 274:26369,
1999) and to specific nuclear proteins (Scaggiante et al. Eur. J.
Biochem. 252:207, 1998), and to inhibit proliferation of cancer
cells. Synthetic oligonucleotides have not been reported to possess
differentiating activity on acute and/or chronic pro-myelocytic
cells and/or myeloid leukemia cells. Synthetic phosphorothioate
oligonucleotides having a CpG motif (5'purine-purine-cytosine
(C)-guanine (G)-pyrimidine-pyrimidine3') have been shown to induce
the proliferation of B-cell chronic lymphocytic leukemia (Decker et
al., Blood 95:999, 2000). Synthetic 27 base sequences containing G
and variable amounts of thymine (T), hereinafter oligonucleotide
GTn, wherein n is .gtoreq.1 or .ltoreq.7 Ts (Scaggiante et al.,
Eur. J. Biochem. 252:207, 1998), and wherein the number of bases is
>20 (Morassutti et al., Nucleosides and Nucleotides 18:1711,
1999), have been reported to inhibit growth of leukemia cells by
sequence specific binding to a 45 kDa nuclear protein. In contrast,
GTn sequences, wherein the total number of bases is less than 15,
are reported to be inactive against these cells (Morassutti et al.
Nucleosides and Nucleotides 18:1711, 1999). Chimeric
methylphosphonodiester/phosphodiester oligonucleotides of sequence
type SEQ ID NO:5 CGNNN (N=A, C, G or T), introduced into the
cytoplasm of cells by 10 minutes of reversible permeabilization
with streptolysin O, induce apoptosis of human T cell leukemia
cells. Nevertheless, the CGNNN oligonucleotides are reported to be
inactive against three CML cell lines (K562, LAMA84 and KYO1),
showing no significant effect on the growth and survival of these
cells (Tidd et al., Nucleic Acid Res. 28:2242, 2000).
[0008] Depletion of bone marrow derived cells is observed in
several conditions, including depletion following radiation therapy
or chemotherapy. Insufficient production of cells destined to
become erythrocytes or granulocytes is associated with numerous
problems, including but not limited to, reduced delivery of oxygen
to cells, decreased immune function, and clotting abnormalities.
Various therapies, including expensive chemotherapies, are often
required to stimulate production of red and white cells.
[0009] Most prior art differentiating therapies have proven to be
less than adequate for clinical applications. Many of these
therapies are inefficient or toxic, have significant adverse
effects and are debilitating for the recipient. Therefore, there is
a continuing need for novel compositions and methods that induce
differentiation of cells such as myeloid-derived leukemia cells.
What is also needed are new therapeutic compositions and methods
that stimulate production and differentiation of pluripotent cells
such as bone-marrow derived cells. Also needed are new therapeutic
compositions that induce differentiation of cells. What is also
needed are compositions and methods that may be used to treat
diseases and conditions characterized by insufficient
differentiation of cells or insufficient production of marrow
derived cells.
SUMMARY OF THE INVENTION
[0010] The present invention fulfills these needs by providing a
method comprising administration of a composition comprising a
3'-OH, 5'-OH, chemically unmodified, synthetic phosphodiester
oligonucleotide sequence (hereinafter sequence) selected from the
group consisting of SEQ ID NO: 1 (5'GTG3'), SEQ ID NO: 2
(5'GGGTGG3'), SEQ ID NO: 3 (5'GGGAGG3') and SEQ ID NO: 4
(5'CCACCC3') and a pharmaceutically acceptable carrier, wherein the
composition induces differentiation of cells. The present invention
provides a method to treat diseases associated with growth of cells
that are not differentiated to a mature phenotype. Terminal
differentiation of cells may include one or more responses selected
from the group consisting of induction of erythrocyte-like
phenotype, monocyte-like phenotype, megakaryocyte-like phenotype,
inhibition of proliferation of leukemia cells and induction of
hemoglobin synthesis.
[0011] The compositions of the present invention may be used to
treat diseases related to insufficient differentiation of cells.
Such diseases include but are not limited to leukemia, lymphoma,
and non-malignant blood disorders such as hemoglobinopathies,
sickle cell disease or myelodysplastic syndrome. The compositions
of the present invention are believed to be useful for treatment of
pancytopenia, anemia, thrombocytopenia and leukopenia. Other
conditions that may be treated with the compositions of the present
invention include lymphoma and nonmalignant blood disorders,
including but not limited to hemoglobinopathies, sickle cell
disease and myelodysplastic syndromes. In a preferred embodiment,
the compositions of the present invention are administered to an
animal or a human with leukemia in an amount effective to treat the
leukemia.
[0012] The compositions of the present invention may also be
administered to an animal or human together with other therapies as
a combination therapy. These therapies may include administration
of therapeutic compounds or radiation therapy. The compositions of
the present invention may be administered before, after, or
concomitantly with the other therapy. Such combination therapy may
augment the net therapeutic effect on the animal or human. The
compositions of the present invention may be administered alone, or
in combination with other therapeutic modalities including, but not
limited to, chemotherapeutic agents, differentiating agents,
immunotherapeutic agents, antimicrobial agents, antiviral agents or
in combination with radiation therapy.
[0013] The compositions of the present invention may be
administered to a recipient to stimulate production of cells after
other therapies administered to the recipient have depleted such
cells. One non-limiting example involves depletion of cells derived
from bone marrow following radiation therapy or chemotherapy. The
compositions of the present invention may also be administered to a
recipient to stimulate production and differentiation of other
cells such as pluripotent stem cells, myeloid stem cells, lymphoid
stem cells, progenitor cells, immune cell precursors, and/or other
cells derived from these pluripotent stem cells, myeloid stem
cells, lymphoid stem cells, progenitor cells, and immune cell
precursors. The compositions of the present invention may also be
administered to a recipient to stimulate production and
differentiation of cells from numerous sources, including but not
limited to, bone marrow, liver, spleen, lymph nodes, thymus and
cord blood.
[0014] The compositions of the present invention may also be
administered in vitro to affect differentiation of cells such as
pluripotent stem cells, myeloid stem cells, lymphoid stem cells,
progenitor cells, immune cell precursors, and/or other cells
derived from these pluripotent stem cells, myeloid stem cells,
lymphoid stem cells, progenitor cells, and immune cell
precursors.
[0015] The unexpected and surprising ability of the composition of
the present invention to induce differentiation of bone-marrow
derived cells, including leukemia cells, addresses a long-felt,
unfulfilled need in the medical arts and provides an important
benefit for animals and humans.
[0016] Accordingly, it is an object of the present invention to
provide a method comprising administration of a composition
comprising a 3'-OH, 5'-OH, chemically unmodified, synthetic
phosphodiester oligonucleotide sequence (hereinafter sequence)
selected from the group consisting of SEQ ID NO: 1 (5'GTG3'), SEQ
ID NO: 2 (5'GGGTGG3'), SEQ ID NO: 3 (5'GGGAGG3') and SEQ ID NO: 4
(5'CCACCC3') and a pharmaceutically acceptable carrier to treat
disease in animals and humans, wherein the disease is characterized
by insufficient differentiation of cells.
[0017] Another object of the present invention is to provide a
method comprising administration of a composition comprising a
3'-OH, 5'-OH, chemically unmodified, synthetic phosphodiester
oligonucleotide sequence (hereinafter sequence) selected from the
group consisting of SEQ ID NO: 1 (5'GTG3'), SEQ ID NO: 2
(5'GGGTGG3'), SEQ ID NO: 3 (5'GGGAGG3') and SEQ ID NO: 4
(5'CCACCC3') and a pharmaceutically acceptable carrier to induce
progenitor cell maturation and differentiation in animals and
humans.
[0018] Another object of the present invention is to provide a
composition and method to treat leukemia.
[0019] Yet another object of the present invention is to provide a
method that inhibits proliferation of leukemic cells and induces
differentiation of leukemic cells.
[0020] Another object of the present invention is to provide a
method to treat lymphoma.
[0021] Yet another object of the present invention is to provide a
method to treat non-malignant blood disorders.
[0022] Still another object of the present invention is to provide
a method to treat hemoglobinopathies.
[0023] A further object of the present invention is to provide a
method to treat sickle cell disease.
[0024] Yet another object of the present invention is to provide a
method to treat myelodysplastic syndrome.
[0025] Another object of the present invention is to provide a
method to treat pancytopenia.
[0026] Yet another object of the present invention is to provide a
method to treat anemia.
[0027] A further object of the present invention is to provide a
method to treat thrombocytopenia.
[0028] Another object of the present invention is to provide a
method to treat leukopenia.
[0029] Still another object of the present invention is to provide
a method to induce progenitor cell maturation and
differentiation.
[0030] Yet another object of the present invention is to provide a
method to induce maturation and differentiation of cells including
but not limited to pluripotent stem cells, myeloid stem cells,
lymphoid stem cells, progenitor cells, immune cell precursors,
and/or other cells derived from these pluripotent stem cells,
myeloid stem cells, lymphoid stem cells, progenitor cells, and
immune cell precursors.
[0031] Still another object of the present invention is to provide
a method to induce bone marrow-derived progenitor cell
maturation.
[0032] Another object of the present invention is to provide a
method that increases the number of bone marrow derived-cells
following treatment with therapeutic agents.
[0033] Yet another object of the present invention is to provide a
method that increases the number of bone marrow derived-cells
following treatment with chemotherapeutic agents.
[0034] Another object of the present invention is to provide a
method that restores the number of bone marrow derived-cells
following treatment with radiotherapy.
[0035] Still another object of the present invention is to provide
a method that restores the number of bone marrow derived-cells
following treatment with immunosuppressive agents.
[0036] Still another object of the present invention is to provide
a composition that is minimally toxic to the recipient.
[0037] These and other objects, features and advantages of the
present invention will become apparent after a review of the
following detailed description of the disclosed embodiments and the
appended claims.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention may be understood more readily by
reference to the following detailed description of specific
embodiments included herein.
[0039] The present invention comprises a method comprising
administration of a composition comprising a 3'-OH, 5'-OH,
chemically unmodified, synthetic phosphodiester oligonucleotide
sequences (hereinafter sequence) selected from the group consisting
of SEQ ID NOs: 1, 2, 3, or 4, and a pharmaceutically acceptable
carrier, to an animal or a human in an amount effective to induce
differentiation of cells. The compositions of the present invention
may be used to treat disease related to insufficient
differentiation of cells. Such diseases include but are not limited
to leukemia, lymphoma, and non-malignant blood disorders such as
hemoglobinopathies, sickle cell disease and myelodysplastic
syndrome. The compositions of the present invention are believed to
be useful for treatment of pancytopenia, anemia, thrombocytopenia
and leukopenia. Other conditions that may be treated with the
compositions of the present invention include lymphoma and
nonmalignant blood disorders, including but not limited to
hemoglobinopathies, sickle cell disease and myelodysplastic
syndromes. The unexpected and surprising ability of these
compositions to induce differentiation and to inhibit proliferation
of leukemia cells addresses a long felt unfulfilled need in the
medical arts and provides an important benefit for animals and
humans.
[0040] The present invention also comprises a method comprising
administration of a composition comprising a 3'-OH, 5'-OH,
chemically unmodified, synthetic phosphodiester oligonucleotide
sequence (hereinafter sequence) selected from the group consisting
of SEQ ID NO: 1 (5'GTG3'), SEQ ID NO: 2 (5'GGGTGG3'), SEQ ID NO: 3
(5'GGGAGG3') and SEQ ID NO: 4 (5'CCACCC3') and a pharmaceutically
acceptable carrier, to induce maturation of progenitor cells in
animals and humans. Such cells include but are not limited to
pluripotent stem cells, myeloid stem cells, lymphoid stem cells,
immune cell precursors, and/or other cells derived from these
pluripotent stem cells, myeloid stem cells, lymphoid stem cells and
immune cell precursors. The compositions of the present invention
may also be administered to an animal or human to stimulate
production and differentiation of cells from numerous sources,
including but not limited to, bone marrow, liver, spleen, lymph
nodes, thymus and cord blood.
[0041] As used herein, the word "sequence" refers a sequence
comprising a 3'-OH, 5'-OH chemically unmodified, synthetic
phosphodiester nucleotide sequence selected from the group
consisting of SEQ ID NO: 1 (5'GTG3'), SEQ ID NO: 2 (5'GGGTGG3'),
SEQ ID NO: 3 (5'GGGAGG3') and SEQ ID NO: 4 (5'CCACCC3').
[0042] The word "response", as used herein refers to one or more of
the following non-limiting examples of responses: induced
differentiation of pluripotent stem cells, myeloid stem cells,
lymphoid stem cells, immune cell precursors, and/or other cells
derived from these pluripotent stem cells, myeloid stem cells,
lymphoid stem cells and immune cell precursors; induced
differentiation of erythrocyte-like cells, monocyte-like cells or
megakaryocyte-like cells; inhibition of cellular proliferation due
to the induction of terminal differentiation; induction of
hemoglobin synthesis; and stimulation of hemoglobin synthesis.
[0043] As used herein, the phrase "effective in responsive cells"
refers to the ability of the compositions of the present invention
to induce differentiation and/or inhibition of proliferation and/or
synthesis of hemoglobin.
[0044] As used herein, the phrases "therapeutic treatment",
"effective amount" and "amount effective to" refer to an amount of
a sequence effective to induce differentiation of cells, to inhibit
proliferation of cells or to stimulate production of pluripotent
cells such as bone marrow-derived cells.
[0045] The word "disease", as used herein, relates to a condition
wherein bodily health is impaired. As used herein, the phrase
"chemotherapeutic" is any agent approved by a regulatory agency of
a country or a state government or listed in the U.S.
[0046] Pharmacopoeia or other generally recognized pharmacopoeia
for use to treat cancer in an animal or human. As used herein, the
phrase "chemotherapeutic" includes immunosuppressive agents.
Administration of an effective amount of the composition of the
present invention to an animal or a human is a therapeutic
treatment that prevents, treats or eliminates a disease including,
but not limited to, leukemia, pancytopenia, anemia,
thrombocytopenia, leukopenia, lymphoma, and non-malignant blood
disorders such as hemoglobinopathies, sickle cell disease or
myelodysplastic syndrome. Types of leukemia include, but are not
limited to APL, AML, CPL and CML. Administration of an effective
amount of the composition of the present invention to an animal or
a human is also a therapeutic treatment that stimulates production
of progenitor cells, including but not limited to pluripotent stem
cells, myeloid stem cells, lymphoid stem cells, immune cell
precursors, and/or other cells derived from these pluripotent stem
cells, myeloid stem cells, lymphoid stem cells and immune cell
precursors. In a preferred embodiment, the present invention
provides a method to stimulate production and differentiation of
marrow derived cells. The compositions of the present invention may
also be administered to an animal or human to stimulate production
and differentiation of cells from numerous sources, including but
not limited to, bone marrow, liver, spleen, lymph nodes, thymus and
cord blood.
[0047] The terms "pharmaceutically acceptable carrier" or
"pharmaceutically acceptable vehicle" are used herein to mean,
without limitation, any liquid, solid or semi-solid, including, but
not limited to, water or saline, a gel, cream, salve, solvent,
diluent, fluid ointment base, ointment, paste, implant, liposome,
micelle, giant micelle, and the like, which is suitable for use in
contact with living animal or human tissue without causing adverse
physiological responses, and which does not interact with the other
components of the composition in a deleterious manner. Other
pharmaceutically acceptable carriers or vehicles known to one of
skill in the art may be employed to make compositions for
delivering the oligonucleotide sequences of the present
invention.
[0048] The oligonucleotide sequences of the present invention may
be combined with pharmaceutically acceptable carriers and
administered as compositions in vitro or in vivo. Forms of
administration include, but are not limited to, injections,
solutions, creams, gels, implants, pumps, ointments, emulsions,
suspensions, microspheres, particles, microparticles,
nanoparticles, liposomes, pastes, patches, tablets, transdermal
delivery devices, sprays, aerosols, or other means familiar to one
of ordinary skill in the art. Such pharmaceutically acceptable
carriers are commonly known to one of ordinary skill in the art.
Pharmaceutical formulations of the present invention can be
prepared by procedures known in the art using well known and
readily available ingredients. For example, the compounds can be
formulated with common excipients, diluents, or carriers, and
formed into tablets, capsules, suspensions, powders, and the like.
Examples of excipients, diluents, and carriers that are suitable
for such formulations include the following: fillers and extenders
(e.g., starch, sugars, mannitol, and silicic derivatives); binding
agents (e.g., carboxymethyl cellulose and other cellulose
derivatives, alginates, gelatin, and polyvinyl-pyrrolidone);
moisturizing agents (e.g., glycerol); disintegrating agents (e.g.,
calcium carbonate and sodium bicarbonate); agents for retarding
dissolution (e.g., paraffin); resorption accelerators (e.g.,
quaternary ammonium compounds); surface active agents (e.g., cetyl
alcohol, glycerol monostearate); adsorptive carriers (e.g., kaolin
and bentonite); emulsifiers; preservatives; sweeteners;
stabilizers; coloring agents; perfuming agents; flavoring agents;
lubricants (e.g., talc, calcium and magnesium stearate); solid
polyethyl glycols; and mixtures thereof.
[0049] The formulations can be so constituted that they release the
active ingredient only or preferably in a particular location,
possibly over a period of time. Such combinations provide yet a
further mechanism for controlling release kinetics. The coatings,
envelopes, and protective matrices may be made, for example, from
polymeric substances or waxes.
[0050] Compositions comprising one or more sequences and a
pharmaceutically acceptable carrier are prepared by uniformly and
intimately bringing into association the sequence and the
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers include liquid carriers, solid carriers or both. Liquid
carriers are aqueous carriers, non-aqueous carriers or both, and
include, but are not limited to, aqueous suspensions, oil
emulsions, water-in-oil emulsions, water-in-oil-in-water emulsions,
site-specific emulsions, long-residence emulsions,
sticky-emulsions, microemulsions and nanoemulsions. Solid carriers
are biological carriers, chemical carriers or both and include, but
are not limited to, viral vector systems, particles,
microparticles, nanoparticles, microspheres, nanospheres,
minipumps, bacterial cell wall extracts and biodegradable or
non-biodegradable natural or synthetic polymers that allow for
sustained release of the oligonucleotide compositions. Emulsions,
minipumps and polymers can be implanted in the vicinity of where
delivery is required (Brem et al. J. Neurosurg. 74: 441, 1991).
Methods used to complex an oligonucleotide sequence(s) to a solid
carrier include, but are not limited to, direct adsorption to the
surface of the solid carrier, covalent coupling to the surface of
the solid carrier, either directly or via a linking moiety, and
covalent coupling to the polymer used to make the solid carrier.
Optionally, a sequence(s) can be stabilized by the addition of
non-ionic or ionic polymers such as polyoxyethylenesorbitan
monooleates (TWEENs) or hyaluronic acid.
[0051] Preferred aqueous carriers include, but are not limited to,
water, saline and pharmaceutically acceptable buffers. Preferred
non-aqueous carriers include, but are not limited to, a mineral oil
or a neutral oil including, but not limited to, a diglyceride, a
triglyceride, a phospholipid, a lipid, an oil and mixtures thereof,
wherein the oil contains an appropriate mix of polyunsaturated and
saturated fatty acids. Examples include, but are not limited to,
soybean oil, canola oil, palm oil, olive oil and myglyol, wherein
the fatty acids can be saturated or unsaturated. Optionally,
excipients may be included regardless of the pharmaceutically
acceptable carrier used to present the oligonucleotide compositions
to cells. These excipients include, but are not limited to,
anti-oxidants, buffers, and bacteriostats, and may include
suspending agents and thickening agents.
[0052] One or more sequences may be administered alone, or in
combination with other therapeutic modalities including, but not
limited to, chemotherapeutic agents, differentiating agents,
immunotherapeutic agents, antimicrobial agents, antiviral agents or
in combination with radiation therapy. Differentiating agents
include, but are not limited to, hemin, butyric acid,
5-azacytidine, cytosine arabinoside, hydroxyurea, guanosine,
guanine, retinoic acid, trimidox, gamma-irradiation, mithramycin
and chromomycin. Chemotherapeutic agents include, but are not
limited to, anti-metabolites, DNA damaging, microtubule
destabilizing, microtubule stabilizing, actin depolymerizing,
growth inhibiting, topoisomerase inhibiting, HMG-CoA inhibiting,
purine inhibiting, pyrimidine inhibiting, metalloproteinase
inhibiting, CDK inhibiting, angiogenesis inhibiting,
differentiation enhancing and immunotherapeutic agents. Dosages and
methods of administration of these other therapeutic modalities are
known to one of ordinary skill in the art.
[0053] Methods of in vivo administration of the compositions of the
present invention, or of formulations comprising such compositions
and other materials such as carriers of the present invention that
are particularly suitable for various forms include, but are not
limited to the following types of administration, oral (e.g. buccal
or sublingual), anal, rectal, as a suppository, topical,
parenteral, aerosol, inhalation, intrathecal, intraperitoneal,
intravenous, intraarterial, transdermal, intradermal, subdermal,
intramuscular, intrauterine, vaginal, into a body cavity, surgical
administration at the location of a tumor or internal injury,
directly into tumors, into the lumen or parenchyma of an organ, and
into bone marrow. Techniques useful in the various forms of
administrations mentioned above include but are not limited to,
topical application, ingestion, surgical administration,
injections, sprays, transdermal delivery devices, osmotic pumps,
electrodepositing directly on a desired site, or other means
familiar to one of ordinary skill in the art. Sites of application
can be external, such as on the epidermis, or internal, for example
a gastric ulcer, a surgical field, or elsewhere.
[0054] The compositions of the present invention can be applied in
the form of creams, gels, solutions, suspensions, liposomes,
particles, or other means known to one of skill in the art of
formulation and delivery of the compositions. Ultrafine particle
sizes can be used for inhalation delivery of therapeutics. Some
examples of appropriate formulations for subcutaneous
administration include but are not limited to implants, depot,
needles, capsules, and osmotic pumps. Some examples of appropriate
formulations for vaginal administration include but are not limited
to creams and rings. Some examples of appropriate formulations for
oral administration include but are not limited to: pills, liquids,
syrups, and suspensions. Some examples of appropriate formulations
for transdermal administration include but are not limited to gels,
creams, pastes, patches, sprays, and gels. Some examples of
appropriate delivery mechanisms for subcutaneous administration
include but are not limited to implants, depots, needles, capsules,
and osmotic pumps. Formulations suitable for parenteral
administration include but are not limited to aqueous and
non-aqueous sterile injection solutions which may contain
anti-oxidants, buffets, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient, and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets commonly used by one of ordinary skill in the
art.
[0055] Embodiments in which the compositions of the invention are
combined with, for example, one or more pharmaceutically acceptable
carriers or excipients may conveniently be presented in unit dosage
form and may be prepared by conventional pharmaceutical techniques.
Such techniques include the step of bringing into association the
compositions containing the active ingredient and the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredient with liquid carriers. Preferred
unit dosage formulations are those containing a dose or unit, or an
appropriate fraction thereof, of the administered ingredient. It
should be understood that in addition to the ingredients
particularly mentioned above, formulations comprising the
compositions of the present invention may include other agents
commonly used by one of ordinary skill in the art.
[0056] The volume of administration will vary depending on the
route of administration. Such volumes are known to one of ordinary
skill in the art of administering compositions to animals or
humans. Depending on the route of administration, the volume per
dose is preferably about 0.001 to 100 ml per dose, more preferably
about 0.01 to 50 ml per dose and most preferably about 0.1 to 30 ml
per dose. For example, intramuscular injections may range in volume
from about 0.1 ml to 1.0 ml. The oligonucleotide compositions
administered alone, or together with other therapeutic agent(s),
can be administered in a single dose treatment, in multiple dose
treatments, or continuously infused on a schedule and over a period
of time appropriate to the disease being treated, the condition of
the recipient and the route of administration. Moreover, the other
therapeutic agent can be administered before, at the same time as,
or after administration of the oligonucleotide compositions.
[0057] Preferably, the amount of oligonucleotide composition
administered per dose is from about 0.0001 to 100 mg/kg, more
preferably from about 0.001 to 10 mg/kg and most preferably from
about 0.01 to 5 mg/kg. In a preferred embodiment, the
oligonucleotide compositions in combination with a chemotherapeutic
agent is administered to an animal or human having leukemia in an
amount effective to add to, synergize with or potentiate the
anti-neoplastic effect of the chemotherapeutic agent. Preferably,
the amount of therapeutic agent administered per dose is from about
0.001 to 1000 mg/kg, more preferably from about 0.01 to 500 mg/kg
and most preferably from about 0.1 to 100 mg/kg. The particular
sequence and the particular therapeutic agent administered, the
amount per dose, the dose schedule and the route of administration
should be decided by the practitioner using methods known to those
skilled in the art and will depend on the type of disease, the
severity of the disease, the location of the disease and other
clinical factors such as the size, weight and physical condition of
the recipient. In addition, in vitro assays may optionally be
employed to help identify optimal ranges for sequence and for
sequence plus therapeutic agent administration.
[0058] The compositions of the present invention may also be
administered in vitro to affect differentiation of cells such as
pluripotent stem cells, myeloid stem cells, lymphoid stem cells,
progenitor cells, immune cell precursors, and/or other cells
derived from these pluripotent stem cells, myeloid stem cells,
lymphoid stem cells, progenitor cells, and immune cell
precursors.
[0059] The present invention is further illustrated by the
following examples, which are not to be construed in any way as
imposing limitations upon the scope thereof. On the contrary, it is
to be clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof, which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention.
EXAMPLE 1
Preparation of Sequences
[0060] Phosphodiester nucleotide sequences (SEQ ID NOs: 1, 2, 3 and
4) were prepared by Sigma-Genosys (Woodlands, Tex.) using Abacus
Segmented Synthesis Technology. Unless stated otherwise, the
sequences were dispersed in autoclaved deionized water or in a
pharmaceutically acceptable buffer such as, but not limited to,
saline immediately prior to use.
EXAMPLE 2
Cells
[0061] The K562 cell line derived from the leukemic cells of a CML
patient in blastic crisis is used as the standard model for
determining, in vitro, the therapeutic potential of new
differentiating compounds (Rutherford et al., Nature, 280:164,
1979; Drexler et al. DSMZ Catalogue of Human and Animal Cell Lines,
6.sup.th ed., Braunschweig, Germany: DSMZ, 1997). K562 cells were
obtained from the American Type Culture Collection (ATCC,
Rockville, Md.) and were cultured in the medium recommended by the
ATCC.
EXAMPLE 3
Hemoglobin Synthesis by K562 Cells Cultured with SEQ ID NO. 1, SEQ
ID NO. 2, SEQ ID NO: 3 and SEQ ID NO: 4.
[0062] K562 cells were seeded in 1.0 ml at 2.0.times.10.sup.5
cells/ml in 6-well flat-bottomed tissue culture plates for 72 hours
with 100 .mu.g of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ
ID NO: 4. For hemoglobin determination, the cells were washed twice
by centrifugation in phosphate-buffered saline (PBS), stained with
0.2% benzidine (Sigma-Aldrich Canada, Oakville, Ontario) in 0.5 M
acetic acid activated with 10% H.sub.2O.sub.2 (Gambari et al.,
Experimentia 41:673, 1985). After 10 minutes incubation in the
dark, the percentages of benzidine positive cells (hemoglobin
positive cells) were determined by light microscopy using an
hemocytometer. Approximately 500 cells were counted for the
determination of the percentages of benzidine positive cells. Cell
size was also determined by light microscopy. Hemin (20 .mu.g) was
added to K562 cells for 72 hours as a control for hemoglobin
synthesis. Hemin was obtained from Sigma-Aldrich Canada.
TABLE-US-00001 TABLE 1 Evaluation of hemoglobin synthesis
(benzidine-positivity) and cell size of K562 cells SEQUENCE % of
benzidine-positive cells Cell size None 5.4 normal SEQ ID NO: 1
17.4 increased SEQ ID NO: 2 35.8 increased SEQ ID NO: 3 11.4
increased SEQ ID NO: 4 10.1 increased Hemin 17.8 normal
As shown in Table 1, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and
SEQ ID NO: 4 induce the synthesis of hemoglobin by K562 cells and
an increase in their cell size, two measures of erythroid
differentiation.
EXAMPLE 4
[0063] Upregulation of Rh D in K562 cells cultured with SEQ ID NO.
2 or SEQ ID NO: 3.
[0064] The Rh D antigen is the most important antigen of the Rh
blood group system. In humans, the Rh D antigen is expressed solely
on erythrocytes (Cartron, Blood Rev. 6:199, 1994). K562 cells were
seeded in 1.0 ml at 2.0.times.10.sup.5 cells/ml in 6-well
flat-bottomed tissue culture plates for 72 hours with 2.5, 10.0,
25.0, 50.0 or 100.0 .mu.g of SEQ ID NO: 2 or SEQ ID NO: 3. The
expression of Rh D at the cell surface was monitored by flow
cytometry. After incubation, K562 cells were washed twice by
centrifugation with PBS and labeled with phycoerythrin
(PE)-conjugated anti-Rh D monoclonal antibody (IBGRL research
product, Bristol, Netherlands) for 30 min at 4.degree. C. After
washing twice with PBS-1% bovine serum albumin, cellular
fluorescence was then determined. Flow cytometry was carried out on
a FACSCalibur cell sorter (Becton Dickinson, San Jose, Calif., USA)
and analyzed using the program CELLQuest (Becton Dickinson). The
fold increase in Rh D level over control (0 .mu.g oligonucleotide)
was determined. Untreated K562 cells were essentially negative for
this marker. TABLE-US-00002 TABLE 2 Fold increase in Rh D level
over control in treated K562 with SEQ ID NO: 2 and SEQ ID NO: 3
Concentration (.mu.g/ml) SEQUENCE 2.5 10 25 50 100 SEQ ID NO: 2 1.7
x 2.5 x 4.6 x 9.8 x 15.4 x SEQ ID NO: 3 2.0 x 2.2 x 2.9 x 6.5 x 4.7
x
As shown in Table 2, SEQ ID NO: 2 and SEQ ID NO: 3 induced the
expression of Rh D antigen at the cell surface of K562, a measure
of erythroid differentiation.
EXAMPLE 5
Upregulation of CD41a Antigen in K562 Cells Cultured with SEQ ID
NO. 2 or SEQ ID NO. 3.
[0065] The CD41a antigen, also named GpIIb/IIIa, is expressed on
platelets and megakaryocytes (Gruel et al., Blood 68:488, 1986).
K562 cells were seeded in 1.0 ml at 2.0.times.10.sup.5 cells/ml in
6-well flat-bottomed tissue culture plates for 72 hours with 2.5,
10.0, 25.0, 50.0 or 100.0 .mu.g of SEQ ID NO: 2 or SEQ ID NO: 3.
The expression of CD41a at the cell surface was monitored by flow
cytometry. After incubation, K562 cells were washed twice by
centrifugation with PBS and labeled with phycoerythrin
(PE)-conjugated anti-CD41a monoclonal antibody (BD Pharmingen,
Mississauga, Ontario, Canada) for 30 min at 4.degree. C. After
washing twice with PBS-1% bovine serum albumin, cellular
fluorescence was then determined. Flow cytometry was carried out on
a FACSCalibur cell sorter (Becton Dickinson) and analyzed using the
program CELLQuest (Becton Dickinson). The fold increase in CD41a
level over control (0 .mu.g oligonucleotide) was determined.
Untreated K562 cells were essentially negative for this marker.
TABLE-US-00003 TABLE 3 Fold increase in CD41a level over control in
treated K562 cells with SEQ ID NO: 2 or SEQ ID NO: 3 Concentration
(.mu.g/ml) SEQUENCE 2.5 10 25 50 100 SEQ ID NO: 2 3.0 x 3.9 x 12.3
x 20.6 x 18.9 x SEQ ID NO: 3 3.2 x 3.7 x 8.3 x 13.4 x 11.7 x
As shown in Table 3, SEQ ID NO: 2 and SEQ ID NO: 3 induced the
expression of CD41a antigen, a measure of megakaryocyte
differentiation, at the cell surface of K562 cells.
EXAMPLE 6
Upregulation of CD14 in K562 Cells Cultured with SEQ ID NO. 2 or
SEQ ID NO. 3.
[0066] The CD14 antigen is expressed at high levels on monocytes.
Additionally, CD14 is expressed on interfollicular macrophages,
reticular dendritic cells and some Langherans cells (Wright et al.,
Science 249:1434, 1990). K562 cells were seeded in 1.0 ml at
2.0.times.10.sup.5 cells/ml in 6-well flat-bottomed tissue culture
plates for 72 hours with 2.5, 10.0, 25.0, 50.0 or 100.0 .mu.g of
SEQ ID NO: 2 or SEQ ID NO: 3. The expression of CD14 at the cell
surface was monitored by flow cytometry. After incubation, K562
cells were washed twice by centrifugation with PBS and labeled with
fluorescein isothiocyanate (FITC)-conjugated anti-CD14 monoclonal
antibody (BD Pharmingen) for 30 min at 4.degree. C. After washing
twice with PBS-1% bovine serum albumin, cellular fluorescence was
then determined. Flow cytometry was carried out on a FACSCalibur
cell sorter (Becton Dickinson) and analyzed using the program
CELLQuest (Becton Dickinson). The fold increase in CD14 level over
control (0 .mu.g oligonucleotide) was determined. Untreated K562
cells were essentially negative for this marker. TABLE-US-00004
TABLE 4 Fold increase in CD14 level over control in K562 cells
treated with SEQ ID NO: 2 or SEQ ID NO: 3 Concentration (.mu.g/ml)
SEQUENCE 2.5 10 25 50 100 SEQ ID NO: 2 1.8 x 1.9 x 3.9 x 7.2 x 9.8
x SEQ ID NO: 3 1.9 x 2.8 x 3.8 x 7.2 x 3.7 x
As shown in Table 4, SEQ ID NO: 2 and SEQ ID NO: 3 induced the
expression of CD14 antigen, a measure of monocyte differentiation,
at the cell surface of K562 cells.
EXAMPLE 7
Induction of CD14.sup.+Rh D.sup.+, CD14.sup.+CD41a.sup.+ and Rh
D.sup.+CD41a.sup.+ Phenotype in K562 Cells Cultured with SEQ ID NO.
2.
[0067] K562 cells were seeded in 1.0 ml at 2.0.times.10.sup.5
cells/ml in 6-well flat-bottomed tissue culture plates for 72 hours
with 2.5, 10.0, 25.0, 50.0 or 100.0 .mu.g of SEQ ID NO: 2. The
expression of CD14, CD41a and Rh D at the cell surface was
monitored by two dimensional flow cytometry. After incubation, K562
cells were washed twice by centrifugation with PBS and labeled with
FITC-conjugated anti-CD14, PE-conjugated anti-CD41a and/or
PE-conjugated anti-Rh D monoclonal antibody (BD Pharmingen) for 30
min at 4.degree. C. After washing twice with PBS-1% bovine serum
albumin, cellular fluorescence was determined. Flow cytometry was
carried out on a FACSCalibur cell sorter (Becton Dickinson) and
analyzed using the program CELLQuest (Becton Dickinson). The fold
increase in CD14.sup.+Rh D.sup.+, CD14.sup.+CD41a.sup.+ and Rh
D.sup.+CD41a.sup.+ level over control (0 .mu.g oligonucleotide) was
determined. Untreated K562 cells were essentially negative for
these markers. TABLE-US-00005 TABLE 5 Fold increase in CD14.sup.+Rh
D.sup.+, CD14.sup.+CD41a.sup.+ and Rh D.sup.+CD41a.sup.+ levels in
K562 treated with SEQ ID NO: 2 Concentration (.mu.g/ml) PHENOTYPE
2.5 10 25 50 100 CD14.sup.+Rh D.sup.+ 4.0 x 7.0 x 16.0 x 34.0 x
41.0 x CD14.sup.+CD41a.sup.+ 2.0 x 2.0 x 4.0 x 11.0 x 22.0 x Rh
D.sup.+CD41a.sup.+ 20.0 x 22.0 x 45.0 x 87.0 x 100.7 x
As shown in Table 5, SEQ ID NO: 2 induced the differentiation of
K562 cells into cells with heterogeneous phenotypes.
EXAMPLE 8
Induction of CD14.sup.+Rh D.sup.+, CD14.sup.+CD41a.sup.+ and Rh
D.sup.+CD41a.sup.+ Phenotype in K562 Cells Cultured with SEQ ID NO:
3.
[0068] K562 cells were seeded in 1.0 ml at 2.0.times.10.sup.5
cells/ml in 6-well flat-bottomed tissue culture plates for 72 hours
with 2.5, 10.0, 25.0, 50.0 or 100.0 .mu.g of SEQ ID NO: 3. The
expression of CD14, CD41a and Rh D at the cell surface was
monitored by two dimensional flow cytometry. After incubation, K562
cells were washed twice by centrifugation with PBS and labeled with
FITC-conjugated anti-CD14, PE-conjugated anti-CD41a and/or
PE-conjugated anti-Rh D monoclonal antibody (BD Pharmingen) for 30
min at 4.degree. C. After washing twice with PBS-1% bovine serum
albumin, cellular fluorescence was then determined. Flow cytometry
was carried out on a FACSCalibur cell sorter (Becton Dickinson) and
analyzed using the program CELLQuest (Becton Dickinson). The fold
increase in CD14.sup.+Rh D.sup.+, CD14.sup.+CD41a.sup.+ and Rh
D.sup.+CD41a.sup.+ level over control (0 .mu.g oligonucleotide) was
determined. Untreated K562 cells were essentially negative for
these markers. TABLE-US-00006 TABLE 6 Fold increase in CD14.sup.+Rh
D.sup.+, CD14.sup.+CD41a.sup.+ and Rh D.sup.+CD41a.sup.+ levels in
K562 treated with SEQ ID NO: 3 Concentration (.mu.g/ml) PHENOTYPE
2.5 14 25 50 100 CD14.sup.+Rh D.sup.+ 7.0 x 8.0 x 9.0 x 11.0 x 10.0
x CD14.sup.+CD41a.sup.+ 2.0 x 3.0 x 4.0 x 4.0 x 3.0 x Rh
D.sup.+CD41a.sup.+ 6.0 x 4.0 x 7.0 x 29.0 x 31.0 x
As shown in Table 6, SEQ ID NO: 3 induced the differentiation of
K562 cells into cells with heterogeneous phenotypes.
EXAMPLE 9
Inhibition of K562 Cell Growth by SEQ ID NO. 2 and SEQ ID NO.
3.
[0069] Terminal differentiation of K562 cells has been reported to
stop their cellular growth (Bianchi et al., Biochem. Pharmacol.
60:31, 2000). K562 cells were seeded in 1.0 ml at
2.0.times.10.sup.5 cells/ml in 6-well flat-bottomed tissue culture
plates for 72 hours with 1.0, 10.0 or 100.0 .mu.g of SEQ ID NO: 2
or SEQ ID NO: 3. Cells were counted after 24, 48 and 72 hours of
incubation using light microscopy and trypan blue dye.
TABLE-US-00007 TABLE 7 Number of K562 cells (.times.10.sup.5) after
treatment with SEQ ID NO: 2 or SEQ ID NO: 3 SEQ ID NO: 2 SEQ ID NO:
3 No oligo- 1.0 10.0 100.0 1.0 10.0 100.0 Hours nucleotide .mu.g/ml
.mu.g/ml .mu.g/ml .mu.g/ml .mu.g/ml .mu.g/ml 24 3.6 3.1 2.5 2.6 3.3
2.7 3.2 48 7.4 7.8 6.4 2.2 8.4 7.1 3.4 72 11.5 11.3 8.5 1.7 11.2
8.4 3.3
As shown in Table 7, SEQ ID NO: 2 and SEQ ID NO: 3 inhibited the
cellular growth of K562 cells in a dose-dependent manner. The
trypan blue exclusion assay demonstrated that treatment of K562
cells with SEQ ID NO: 2 or SEQ ID NO: 3 was not cytotoxic since no
trypan blue dye was incorporated by K562 cells.
EXAMPLE 10
Differentiation of Human Committed Erythroid Precursor by SEQ ID
NO. 2
[0070] Glycophorin A is the major glycoprotein of the human
erythrocyte membrane. Maturation of committed human erythroid
precursors is characterized by the expression of glycophorin A at
the cell surface and by an increase in intracellular granulosity
(Daniel and Greens, Vox Sang. S2:149, 2000; Wheater et al.,
Functional Histology, a text and color atlas, 2.sup.nd edition,
Churchill Livingstone, U.K., 1987). Human committed erythroid
precursors defined by the cell surface glycoprotein CD36 were
isolated from expanded human cord blood CD34+ progenitors by
positive immunoselection of CD36+ cells (Clonetics, San Diego,
Calif., USA). Human committed erythroid cells were seeded in 1.0 ml
at 1.5.times.10.sup.5 cells/ml in 6-well flat-bottomed tissue
culture plates for 96 hours with 100.0 .mu.g of SEQ ID NO: 2 or SEQ
ID NO: 3. The expression of glycophorin A at the cell surface and
the intracellular granulosity were monitored by flow cytometry.
After 48 and 96 hours of incubation, human committed erythroid
cells were washed twice by centrifugation with PBS and labeled with
PE-conjugated glycophorin A monoclonal antibody (Caltag
Laboratories, Burlingame, Calif., USA) for 30 min at 4.degree. C.
After washing twice with PBS-1% bovine serum albumin, cellular
fluorescence was determined. The intracellular granulosity was
determined by the measure of side light scatter (SSC) using a flow
cytometer. Flow cytometry was carried out on a FACSCalibur (Becton
Dickinson) and analyzed using the program CELLQuest (Becton
Dickinson). The percentages of cells in SSC.sup.hi glycophorin
A.sup.+ and in SSC.sup.lo glycophorin A.sup.+ were determined.
SSC.sup.hi is defined as >450 units; SSC.sup.lo is defined as
<450 units. TABLE-US-00008 TABLE 8 Percentages of human
committed erythroid precursor cells in SSC.sup.hi glycophorin
A.sup.+ and in SSC.sup.lo glycophorin A.sup.+ after treatment with
SEQ ID NO: 2 or SEQ ID NO: 3 SEQ ID SEQ ID None NO: 2 NO: 3 48 h 96
h 48 h 96 h 48 h 96 h SSC.sup.higlycophorinA.sup.+ 0.9 1.2 8.0 12.8
0.6 0.4 SSC.sup.loglycophorinA.sup.+ 9.1 3.8 9.3 18.1 7.4 2.2
As shown in Table 8, SEQ ID NO: 2 induced the differentiation of
human committed erythroid precursor cells in SSC.sup.hi glycophorin
A.sup.+ and in SSC.sup.lo glycophorin A.sup.+ cells.
EXAMPLE 11
[0071] Effect of SEQ ID NO. 2 on Human Disseminated Chronic Myeloid
Leukemia K562 Cells in Severe Combined Immunodeficiency Mice
[0072] Forty female severe combined immunodeficiency mice (SCID
mice) were exposed to 1.8 Gy of radiation (rate: 7.5 Gy/h) from a
.gamma. source. Twenty-four hours after whole body irradiation (day
0), the 40 female SCID mice were weighed and randomized to form 4
groups (10 mice/group). The mean body weight of each group was not
statistically different from the others (analysis of variance).
Mice were injected intraperitoneally (ip) with 2.0.times.10.sup.7
K562 cells in 0.5 ml of RPMI-1640 medium. SEQ ID NO: 2 (5'GGGTGG3')
resuspended in 0.9% sodium chloride USP was administrated ip at
0.01, 0.1 and 1 mg per mouse per day from day 1 to day 29 (30
days). A vehicle group was ip injected with vehicle (0.9% sodium
chloride USP) following the same schedule. The treatment schedule
is summarized in the table below: TABLE-US-00009 TABLE 9 Dose/inj.
(mg/ Vol./ Mice/ Route mouse/ inj. Treatment Group Treatment group
Admin. inj.) (ml) schedule 1 Vehicle 10 ip 0 0.250 Q1DX30 2 SEQ ID
NO: 2 10 ip 0.01 0.250 Q1DX30 3 SEQ ID NO: 2 10 ip 0.1 0.250 Q1DX30
4 SEQ ID NO: 2 10 ip 1 0.250 Q1DX30
The experiment was stopped at 120 days when mice were sacrificed.
Survival was recorded two times per week.
[0073] The test evaluation expressed as a percentage (T/C %) and as
the increased life span value (ILS %) of the control evaluation was
determined. These are measures of the effectiveness of the
compounds tested. Survival systems indicate a degree of success
when T/C percentages exceed 125 and ILS percentages exceed 25. T is
the median survival times of animals treated with drugs and C is
the median survival time of control animals. T/C % and ILS % is
expressed as following: ILS %=[(T-C)/C].times.100 T/C
%=[T/C].times.100
[0074] Statistical analysis was performed using StatView.RTM.
(Abacus Concept, Berkeley, USA). Statistical analysis of the
efficiency of the treatment was performed using the Bonferroni/Dunn
test (ANOVA comparison). TABLE-US-00010 TABLE 10 Survival time of
SCID mice having human disseminated chronic myeloid K562 leukemia
treated with SEQ ID NO: 2 TREATMENT Group 4: 1 mg Group 2: Group 3:
SEQ ID Group 1: 0.01 mg 0.1 mg NO: 2 vehicle SEQ ID NO: 2 SEQ ID
NO: 2 Survival Survival Survival time Survival time time time
(days) (days) (days) (days) 38 120 85 49 54 57 34 120 46 54 57 41
54 75 75 57 34 120 34 57 38 120 31 99 34 120 64 61 54 61 54 61 75
61 64 46 54 34 41 46 34 38 34 49 Mean .+-. sd 48.6 .+-. 12.2 78.2
.+-. 34.9 52.1 .+-. 18.7 62.4 .+-. 24.6 median 46 61 41 49 ILS % --
32.6 -12.2 6.5 T/C % -- 132.6 89.1 106.5
[0075] As shown in Table 10, SEQ ID NO: 2, at 0.01 mg/mouse/day,
significantly increased the life span of SCID mice having human
disseminated chronic myeloid K562 leukemia (p<0.05). After 120
days, 4 of 11 mice treated with SEQ ID NO:2 at 0.01 mg/mouse/day
were alive while none of the 11 untreated mice was alive.
EXAMPLE 12
Effect of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO. 3 and SEQ ID NO: 4
on Differentiation of Bone Marrow-Derived Cells from Mice
[0076] The C57BL/6 mice are divided into 5 groups of 10 mice. Mice
receive gamma-irradiation to induce a reduction in the number of
bone marrow derived-cells and bone marrow precursor cells. On day
0, group 1 mice receive saline, group 2 receive SEQ ID NO: 1, group
3 receive SEQ ID NO: 2, group 4 receive SEQ ID NO: 3, group 5
receive SEQ ID NO: 4. Sequences resuspended in 0.9% sodium chloride
USP are administrated ip at 0.01 mg per mouse per day for 7
days.
[0077] After 7 days of treatment, the mice are sacrificed. Cells
present in peripheral blood and in bone marrow are counted and
their phenotype determined by flow cytometry. Hemoglobin levels are
also determined. Mice in groups 2, 3, 4 and 5 have more bone
marrow-derived cells than the mice in group 1. Mice in groups 2, 3,
4 and 5 have more mature bone marrow-derived cells than the mice in
group 1. The levels of hemoglobin are more elevated in mice in
groups 2, 3, 4 than in mice in group 1.
EXAMPLE 13
Effect of SEQ ID NO: 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4
on CML in SCID Mice
[0078] K562 cells (2.times.10.sup.7 cells) are inoculated into SCID
mice (severe combined immunodeficiency mice) as previously
described (Beran et al., Hematol. Pathol. 8:135, 1994). The mice
are divided into 5 groups of 10 mice. On day 0, group 1 mice
receive saline, group 2 mice receive SEQ ID NO: 1, group 3 mice
receive SEQ ID NO: 2, group 4 receive SEQ ID NO: 3, group 5 mice
receive SEQ ID NO: 4. Sequences resuspended in 0.9% sodium chloride
USP are administrated ip at 0.01 mg per mouse per day for 30
days.
[0079] After 30 days, the mice are sacrificed. Leukemic
dissemination, leukemic cell phenotype and hemoglobin levels are
analyzed. Mice in Group 1 have the most leukemia cells and
dissemination. Mice in groups 2, 3, 4 and 5 have less leukemia
cells and dissemination. Mice in groups 2, 3, 4 and 5 show a higher
number of differentiated K562 cells than mice in group 1. Mice in
groups 2, 3, 4 and 5 show more hemoglobin synthesis than the mice
in group 1.
[0080] All patents, publications and abstracts cited above are
incorporated herein by reference in their entirety. It should be
understood that the foregoing relates only to preferred embodiments
of the present invention and that numerous modifications or
alterations may be made therein without departing from the spirit
and the scope of the present invention as defined in the following
claims.
Sequence CWU 1
1
5 1 3 DNA Artificial Sequence synthetic oligonucleotide 1 gtg 3 2 6
DNA Artificial Sequence synthetic oligonucleotide 2 gggtgg 6 3 6
DNA Artificial Sequence synthetic oligonucleotide 3 gggagg 6 4 6
DNA Artificial Sequence synthetic oligonucleotide 4 ccaccc 6 5 5
DNA Artificial Sequence synthetic oligonucleotide misc_feature
(3)..(5) Any "n" = any nucleotide 5 cgnnn 5
* * * * *