U.S. patent application number 14/568736 was filed with the patent office on 2015-04-30 for amphoteric liposome formulation.
The applicant listed for this patent is Novosom AG, ProNAi Therapeutics, Inc.. Invention is credited to Gerold Endert, Neal Clifford Goodwin, Natalie Herzog, Yvonne Kerwitz, Steffen Panzner, Wendi Rodrigueza.
Application Number | 20150118291 14/568736 |
Document ID | / |
Family ID | 37983382 |
Filed Date | 2015-04-30 |
United States Patent
Application |
20150118291 |
Kind Code |
A1 |
Goodwin; Neal Clifford ; et
al. |
April 30, 2015 |
Amphoteric Liposome Formulation
Abstract
The invention relates to compositions and methods to inhibit
gene expression. In particular, the invention provides DNAi
oligonucleotides sequestered by amphoteric liposomes for the
treatment of cancer.
Inventors: |
Goodwin; Neal Clifford;
(Plainwell, MI) ; Endert; Gerold; (Halle, DE)
; Herzog; Natalie; (Cottbus, DE) ; Kerwitz;
Yvonne; (Nordhausen, DE) ; Panzner; Steffen;
(Halle, DE) ; Rodrigueza; Wendi; (Roslindale,
MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ProNAi Therapeutics, Inc.
Novosom AG |
Kalamazoo
Halle |
MI |
US
DE |
|
|
Family ID: |
37983382 |
Appl. No.: |
14/568736 |
Filed: |
December 12, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13751536 |
Jan 28, 2013 |
|
|
|
14568736 |
|
|
|
|
12085893 |
Mar 19, 2009 |
8367628 |
|
|
PCT/US2006/045955 |
Dec 1, 2006 |
|
|
|
13751536 |
|
|
|
|
60741192 |
Dec 1, 2005 |
|
|
|
60778473 |
Mar 2, 2006 |
|
|
|
Current U.S.
Class: |
424/450 ;
435/375; 514/44A |
Current CPC
Class: |
A61K 9/1272 20130101;
A61K 31/711 20130101; A61K 9/1271 20130101; C12N 15/111 20130101;
A61K 9/127 20130101; C12N 15/1135 20130101; C12N 2320/32 20130101;
A61P 35/00 20180101; C12N 2310/14 20130101; C12N 15/113
20130101 |
Class at
Publication: |
424/450 ;
514/44.A; 435/375 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 9/127 20060101 A61K009/127 |
Claims
1. A mixture comprising an amphoteric liposome and a DNAi
oligonucleotide.
2. The mixture of claim 1, wherein the amphoteric liposome has an
isoelectric point between 4 and 8.
3. The mixture of claim 1 or 2, wherein the amphoteric liposome has
a net negative charge or are neutral at pH 7.4 and have a net
positive charge at pH 4.
4. The mixture of claim 1, 2 or 3, wherein the amphoteric liposome
a is formed from a lipid phase comprising an amphoteric lipid.
5. The mixture of claim 4, wherein the lipid phase comprises 5 to
30 mol. % of the amphoteric lipid.
6. The mixture of claim 4 or 5, wherein the amphoteric lipid is
selected from the group consisting of HistChol, HistDG,
isoHistSuccDG, Acylcarnosine and HCChol.
7. The mixture of claim 1, 2 or 3, wherein the amphoteric liposome
is formed from a lipid phase comprising a mixture of lipid
components with amphoteric properties.
8. The mixture of claim 7, wherein the mixture of lipid components
comprises anionic or cationic components, wherein at least one such
component is pH responsive.
9. The mixture of claim 8, wherein the mixture of lipid components
are selected from the group consisting of (i) a stable cationic
lipid and a chargeable anionic lipid, (ii) a chargeable cationic
lipid and chargeable anionic lipid and (iii) a stable anionic lipid
and a chargeable cationic lipid.
10. The mixture of claim 9, wherein the lipid components comprise
one or more anionic lipids selected from the group consisting of
DOGSucc, POGSucc, DMGSucc, DPGSucc, DGSucc, DMPS, DPPS, DOPS, POPS,
DMPG, DPPG, DOPG, POPG, DMPA, DPPA, DOPA, POPA, CHEMS and
Cet-P.
11. The mixture of claim 9 or claim 10, wherein the lipid
components comprise one or more anionic lipids selected from the
group consisting of DMGSucc, DOPA, CHEMS and Cet-P.
12. The mixture of any of claims 8 to 11, wherein the lipid
components comprise one or more cationic lipids selected from the
group consisting of DMTAP, DPTAP, DOTAP, DC-Chol, MoChol, HisChol,
DPIM, CHIM, DORIE, DDAB, DAC-Chol, TC-Chol, DOTMA, DOGS,
(C18).sub.2Gly.sup.+N,N-dioctadecylamido-glycine, CTAP, CPyC, DODAP
and DOEPC.
13. The mixture of any of claims 8 to 11, wherein the lipid
components comprise one or more cationic lipids selected from the
group consisting of DOTAP, DC-Chol, MoChol and HisChol.
14. The mixture of claim 7 wherein the anionic lipid is CHEMS and
the cationic lipid is MoChol.
15. The mixture of claim 7 wherein the anionic lipid is CHEMS and
the cationic lipid is DOTAP.
16. The mixture of claim 7 wherein the anionic lipid is Cet-P and
the cationic lipid is MoChol.
17. The mixture of claim 7 wherein the anionic lipid is DMGSucc and
the cationic lipid is MoChol.
18. The mixture of any of claims 4 to 17, wherein the lipid phase
further comprises neutral lipids.
19. The mixture of claim 18 wherein the neutral lipids comprise
sterols and derivatives thereof.
20. The mixture of claim 18 wherein the neutral lipids comprise
neutral phospholipids.
21. The mixture of claim 20 wherein the neutral phospholipids
comprise phosphatidylcholines.
22. The mixture of claim 21 wherein the phosphatidylcholines are
selected from the group consisting of POPC, OPPC, natural or
hydrogenated soy bean PC, natural or hydrogenated egg PC, DMPC,
DPPC or DOPC and derivatives thereof.
23. The mixture of claim 22 wherein the phosphatidylcholine is
POPC.
24. The mixture of claim 20 wherein the neutral phospholipids
comprise mixtures of phosphatidylcholines and
phosphoethanolamines.
25. The mixture of claim 24 wherein the phosphatidylcholines are
selected from the group consisting of POPC, OPPC, natural or
hydrogenated soy bean PC, natural or hydrogenated egg PC, DMPC,
DPPC or DOPC and derivatives thereof and the
phosphatidylethanolamines are selected from the group consisting of
DOPE, DMPE, DPPE and derivatives thereof.
26. The mixture of claim 25 wherein the phosphatidylcholine is POPC
and the phosphatidylethanolamine is DOPE.
27. The mixture of claim 18 wherein the neutral lipids are POPC and
cholesterol.
28. The mixture of claim 24 wherein the amphoteric liposomes
comprise DOPE, POPC, CHEMS and MoChol.
29. The mixture of claim 24 wherein the amphoteric liposome
comprises POPC, DOPE, MoChol and DMGSucc.
30. The mixture of claim 27 wherein the amphoteric liposome
comprises POPC, Chol, CHEMS and DOTAP.
31. The mixture of claim 27 wherein the amphoteric liposome
comprises POPC, Chol, CetP and MoChol.
32. A mixture comprising an amphoteric liposome comprising PNT-100
(SEQ ID NO:1251); a mixture of lipid components with amphoteric
properties wherein the mixture of lipid components comprises
anionic and cationic components, wherein at least one such
component is pH responsive and neutral lipids, wherein the neutral
lipids comprise phosphatidylcholines, phosphatidylethanolamines or
sterols and the mixture contains 30 to 70 mole % amphoteric lipids
and 30 to 60 mole % neutral lipids.
33. The mixture of claim 32, wherein the cationic lipids are
selected from the group consisting of DMTAP, DPTAP, DPTAP, DOTAP,
DC-Chol, TC-Chol, MoChol, HisChol, DPIM, CHIM, DORIE, DDAB,
DAC-Chol, TC-Chol, DOTMA, DOGS,
(C18).sub.2Gly.sup.+N,N-dioctadecylamido-glycine, CTAB, CPyC,
DODAP, DOEPC and derivatives thereof, and the anionic lipids are
selected from the group consisting of DOGSucc, POGSucc, DMGSucc,
DPGSucc, DGSucc, DMPS, DPPS, DOPS, POPS, DMPG, DPPG, DOPG, POPG,
DMPA, DPPA, DOPA, POPA, CHEMS, Cet-P and derivatives thereof, and
wherein the phosphatidylcholines are selected from the group
consisting of POPC, OPPC, natural or hydrogenated soy bean PC,
natural or hydrogenated egg PC, DMPC, DPPC or DOPC and derivatives
thereof, the phosphatidylethanolamines are selected from the group
consisting of DOPE, DMPE, DPPE and derivatives thereof, and the
sterol is cholesterol or a derivative thereof.
34. The mixture of claim 32, wherein the cationic lipids are
selected from the group consisting of DOTAP, DC-Chol, MoChol and
HisChol, and the anionic lipids are selected from the group
consisting of DMGSucc, DOPA, CHEMS and Cet-P.
35. The mixture of claim 32, wherein the cationic lipid is MoChol
and the anionic lipid is CHEMS.
36. The mixture of claim 35, wherein the mixture comprises 10 to 60
mole % of MoChol and 10 to 30 mole % of CHEMS.
37. The mixture of claim 32, wherein the cationic lipid is DOTAP
and the anionic lipid is CHEMS.
38. The mixture of claim 37, wherein the mixture comprises 10 to 30
mole % of CHEMS and 5 to 40 mole % of DOTAP.
39. The mixture of claim 32, wherein the cationic lipid is MoChol
and the anionic lipid is Cet-P.
40. The mixture of claim 39, wherein the mixture comprises 10 to 60
mole % of MoChol and 5 to 30 mole % of Cet-P.
41. The mixture of claim 32, wherein the cationic lipid is MoChol
and the anionic lipid is DMGSucc.
42. The mixture of claim 41, wherein the mixture comprises 20 to 60
mole % of MoChol and 20 to 60 mole % of DMGSucc.
43. The mixture of claim 32, wherein the neutral lipids comprise
POPC and DOPE.
44. The mixture of claim 44, wherein the mixture comprises 5 to 40
mole % of POPC and 20 to 50 mole % of DOPE.
45. The mixture of claim 32, wherein the neutral lipids comprise
POPC and Chol.
46. The mixture of claim 45, wherein the mixture comprises 10 to 50
mole % of POPC and 30 to 50 mole % of Chol.
47. The mixture of claim 32, wherein the amphoteric liposome
comprises POPC, DOPE, MoChol and CHEMS.
48. The mixture of claim 47, wherein the mixture comprises 3 to 20
mole % of POPC, 10 to 60 mole % of DOPE, 10 to 60 mole % of MoChol
and 10 to 60 mole % of CHEMS.
49. The mixture of claim 47, wherein the molar ratio of
POPC/DOPE/MoChol/CHEMS is about 6/24/47/23.
50. The mixture of claim 47, wherein the molar ratio of
POPC/DOPE/MoChol/CHEMS is 15/45/20/20.
51. The mixture of claim 32, wherein the amphoteric liposome
comprises POPC, DOPE, DMGSucc and MoChol.
52. The mixture of claim 51, wherein the mixture comprises 3 to 20
mole % of POPC, 10 to 40 mole % of DOPE, 15 to 60 mole % of DMGSucc
and 15 to 60 mole % of MoChol.
53. The mixture of claim 52, wherein the molar ratio of
POPC/DOPE/DMGSucc/MoChol is about 6/24/23/47.
54. The mixture of claim 52, wherein the molar ratio of
POPC/DOPE/DMGSucc/MoChol is about 6/24/47/23.
55. The mixture of claim 32, wherein the amphoteric liposome
comprises POPC, Chol, CHEMS and DOTAP.
56. The mixture of claim 55, wherein the mixture comprises 10 to 50
mole % of POPC, 20 to 60 mole % of Chol, 10 to 40 mole % of CHEMS
and 5 to 20 mole % of DOTAP.
57. The mixture of claim 56, wherein the molar ratio of
POPC/Chol/CHEMS/DOTAP is about 30/40/20/10.
58. The mixture of claim 32, wherein the amphoteric liposome
comprises POPC, Chol, Cet-P and MoChol.
59. The mixture of claim 58, wherein the mixture comprises 10 to 40
mole % of POPC, 20 to 50 mole % of Chol, 5 to 30 mole % of Cet-P
and 10 to 40 mole % of MoChol.
60. The mixture of claim 59, wherein the molar ratio of
POPC/Chol/Cet-P/MoChol is about 35/35/10/20.
61. The mixture of any one of the preceding claims wherein the
amphoteric liposomes comprise a size between 50 and 500 .eta.m.
62. The mixture of any of claims 1-61 wherein the amphoteric
liposome comprises a size between 80 and 300 .eta.m.
63. The mixture of any of claims 1-61 wherein the amphoteric
liposome comprises a size between 90 and 200 .eta.m.
64. The mixture of any of claims 1-63 wherein the amphoteric
liposome has a DNAi oligonucleotide concentration of at least 2
mg/ml at a lipid concentration of about 10 to 100 mM or less.
65. The mixture of any of claims 1-64 wherein the DNAi
oligonucleotide comprises a DNAi oligomer that hybridizes under
physiological conditions to nucleotides 500-2026 of SEQ ID NO:1249
or the complement thereof.
66. The mixture of any of claims 1-64 wherein the DNAi
oligonucleotide comprises a DNAi oligomer that hybridizes under
physiological conditions to nucleotides 500-1525 of SEQ ID NO:1249
or the complement thereof.
67. The mixture of any of claims 1-64 wherein the DNAi
oligonucleotide comprises a DNAi oligomer that hybridizes under
physiological conditions to nucleotides 800-1225 of SEQ ID NO:1249
or the complement thereof.
68. The mixture of any of claims 1-64 wherein the DNAi
oligonucleotide comprises a DNAi oligomer that hybridizes under
physiological conditions to nucleotides 900-1125 of SEQ ID NO:1249
or the complement thereof.
69. The mixture of any of claims 1-64 wherein the DNAi
oligonucleotide comprises a DNAi oligomer that hybridizes under
physiological conditions to nucleotides 950-1075 of SEQ ID NO:1249
or the complement thereof.
70. The mixture of any of claims 1-64 wherein the DNAi
oligonucleotide comprises a DNAi oligomer that hybridizes under
physiological conditions to nucleotides 970-1045 of SEQ ID NO:1249
or the complement thereof.
71. The mixture of any of claims 1-64 wherein the DNAi
oligonucleotide comprises a DNAi oligomer selected from the group
consisting of SEQ ID NOs:1250, 1251, 1252, 1253, 1267-1447 and the
complements thereof.
72. The mixture of any of claims 1-64 wherein the DNAi
oligonucleotide comprises a DNAi oligomer selected from the group
consisting of SEQ ID NOs 1250, 1251, 1267, 1268, 1276, 1277, 1285,
1286 and the complements thereof.
73. The mixture of any of claims 1-64 wherein the DNAi
oligonucleotide comprises a DNAi oligomer selected from the group
consisting of SEQ ID NOs 1250, 1251, 1289-1358 and the complements
thereof.
74. The mixture of any of claims 1-64, wherein the DNAi
oligonucleotide comprises SEQ ID NO:1250 or 1251.
75. A method comprising (a) providing the mixture of any of claims
1-64 and a cell or animal capable of expressing the bcl-2 gene, and
(b) introducing the mixture into the cell or animal.
76. The method of claim 75, wherein introducing the composition
results in a reduction of proliferation of the cell, or induces
cell death.
77. The method of claim 75, wherein the cell is a cancer cell.
78. The method of claim 75, wherein the animal is a non-human
animal.
79. The method of claim 75, wherein the animal is a human.
80. The method of claim 78, wherein the mixture is introduced to
the animal at a dosage of between 0.01 mg to 100 mg per kg of body
weight.
81. The method of claim 78, wherein the mixture is introduced to
the animal one or more times per day.
82. The method of claim 78, wherein the mixture is introduced to
the animal continuously.
83. The method of claim 78, wherein the mixture is introduced to
the animal by one or more routes of administration selected from
the group consisting of topical, pulmonary, intraocular,
intranasal, parenteral, and a medical device.
84. The method of claim 74, wherein the cell is in cell
culture.
85. The method of claim 74, further comprising the step of
introducing a test compound to the cell or animal.
86. The method of claim 85, wherein the test compound is a
chemotherapy agent.
87. The method of claim 86, wherein animal has cancer which is
selected from the group consisting of pancreatic cancer, colon
cancer, breast cancer, bladder cancer, lung cancer, leukemia,
prostate cancer, lymphoma, ovarian cancer and melanoma.
88. A pharmaceutical composition comprising the mixture of any of
claims 1 to 74.
89. A mixture comprising an amphoteric liposome and a DNAi
oligonucleotide comprising SEQ ID NO:1251 or 1250 wherein the
liposome comprises POPC, DOPE, MoChol and CHEMS in the molar ratio
of POPC/DOPE/MoChol/CHEMS of 15/45/20/20.
90. A mixture comprising amphoteric liposome and a DNAi
oligonucleotide comprising SEQ ID NO:1251 or 1250 wherein the
liposome comprises POPC, DOPE, MoChol and CHEMS in the molar ratio
of POPC/DOPE/MoChol/CHEMS of 6/24/47/23.
Description
CLAIM OF PRIORITY
[0001] This application is a continuation of, and claims priority
under U.S.C. .sctn.120, to U.S. Non-Provisional patent application
Ser. No. 13/751,536 filed on Jan. 28, 2013, which is a divisional
of U.S. Non-Provisional patent application Ser. No. 12/085,893,
filed on Jun. 2, 2008, which is the U.S. National phase of
International patent application No. PCT/US2006/045955 filed on
Dec. 1, 2006, which claims priority to U.S. Provisional patent
application No. 60/741,192, filed on Dec. 1, 2005 and to U.S.
Provisional patent application No. 60/778,473, filed on Mar. 2,
2006, all of which are herein incorporated by reference in their
entireties.
SEQUENCE LISTING
[0002] This application incorporates by reference in its entirety
the Sequence Listing entitled "Amphoteric_Sequence_ST25.txt" (701
kilobytes) which was last modified on Dec. 12, 2014 and filed
electronically herewith.
BRIEF DESCRIPTION OF THE INVENTION
[0003] The invention relates to compositions and methods of using
the same to treat cancer. In particular, the invention relates to
DNAi oligonucleotides sequestered with amphoteric liposomes for the
treatment of cancer.
BACKGROUND
[0004] Oncogenes have become the central concept in understanding
cancer biology and may provide valuable targets for therapeutic
drugs. In many types of human tumors, including lymphomas and
leukemias, the oncogenes are overexpressed, and may be associated
with tumorigenicity (Tsujimoto et al., Science 228:1440-1443
(1985)). For instance, high levels of expression of the human bcl-2
gene have been found in all lymphomas with a t(14; 18) chromosomal
translocations including most follicular B cell lymphomas and many
large cell non-Hodgkin's lymphomas. High levels of bcl-2 gene
expression have also been found in certain leukemias that do not
have a t(14; 18) chromosomal translation, including most cases of
chronic lymphocytic leukemia acute, many lymphocytic leukemias of
the pre-B cell type, neuroblastomas, nasophryngeal carcinomas, and
many adenocarcinomas of the prostate, breast and colon. (Reed et
al., Cancer Res. 51:6529 [1991]; Yunis et al., New England J. Med.
320:1047; Campos et al., Blood 81:3091-3096 [1993]; McDonnell et
al., Cancer Res. 52:6940-6944 [1992); Lu et al., Int. J Cancer
53:29-35 [1993]; Bonner et al., Lab Invest. 68:43A [1993]. Other
oncogenes include TGF-.alpha., c-ki-ras, ras, Her-2, and c-myc.
[0005] The expression of oncogenes may be inhibited by single
stranded DNAi oligonucleotides. Nucleic acid therapeutics, however,
often lack therapeutic efficacy due to instability in body fluids
or inefficient uptake into cells.
[0006] There is therefore a need for a stable and efficient
delivery of such DNAi oligonucleotides in body fluids and cells for
the treatment of cancer.
SUMMARY OF THE INVENTION
[0007] The invention provides compositions and methods for
preparing and using amphoteric liposomes for the delivery of DNAi
oligonucleotides for the treatment of cancer. Such amphoteric
liposomes may, for example, have an anionic or neutral charge at
physiological pH and a cationic charge at an acidic pH of about 4.
Advantageously, the compositions of the present invention sequester
high amounts of DNAi oligonucleotides, between about 1 to 4 mg/ml
(e.g., about 2 mg/ml) at a lipid concentration of about 10 to 100
mM or less; exhibit colloidal and serum stability; enhanced uptake
into cells and tumors due to average liposome sizes of less than
200 .eta.m; and low toxicity relative to liposomes formed with
cationic lipids that are used in conventional transfection
reagents.
[0008] In a first aspect, the invention provides a mixture
comprising amphoteric liposomes and a DNAi oligonucleotide. In an
embodiment of the first aspect, the amphoteric liposomes have an
isoelectric point of between 4 and 8. In a further embodiment, the
amphoteric liposomes are negatively charged or neutral at pH 7.4
and positively charged at pH 4.
[0009] In another embodiment of the first aspect, the amphoteric
liposomes include amphoteric lipids. In a further embodiment, the
amphoteric lipids can be HistChol, HistDG, isoHistSucc DG,
Acylcarnosine, HCChol or combinations thereof. In another
embodiment, the amphoteric liposomes include a mixture of one or
more cationic lipids and one or more anionic lipids. In yet another
embodiment, the cationic lipids can be DMTAP, DPTAP, DOTAP,
DC-Chol, MoChol or HisChol, or combinations thereof, and the
anionic lipids can be CHEMS, DGSucc, Cet-P, DMGSucc, DOGSucc,
POGSucc, DPGSucc, DG Succ, DMPS, DPPS, DOPS, POPS, DMPG, DPPG,
DOPG, POPG, DMPA, DPPA, DOPA, POPA or combinations thereof.
[0010] In yet another embodiment, the liposomes also include
neutral lipids. In a further embodiment, the neutral lipids include
sterols and derivatives thereof. In an even further embodiment, the
sterols comprise cholesterol and derivatives thereof. The neutral
lipids may also include neutral phospholipids. In one embodiment,
the phospholipids include phosphatidylcholines or
phosphatidylcholines and phosphoethanolamines. In another
embodiment, the phosphatidylcholines are POPC, OPPC, natural or
hydrogenated soy bean PC, natural or hydrogenated egg PC, DMPC,
DPPC or DOPC and derivatives thereof and the
phosphatidylethanolamines are DOPE, DMPE, DPPE or derivatives and
combinations thereof. In a further embodiment, the
phosphatidylcholine is POPC, OPPC, soy bean PC or egg PC and the
phosphatidylethanolamines is DOPE.
[0011] In an even further embodiment, the lipids of the amphoteric
liposomes include DOPE, POPC, CHEMS and MoChol; POPC, Chol, CHEMS
and DOTAP; POPC, Chol, Cet-P and MoChol, or POPC, DOPE, MoChol and
DMGSucc.
[0012] In a second aspect, the amphoteric liposomes of the mixture
of the invention can be formed from a lipid phase comprising a
mixture of lipid components with amphoteric properties, wherein the
total amount of charged lipids in the liposome can vary from 5 mole
% to 70 mole %, the total amount of neutral lipids may vary from 20
mole % to 70 mole %, and a DNAi oligonucleotide. In an embodiment
of the first aspect, the amphoteric liposomes include 3 to 20 mole
% of POPC, 10 to 60 mole % of DOPE, 10 to 60 mole % of MoChol and
10 to 50 mole % of CHEMS. In a further embodiment, the liposomes
include POPC, DOPE, MoChol and CHEMS in the molar ratios of
POPC/DOPE/MoChol/CHEMS of about 6/24/47/23 or 15/45/20/20. In yet
another embodiment, the liposomes include 3 to 20 mole % of POPC,
10 to 40 mole % of DOPE, 15 to 60 mole % of MoChol and 15 to 60
mole % of DMGSucc. In a further embodiment, the liposomes include
POPC, DOPE, DMGSucc and MoChol in the molar ratios of
POPC/DOPE/DMGSucc/MoChol of about 6/24/47/23 or 6/24/23/47. In
still another embodiment, the liposomes include 10 to 50 mole % of
POPC, 20 to 60 mole % of Chol, 10 to 40 mole % of CHEMS and 5 to 20
mole % of DOTAP. In a further embodiment, the liposomes include
POPC, Chol, CHEMS and DOTAP in the molar ratio of
POPC/Chol/CHEMS/DOTAP of about 30/40/20/10. In yet another
embodiment the liposomes include 10 to 40 mole % of POPC, 20 to 50
mole % of Chol, 5 to 30 mole % of Cet-P and 10 to 40 mole % of
MoChol. In a further embodiment, the molar ratio of
POPC/Chol/Cet-P/MoChol is about 35/35/10/20.
[0013] In a third aspect, the DNAi oligonucleotide contained in the
amphoteric liposomal mixture comprises a DNAi oligonucleotide that
hybridizes to SEQ ID NO:1249 or portions thereof. In another
embodiment, the DNAi oligonucleotide can be SEQ ID NO:1250, 1251,
1252, 1253, 1267-1447 or the complement thereof. In yet another
embodiment the DNAi oligonucleotide can be SEQ ID NO:1250 or 1251
or the complement thereof.
[0014] The amphoteric liposomal mixture of this invention may
further include an additional DNAi oligonucleotide, e.g.,
comprising one of SEQ ID NOs: 1250-1253 and 1270-1477, or selected
from the group consisting of SEQ ID NOs: 2-281, 283-461, 463-935,
937-1080, 1082-1248 and the complements thereof.
[0015] In another embodiment, the DNAi oligonucleotides contained
in the liposomal mixture are between 15 and 35 base pairs in
length.
[0016] In a fourth aspect, the amphoteric liposome-DNAi
oligonucleotide mixture includes the DNAi oligonucleotides SEQ ID
NO:1250 or 1251 and amphoteric liposomes comprising POPC, DOPE,
MoChol and CHEMS in the molar ratio of POPC/DOPE/MoChol/CHEMS of
about 6/24/47/23.
[0017] In a fifth aspect, the amphoteric liposome-DNAi
oligonucleotide mixture includes the DNAi oligonucleotide, PNT-100
(SEQ ID NO:1251), and amphoteric liposomes comprising POPC, DOPE,
MoChol and CHEMS in the molar ratio of POPC/DOPE/MoChol/CHEMS of
about 15/45/20/20.
[0018] In a sixth aspect, the amphoteric liposomes of the mixture
can include a size between 50 and 500 .eta.m. In one embodiment,
the size is between 80 and 300 .eta.m and in another embodiment the
size is between 90 and 200 .eta.m.
[0019] In a seventh aspect, the amphoteric liposomes may have an
isoelectric point between 4 and 8. In an embodiment of the sixth
aspect, the amphoteric liposomes may be negatively charged or
neutral at pH 7.4 and positively charged at pH 4.
[0020] In an eighth aspect, the amphoteric liposomes have a DNAi
oligonucleotide concentration of at least about 2 mg/ml at a lipid
concentration of 10 to 100 mM or less.
[0021] In a ninth aspect, the invention provides a method of
preparing amphoteric liposomes containing a DNAi oligonucleotide.
In one embodiment, the method includes using an active loading
procedure and in another, a passive loading procedure. In a further
embodiment, the method produces liposomes using manual extrusion,
machine extrusion, homogenization, microfluidization or ethanol
injection. In yet another embodiment, the method has an
encapsulation efficiency of at least 35%.
[0022] In a tenth aspect, the invention provides a method of
introducing the DNAi oligonucleotide-amphoteric liposome mixture to
cells or an animal. In one embodiment, the method includes
administering the mixture to mammal to treat cancer. The
administered mixtures can reduce or stop tumor growth in mammals.
In another embodiment, the introduction of the mixture results in a
reduction of cell proliferation. In another embodiment, the mixture
is administered to a cancer cell, a non-human animal or a human. In
a further embodiment, the mixture is introduced to an animal at a
dosage of between 0.01 mg to 100 mg per kg of body weight. In yet
another embodiment, the mixture is introduced to the animal one or
more times per day or continuously. In still another embodiment,
the mixture is introduced to the animal via topical, pulmonary or
parenteral administration or via a medical device. In an even
further embodiment, the mixture administered to the animal or cells
further includes a chemotherapy agent, and/or a cell targeting
component.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows the effect of SEQ ID NO:1251 sequestered in
amphoteric liposomes on the size of tumors from non-Hodgkin's
Lymphoma WSU-DLCL2 xenografts in SCID mice.
[0024] FIG. 2 shows the effect of different lots of SEQ ID NO:1251
sequestered in amphoteric liposomes on the size of tumors from
non-Hodgkin's Lymphoma WSU-DLCL2 xenografts in SCID mice.
[0025] FIG. 3 shows the tumor burden in mice carrying non-Hodgkin's
Lymphoma WSU-DLCL2 xenografts treated with SEQ ID NO:1251
sequestered in amphoteric liposomes.
[0026] FIG. 4 shows a dose response evaluation of two formulations
of SEQ ID NO:1251 sequestered in amphoteric liposomes on WSU-DLCL2
xenograft bearing mice.
[0027] FIG. 5 shows an enlarged view of a dose response evaluation
of two formulations of SEQ ID NO:1251 sequestered in amphoteric
liposomes on WSU-DLCL2 xenograft bearing mice.
[0028] FIG. 6 shows a dose response animal body weight evaluation
in WSU-DLCL2 xenograft bearing mice treated with two formulations
of SEQ ID NO:1251 sequestered in amphoteric liposomes.
[0029] FIG. 7 shows the effect of SEQ ID NO:1251 sequestered in
amphoteric liposomes on the size of tumors from PC-3 xenografts in
nude mice.
[0030] FIG. 8 shows the effect of SEQ ID NO:1251 sequestered in
amphoteric liposomes on the growth rate of tumors from PC-3
xenografts in nude mice.
[0031] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE SEQUENCES
[0032] SEQ ID NO:1 c-erb-2 (her-2) upstream region [0033] SEQ ID
NOs:2-281 c-erb-2 (her-2) DNAi oligonucleotides [0034] SEQ ID
NO:282 c-ki-ras upstream region [0035] SEQ ID NOs:283-461 c-ki-ras
DNAi oligonucleotides [0036] SEQ ID NO:462 c-Ha-ras upstream region
[0037] SEQ ID NOs:463-935 c-Ha-ras DNAi oligonucleotides [0038] SEQ
ID NO:936 c-myc upstream region [0039] SEQ ID NOs:937-1080 c-myc
DNAi oligonucleotides [0040] SEQ ID NO:1081 TGF-.alpha. upstream
region [0041] SEQ ID NOs:1082-1248 TGF-.alpha. DNAi
oligonucleotides [0042] SEQ ID NO:1249 bcl-2 upstream region [0043]
SEQ ID NO:1250 PNT-100 DNAi oligomer methylated [0044] SEQ ID
NO:1251 PNT-100 DNAi oligomer [0045] SEQ ID NO:1252 DNAi oligomer
methylated [0046] SEQ ID NO:1253 DNAi oligomer [0047] SEQ ID
NO:1255 bcl-2 secondary promoter sequence [0048] SEQ ID
NOs:1256-1266 bcl-2 sequences [0049] SEQ ID NOs:1267-1477 [0050]
and 1250-1254 bcl-2 DNAi oligomers
DETAILED DESCRIPTION
I. Definitions
[0051] To facilitate understanding of the invention, a number of
terms are defined below.
[0052] As used herein, "amphoter" or "amphoteric" character refers
to a structure, being a single substance (e.g., a compound) or a
mixture of substances (e.g., a mixture of two or more compounds) or
a supramolecular complex (e.g., a liposome) comprising charged
groups of both anionic and cationic character wherein [0053] (i) at
least one of the charged groups has a pK between 4 and 8, [0054]
(ii) the cationic charge prevails at pH 4 and [0055] (iii) the
anionic charge prevails at pH 8, resulting in an isoelectric point
of neutral net charge between pH 4 and pH 8. Amphoteric character
by that definition is different from zwitterionic character, as
zwitterions do not have a pK in the range mentioned above.
Consequently, zwitterions are essentially neutrally charged over a
range of pH values. Phosphatidylcholine or
phosphatidylethanolamines are neutral lipids with zwitterionic
character.
[0056] As used herein, "Amphoter I Lipid Pairs" refers to lipid
pairs containing a stable cation and a chargeable anion. Examples
include without limitation DDAB/CHEMS, DOTAP/CHEMS and DOTAP/DOPS.
In some aspects, the ratio of the percent of cationic lipids to
anionic lipids is lower than 1.
[0057] As used herein, "Amphoter II Lipid Pairs" refers to lipid
pairs containing a chargeable cation and a chargeable anion.
Examples include without limitation Mo-Chol/CHEMS, DPIM/CHEMS or
DPIM/DG-Succ. In some aspects, the ratio of the percent of cationic
lipids to anionic lipids is between about 5 and 0.2.
[0058] As used herein, "Amphoter III Lipid Pairs" refers to lipid
pairs containing a chargeable cation and stable anion. Examples
include without limitation Mo-Chol/DOPG or Mo-Chol/Chol-SO.sub.4.
In one embodiment, the ratio of the percent of cationic lipids to
anionic lipids is higher than 1.
[0059] As used herein, "liposome" refers to one or more lipids
forming a complex, usually surrounded by an aqueous solution.
Liposomes are generally spherical structures comprising lipids
fatty acids, lipid bilayer type structures, unilamellar vesicles
and amorphous lipid vesicles. Generally, liposomes are completely
closed lipid bilayer membranes containing an entrapped aqueous
volume. The liposomes may be unilamellar vesicles (possessing a
single bilayer membrane), oligolamellar or multilamellar (an
onion-like structure characterized by multiple membrane bilayers,
each separated from the next by an aqueous layer). Liposomes of the
present invention also include a DNAi oligonucleotide as defined
below, either bound to the liposomes or sequestered in or on the
liposomes. The molecules include, without limitation, DNAi
oligonucleotides and/or other agents used to treat diseases such as
cancer.
[0060] As used herein, an "amphoteric liposome" is a liposome with
an amphoteric character, as defined above.
[0061] As used herein, sequestered, sequestering, or sequester
refers to encapsulation, incorporation, or association of a DNAi
oligonucleotide, with the lipids of a liposome. The DNAi
oligonucleotide may be associated with the lipid bilayer or present
in the aqueous interior of the liposome or both. It includes
encapsulation in the aqueous core of the liposome. It also
encompasses situations in which part or all of the DNAi
oligonucleotide is located in the aqueous core of the liposome and
part outside of the liposome in the aqueous phase of the liposomal
suspension, where part of the DNAi oligonucleotide is located in
the aqueous core of the liposome and part in the lipid portion of
the liposome, or part sticking out of the liposomal exterior, where
DNAi oligonucleotides are partially or totally embedded in the
lipid portion of the liposome, and includes DNAi oligonucleotides
associated with the liposomes, with all or part of the DNAi
oligonucleotide associated with the exterior of the liposome.
[0062] As used herein, a Passive Loading Procedure (PLP) is a
process wherein liposomes are charged with DNAi oligonucleotides
and/or other molecules where the charges of the lipids are not
useful for binding the oligonucleotides.
[0063] Advanced Loading Procedure (ALP) is an ion exchange process
taking advantage of the positive charge of one lipid at acidic pH
to bind the DNAi oligonucleotides.
[0064] As used herein, the term "non-human animals" refers to all
non-human animals including, without limitation, vertebrates such
as rodents, non-human primates, ovines, bovines, ruminants,
lagomorphs, porcines, caprines, equines, canines, felines, ayes,
etc.
[0065] As used herein, the term "nucleic acid molecule", "nucleic
acid sequence" or "polynucleotide" refers to any nucleic acid
containing molecule, including without limitation, DNA or RNA. The
term polynucleotide(s) generally refers to any polyribonucleotide
or polydeoxyribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. Thus, for instance, polynucleotides as used
herein refers to, among others, single- and double-stranded DNA,
DNA that is a mixture of single- and double-stranded regions,
single- and double-stranded RNA, and RNA that is mixture of single-
and double-stranded regions, hybrid molecules comprising DNA and
RNA that may be single-stranded or, more typically, double-stranded
or a mixture of single- and double-stranded regions.
[0066] In addition, "polynucleotide" as used herein refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more of
the molecules, but more typically involve only a region of some of
the molecules. One of the molecules of a triple-helical region
often is an oligonucleotide.
[0067] The term "polynucleotide," "nucleic acid molecule" or
"nucleic acid sequence" includes DNAs or RNAs that contain one or
more modified bases. Thus, DNAs or RNAs with backbones modified for
stability or for other reasons are "polynucloeotides," "nucleic
acid molecules" or "nucleic acid sequences" as those terms are
intended herein. The terms also encompass sequences that include
any of the known base analogs of DNA and RNA.
[0068] It will be appreciated that a great variety of modifications
have been made to DNA and RNA that serve many useful purposes known
to those of skill in the art. The term "polynucleotide" as it is
employed herein embraces such chemically, enzymatically or
metabolically modified forms of polynucleotides, as well as the
chemical forms of DNA and RNA characteristic of viruses and cells,
including simple and complex cells, among others.
[0069] By "isolated nucleic acid sequence" is meant a
polynucleotide that is not immediately contiguous with either of
the coding sequences with which it is immediately contiguous (one
on the 5' end and one on the 3' end) in the naturally occurring
genome of the organism from which it is derived. The term therefore
includes, for example, a recombinant DNA which is incorporated into
a vector; into an autonomously replicating plasmid or virus; or
into the genomic DNA of a prokaryote or eukaryote, or which exists
as a separate molecule (e.g., a cDNA) independent of other
sequences.
[0070] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that includes coding sequences necessary for the
production of a polypeptide, precursor or RNA (e.g., rRNA, tRNA).
The polypeptide can be encoded by a full length coding sequence or
by any portion of the coding sequence so long as the desired
activity or functional properties (e.g., enzymatic activity, ligand
binding, signal transduction, immunogenicity, etc.) of the
full-length or fragment is retained.
[0071] The term also encompasses the coding region of a structural
gene and the sequences preceding and following the coding region,
(leader and trailer) as well as intervening sequences (introns)
between individual coding segments (exons). Sequences located 5' of
the coding region and present on the mRNA are referred to as 5'
non-translated sequences. Sequences located 3' or downstream of the
coding region and present on the mRNA are referred to as 3'
non-translated sequences. The term "gene" encompasses both cDNA and
genomic forms of a gene. A genomic form or clone of a gene contains
the coding region interrupted with non-coding sequences termed
"introns" or "intervening regions" or "intervening sequences."
Introns are segments of a gene that are transcribed into nuclear
RNA (hnRNA); introns may contain regulatory elements such as
enhancers. Introns are removed or "spliced out" from the nuclear or
primary transcript; introns therefore are absent in the messenger
RNA (mRNA) transcript. The mRNA functions during translation to
specify the sequence or order of amino acids in a nascent
polypeptide.
[0072] As used herein, the term "gene expression" refers to the
process of converting genetic information encoded in a gene into
RNA (e.g., mRNA, rRNA, tRNA, micro RNA or snRNA) through
"transcription" of the gene (i.e., via the enzymatic action of an
RNA polymerase), and for protein encoding genes, into protein
through "translation" of mRNA. Gene expression can be regulated at
many stages in the process. "Up-regulation" or "activation" refers
to regulation that increases the production of gene expression
products (i.e., RNA or protein), while "down-regulation" or
"repression" refers to regulation that decrease production.
Molecules (e.g., transcription factors) that are involved in
up-regulation or down-regulation are often called "activators" and
"repressors," respectively.
[0073] In addition to containing introns, genomic forms of a gene
may also include sequences located on both the 5' and 3' end of the
sequences that are present on the RNA transcript. These sequences
are referred to as "flanking" sequences or regions (these flanking
sequences are located 5' or 3' to the non-translated sequences
present on the mRNA transcript). The 5' flanking region (or
upstream region) may contain regulatory sequences such as promoters
and enhancers that control or influence the transcription of the
gene. The 3' flanking region may contain sequences that direct the
termination of transcription, post-transcriptional cleavage and
polyadenylation.
[0074] As used herein, the terms "nucleic acid molecule encoding,"
"DNA sequence encoding," and "DNA encoding" refer to the order or
sequence of deoxyribonucleotides along a strand of deoxyribonucleic
acid. The order of these deoxyribonucleotides determines the order
of amino acids along the polypeptide (protein) chain. The DNA
sequence thus codes for the amino acid sequence.
[0075] The term "oligonucleotide" as used herein is defined as a
molecule with two or more deoxyribonucleotides or ribonucleotides,
often more than three, and usually more than ten. The exact size of
an oligonucleotide will depend on many factors, including the
ultimate function or use of the oligonucleotide. Oligonucleotides
can be prepared by any suitable method, including, for example,
cloning and restriction of appropriate sequences and direct
chemical synthesis by a method such as the phosphotriester method
of Narang et al., 1979, Meth. Enzymol., 68:90-99; the
phosphodiester method of Brown et al., 1979, Method Enzymol.,
68:109-151, the diethylphosphoramidite method of Beaucage et al.,
1981, Tetrahedron Lett., 22:1859-1862; the triester method of
Matteucci et al., 1981, J. Am. Chem. Soc., 103:3185-3191, or
automated synthesis methods; and the solid support method of U.S.
Pat. No. 4,458,066.
[0076] As used herein, a "DNAi oligonucleotide" or "DNAi" refers to
a single stranded nucleic acid oligonucleotide or derivative
thereof, whose sequence is complementary, in part, to a portion of
the longest non-transcribed region of a gene in which the
oligonucleotide affects indirectly or directly the expression,
regulation or production of the same or different gene, wherein the
longest non-transcribed region includes any portion of the gene
that is not transcribed when the transcriptional start site is the
site closest to the translation start site. DNAi does not include
RNAi and antisense oligonucleotides that base pair only with mRNAs
or pre-mRNAs and interfere with RNA processing and/or message
translation.
[0077] In some embodiments utilizing methylated DNAi
oligonucleotides, the nucleotide, dC is replaced by 5-methyl-dC
where appropriate, as taught by the present invention.
[0078] The DNAi oligonucleotides may comprise, without limitation,
oligonucleotide mimetics such as are described below. The DNAi
oligonucleotide compounds in accordance with this invention may
comprise from about 15 to about 35 nucleobases (i.e., from about 15
to about 35 linked bases), although both longer and shorter
sequences may find use with the present invention.
[0079] Oligonucleotides may also include nucleobase (often referred
to in the art simply as "base") modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include the
purine bases adenine (A) and guanine (G), and the pyrimidine bases
thymine (T), 16182651.1 cytosine (C) and uracil (U). Modified
nucleobases include other synthetic and natural nucleobases.
[0080] In some embodiments, the DNAi oligonucleotides may
hybridizes to the promoter region of a gene. In some embodiments,
the hybridization of the DNAi oligonucleotide to the promoter
inhibits expression of the gene.
[0081] By "promoter" is meant a sequence sufficient to direct
transcription, including promoter elements that are sufficient to
render promoter-dependent gene expression controllable for
cell-type specific, tissue-specific, or inducible by external
signals or agents; such elements may be located in the 5' or 3'
regions of the gene. Both constitutive and inducible promoters, are
included in the definition (see e.g., Bitter et al., Methods in
Enzymology 153:516-544, 1987). For example, when cloning in
bacterial systems, inducible promoters such as pL of bacteriophage
.gamma., plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like
may be used. When cloning in mammalian cell systems, promoters
derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses (e.g., the retrovirus long
terminal repeat; the adenovirus late promoter; the vaccinia virus
7.5K promoter) may be used. Promoters produced by recombinant DNA
or synthetic techniques are also defined as promoters.
[0082] As used herein, the "regulatory region" of a gene is any
part of a gene that regulates the expression of a gene, including,
without limitation, transcriptional and translational regulation.
The regions include without limitation the 5' and 3' regions of
genes, binding sites for regulatory factors, including without
limitation transcription factor binding sites. The regions also
include regions that are as long as 20,000 or more base pairs
upstream or downstream of translational start sites, so long as the
region is involved in any way in the regulation of the expression
of the gene. The region may be as short as 20 base pairs or as long
as thousands of base pairs.
[0083] By "transformation" or "transfection" is meant a permanent
or transient genetic change induced in a cell following
incorporation of new DNA (i.e., DNA exogenous to the cell). Where
the cell is a mammalian cell, a permanent genetic change is
generally achieved by introduction of the DNA into the genome of
the cell.
[0084] By "transformed cell" or "host cell" is meant a cell (e.g.,
prokaryotic or eukaryotic) into which (or into an ancestor of
which) has been introduced, by means of recombinant DNA techniques,
a DNA molecule encoding a polypeptide of the invention (i.e., a
Methuselah polypeptide), or fragment thereof.
[0085] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method by procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell or by electroporation.
[0086] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides) related by the base-pairing rules. For
example, for the sequence "A-G-T," is complementary to the sequence
"T-C-A." Complementarity may be "partial," in which only some of
the nucleic acids' bases are matched according to the base pairing
rules. Or, there may be "complete" or "total" complementarity
between the nucleic acids. The degree of complementarity between
nucleic acid strands has significant effects on the efficiency and
strength of hybridization between nucleic acid strands.
[0087] As used herein, the term "completely complementary," for
example when used in reference to a DNAi oligonucleotide of the
present invention refers to an oligonucleotide where all of the
nucleotides are complementary to a target sequence (e.g., a
gene).
[0088] As used herein, the term "partially complementary," refers
to a sequence where at least one nucleotide is not complementary to
the target sequence. Preferred partially complementary sequences
are those that can still hybridize to the target sequence under
physiological conditions. The term "partially complementary" refers
to sequences that have regions of one or more non-complementary
nucleotides both internal to the sequence or at either end.
Sequences with mismatches at the ends may still hybridize to the
target sequence.
[0089] The term "homology" refers to a degree of complementarity.
There may be partial homology or complete homology (i.e.,
identity). A partially complementary sequence is a nucleic acid
molecule that at least partially inhibits a completely
complementary nucleic acid molecule from hybridizing to a target
nucleic acid is "substantially homologous." The inhibition of
hybridization of the completely complementary sequence to the
target sequence may be examined using a hybridization assay
(Southern or northern blot, solution hybridization and the like)
under conditions of low stringency. A substantially homologous
sequence or probe will compete for and inhibit the binding (i.e.,
the hybridization) of a completely homologous nucleic acid molecule
to a target under conditions of low stringency. Likewise, a
substantially complementary sequence or probe will compete for and
inhibit the binding (i.e., the hybridization) of a completely
complementary nucleic acid molecule to a target under conditions of
low stringency. This is not to say that conditions of low
stringency are such that non-specific binding is permitted; low
stringency conditions require that the binding of two sequences to
one another be a specific (i.e., selective) interaction. The
absence of non-specific binding may be tested by the use of a
second target that is substantially non-complementary (e.g., less
than about 30% identity); in the absence of non-specific binding
the probe will not hybridize to the second non-complementary
target.
[0090] When used in reference to a double-stranded nucleic acid
sequence such as a cDNA or genomic clone, the term "substantially
homologous" refers to any probe that can hybridize to either or
both strands of the double-stranded nucleic acid sequence under
conditions of low stringency as described above.
[0091] When used in reference to a single-stranded nucleic acid
sequence, the term "substantially homologous" refers to any probe
that can hybridize (i.e., it is the complement of) the
single-stranded nucleic acid sequence under conditions of low
stringency as described above.
[0092] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids.
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acids) is impacted by such
factors as the degree of complementary between the nucleic acids,
stringency of the conditions involved, the T.sub.m of the formed
hybrid, and the G:C ratio within the nucleic acids. A single
molecule that contains pairing of complementary nucleic acids
within its structure is said to be "self-hybridized."
[0093] As used herein, the term "T.sub.m" is used in reference to
the "melting temperature." The melting temperature is the
temperature at which a population of double-stranded nucleic acid
molecules becomes half dissociated into single strands. The
equation for calculating the T.sub.m of nucleic acids is well known
in the art. As indicated by standard references, a simple estimate
of the T.sub.m value may be calculated by the equation
T.sub.m=81.5+0.41(% G+C), when a nucleic acid is in aqueous
solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative
Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other
references include more sophisticated computations that take
structural as well as sequence characteristics into account for the
calculation of T.sub.m.
[0094] Inhibition of hybridization of the completely complementary
sequence to the target sequence may be examined using a
hybridization assay (Southern or northern blot, solution
hybridization and the like) under conditions of low stringency. A
substantially homologous sequence or probe will compete for and
inhibit the binding (i.e., the hybridization) of a completely
homologous nucleic acid molecule to a target under conditions of
low stringency. This is not to say that conditions of low
stringency are such that non-specific binding is permitted; low
stringency conditions require that the binding of two sequences to
one another be a specific (i.e., selective) interaction. The
absence of non-specific binding may be tested by the use of a
second target that is substantially non-complementary (e.g., less
than about 30% identity); in the absence of non-specific binding
the probe will not hybridize to the second non-complementary
target.
[0095] When used in reference to a double-stranded nucleic acid
sequence such as a cDNA or genomic clone, the term "substantially
homologous" refers to any probe that can hybridize to either or
both strands of the double-stranded nucleic acid sequence under
conditions of low stringency as described above.
[0096] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength and the presence of
other compounds such as organic solvents, under which nucleic acid
hybridizations are conducted. Under "low stringency conditions" a
nucleic acid sequence of interest will hybridize to its exact
complement, sequences with single base mismatches, closely related
sequences (e.g., sequences with 90% or greater homology), and
sequences having only partial homology (e.g., sequences with 50-90%
homology). Under "medium stringency conditions," a nucleic acid
sequence of interest will hybridize only to its exact complement,
sequences with single base mismatches, and closely relation
sequences (e.g., 90% or greater homology). Under "high stringency
conditions," a nucleic acid sequence of interest will hybridize
only to its exact complement, and (depending on conditions such a
temperature) sequences with single base mismatches. In other words,
under conditions of high stringency the temperature can be raised
so as to exclude hybridization to sequences with single base
mismatches.
[0097] As used herein, the term "physiological conditions" refers
to specific stringency conditions that approximate or are
conditions inside an animal (e.g., a human). Exemplary
physiological conditions for use in vitro include, but are not
limited to, 37.degree. C., 95% air, 5% CO.sub.2, commercial medium
for culture of mammalian cells (e.g., DMEM media available from
Gibco, Md.), 5-10% serum (e.g., calf serum or horse serum),
additional buffers, and optionally hormone (e.g., insulin and
epidermal growth factor).
[0098] The term "isolated" means altered "by the hand of man" from
its natural state; i.e., if it occurs in nature, it has been
changed or removed from its original environment or both. For
example, when used in relation to a nucleic acid, as in "an
isolated nucleotide" or "isolated polynucleotide" refers to a
nucleic acid sequence that is identified and separated from at
least one component or contaminant with which it is ordinarily
associated in its natural source. Isolated nucleic acid as such
present in a form or setting that is different from that in which
it is found in nature. In contrast, non-isolated nucleic acids are
nucleic acids such as DNA and RNA found in the state they exist in
nature. For example, a given DNA sequence (e.g., a gene) is found
on the host cell chromosome in proximity to neighboring genes; RNA
sequences, such as a specific mRNA sequence encoding a specific
protein, are found in the cell as a mixture with numerous other
mRNAs that encode a multitude of proteins. However, isolated
nucleic acid encoding a given protein includes, by way of example,
such nucleic acid in cells ordinarily expressing the given protein
where the nucleic acid is in a chromosomal location different from
that of natural cells, or is otherwise flanked by a different
nucleic acid sequence than that found in nature. The isolated
nucleic acid, oligonucleotide or polynucleotide may be present in
single-stranded or double-stranded form. When an isolated nucleic
acid, oligonucleotide or polynucleotide is to be used to express a
protein, the oligonucleotide or polynucleotide will contain at a
minimum the sense or coding strand (i.e., the oligonucleotide or
polynucleotide may be single-stranded), but may contain both the
sense and anti-sense strands (i.e., the oligonucleotide or
polynucleotide may be double-stranded).
[0099] As used herein, the term "purified" or "to purify" refers to
removing components (e.g., contaminants) from a sample. For
example, recombinant polypeptides are expressed in bacterial host
cells and the polypeptides are purified by removing host cell
proteins; the percent of recombinant polypeptides is thereby
increased in the sample.
[0100] As used herein, the term "cell culture" refers to any in
vitro culture of cells. Included within this term are continuous
cell lines (e.g., with an immortal phenotype), primary cell
cultures, transformed cell lines, finite cell lines (e.g.,
non-transformed cells), and any other cell population maintained in
vitro.
[0101] As used, the term "eukaryote" refers to organisms
distinguishable from "prokaryotes." It is intended that the term
encompass all organisms with cells that exhibit the usual
characteristics of eukaryotes, such as the presence of a true
nucleus bounded by a nuclear membrane, within which lie the
chromosomes, the presence of membrane-bound organelles and other
characteristics commonly observed in eukaryotic organisms. Thus,
the term includes, but is not limited to such organisms as fungi,
protozoa and animals (e.g., humans).
[0102] As used herein, the term "in vitro" refers to an artificial
environment and to processes or reactions that occur within an
artificial environment. In vitro environments can consist of, but
are not limited to, test tubes and cell culture. The term "in vivo"
refers to the natural environment (e.g., an animal or a cell) and
to processes or reaction that occur within a natural
environment.
[0103] As used herein, the term "under conditions such that
expression of a gene is inhibited" refers to conditions where a
DNAi oligonucleotide of the present invention hybridizes to a gene
(e.g., the promoter region of the gene) and inhibits transcription
of the gene by at least 10%, at least 25%, at least 50% or at least
90% relative to the level of transcription in the absence of the
oligonucleotide.
[0104] As used herein, the term "under conditions such that growth
of a cell is reduced" refers to conditions where a DNAi
oligonucleotide of the present invention, when administered to a
cell (e.g., a cancer) reduces the rate of growth of the cell by at
least 10%, at least 25%, at least 50% or at least 90% relative to
the rate of growth of the cell in the absence of the
oligonucleotide.
[0105] The terms "test compound" and "candidate compound" refer to
any chemical entity, pharmaceutical, drug, biologic and the like
that is a candidate for use to treat or prevent a disease, illness,
sickness, or disorder of bodily function (e.g., cancer). Test
compounds include both known and potential therapeutic compounds. A
test compound can be determined to be therapeutic by screening
using the screening methods of the present invention. In some
embodiments of the present invention, the mixture includes a DNAi
oligonucleotide a test compound such as an antisense compound or a
chemotherapy agent.
[0106] As used herein, the term "chemotherapeutic agents" refers to
compounds that can be useful in the treatment of disease (e.g.,
cancer). Exemplary chemotherapeutic agents affective against cancer
include, without limitation, daunorubicin, dactinomycin,
doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil,
melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine,
cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR),
methotrexate (MTX), colchicine, taxotere, vincristine, vinblastine,
etoposide, teniposide, cisplatin and diethylstilbestrol (DES).
[0107] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 75.sup.th Ed.
Additionally, general principles of organic chemistry are described
in "Organic Chemistry", Thomas Sorrell, University Science Books,
Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed.,
Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York:
2001.
[0108] As used herein the term "aliphatic" encompasses the terms
alkyl, alkenyl, alkynyl, each of which being optionally substituted
as set forth below.
[0109] As used herein, an "alkyl" group refers to a saturated
aliphatic hydrocarbon group containing 1-8 (e.g., 1-6 or 1-4)
carbon atoms. An alkyl group can be straight or branched. Examples
of alkyl groups include, but are not limited to, methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,
n-pentyl, n-heptyl or 2-ethylhexyl. An alkyl group can be
substituted (i.e., optionally substituted) with one or more
substituents such as halo, cycloaliphatic, heterocycloaliphatic,
aryl, heteroaryl, alkoxy, aroyl, heteroaroyl,
cycloaliphaticcarbonyl, (heterocycloaliphatic)carbonyl, nitro,
cyano, amino, amido, acyl, sulfonyl, sulfinyl, sulfanyl, sulfoxy,
urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl,
cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy, heteroaryloxy,
aralkyloxy, heteroarylalkoxy, or hydroxy. Without limitation, some
examples of substituted alkyls include carboxyalkyl (such as
HOOC-alkyl, alkoxycarbonylalkyl and alkylcarbonyloxyalkyl),
cyanoalkyl, hydroxyalkyl, alkoxyalkyl, acylalkyl, hydroxyalkyl,
aralkyl, (alkoxyaryl)alkyl, (sulfonylamino)alkyl (such as
(alkylsulfonylamino)alkyl), aminoalkyl, amidoalkyl,
(cycloaliphatic)alkyl, cyanoalkyl, or haloalkyl.
[0110] As used herein, an "alkenyl" group refers to an aliphatic
carbon group that contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and
at least one double bond. Like an alkyl group, an alkenyl group can
be straight or branched. Examples of an alkenyl group include, but
are not limited to, allyl, isoprenyl, 2-butenyl and 2-hexenyl. An
alkenyl group can be optionally substituted with one or more
substituents such as halo, cycloaliphatic, heterocycloaliphatic,
aryl, heteroaryl, alkoxy, aroyl, heteroaroyl,
(cycloaliphatic)carbonyl, (heterocycloaliphatic)carbonyl, nitro,
cyano, amino, amido, acyl, sulfonyl, sulfinyl, sulfanyl, sulfoxy,
urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl,
(cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy,
heteroaryloxy, aralkyloxy, (heteroaryl)alkoxy, or hydroxy.
[0111] As used herein, an "alkynyl" group refers to an aliphatic
carbon group that contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and
has at least one triple bond. An alkynyl group can be straight or
branched. Examples of an alkynyl group include, but are not limited
to, propargyl and butynyl. An alkynyl group can be optionally
substituted with one or more substituents such as halo,
cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, alkoxy,
aroyl, heteroaroyl, (cycloaliphatic)carbonyl,
(heterocycloaliphatic)carbonyl, nitro, cyano, amino, amido, acyl,
sulfonyl, sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl,
sulfamide, oxo, carboxy, carbamoyl, (cycloaliphatic)oxy,
(heterocycloaliphatic)oxy, aryloxy, heteroaryloxy, aralkyloxy,
(heteroaryl)alkoxy, or hydroxy.
[0112] As used herein, an "amido" encompasses both "aminocarbonyl"
and "carbonylamino". These terms when used alone or in connection
with another group refers to an amido group such as
N(R.sup.X).sub.2--C(O)-- or R.sup.YC(O)--N(R.sup.X).sub.2-- when
used terminally and --C(O)--N(R.sup.X)-- or --N(R.sup.X)--C(O)--
when used internally, wherein R.sup.X and R.sup.Y are defined
below. Examples of amido groups include alkylamido (such as
alkylcarbonylamino and alkylcarbonylamino),
(heterocycloaliphatic)amido, (heteroaralkyl)amido,
(heteroaryl)amido, (heterocycloalkyl)alkylamido, arylamido,
aralkylamido, (cycloalkyl)alkylamido, and cycloalkylamido.
[0113] As used herein, an "amino" group refers to --NR.sup.XR.sup.Y
wherein each of R.sup.X and R.sup.Y is independently hydrogen,
alkyl, cycloaliphatic, (cycloaliphatic)aliphatic, aryl,
araliphatic, heterocycloaliphatic, (heterocycloaliphatic)aliphatic,
heteroaryl, carboxy, sulfanyl, sulfinyl, sulfonyl,
(aliphatic)carbonyl, (cycloaliphatic)carbonyl,
((cycloaliphatic)aliphatic)carbonyl, arylcarbonyl,
(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,
((heterocycloaliphatic)aliphatic)carbonyl, (heteroaryl)carbonyl, or
(heteroaraliphatic)carbonyl, each of which being defined herein and
being optionally substituted. Examples of amino groups include
alkylamino, dialkylamino, and arylamino.
[0114] When the term "amino" is not the terminal group (e.g.,
alkylcarbonylamino), it is represented by --NR.sup.X--. R.sup.X has
the same meaning as defined above.
[0115] As used herein, an "aryl" group used alone or as part of a
larger moiety as in "aralkyl", "aralkoxy", or "aryloxyalkyl" refers
to monocyclic (e.g., phenyl); bicyclic (e.g., indenyl,
naphthalenyl, tetrahydronaphthyl, tetrahydroindenyl); and tricyclic
(e.g., fluorenyl tetrahydrofluorenyl, or tetrahydroanthracenyl,
anthracenyl). The bicyclic and tricyclic groups include benzofused
2-3 membered carbocyclic rings. For example, a benzofused group
includes phenyl fused with two or more C.sub.4-8 carbocyclic
moieties. An aryl is optionally substituted with one or more
substituents including aliphatic [e.g., alkyl, alkenyl, or
alkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic;
heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl;
heteroaryl; alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy;
aryloxy; heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy;
aroyl; heteroaroyl; amino; oxo (on a non-aromatic carbocyclic ring
of a benzofused bicyclic or tricyclic aryl); nitro; carboxy; amido;
acyl [e.g., aliphaticcarbonyl; (cycloaliphatic)carbonyl;
((cycloaliphatic)aliphatic)carbonyl; (araliphatic)carbonyl;
(heterocycloaliphatic)carbonyl; ((heterocycloaliphatic)
aliphatic)carbonyl; and (heteroaraliphatic)carbonyl]; sulfonyl
[e.g., aliphaticsulfonyl and aminosulfonyl]; sulfinyl [e.g.,
aliphaticsulfinyl]; sulfanyl [e.g., aliphaticsulfanyl]; nitro;
cyano; halo; hydroxyl; mercapto; sulfoxy; urea; thiourea;
sulfamoyl; sulfamide; and carbamoyl. Alternatively, an aryl can be
unsubstituted.
[0116] Non-limiting examples of substituted aryls include haloaryl
[e.g., mono-, di (such as p,m-dihaloaryl), and (trihalo)aryl];
(carboxy)aryl [e.g., (alkoxycarbonyl)aryl,
((aralkyl)carbonyloxy)aryl, and (alkoxycarbonyl)aryl]; (amido)aryl
[e.g., (aminocarbonyl)aryl, (((alkylamino)alkyl)aminocarbonyl)aryl,
(alkylcarbonyl)aminoaryl, (arylaminocarbonyl)aryl, and
(((heteroaryl)amino)carbonyl)aryl]; aminoaryl [e.g.,
((alkylsulfonyl)amino)aryl and ((dialkyl)amino)aryl];
(cyanoalkyl)aryl; (alkoxy)aryl; (sulfamoyl)aryl [e.g.,
(aminosulfonyl)aryl]; (alkylsulfonyl)aryl; (cyano)aryl;
(hydroxyalkyl)aryl; ((alkoxy)alkyl)aryl; (hydroxyl)aryl,
((carboxy)alkyl)aryl; (((dialkyl)amino)alkyl)aryl;
(nitroalkyl)aryl; (((alkylsulfonyl)amino)alkyl)aryl;
((heterocycloaliphatic)carbonyl)aryl; ((alkylsulfonyl)alkyl)aryl;
(cyanoalkyl)aryl; (hydroxyalkyl)aryl; (alkylcarbonyl)aryl;
alkylaryl; (trihaloalkyl)aryl; p-amino-m-alkoxycarbonylaryl;
p-amino-m-cyanoaryl; p-halo-m-aminoaryl; and
(m-(heterocycloaliphatic)-o-(alkyl))aryl.
[0117] As used herein, an "araliphatic" such as an "aralkyl" group
refers to an aliphatic group (e.g., a C.sub.1-4 alkyl group) that
is substituted with an aryl group. "Aliphatic," "alkyl," and "aryl"
are defined herein. An example of an araliphatic such as an aralkyl
group is benzyl.
[0118] As used herein, a "bicyclic ring system" includes 8-12
(e.g., 9, 10, or 11) membered structures that form two rings,
wherein the two rings have at least one atom in common (e.g., 2
atoms in common). Bicyclic ring systems include bicycloaliphatics
(e.g., bicycloalkyl or bicycloalkenyl), bicycloheteroaliphatics,
bicyclic aryls, and bicyclic heteroaryls.
[0119] As used herein, a "cycloaliphatic" group encompasses a
"cycloalkyl" group and a "cycloalkenyl" group, each of which being
optionally substituted as set forth below.
[0120] As used herein, a "cycloalkyl" group refers to a saturated
carbocyclic mono- or bicyclic (fused or bridged) ring of 3-10
(e.g., 5-10) carbon atoms. Examples of cycloalkyl groups include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
adamantyl, norbornyl, cubyl, octahydro-indenyl, decahydro-naphthyl,
bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl,
bicyclo[3.3.2.]decyl, bicyclo[2.2.2]octyl, adamantyl,
azacycloalkyl, or ((aminocarbonyl)cycloalkyl)cycloalkyl. A
"cycloalkenyl" group, as used herein, refers to a non-aromatic
carbocyclic ring of 3-10 (e.g., 4-8) carbon atoms having one or
more double bonds. Examples of cycloalkenyl groups include
cyclopentenyl, 1,4-cyclohexa-di-enyl, cycloheptenyl, cyclooctenyl,
hexahydro-indenyl, octahydro-naphthyl, cyclohexenyl, cyclopentenyl,
bicyclo[2.2.2]octenyl, and bicyclo[3.3.1]nonenyl.
[0121] A cycloalkyl or cycloalkenyl group can be optionally
substituted with one or more substituents such as aliphatic [e.g.,
alkyl, alkenyl, or alkynyl], cycloaliphatic, (cycloaliphatic)
aliphatic, heterocycloaliphatic, (heterocycloaliphatic) aliphatic,
aryl, heteroaryl, alkoxy, (cycloaliphatic)oxy,
(heterocycloaliphatic)oxy, aryloxy, heteroaryloxy,
(araliphatic)oxy, (heteroaraliphatic)oxy, aroyl, heteroaroyl,
amino, amido [e.g., (aliphatic)carbonylamino,
(cycloaliphatic)carbonylamino,
((cycloaliphatic)aliphatic)carbonylamino, (aryl)carbonylamino,
(araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino,
((heterocycloaliphatic)aliphatic)carbonylamino,
(heteroaryl)carbonylamino, and (heteroaraliphatic)carbonylamino],
nitro, carboxy [e.g., HOOC--, alkoxycarbonyl, and
alkylcarbonyloxy], acyl [e.g., (cycloaliphatic)carbonyl,
((cycloaliphatic) aliphatic)carbonyl, (araliphatic)carbonyl,
(heterocycloaliphatic)carbonyl,
((heterocycloaliphatic)aliphatic)carbonyl, and
(heteroaraliphatic)carbonyl], nitro, cyano, halo, hydroxy,
mercapto, sulfonyl [e.g., alkylsulfonyl and arylsulfonyl], sulfinyl
[e.g., alkylsulfinyl], sulfanyl [e.g., alkylsulfanyl], sulfoxy,
urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.
[0122] As used herein, "cyclic moiety" includes cycloaliphatic,
heterocycloaliphatic, aryl, or heteroaryl, each of which has been
defined previously.
[0123] As used herein, the term "heterocycloaliphatic" encompasses
a heterocycloalkyl group and a heterocycloalkenyl group, each of
which being optionally substituted as set forth below.
[0124] As used herein, a "heterocycloalkyl" group refers to a 3-10
membered mono- or bicylic (fused or bridged) (e.g., 5- to
10-membered mono- or bicyclic) saturated ring structure, in which
one or more of the ring atoms is a heteroatom (e.g., N, O, S, or
combinations thereof). Examples of a heterocycloalkyl group include
piperidyl, piperazyl, tetrahydropyranyl, tetrahydrofuryl,
1,4-dioxolanyl, 1,4-dithianyl, 1,3-dioxolanyl, oxazolidyl,
isoxazolidyl, morpholinyl, thiomorpholyl, octahydro-benzofuryl,
octahydro-chromenyl, octahydro-thiochromenyl, octahydro-indolyl,
octahydro-pyrindinyl, decahydro-quinolinyl,
octahydro-benzo[b]thiopheneyl, 2-oxa-bicyclo[2.2.2]octyl,
1-aza-bicyclo[2.2.2]octyl, 3-aza-bicyclo[3.2.1]octyl, and
2,6-dioxa-tricyclo[3.3.1.0.sup.3,7]nonyl. A monocyclic
heterocycloalkyl group can be fused with a phenyl moiety such as
tetrahydroisoquinoline. A "heterocycloalkenyl" group, as used
herein, refers to a mono- or bicylic (e.g., 5- to 10-membered mono-
or bicyclic) non-aromatic ring structure having one or more double
bonds, and wherein one or more of the ring atoms is a heteroatom
(e.g., N, O, or S). Monocyclic and bicycloheteroaliphatics are
numbered according to standard chemical nomenclature.
[0125] A heterocycloalkyl or heterocycloalkenyl group can be
optionally substituted with one or more substituents such as
aliphatic [e.g., alkyl, alkenyl, or alkynyl], cycloaliphatic,
(cycloaliphatic) aliphatic, heterocycloaliphatic,
(heterocycloaliphatic) aliphatic, aryl, heteroaryl, alkoxy,
(cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy,
heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl,
heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino,
(cycloaliphatic)carbonylamino, ((cycloaliphatic)
aliphatic)carbonylamino, (aryl)carbonylamino,
(araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino,
((heterocycloaliphatic) aliphatic)carbonylamino,
(heteroaryl)carbonylamino, and (heteroaraliphatic)carbonylamino],
nitro, carboxy [e.g., HOOC--, alkoxycarbonyl, and
alkylcarbonyloxy], acyl [e.g., (cycloaliphatic)carbonyl,
((cycloaliphatic) aliphatic)carbonyl, (araliphatic)carbonyl,
(heterocycloaliphatic)carbonyl,
((heterocycloaliphatic)aliphatic)carbonyl, and
(heteroaraliphatic)carbonyl], nitro, cyano, halo, hydroxy,
mercapto, sulfonyl [e.g., alkylsulfonyl and arylsulfonyl], sulfinyl
[e.g., alkylsulfinyl], sulfanyl [e.g., alkylsulfanyl], sulfoxy,
urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.
[0126] A "heteroaryl" group, as used herein, refers to a
monocyclic, bicyclic, or tricyclic ring structure having 4 to 15
ring atoms wherein one or more of the ring atoms is a heteroatom
(e.g., N, O, S, or combinations thereof) and wherein one ore more
rings of the bicyclic or tricyclic ring structure is aromatic. A
heteroaryl group includes a benzofused ring system having 2 to 3
rings. For example, a benzofused group includes benzo fused with
one or two 4 to 8 membered heterocycloaliphatic moieties (e.g.,
indolizyl, indolyl, isoindolyl, 3H-indolyl, indolinyl,
benzo[b]furyl, benzo[b]thiophenyl, quinolinyl, or isoquinolinyl).
Some examples of heteroaryl are azetidinyl, pyridyl, 1H-indazolyl,
furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl,
tetrazolyl, benzofuryl, isoquinolinyl, benzthiazolyl, xanthene,
thioxanthene, phenothiazine, dihydroindole, benzo[1,3]dioxole,
benzo[b]furyl, benzo[b]thiophenyl, indazolyl, benzimidazolyl,
benzthiazolyl, puryl, cinnolyl, quinolyl, quinazolyl, cinnolyl,
phthalazyl, quinazolyl, quinoxalyl, isoquinolyl, 4H-quinolizyl,
benzo-1,2,5-thiadiazolyl, or 1,8-naphthyridyl.
[0127] Without limitation, monocyclic heteroaryls include furyl,
thiophenyl, 2H-pyrrolyl, pyrrolyl, oxazolyl, thazolyl, imidazolyl,
pyrazolyl, isoxazolyl, isothiazolyl, 1,3,4-thiadiazolyl,
2H-pyranyl, 4-H-pranyl, pyridyl, pyridazyl, pyrimidyl, pyrazolyl,
pyrazyl, or 1,3,5-triazyl. Monocyclic heteroaryls are numbered
according to standard chemical nomenclature.
[0128] Without limitation, bicyclic heteroaryls include indolizyl,
indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl,
benzo[b]thiophenyl, quinolinyl, isoquinolinyl, indazolyl,
benzimidazyl, benzthiazolyl, purinyl, 4H-quinolizyl, quinolyl,
isoquinolyl, cinnolyl, phthalazyl, quinazolyl, quinoxalyl,
1,8-naphthpidyl, or pteridyl. Bicyclic heteroaryls are numbered
according to standard chemical nomenclature.
[0129] A heteroaryl is optionally substituted with one or more
substituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl];
cycloaliphatic; (cycloaliphatic)aliphatic; heterocycloaliphatic;
(heterocycloaliphatic)aliphatic; aryl; heteroaryl; alkoxy;
(cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy;
heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl;
heteroaroyl; amino; oxo (on a non-aromatic carbocyclic or
heterocyclic ring of a bicyclic or tricyclic heteroaryl); nitro;
carboxy; amido; acyl [e.g., aliphaticcarbonyl;
(cycloaliphatic)carbonyl; ((cycloaliphatic)aliphatic)carbonyl;
(araliphatic)carbonyl; (heterocycloaliphatic)carbonyl;
((heterocycloaliphatic) aliphatic)carbonyl; and
(heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphaticsulfonyl and
aminosulfonyl]; sulfinyl [e.g., aliphaticsulfinyl]; sulfanyl [e.g.,
aliphaticsulfanyl]; nitro; cyano; halo; hydroxyl; mercapto;
sulfoxy; urea; thiourea; sulfamoyl; sulfamide; or carbamoyl.
Alternatively, a heteroaryl can be unsubstituted.
[0130] Non-limiting examples of substituted heteroaryls include
(halo)heteroaryl [e.g., mono- and di-(halo)heteroaryl];
(carboxy)heteroaryl [e.g., (alkoxycarbonyl)heteroaryl];
cyanoheteroaryl; aminoheteroaryl [e.g.,
((alkylsulfonyl)amino)heteroaryl and ((dialkyl)amino)heteroaryl];
(amido)heteroaryl [e.g., aminocarbonylheteroaryl,
((alkylcarbonyl)amino)heteroaryl,
((((alkyl)amino)alkyl)aminocarbonyl)heteroaryl,
(((heteroaryl)amino)carbonyl)heteroaryl,
((heterocycloaliphatic)carbonyl)heteroaryl, and
((alkylcarbonyl)amino)heteroaryl]; (cyanoalkyl)heteroaryl;
(alkoxy)heteroaryl; (sulfamoyl)heteroaryl [e.g.,
(aminosulfonyl)heteroaryl]; (sulfonyl)heteroaryl [e.g.,
(alkylsulfonyl)heteroaryl]; (hydroxyalkyl)heteroaryl;
(alkoxyalkyl)heteroaryl; (hydroxyl)heteroaryl;
((carboxy)alkyl)heteroaryl; [((dialkyl)amino)alkyl]heteroaryl;
(heterocycloaliphatic)heteroaryl; (cycloaliphatic)heteroaryl;
(nitroalkyl)heteroaryl; (((alkylsulfonyl)amino)alkyl)heteroaryl;
((alkylsulfonyl)alkyl)heteroaryl; (cyanoalkyl)heteroaryl;
(acyl)heteroaryl [e.g., (alkylcarbonyl)heteroaryl];
(alkyl)heteroaryl, and (haloalkyl)heteroaryl [e.g.,
trihaloalkylheteroaryl].
[0131] A "heteroaraliphatic (such as a heteroaralkyl group) as used
herein, refers to an aliphatic group (e.g., a C.sub.1-4 alkyl
group) that is substituted with a heteroaryl group. "Aliphatic,"
"alkyl," and "heteroaryl" have been defined above.
[0132] As used herein, an "acyl" group refers to a formyl group or
R.sup.X--C(O)-- (such as -alkyl-C(O)--, also referred to as
"alkylcarbonyl") where R.sup.X and "alkyl" have been defined
previously. Acetyl and pivaloyl are examples of acyl groups.
[0133] As used herein, an "alkoxy" group refers to an alkyl-O--
group where "alkyl" has been defined previously.
[0134] As used herein, a "carbamoyl" group refers to a group having
the structure --O--CO--NR.sup.XR.sup.Y or
--NR.sup.X--CO--O--R.sup.Z wherein R.sup.X and R.sup.Y have been
defined above and R.sup.Z can be aliphatic, aryl, araliphatic,
heterocycloaliphatic, heteroaryl, or heteroaraliphatic.
[0135] As used herein, a "carboxy" group refers to --COOH,
--COOR.sup.X, --OC(O)H, --OC(O)R.sup.X when used as a terminal
group or --OC(O)-- or --C(O)O--; when used as an internal
group.
[0136] As used herein, a "haloaliphatic" group refers to an
aliphatic group substituted with 1-3 halogen. For instance, the
term haloalkyl includes the group --CF.sub.3.
[0137] As used herein, a "mercapto" group refers to --SH.
[0138] As used herein, a "sulfo" group refers to --SO.sub.3H or
--SO.sub.3R.sup.X when used terminally or --S(O)3- when used
internally.
[0139] As used herein, a "sulfamide" group refers to the structure
--NR.sup.X--S(O).sub.2--NR.sup.YR.sup.Z when used terminally and
--NRX--S(O).sub.2--NR.sup.Y-- when used internally, wherein
R.sup.X, R.sup.Y, and R.sup.Z have been defined above.
[0140] As used herein, a "sulfamoyl" group refers to the structure
--S(O).sub.2--NR.sup.XR.sup.Y or --NR.sup.X--S(O).sub.2--R.sup.Z
when used terminally or --S(O).sub.2--NR.sup.X-- or
--NR.sup.X--S(O).sub.2-- when used internally, wherein R.sup.X,
R.sup.Y, and R.sup.Z are defined above.
[0141] As used herein a "sulfanyl" group refers to --S--R.sup.X
when used terminally and --S-- when used internally, wherein
R.sup.X has been defined above. Examples of sulfanyls include
alkylsulfanyl.
[0142] As used herein a "sulfinyl" group refers to --S(O)--R.sup.X
when used terminally and --S(O)-- when used internally, wherein
R.sup.X has been defined above.
[0143] As used herein, a "sulfonyl" group refers to
--S(O).sub.2--R.sup.X when used terminally and --S(O).sub.2-- when
used internally, wherein R.sup.X has been defined above.
[0144] As used herein, a "sulfoxy" group refers to --O--SO--R.sup.X
or --SO--O--R.sup.X, when used terminally and --O--S(O)-- or
--S(O)--O-- when used internally, where R.sup.X has been defined
above.
[0145] As used herein, a "halogen" or "halo" group refers to
fluorine, chlorine, bromine or iodine.
[0146] As used herein, an "alkoxycarbonyl," which is encompassed by
the term carboxy, used alone or in connection with another group
refers to a group such as alkyl-O--C(O)--.
[0147] As used herein, an "alkoxyalkyl" refers to an alkyl group
such as alkyl-O-alkyl-, wherein alkyl has been defined above.
[0148] As used herein, a "carbonyl" refers to --C(O)--.
[0149] As used herein, an "oxo" refers to .dbd.O.
[0150] As used herein, an "aminoalkyl" refers to the structure
(R.sup.X).sub.2N-alkyl-.
[0151] As used herein, a "cyanoalkyl" refers to the structure
(NC)-alkyl-
[0152] As used herein, a "urea" group refers to the structure
--NR.sup.X--CO--NR.sup.YR.sup.Z and a "thiourea" group refers to
the structure --NR.sup.X--CS--NR.sup.YR.sup.Z when used terminally
and --NR.sup.X--CO--NR.sup.Y-- or --NR.sup.X--CS--NR.sup.Y-- when
used internally, wherein R.sup.X, R'', and R.sup.Z have been
defined above.
[0153] As used herein, a "guanidine" group refers to the structure
--N.dbd.C(N(R.sup.XR.sup.Y))N(R.sup.XR.sup.Y) wherein R.sup.X and
R.sup.Y have been defined above.
[0154] As used herein, the term "amidino" group refers to the
structure --C.dbd.(NR.sup.X)N(R.sup.XR.sup.Y) wherein R.sup.X and
R.sup.Y have been defined above.
[0155] The terms "terminally" and "internally" refer to the
location of a group within a substituent. A group is terminal when
the group is present at the end of the substituent not further
bonded to the rest of the chemical structure. Carboxyalkyl, i.e.,
R.sup.XO(O)C-alkyl is an example of a carboxy group used
terminally. A group is internal when the group is present in the
middle of a substituent to at the end of the substituent bound to
the to the rest of the chemical structure. Alkylcarboxy (e.g.,
alkyl-C(O)O-- or alkyl-OC(O)--) and alkylcarboxyaryl (e.g.,
alkyl-C(O)O-aryl- or alkyl-O(CO)-aryl-) are examples of carboxy
groups used internally.
[0156] The phrase "optionally substituted" is used interchangeably
with the phrase "substituted or unsubstituted." As described
herein, compounds of the invention can optionally be substituted
with one or more substituents, such as are illustrated generally
above, or as exemplified by particular classes, subclasses, and
species of the invention. As described herein, the variables
contained herein encompass specific groups, such as alkyl and aryl.
Unless otherwise noted, each of the specific groups for the
variables contained herein can be optionally substituted with one
or more substituents described herein. Each substituent of a
specific group is further optionally substituted with one to three
of halo, cyano, oxoalkoxy, hydroxyl, amino, nitro, aryl, haloalkyl,
and alkyl. For instance, an alkyl group can be substituted with
alkylsulfanyl and the alkylsulfanyl can be optionally substituted
with one to three of halo, cyano, oxoalkoxy, hydroxyl, amino,
nitro, aryl, haloalkyl, and alkyl. As an additional example, the
cycloalkyl portion of a (cycloalkyl)carbonylamino can be optionally
substituted with one to three of halo, cyano, alkoxy, hydroxyl,
nitro, haloalkyl, and alkyl. When two alkoxy groups are bound to
the same atom or adjacent atoms, the two alkoxy groups can form a
ring together with the atom(s) to which they are bound.
[0157] In general, the term "substituted," whether preceded by the
term "optionally" or not, refers to the replacement of hydrogen
radicals in a given structure with the radical of a specified
substituent. Specific substituents are described above in the
definitions and below in the description of compounds and examples
thereof. Unless otherwise indicated, an optionally substituted
group can have a substituent at each substitutable position of the
group, and when more than one position in any given structure can
be substituted with more than one substituent selected from a
specified group, the substituent can be either the same or
different at every position. A ring substituent, such as a
heterocycloalkyl, can be bound to another ring, such as a
cycloalkyl, to form a spiro-bicyclic ring system, e.g., both rings
share one common atom. As one of ordinary skill in the art will
recognize, combinations of substituents envisioned by this
invention are those combinations that result in the formation of
stable or chemically feasible compounds.
[0158] The phrase "stable or chemically feasible," as used herein,
refers to compounds that are not substantially altered when
subjected to conditions to allow for their production, detection,
and preferably their recovery, purification, and use for one or
more of the purposes disclosed herein.
[0159] As used herein, an effective amount is defined as the amount
required to confer a therapeutic effect on the treated patient, and
is typically determined based on age, surface area, weight and
condition of the patient. The interrelationship of dosages for
animals and humans (based on milligrams per meter squared of body
surface) is described by Freireich et al., Cancer Chemother. Rep.,
50: 219 (1966). Body surface area can be approximately determined
from height and weight of the patient. See, e.g., Scientific
Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970).
[0160] Unless otherwise stated, structures depicted herein are also
meant to include all isomeric (e.g., enantiomeric, diastereomeric,
and geometric (or conformational)) forms of the structure; for
example, the R and S configurations for each asymmetric center, (Z)
and (E) double bond isomers, and (Z) and (E) conformational
isomers. Therefore, single stereochemical isomers as well as
enantiomeric, diastereomeric, and geometric (or conformational)
mixtures of the present compounds are within the scope of the
invention. Unless otherwise stated, all tautomeric forms of the
compounds of the invention are within the scope of the invention.
Additionally, unless otherwise stated, structures depicted herein
are also meant to include compounds that differ only in the
presence of one or more isotopically enriched atoms. For example,
compounds having the present structures except for the replacement
of hydrogen by deuterium or tritium, or the replacement of a carbon
by a .sup.13C- or .sup.14C-enriched carbon are within the scope of
this invention. Such compounds are useful, for example, as
analytical tools or probes in biological assays.
II. Oncogene Targets
[0161] In some embodiments, the present invention provides antigene
inhibitors of oncogenes. The present invention is not limited to
the inhibition of a particular oncogene. Indeed, the present
invention encompasses antigene inhibitors to any number of
oncogenes including, but not limited to, those disclosed
herein.
[0162] A. Ras
[0163] One gene which has captured the attention of many scientists
is the human proto-oncogene, c-Ha-ras. This gene acts as a central
dispatcher, relaying chemical signals into cells and controlling
cell division. Ras gene alteration may cause the gene to stay in
the "on" position. The ras oncogene is believed to underlie up to
30% of cancer, including colon cancer, lung cancer, bladder and
mammary carcinoma (Bos, Cancer Res. 49:4682-4689 [1989]). The ras
oncogene has therefore become a target for therapeutic drugs.
[0164] There are several reports showing that oligonucleotides
complementary to various sites of ras mRNA can inhibit synthesis of
ras protein (p21), which decreases the cell proliferation rate in
cell culture (U.S. Pat. No. 5,576,208; U.S. Pat. No. 5,582,986;
Daska et al., Oncogene Res. 5:267-275 [1990]; Brown et al.,
Oncogene Res. 4:243-252 [1989]; Saison-Behmoaras et al., EMBO J.
10:1111-1116 [1991)]. Oligonucleotides complementary to the 5'
flanking region of the c-Ha-ras RNA transcript have shown to
inhibit tumor growth in nude mice for up to 14 days (Gray et al.,
Cancer Res. 53:577-580 [1993]). It was recently reported that an
antisense oligonucleotide directed to a point mutation (G>C) in
codon 12 of the c-Ha-ras mRNA inhibited cell proliferation as well
as tumor growth in nude mice when it was injected subcutaneously
(U.S. Pat. No. 5,576,208; U.S. Pat. No. 5,582,986; Schwab et al.,
Proc. Natl. Acad. Sci. USA 91:10460-10464 [1994]; each of which is
herein incorporated by reference). Researchers have also reported
that antisense drugs shrank ovarian tumors in small clinical trials
(Roush et al., Science 276:1192-1194 [1997]).
[0165] B. Her-2
[0166] The -her-2 (also known as neu oncogene or erbB-2) oncogene
encodes a receptor-like tyrosine kinase (RTK) that has been
extensively investigated because of its role in several human
carcinomas (Hynes and Stern, Biochim. et Biophy. Acta 1198:165-184
[1994]; Dougall et al., Oncogene 9:2109-2123 [1994]) and in
mammalian development (Lee et al., Nature 378:394-398 [1995]).
Her-2 is one of the most frequently altered genes in cancer. It
encodes a transmembrane receptor (also known as p185) with tyrosine
kinase activity and is a member of the epidermal growth factor
(EGF) family, and thus is related to the epidermal growth factor
receptor (EGFR or HER-1). Aberrant her-2 gene expression is present
in a wide variety of cancers and is most common in breast, ovarian
and gastric cancers. HER-2 is overexpressed in 25-30% of all human
breast and ovarian cancers. Levels of HER-2 overexpression
correlate well with clinical stage of breast cancer, prognosis and
metastatic potential. Overexpression of HER-2 is associated with
lower survival rates, increased relapse rates and increased
metastatic potential. Tan et al., (Cancer Res., 57:1199 [1997])
have shown that overexpression of the HER-2 gene increases the
metastatic potential of breast cancer cells without increasing
their transformation ability.
[0167] Aberrant expression of HER-2 includes both increased
expression of normal HER-2 and expression of mutant HER-2.
Activation of the her-2 proto-oncogene can occur by any of three
mechanisms--point mutation, gene amplification and overexpression.
Gene amplification is the most common mechanism. Unlike the other
EGF family members for whom ligand activation is necessary for
promoting transformation, overexpression of HER-2 alone is
sufficient for transformation (Cohen, et al., J. Biol. Chem.,
271:30897 [1996]).
[0168] Several therapeutic approaches have been used to reduce
levels of the her-2 gene product. The adenovirus type 5 gene
product E1A has been studied as a potential therapeutic using a
breast cancer model in nude mice. This gene product can repress
her-2/neu overexpression by repressing her-2/neu promoter activity,
and suppress the tumorigenic potential of her-2/neu-overexpressing
ovarian cancer cells. In mice bearing her-2/neu-overexpressing
breast cancer xenografts, E1A delivered either by adenovirus or
liposome significantly inhibited tumor growth and prolonged mouse
survival compared with the controls (Chang et al., Oncogene 14:561
[1997]). Clinical trials have been conducted to evaluate a
bispecific antibody which targets the extracellular domains of both
the HER-2/neu protein product and Fc gamma RIII (CD16), the Fc
gamma receptor expressed by human natural killer cells,
neutrophils, and differentiated mononuclear phagocytes (Weiner et
al., J. Hematotherapy, 4:471 [1995]).
[0169] Overexpression of HER-2 has also been found to be associated
with increased resistance to chemotherapy. Thus, patients with
elevated levels of HER-2 respond poorly to many drugs. Methods used
to inhibit HER-2 expression have been combined with commonly used
chemotherapeutic agents (Ueno et al., Oncogone 15:953 [1997]).
Combining the adenovirus type 5 gene product, E1A, with taxol
showed a synergistic effect in human breast cancer cells. Zhang et
al., (Oncogene, 12:571 [1996]) demonstrated that emodin, a
tyrosine-specific inhibitor, sensitized non-small cell lung cancer
(NSCLC) cells to a variety of chemotherapeutic drugs, including
cisplatin, doxorubicin and etoposide. A HER-2 antibody was found to
increase the efficacy of tamoxifen in human breast cancer cells
(Witters et al., Breast Cancer Res. and Treatment, 42:1
[1997]).
[0170] Oligonucleotides have also been used to study the function
of HER-2. A triplex-forming oligonucleotide targeted to the her-2
promoter, 42 to 69 nucleotides upstream of the mRNA transcription
start site was found to inhibit HER-2 expression in vitro
(Ebbinghaus et al., J. Clin. Invest., 92:2433 [1993]). Porumb et
al. (Cancer Res., 56:515 [1996]) also used a triplex-forming
oligonucleotide targeted to the same her-2 promoter region.
Decreases in her-2 mRNA and protein levels were seen in cultured
cells. Juhl et al. (J. Biol. Chem., 272:29482 [1997]) used
anti-her-2 ribozymes targeted to a central region of the her-2 RNA
just downstream of the transmembrane region of the protein to
demonstrate a reduction in her-2 mRNA and protein levels in human
ovarian cancer cells. A reduction in tumor growth in nude mice was
also seen.
[0171] An antisense approach has been used as a potential
therapeutic for HER-2 overexpressing cancers. Pegues et al. (Cancer
Lett., 117:73 [1997]) cloned a 1.5 kb fragment of her-2 in an
antisense orientation into an expression vector; transfecting of
this construct into ovarian cancer cells resulted in a reduction of
anchorage-independent growth. Casalini et al. (Int. J. Cancer
72:631 [1997]) used several human her-2 antisense vector
constructs, containing her-2 fragments from 151 by to 415 by in
length, to demonstrate reduction in HER-2 protein levels and
anchorage-independent growth in lung adenocarcinoma cells. Colomer
et al. (Br. J. Cancer, 70:819 [1994]) showed that phosphodiester
antisense oligonucleotides targeted at or immediately downstream
of, the translation initiation codon inhibited proliferation of
human breast cancer cells by up to 60%. Wiechen et al. (Int. J.
Cancer 63:604 [1995]) demonstrated that an 18-nucleotide
phosphorothioate oligonucleotide targeted to the coding region, 33
nucleotides downstream of the translation initiation codon, of
her-2 reduced anchorage-independent growth of ovarian cancer cells.
Bertram et al. (Biochem. Biophys. Res. Commun., 200:661 [1994])
used antisense phosphorothioate oligonucleotides targeted to the
translation initiation region and a sequence at the 3' part of the
translated region of the mRNA which has high homology to a tyrosine
kinase consensus sequence, and demonstrated a 75% reduction in
HER-2 protein levels in human breast cancer cells. Liu et al.,
(Antisense and Nucleic Acid Drug Develop., 6:9 [1996]) used
antisense phosphorothioate oligonucleotides targeted to the 5' cap
site and coding region. The most effective oligonucleotide,
targeted to the 5' cap site, reduced HER-2 protein expression by
90%. Cell proliferation was also reduced by a comparable amount.
Vaughn et al. (Nuc. Acids Res., 24:4558 [1996]) used
phosphorothioate, phosphorodithioate and chimeric antisense
oligonucleotides targeted at or adjacent to (either side) the
translation initiation region of her-2. An alternating
dithioate/diester oligonucleotide targeted to the translation
initiation region worked slightly better than an all
phosphorothioate oligonucleotide. Brysch et al. (Cancer Gene Ther.,
1: 99 [1994]) used chemically modified antisense oligonucleotides
targeted to the translation initiation codon of HER-2 to reduce
protein levels and cause growth arrest of human breast cancer cell
line.
[0172] C. C-Myc
[0173] The c-myc gene product is encoded by an immediate early
response gene, the expression of which can be induced by various
mitogens. C-myc expression is involved in signal transduction
pathways leading to cell division. Studies have demonstrated that
proliferating cells have higher levels of c-myc mRNA and c-myc
protein than do quiescent cells. Antibodies directed against the
human c-myc protein are known to inhibit DNA synthesis in nuclei
isolated from human cells. Conversely, constitutive expression of
c-myc produced by gene transfer inhibits induced differentiation of
several cell lines. Constitutive expression of c-myc predisposes
transgenic mice to the development of tumors.
[0174] Some studies have suggested that the c-myc gene product may
play a proliferative role in smooth muscle cells (SMCs). Balloon
de-endothelialization and injury of rat aortas is known to increase
c-myc mRNA expression of vascular SMC prior to their subsequent
proliferation and migration. Also, SMCs in culture proliferate when
exposed to several mitogens, including PDGF, FGF, EGF, IGF-1 and to
serum. Each of these mitogens has been found to be capable of
increasing the expression in other cell lines of either c-myc
protein, c-myc mRNA, or both. Additionally, blood serum has been
found to increase c-myc mRNA levels in SMCs.
[0175] Harel-Bellan et al. (J. Immun. 140; 2431-2435 (1988))
demonstrated that antisense oligonucleotides complementary to c-myc
mRNA effectively inhibited the translation thereof in human T
cells. These T cells were prevented from entering the S phase of
cell division. c-myc proto-oncogene sequences are described in
Marcu et al., Ann. Rev. Biochem., 61:809-860 [1992]; Watt et al.,
Nature, 303:725-728 [1983)]; Battey et al., Cell, 34:779-787
(1983); and Epstein et al, NTIS publication PB93-100576
[0176] D. Bcl-2
[0177] In many types of human tumors, including lymphomas and
leukemias, the bcl-2 gene is overexpressed, and may be associated
with tumorigenicity (Tsujimoto et al., Science 228:1440-1443
[1985]). High levels of expression of the bcl-2 gene have been
found in all lymphomas with t (14; 18) chromosomal translocations
including most follicular B cell lymphomas and many large cell
non-Hodgkin's lymphomas. High levels of expression of the bcl-2
gene have also been found in certain leukemias that do not have a
t(14; 18) chromosomal translation, including most cases of chronic
lymphocytic leukemia acute, many lymphocytic leukemias of the pre-B
cell type, neuroblastomas, nasopharyngeal carcinomas, and many
adenocarcinomas of the prostate, breast and colon. (Reed et al.,
Cancer Res. 51:6529 [1991]; Yunis et al., New England J. Med.
320:1047; Campos et al., Blood 81:3091-3096 [1993]; McDonnell et
al., Cancer Res. 52:6940-6944 [1992); Lu et al., Int. J Cancer
53:29-35 [1993]; Bonner et al., Lab Invest. 68:43A [1993]).
[0178] E. TGF-.alpha.
[0179] Transforming Growth Factor Alpha (TGF-.alpha.) is a
polypeptide of 50 amino acids. It was first isolated from a
retrovirus-transformed mouse cell line and subsequently was
identified in human tumor cells, in early rat embryo cells and in
cell cultures from the human pituitary gland. TGF-.alpha. is
closely related to Epidermal Growth Factor (EGF), both structurally
and functionally, and both bind to the same receptor, i.e.,
Epidermal Growth Factor Receptor (EGFR). The sequence and three
dimensional structure of both EGF and TGF-.alpha. have been
determined (Campbell et al., Prog. Growth Factor Res. 1:13 [1989]).
TGF-.alpha. is a 50 amino acid polypeptide having about 40%
homology of residues with EGF. Both peptides are characterized by
three well defined loops (denoted A, B and C) and have three
intramolecular disulphide bonds.
[0180] Several growth factors, including TGF-.alpha. and EGF, are
believed to exert their biological effects via interaction with the
Epidermal Growth Factor Receptor (EGF Receptor). The EGF Receptor
is a Type 1 receptor tyrosine kinase. The EGF Receptor and its
ligands are of interest for their roles in normal physiological
processes as well as in hyperproliferative and neoplastic
diseases.
[0181] The in vivo precursor of TGF-.alpha. is a 160 amino acid
residue membrane-bound protein (pro-TGF-.alpha.) that is cleaved to
yield a soluble compound (Massague, J. Biol. Chem., 265:21393-21396
[1990]). This cleavage removes an extracellular portion comprised
of 50 amino acids with a molecular weight of 6 Kd and is considered
to be an important regulatory event (Pandiella et al., Proc. Natl.
Acad. Sci. USA, 88:1726-1730 [1990]) that can be stimulated by
phorbol esters acting via protein kinase C (Pandiella et al., J.
Biol. Chem., 266:5769-5773 [1991]).
[0182] Cultured human prostatic tumor lines contain elevated levels
of TGF-.alpha. mRNA and proliferate in response to TGF-.alpha.
(Wilding et al., The Prostate, 15:1-12 [1989]). TGF-.alpha. appears
to have both autocrine and paracrine function, stimulating
physiologic activities such as cell division and angiogenesis. When
induced in transgenic mice, TGF-.alpha. produced epithelial
hyperplasia and focal dysplastic changes that resembled carcinoma
in situ (Sandgren et al., Cell, 61:1121-1135 [1990]).
[0183] F. c-ki-Ras
[0184] The c-Ki-Ras (KRAS) oncogene is expressed ubiquitously.
KRAS, with a length of more than 30 kb, is much larger than HRAS or
NRAS. Although the 3 ras genes, HRAS, KRAS, and NRAS, have
different genetic structures, all code for proteins of 189 amino
acid residues, generically designated p21. These genes acquire
malignant properties by single point mutations that affect the
incorporation of the 12th or 61st amino acid residue of their
respective p21. KRAS is involved in malignancy much more often than
is HRAS. In a study of 96 human tumors or tumor cell lines in the
NIH 3T3 transforming system, (Pulciani et al., Nature 300: 539
(1982) found a mutated HRAS locus only in T24 bladder cancer cells,
whereas transforming KRAS genes were identified in 8 different
carcinomas and sarcomas.
[0185] In a serous cystadenocarcinoma of the ovary, Feig et al.
(Science 223: 698 (1984)) showed the presence of an activated KRAS
oncogene not activated in normal cells of the same patient. The
transforming gene product displayed an electrophoretic mobility in
SDS-polyacrylamide gels that differed from the mobility of KRAS
transforming proteins in other tumors. Thus, a previously
undescribed mutation was responsible for activation of KRAS in this
ovarian carcinoma. To study the role of oncogenes in lung cancer,
Rodenhuis et al. (New Eng. J. Med. 317: 929 (1987)) used an assay
based on oligonucleotide hybridization following an in vitro
amplification step. Genomic DNA was examined from 39 tumor
specimens obtained at thoracotomy. The KRAS gene was found to be
activated by point mutations in codon 12 in 5 of 10
adenocarcinomas. Two of these tumors were less than 2 cm in size
and had not metastasized. No HRAS, KRAS, or NRAS mutations were
observed in 15 squamous cell carcinomas, 10 large cell carcinomas,
1 carcinoid, 2 metastatic adenocarcinomas from primary tumors
outside the lung, and 1 small cell carcinoma. An approximately
20-fold amplification of the unmutated KRAS gene was observed in a
tumor that proved to be a solitary lung metastasis of a rectal
carcinoma. Yanez et al. (Oncogene 1:315 (1987)) found mutations in
codon 12 of the KRAS gene in 4 of 16 colon cancers, 2 of 27 lung
cancers, and 1 of 8 breast cancers; no mutations were found at
position 61. Of the 6 possible amino acid replacements in codon 12,
all but one were represented in the 7 mutations identified.
[0186] G. Other Oncogene Targets
[0187] The present invention is not limited to the oncogenes
described above. The methods of the present invention are suitable
for use with any oncogene with a known promoter region. Exemplary
oncogenes included, but are not limited to, BCR/ABL, ABL1/BCR, ABL,
BCL1, CD24, CDK4, EGFR/ERBB-1, HSTF1, INT1/WNT1, INT2, MDM2, MET,
MYB, MYC, MYCN, MYCL1, RAF1, NRAS, REL, AKT2, APC, BCL2-ALPHA,
BCL2-BETA, BCL3, BCR, BRCA1, BRCA2, CBL, CCND1, CDKN1A, CDKN1C,
CDKN2A, CDKN2B, CRK, CRK-II, CSF1R/FMS, DBL, DDOST, DCC,
DPC4/SMAD4, E-CAD, E2F1/RBAP, ELK1, ELK3, EPH, EPHA1, E2F1, EPHA3,
ERG, ETS1, ETS2, FER, FGR, FLI1/ERGB2, FOS, FPS/FES, FRA1, FRA2,
FYN, HCK, HEK, HER3/ERBB-2, ERBB-3, HER4/ERBB-4, HST2, INK4A,
INK4B, JUN, JUNB, JUND, KIP2, KIT, KRAS2A, KRAS2B, LCK, LYN, MAS,
MAX, MCC, MLH1, MOS, MSH2, MYBA, MYBB, NF1, NF2, P53, PDGFB, PIM1,
PTC, RB1, RET, ROS1, SKI, SRC1, TAL1, TGFBR2, THRA1, THRB, TIAM1,
TRK, VAV, VHL, WAF1, WNT2, WT1, YES1, ALK/NPM1, AMI1, AXL, FMS,
GIP, GLI, GSP, HOX11, HST, IL3, INT2, KS3, K-SAM, LBC, LMO-1,
LMO-2, L-MYC, LYL1, LYT-10, MDM-2, MLH1, MLL, MLM, N-MYC, OST,
PAX-5, PMS-1, PMS-2, PRAD-1, RAF, RHOM-1, RHOM-2, SIS, TAL2, TAN1,
TIAM1, TSC2, TRK, TSC1, STK11, PTCH, MEN1, MEN2, P57/KIP2, PTEN,
HPC1, ATM, XPA/XPG, BCL6, DEK, AKAP13, CDH1, BLM, EWSR1/FLI1, FES,
FGF3, FGF4, FGF6, FANCA, FLI1/ERGB2, FOSL1, FOSL2, GLI, HRAS1,
HRX/MLLT1, HRX/MLLT2, KRAS2, MADH4, MAS1, MCF2, MLLT1/MLL,
MLLT2/HRX, MTG8/RUNX1, MYCLK1, MYH11/CBFB, NFKB2, NOTCH1, NPM1/ALK,
NRG/REL, NTRK1, PBX1/TCF3, PML/RARA, PRCA1, RUNX1, RUNX1/CBFA2T1,
SET, TCF3/PBX1, TGFB1, TLX1, P53, WNT1, WNT2, WT1,
.alpha.v-.beta.3, PKC.alpha., TNF.alpha., Clusterin, Surviving,
TGF.beta., c-fos, c-SRC, and INT-1.
III. Non-Oncogene Targets
[0188] The present invention is not limited to the targeting of
oncogenes. The methods and compositions of the present invention
find use in the targeting of any gene of which it is desirable to
down regulate the expression. For example, in some embodiments, the
genes to be targeted include, but are not limited to, an
immunoglobulin or antibody gene, a clotting factor gene, a
protease, a pituitary hormone, a protease inhibitor, a growth
factor, a somatomedian, a gonadotrophin, a chemotactin, a
chemokine, a plasma protein, a plasma protease inhibitor, an
interleukin, an interferon, a cytokine, a transcription factor, or
a pathogen target (e.g., a viral gene, a bacterial gene, a
microbial gene, a fungal gene).
[0189] Examples of specific genes include, but are not limited to,
ADAMTS4, ADAMTS5, APOA1, APOE, APP, B2M, COX2, CRP, DDX25, DMC1,
FKBP8, GH1, GHR, IAPP, IFNA1, IFNG, ILL 1110, IL12, IL13, IL2, IL4,
IL7, IL8, IPW, MAPK14, Mei1, MMP13, MYD88, NDN, PACE4, PRNP, PSEN1,
PSEN2, RAD51, RAD51C, SAP, SNRPN, TLR4, TLR9, TTR, UBE3A, VLA-4,
and PTP-1B, c-RAF, m-TOR, LDL, VLDL, ApoB-100, HDL, VEGF,
rhPDGF-BB, NADs, ICAM-1, MUC1, 2-dG, CTL, PSGL-1, E2F, NF-kB, HIF,
and GCPRs.
[0190] In other embodiments, a gene from a pathogen is targeted.
Exemplary pathogens include, without limitation, Human
Immunodeficiency virus, Hepatitis B virus, hepatitis C virus,
hepatitis A virus, respiratory syncytial virus, pathogens involved
in severe acute respiratory syndrome, west nile virus, and food
borne pathogens (e.g., E. coli).
IV. Abbreviations
[0191] Abbreviations for lipids refer primarily to standard use in
the literature and are included here as a helpful reference: [0192]
DMPC Dimyristoylphosphatidylcholine [0193] DPPC
Dipalmitoylphosphatidylcholine [0194] DSPC
Distearoylphosphatidylcholine [0195] POPC
Palmitoyl-oleoylphosphatidylcholine [0196] OPPC
1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine [0197] DOPC
Dioleoylphosphatidylcholine [0198] DOPE
Dioleoylphosphatidylethanolamine [0199] DMPE
Dimyristoylphosphatidylethanolamine [0200] DPPE
Dipalmitoylphosphatidylethanolamine [0201] DOPG
Dioleoylphosphatidylglycerol [0202] POPG
Palmitoyl-oleoylphosphatidylglycerol [0203] DMPG
Dimyristoylphosphatidylglycerol [0204] DPPG
Dipalmitoylphosphatidylglycerol [0205] DLPG
Dilaurylphosphatidylglycerol [0206] DSPG
Distraroylphosphatidylglycerol [0207] DMPS
Dimyristoylphosphatidylserine [0208] DPPS
Dipalmitoylphosphatidylserine [0209] DOPS
Dioleoylphosphatidylserine [0210] POPS
Palmitoyl-oleoylphosphatidylserine [0211] DMPA
Dimyristoylphosphatidic acid [0212] DPPA Dipalmitoylphosphatidic
acid [0213] DOPA Dioleoylphosphatidic acid [0214] POPA
Palmitoyl-oleoylphosphatidic acid [0215] DSPA
Distearoylphosphatidic acid [0216] DLPA Dilaurylphosphatidic acid
[0217] CHEMS Cholesterolhemisuccinate [0218] DC-Chol
3-.beta.-[N--(N',N'-dimethylethane)carbamoyl]cholesterol [0219]
Cet-P Cetylphosphate [0220] DODAP
(1,2)-dioleoyloxypropyl)-N,N-dimethylammonium chloride [0221] DOEPC
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine [0222] DAC-Chol
3-.beta.-[N--(N,N'-dimethylethane)carbamoyl]cholesterol [0223]
TC-Chol
3-.beta.-[N--(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol
[0224] DOTMA
(1,2-dioleyloxypropyl)-N,N,N-trimethylammoniumchloride) [0225]
(Lipofectin.RTM.) [0226] DOGS ((C18)2GlySper3+)
N,N-dioctadecylamido-glycyl-spermine (Transfectam.RTM.) [0227] CTAB
Cetyl-trimethylammoniumbromide [0228] CPyC Cetyl-pyridiniumchloride
[0229] DOTAP (1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium salt
[0230] DMTAP (1,2-dimyristoyloxypropyl)-N,N,N-trimethylammonium
salt [0231] DPTAP
(1,2-dipalmitoyloxypropyl)-N,N,N-trimethylammonium salt [0232]
DOTMA (1,2-dioleyloxypropyl)-N,N,N-trimethylammonium chloride)
[0233] DORIE (1,2-dioleyloxypropyl)-3 dimethylhydroxyethyl
ammoniumbromide) [0234] DDAB Dimethyldioctadecylammonium bromide
[0235] DPIM 4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole
[0236] CHIM Histaminyl-Cholesterolcarbamate [0237] MoChol
4-(2-Aminoethyl)-Morpholino-Cholesterolhemisuccinate [0238] HisChol
Histaminyl-Cholesterolhemisuccinate [0239] HCChol
N.alpha.-Histidinyl-Cholesterolcarbamate [0240] HistChol
N.alpha.-Histidinyl-Cholesterol-hemisuccinate [0241] AC
Acylcarnosine, Stearyl- & Palmitoylcarnosine [0242] HistDG
1,2-Dipalmitoylglycerol-hemisuccinat-N_-Histidinyl-hemisuccinate,
& Distearoyl-, Dimyristoyl, Dioleoyl or
palmitoyl-oleoylderivatives [0243] IsoHistSuccDG
1,2-ipalmitoylglycerol-Q-Histidinyl-N.alpha.-hemisuccinat, &
Distearoyl-, Dimyristoyl, Dioleoyl or palmitoyl-oleoylderivatives
[0244] DGSucc 1,2-Dipalmitoyglycerol-3-hemisuccinate &
Distearoyl-, dimyristoyl-Dioleoyl or palmitoyl-oleoylderivatives
[0245] EDTA-Chol cholesterol ester of ethylenediaminetetraacetic
acid [0246] Hist-PS N.alpha.-histidinyl-phosphatidylserine [0247]
BGSC bisguanidinium-spermidine-cholesterol [0248] BGTC
bisguanidinium-tren-cholesterol [0249] DOSPER
(1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide [0250] DOSC
(1,2-dioleoyl-3-succinyl-sn-glyceryl choline ester) [0251] DOGSDO
(1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide
ornithine) [0252] DOGSucc 1,2-Dioleoylglycerol-3-hemisucinate
[0253] POGSucc Palimtolyl-oleoylglycerol-oleoyl-3-hemisuccinate
[0254] DMGSucc 1,2-Dimyristoylglycerol-3-hemisuccinate [0255]
DPGSucc 1,2-Dipalmitoylglycerol-3-hemisuccinate
[0256] The following table provides non-limiting examples of lipids
that are suitable for use in the compositions in accordance with
the present invention. The membrane anchors of the lipids are shown
exemplarily and serve only to illustrate the lipids of the
invention and are not intended to limit the same.
##STR00001## ##STR00002##
V. Amphoteric Liposomal Delivery System
[0257] Amphoteric liposomes represent a recently described class of
liposomes having anionic or neutral charge at about pH 7.5 and
cationic charge at pH 4. PCT International Publication Numbers WO
02/066490, WO 02/066120 and WO 03/070220, each of which is
incorporated by reference, give a detailed description of
amphotheric liposomes and suitable lipids therefor. Using
amphoteric liposomes as carriers of DNAi oligonucleotides according
to the present invention, to treat cancer in cells and in mammals,
such as by inhibiting and/or reducing tumor growth, requires that
the liposomes be stable in the bloodstream and in tissues.
Particularly, after a systemic application, the DNAI
oligonucleotides must be stably sequestered in the liposomes until
eventual uptake in the target tissue or cells. Accordingly, the
guidelines for liposomal formulations of the FDA regulate specific
preclinical tests for liposomal drugs
(http://www.fda.gov/cder/guidance/2191dft.pdf). For example, the
ratio of encapsulated drug to free drug must be determined during
the circulation time in the blood stream.
[0258] After injection of liposomes into the blood stream, serum
components interact with the liposomes and can lead to
permeabilization of the liposomes. However, release of a drug or
molecule that is encapsulated in a liposome depends on molecular
dimensions of the drug or molecule. Consequently, a plasmid of
thousands of base pairs is released much more slowly than smaller
oligonucleotides or other small molecules. For liposomal delivery
of drugs or molecules, it is essential that the release of the drug
during circulation of the liposomes in the bloodstream be as low as
possible.
[0259] The amphoteric liposomes of the mixture according to the
present invention, include one or more amphoteric lipids or
alternatively a mix of anionic and cationic lipid components with
amphoteric properties. Suitable amphoteric lipids are disclosed in
PCT International Publication Number WO02/066489 as well as in PCT
International Publication Number WO03/070735, the contents of both
of which are incorporated herein by reference. Alternatively, the
lipid phase may be formulated using pH-responsive anionic and/or
cationic components, as disclosed in PCT International Publication
Number WO02/066012, the contents of which are incorporated by
reference herein. Cationic lipids sensitive to pH are disclosed in
PCT International Publication Numbers WO02/066490 and WO03/070220,
in Budker, et al. 1996, Nat. Biotechnol. 14(6):760-4, and in U.S.
Pat. No. 6,258,792 the contents of which are incorporated by
reference herein, and can be used in combination with
constitutively charged anionic lipids or with anionic lipids that
are sensitive to pH. Conversely, the cationic charge may also be
introduced from constitutively charged lipids that are known to
those skilled in the art in combination with a pH sensitive anionic
lipid. (See also PCT International Publication Numbers WO05/094783,
WO03/070735, WO04/100928, WO06/48329, WO06/053646 and U.S. Patent
applications 2003/0099697, 2005/0164963, 2004/0120997, 2006/002991,
2006/159737, 2006/0216343, each of which is also incorporated in
its entirety by reference.)
[0260] The mixtures of the present invention include 1) amphoteric
lipids or a mixture of lipid components with amphoteric properties
2) neutral lipids; and 3) one or more DNAi oligonucleotides as
defined above.
[0261] A. Lipids used in Amphoteric Liposomes
[0262] 1. Amphoteric Lipids
[0263] Amphoteric lipids are disclosed in PCT International
Publication Numbers WO02/066489 and WO03/070735, the contents of
both of which are incorporated herein by reference. The overall
molecule assumes its pH-dependent charge characteristics by the
simultaneous presence of cationic and anionic groups in the
"amphoteric substance" molecule portion. More specifically, an
amphoteric substance is characterized by the fact that the sum of
its charge components will be precisely zero at a particular pH
value. This point is referred to as isoelectric point (IP). Above
the IP, the compound has a negative charge, and below the IP it is
to be regarded as a positive cation, the IP of the amphoteric
lipids ranging between 4.5 and 8.5.
[0264] The overall charge of the molecule at a particular pH value
of the medium can be calculated as follows:
z=.SIGMA.ni.times.((qi-1)+(10.sup.(pK-pH)/(1+10.sup.(pK-pH)))
[0265] qi: absolute charge of the ionic group below the pK thereof
(e.g. carboxyl=0, single-nitrogen base=1, di-esterified phosphate
group=-1) [0266] ni: number of such groups in the molecule.
[0267] For example, a compound is formed by coupling the amino
group of histidine to cholesterol hemisuccinate. At a neutral pH
value of 7, the product has a negative charge because the carboxyl
function which is present therein is in its fully dissociated form,
and the imidazole function only has low charge. At an acid pH value
of about 4, the situation is reversed: the carboxyl function now is
largely discharged, while the imidazole group is essentially fully
protonated, and the overall charge of the molecule therefore is
positive.
[0268] In one embodiment, the amphoteric lipid is selected from the
group consisting of HistChol, HistDG, isoHistSuccDG, Acylcarnosine
and HCChol. In another embodiment, the amphoteric lipid is
HistChol.
[0269] Amphoteric lipids can include, without limitation,
derivatives of cationic lipids which include an anionic
substituent. Amphoteric lipids include, without limitation, the
compounds having the structure of the formula:
Z--X--W1-Y--W2-HET [0270] wherein: [0271] Z is a sterol or an
aliphatic; [0272] Sterol is selected from the group consisting of
cholesterol, sitosterol, campesterol, desmosterol, fucosterol,
22-ketosterol, 20-hydroxysterol, sigmasterol,
22-hydroxycholesterol, 25 hydroxycholesterol, lanosterol,
7-dehydrocholesteril, dihydrocholesterol, 19-hydroxycholesterol,
5.alpha.cholest-7-en-3.beta.-ol, 7-hydroxycholesterol,
epicholesterol, ergosterol dehydroergosterol, and derivatives
thereof; [0273] Each W1 is independently an unsubstituted
aliphatic; [0274] Each W2 is independently an aliphatic optionally
substituted with HO(O)C-aliphatic-amino or carboxy; [0275] Each X
and Y is independently absent, --(C.dbd.O)--O--, --(C.dbd.O)--NH--,
--(C.dbd.O)--S--, --O--, --NH--, --S--, --CH.dbd.N--,
--O--(O.dbd.C)--, --S--(O.dbd.C)--, --NH--(O.dbd.C)--, and
--N.dbd.CH--; and [0276] HET is an amino, an optionally substituted
heterocycloaliphatic or an optionally substituted heteroaryl.
[0277] In some aspects, the HET is an optionally substituted
heterocycloaliphatic including at least one nitrogen ring atom, or
an optionally substituted heteroaryl including at least one
nitrogen ring atom. In other aspects, the HET is morpholinyl,
piperidinyl, piperazinlyl, pyrimidinyl, or pyridinyl. In another
aspect, the cationic lipid has the structure
Sterol-X-spacer1-Y-spacer2-morpholinyl or
Sterol-X-spacer1-Y-spacer2-imidazolyl. In still further aspects,
the sterol is cholesterol.
[0278] In other embodiments, amphoteric lipids include, without
limitation, the compounds having the structure of the formula:
Z--X--W1-Y--W2-HET [0279] wherein: [0280] Z is a structure
according to the general formula
[0280] ##STR00003## [0281] wherein R1 and R2 are independently
C8-C30 alkyl or acyl chains with 0.1. pr 2 etju; emoca; u
imsatirated bpmds amd M is selected from the group consisting of
--O--(C.dbd.O); --NH--(C.dbd.O)--; --S--(C.dbd.O)--; --O--; --NH--;
--S--; --N.dbd.CH--; --(O.dbd.C)--O--; --S--(O.dbd.C)--;
--NH--(O.dbd.C)--; --N.dbd.CH-- and/or --S--S--; [0282] Sterol is
selected from the group consisting of cholesterol, sitosterol,
campesterol, desmosterol, fucosterol, 22-ketosterol,
20-hydroxysterol, sigmasterol, 22-hydroxycholesterol, 25
hydroxycholesterol, lanosterol, 7-dehydrocholesterol,
dihydrocholesterol, 19-hydroxycholesterol,
5.alpha.cholest-7-en-3.beta.-ol, 7-hydroxycholesterol,
epicholesterol, ergosterol dehydroergosterol, and derivatives
thereof; [0283] Each W1 is independently an unsubstituted aliphatic
with up to 8 carbon atoms; [0284] Each W2 is independently an
aliphatic, carboxylic acid with up to 8 carbon atoms and 0, 1, or 2
ethylenically unsaturated bonds; [0285] X is absent and Y is
--(C.dbd.O)--O--; --(C.dbd.O)--NH--; --NH--(C.dbd.O)--O--; --O--;
--NH--; --CH.dbd.N--; --O--(O.dbd.C)--; --S--; --(O.dbd.C)--;
--NH--(O.dbd.C)--; --O--(O.dbd.C)--NH--, --N.dbd.CH-- and/or
--S--S--; and [0286] HET is an amino, an optionally substituted
heterocycloaliphatic or an optionally substituted heteroaryl.
[0287] In some aspects, the HET is an optionally substituted
heterocycloaliphatic including at least one nitrogen ring atom, or
an optionally substituted heteroaryl including at least one
nitrogen ring atom. In other aspects, the HET is morpholinyl,
piperidinyl, piperazinlyl, pyrimidinyl, or pyridinyl. In another
aspect, the cationic lipid has the structure
Sterol-X-spacer1-Y-spacer2-morpholinyl or
Sterol-X-spacer1-Y-spacer2-imidazolyl. In still further aspects,
the sterol is cholesterol.
[0288] 2. Mixtures of Lipid Components with Amphoteric
Properties
[0289] Alternatively, the lipid phase can be formulated using
pH-responsive anionic and/or cationic components, as disclosed in
PCT International Publication Number WO02/066012, the contents of
which are incorporated by reference herein. Cationic lipids
sensitive to pH are disclosed in PCT International Publication
Numbers WO02/066490 and WO03/070220, in Budker, et al. (1996), Nat
Biotechnol. 14(6):760-4, and in U.S. Pat. No. 6,258,792, the
contents of all of which are incorporated by reference herein.
Alternatively, the cationic charge may be introduced from
constitutively charged lipids known to those skilled in the art in
combination with a pH sensitive anionic lipid. Combinations of
constitutively (e.g., stable charge over a specific pH range such
as a pH between about 4 and 9) charged anionic and cationic lipids,
e.g. DOTAP and DPPG are not preferred. Thus, in some embodiments,
the mixture of lipid components may comprise (i) a stable cationic
lipid and a chargeable anionic lipid, (ii) a chargeable cationic
lipid and chargeable anionic lipid or (iii) a stable anionic lipid
and a chargeable cationic lipid.
[0290] The charged groups can be divided into the following 4
groups.
[0291] (1) Strongly (e.g., constitutively charged) cationic,
pKa>9, net positive charge: on the basis of their chemical
nature, these are, for example, ammonium, amidinium, guanidium or
pyridinium groups or timely, secondary or tertiary amino
functions.
[0292] (2) Weakly cationic, pKa<9, net positive charge: on the
basis of their chemical nature, these are, in particular, nitrogen
bases such as piperazines, imidazoles and morpholines, purines or
pyrimidines. Such molecular fragments, which occur in biological
systems, are, for example, 4-imidazoles (histamine), 2-, 6-, or
9-purines (adenines, guanines, adenosines or guanosines), 1-, 2- or
4-pyrimidines (uracils, thymines, cytosines, uridines, thymidines,
cytidines) or also pyridine-3-carboxylic acids (nicotinic esters or
amides). Nitrogen bases with preferred pKa values are also formed
by substituting nitrogen atoms one or more times with low molecular
weight alkane hydroxyls, such as hydroxymethyl or hydroxyethyl
groups. For example, aminodihydroxypropanes, triethanolamines,
tris-(hydroxymethyl)methylamines, bis-(hydroxymethyl)methylamines,
tris-(hydroxyethyl)methylamines, bis-(hydroxyethyl)methylamines or
the corresponding substituted ethylamines.
[0293] (3) Weakly anionic, pKa>4, net negative charge: on the
basis of their chemical nature, these are, in particular, the
carboxylic acids. These include the aliphatic, linear or branched
mono-, di- or tricarboxylic acids with up to 12 carbon atoms and 0,
1 or 2 ethylenically unsaturated bonds. Carboxylic acids of
suitable behavior are also found as substitutes of aromatic
systems. Other weakly anionic groups are hydroxyls or thiols, which
can dissociate and occur in ascorbic acid, N-substituted alloxane,
N-substituted barbituric acid, veronal, phenol or as a thiol
group.
[0294] (4) Strongly (e.g., constitutively charged) anionic,
pKa<4, net negative charge: on the basis of their chemical
nature, these are functional groups such as sulfonate or phosphate
esters.
[0295] The amphoteric liposomes contain variable amounts of such
membrane-forming or membrane-based amphiphilic materials, so that
they have an amphoteric character. This means that the liposomes
can change the sign of the charge completely. The amount of charge
carrier of a liposome, present at a given pH of the medium, can be
calculated using the following formula:
z=.SIGMA.ni((qi-1)+10.sup.(pK-pH)/(1+10.sup.(pL-pH)) [0296] in
which [0297] qi is the absolute charge of the individual ionic
groups below their pK (for example, carboxyl=0, simple nitrogen
base=1, phosphate group of the second dissociation step=-1, etc.)
[0298] ni is the number of these groups in the liposome.
[0299] At the isoelectric point, the net charge of the liposome is
0. Structures with a largely selectable isoelectric point can be
produced by mixing anionic and cationic portions.
[0300] In one embodiment, cationic components include DPIM, CHIM,
DORIE, DDAB, DAC-Chol, TC-Chol, DOTMA, DOGS,
(C18).sub.2Gly.sup.+N,N-dioctadecylamido-glycine, CTAB, CPyC, DODAP
DMTAP, DPTAP, DOTAP, DC-Chol, MoChol, HisChol and DOEPC. In another
embodiment, cationic lipids include DMTAP, DPTAP, DOTAP, DC-Chol,
MoChol and HisChol.
[0301] pH sensitive cationic lipids are disclosed in PCT
International Publication Numbers WO 02/066490 as well as in and WO
03/070220, the contents of both of which are incorporated herein by
reference.
[0302] pH sensitive cationic lipids can be compounds having the
structure of the formula
L-X-spacer1-Y-spacer2-HET [0303] wherein: [0304] L is a sterol or
[aliphatic(C(O)O)-].sub.2alkyl-; [0305] Sterol is selected from the
group consisting of cholesterol, sitosterol, campesterol,
desmosterol, fucosterol, 22-ketosterol, 20-hydroxysterol,
sigmasterol, 22-hydroxycholesterol, 25 hydroxycholesterol,
lanosterol, 7-dehydrocholesterol, dihydrocholesterol,
19-hydroxycholesterol, 5.alpha.cholest-7-en-3.beta.-ol,
7-hydroxycholesterol, epicholesterol, ergosterol dehydroergosterol,
and derivatives thereof; [0306] Each spacer 1 and spacer 2 is
independently an unsubstituted aliphatic; [0307] Each X and Y is
independently absent, --(C.dbd.O)--O--, --(C.dbd.O)--NH--,
--(C.dbd.O)--S--, --O--, --NH--, --S--, --CH.dbd.N--,
--O--(O.dbd.C)--, --S--(O.dbd.C)--, --NH--(O.dbd.C)--, .dbd.CH--,
--CH.sub.2--, .dbd.N--O--. .dbd.N--NH--, .dbd.N--NH--(C.dbd.O)--,
NH--SO.sub.2--, S(O).sub.n--, S(O).sub.2--NH-- or --N.dbd.CH--; and
[0308] HET is an amino, an optionally substituted
heterocycloaliphatic or an optionally substituted heteroaryl.
[0309] In some aspects, the HET is an optionally substituted
heterocycloaliphatic including at least one nitrogen ring atom, or
an optionally substituted heteroaryl including at least one
nitrogen ring atom. In other aspects, the HET is morpholinyl,
piperidinyl, piperazinlyl, pyrimidinyl or pyridinyl. In another
aspect, the cationic lipid has the structure
Sterol-X-spacer1-Y-spacer2-morpholinyl or
Sterol-X-spacer1-Y-spacer2-imidazolyl. In still further aspects,
the sterol is cholesterol.
[0310] In one embodiment, X is --O--, Spacer 1 and Spacer 2 are
(CH.sub.2).sub.2, Y is --(C.dbd.O)--NH--, and HET is morpholinyl.
In another embodiment, X is .dbd.CH--, Spacer 1 and Spacer 2 are
(CH.sub.2).sub.2, Y is --(C.dbd.O)--NH--, and HET is morpholinyl.
In yet another embodiment, X is --CH.sub.2--, Spacer 1 and Spacer 2
are (CH.sub.2).sub.2, Y is --(C.dbd.O)--NH--, and HET is
morpholinyl. In still another embodiment, X is .dbd.N--O--, Spacer
1 is --CH.sub.2--, Y is --(C.dbd.O)--NH--, Spacer 2 is
(CH.sub.2).sub.2 HET is morpholinyl. In still yet another
embodiment, X is .dbd.N--NH--, Spacer 1 is --CH.sub.2--, Y is
--(C.dbd.O)--NH--, Spacer 2 is (CH.sub.2).sub.2 and HET is
morpholinyl. In a further embodiment, X is .dbd.N--NH--(C.dbd.O)--,
Spacer 1 is --CH.sub.2--, Y is --(C.dbd.O)--NH--, Spacer 2 is
(CH.sub.2).sub.2 and HET is morpholinyl. In still a further
embodiment, X is --NH--(C.dbd.O)--, Spacer 1 is --CH.sub.2--, Y is
--(C.dbd.O)--NH--, Spacer 2 is (CH.sub.2).sub.2 and HET is
morpholinyl. In an even further embodiment, X is --NH--, Spacer 1
and Spacer 2 are (CH.sub.2).sub.2, Y is --(C.dbd.O)--NH--, and HET
is morpholinyl. In another embodiment, X is
--NH--(SO.sub.2).sub.n--, Spacer 1 is --CH.sub.2--, Y is
--(C.dbd.O)--NH--, Spacer 2 is (CH.sub.2).sub.2 and HET is
morpholinyl, wherein n is 1 or 2. In yet another embodiment, X is
--S(O.sub.2)--NH--, Spacer 1 is --CH.sub.2--, Y is
--(C.dbd.O)--NH--, Spacer 2 is (CH.sub.2).sub.2 and HET is
morpholinyl. The above compounds can be synthesized using syntheses
of 1 or more steps, and can be prepared by one skilled in the
art.
[0311] In another embodiment, pH sensitive cationic lipids can be
compounds having the structure of the formula
L-X-spacer1-Y-spacer2-HET [0312] wherein: [0313] L is a structure
according to the general formula
[0313] ##STR00004## [0314] wherein R1 and R2 are independently
C8-C30 alkyl or acyl chains with 0, 1 or 2 ethylenically
unsaturated bonds and M is absent, --O--(C.dbd.O);
--NH--(C.dbd.O)--; --S--(C.dbd.O)--; --O--; --NH--; --S--;
--N.dbd.CH--; --(O.dbd.C)--O--; --S--(O.dbd.C)--;
--NH--(O.dbd.C)--; --N.dbd.CH-- and/or --S--S--; [0315] Sterol is
selected from the group consisting of cholesterol, sitosterol,
campesterol, desmosterol, fucosterol, 22-ketosterol,
20-hydroxysterol, sigmasterol, 22-hydroxycholesterol, 25
hydroxycholesterol, lanosterol, 7-dehydrocholesterol,
dihydrocholesterol, 19-hydroxycholesterol,
5.alpha.cholest-7-en-3.beta.-ol, 7-hydroxycholesterol,
epicholesterol, ergosterol dehydroergosterol, and derivatives
thereof; [0316] Each spacer 1 and spacer 2 is independently an
unsubstituted aliphatic with 1-8 carbon atoms; [0317] X is absent
and Y is absent, --(C.dbd.O)--O--; --(C.dbd.O)--NH--;
--NH--(C.dbd.O)--O--; --O--; --NH--; --CH.dbd.N--;
--O--(O.dbd.C)--; --S--; --(O.dbd.C)--; --NH--(O.dbd.C)--;
--O--(O.dbd.C)--NH--, --N.dbd.CH-- and/or --S--S--; and [0318] HET
is an amino, an optionally substituted heterocycloaliphatic or an
optionally substituted heteroaryl.
[0319] In some aspects, the HET is an optionally substituted
heterocycloaliphatic including at least one nitrogen ring atom, or
an optionally substituted heteroaryl including at least one
nitrogen ring atom. In other aspects, the HET is morpholinyl,
piperidinyl, piperazinlyl, pyrimidinyl or pyridinyl. In another
aspect, the cationic lipid has the structure
Sterol-X-spacer1-Y-spacer2-morpholinyl or
Sterol-X-spacer1-Y-spacer2-imidazolyl. In still further aspects,
the sterol is cholesterol.
[0320] The amphoteric mixtures further comprise anionic lipids,
either constitutively or conditionally charged in response to pH,
and such lipids are also known to those skilled in the art. In one
embodiment, lipids for use with the invention include DOGSucc,
POGSucc, DMGSucc, DPGSucc, DMPS, DPPS, DOPS, POPS, DMPG, DPPG,
DOPG, POPG, DMPA, DPPA, DOPA, POPA, CHEMS, CetylP, DGSucc, and
combinations thereof.
[0321] 3. Neutral Lipids
[0322] Neutral lipids include any lipid that remains neutrally
charged at a pH between about 4 and 9. Neutral lipids include,
without limitation, cholesterol, other sterols and derivatives
thereof, phospholipids, and combinations thereof and other neutral
lipids. The phospholipids include any one phospholipid or
combination of phospholipids capable of forming liposomes. They
include phosphatidylcholines, phosphatidylethanolamines, lecithin
and fractions thereof, phosphatidic acid, phosphatidylglycerols,
phosphatidylinolitols, phosphatidylserines, plasmalogens and
sphingomyelins. The phosphatidylcholines include, without
limitation, those obtained from egg, soy beans or other plant
sources or those that are partially or wholly synthetic or of
variable lipid chain length and unsaturation, POPC, OPPC, natural
or hydrogenated soy bean PC, natural or hydrogenated egg PC, DMPC,
DPPC, DSPC, DOPC and derivatives thereof. In one embodiment,
phosphatidylcholines are POPC, non-hydrogenated soy bean PC and
non-hydrogenated egg PC. Phosphatidylethanolamines include, without
limitation, DOPE, DMPE and DPPE and derivatives thereof.
Phosphatidylglycerols include, without limitation, DMPG, DLPG,
DPPG, and DSPG. Phosphatidic acids include, without limitation,
DSPA, DMPA, DLPA and DPPA.
[0323] Sterols include cholesterol derivatives such as
3-hydroxy-5,6-cholestene and related analogs, such as
3-amino-5,6-cholestene and 5,6-cholestene, cholestane, cholestanol
and related analogs, such as 3-hydroxy-cholestane; and charged
cholesterol derivatives such as cholesteryl-beta-alanine and
cholesterol hemisuccinate.
[0324] Other neutral lipids include .alpha.-tocopherols and
derivatives, such as a-tocopherol acetate.
[0325] In another embodiment neutral lipids include without
limitation, DOPE, POPC, soy bean PC or egg PC and cholesterol.
[0326] B. DNAi Oligonucleotides 1. Regulatory Regions of the bcl-2
Gene
[0327] The bcl-2 gene has two promoters designated P1 and P2. P1
from which most bcl-2 mRNA is transcribed is located approximately
1.4 kb upstream of the translation initiation site and P2 is 1.3 kb
downstream of P1. (See Seto, M. et al. EMBO J. 7, 123-131 (1988).)
P1 is GC-rich, lacks a TATA box, has many transcription start sites
and includes seven consensus binding sites for the SP1
transcription factor. P2 includes a CCAAT box and a TATA box and
has two different transcription initiation sites. There are
multiple NF-.kappa.B recognition sites and an SV40 enhancer-like
octamer motif within P2. (See Heckman, C. A., et al. Oncogene 21,
3898-3908 (2002).) (See SEQ ID NO:1254). Most human follicular
lymphomas contain t(14; 18) chromosomal translocations that result
from 3'-bcl-2 gene region breakpoints. (See Tsujimoto, Y. et al.
Proc. Natl. Acad. Sci. U.S.A. 84, 1329-1331 (1987).) These
translocations place bcl-2 expression under control of the
immunoglobulin heavy chain (IgH) locus enhancer resulting in
upregulation of bcl-2 expression. Alternatively, there are 5'-bcl-2
breakpoint regions that result from fusions with either the IgH
locus or two different immunoglobulin light chain (IgL) loci that
are found in some DLCL lymphoma patient isolates. (See Yonetani, N.
et al. Jpn. J. Cancer Res. 92, 933-940 (2001).) These 5'-bcl-2
breakpoints have been mapped in separate heterogeneous patient
isolates to a region spanning 378 to 2312 by upstream of the
translation initiation site. (See SEQ ID NOs:1255-1266.) Regions
around the breakpoints may be sequences that can be used for bcl-2
DNAi oligonucleotide design.
[0328] The upstream regions of TGF-.alpha., c-ki-ras, c-myc,
c-erb-2 (Her-2), and c-Ha-ras can also be investigated to find
regions to which DNAi oligonucleotides could bind based on
preferred design criteria.
[0329] 2. DNAi Oligonucleotide Design
[0330] The DNAi oligonucleotides, in some embodiments, are DNA
oligomers that are complementary to either the plus strand or minus
strand of double stranded DNA. The DNAi oligonucleotide may
hybridize to regulatory regions of the c-ki-ras, c-Ha-ras, c-myc,
her-2, TGF-.alpha., or bcl-2 gene. For the purposes of this
invention, those upstream regions are defined as SEQ ID NO:1 (for
her-2, or c-erb-2), SEQ ID NO:282 (for c-ki-ras), SEQ ID NO:462
(for c-Ha-ras), SEQ ID NO:936 (for c-myc), SEQ ID NO:1081 (for
TGF-.alpha.) and SEQ ID NOs:1249 and 1254 (for bcl-2), provided
that the DNAi oligonucleotide is a single stranded nucleic acid
oligonucleotide or derivative thereof, whose sequence is
complementary, in part, to a portion of the longest non-transcribed
region of a gene in which the oligonucleotide affects indirectly or
directly the expression, regulation or production of the same or
different gene, wherein the longest non-transcribed region includes
any portion of the gene that is not transcribed when the
transcriptional start site is the site closest to the translation
start site. DNAi oligonucleotides do not include RNAi and antisense
oligonucleotides that base pair only with mRNAs or pre-mRNAs and
interfere with RNA processing and/or message translation.
[0331] In some embodiments, the DNAi oligonucleotides may be
designed based on certain design criteria. Such DNAi
oligonucleotides can then be tested for efficacy using the methods
disclosed herein. For example, in some embodiments, the DNAi
oligonucleotides are methylated on at least one, two or all of the
CpG islands. In other embodiments, the DNAi oligonucleotides
contain no methylation. The present invention is not limited to a
particular mechanism. Indeed, an understanding of the mechanism is
not necessary to practice the present invention. Nonetheless, it is
contemplated that DNAi oligonucleotides in some embodiments are
those that have at least a 50% GC content and at least two GC
dinucleotides. Also, in some embodiments, the DNAi oligonucleotides
do not self hybridize. In further embodiments, the DNAi
oligonucleotides are designed with at least 1 A or T to minimize
self hybridization. In yet further embodiments, commercially
available computer programs are used to survey the DNAi
oligonucleotides for the ability to self hybridize. In still other
embodiments, the DNAi oligonucleotides are at least 10, or 15
nucleotides and no more than 100 nucleotides in length. In further
embodiments, DNAi oligonucleotides are 18-26 nucleotides in length.
In some embodiments, DNAi oligonucleotides comprise the universal
protein binding sequences CGCCC and CGCG or the complements
thereof.
[0332] In some embodiments, the DNAi oligonucleotides hybridize to
a regulatory region of a gene upstream from the TATA box of the
promoter. In further embodiments, DNAi oligonucleotides are
designed to hybridize to regulatory regions of an oncogene known to
be bound by proteins (e.g., transcription factors). In some
embodiments, the DNAi oligonucleotide compounds are not completely
homologous to other regions of the human genome. The homology of
the DNAi oligonucleotides to other regions of the genome can be
determined using available search tools (e.g., BLAST, available at
the internet site of NCBI).
[0333] The present invention is not limited to the specific DNAi
oligonucleotide sequences described herein. Other suitable DNAi
oligonucleotides may be identified (e.g., using the criteria
described above or other criteria). Candidate DNAi oligonucleotides
may be tested for efficacy using any suitable method. For example,
candidate DNAi oligonucleotides can be evaluated for their ability
to prevent cell proliferation at a variety of concentrations. In
some embodiments, DNAi oligonucleotides inhibit gene expression or
cell proliferation at a low concentration (e.g., less that 20
.mu.M, or 10 .mu.M in in vitro assays).
[0334] 3. DNAi Oligonucleotide Zones
[0335] In some embodiments, regions within the regulatory regions
of the oncogenes are further defined as regions for hybridization
of DNAi oligonucleotides. In some embodiments, these regions are
referred to as "hot zones."
[0336] In some embodiments, hot zones are defined based on DNAi
oligonucleotide compounds that are demonstrated to be effective
(see above section on DNAi oligonucleotides) and those that are
contemplated to be effective based on the criteria for DNAi
oligonucleotides described above. In further embodiments, hot zones
encompass 10 by upstream and downstream of each compound included
in each hot zone and have at least one CG or more within an
increment of 40 by further upstream or downstream of each compound.
In yet further embodiments, hot zones encompass a maximum of 100 bp
upstream and downstream of each oligonucleotide compound included
in the hot zone. In additional embodiments, hot zones are defined
at beginning regions of each promoter. These hot zones are defined
either based on effective sequence(s) or contemplated sequences and
have a preferred maximum length of 200 bp. Based on the above
described criteria, exemplary hot zones were designed. These hot
zones are shown in Table 1.
TABLE-US-00001 TABLE 1 Exemplary Hot Zones Gene Hot Zones Bcl-2
679-720, 930-1050, 1070-1280, 1420-1760 c-erbB-2 205-344, 382-435
c-K-ras 1-289, 432-658 c-Ha-ras 21-220, 233-860, 1411-1530,
1631-1722 c-myc 3-124, 165-629 TGF-.alpha. 1-90, 175-219, 261-367,
431-930, 964-1237
[0337] 4. DNAi Oligomers
[0338] In one aspect, the DNAi oligonucleotides can be any DNAi
oligomer that hybridizes under physiological conditions to the
following sequences: SEQ ID NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ
ID NO:936, SEQ ID NO:1081, SEQ ID NOs:1249 and/or 1254. In another
aspect, the DNAi oligonucleotides can be any DNAi oligomer that
hybridizes under physiological conditions to exemplary hot zones in
SEQ ID NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ ID NO:936, SEQ ID
NO:1081 and SEQ ID NO:1249. Examples of DNai oligomers include,
without limitation, those DNAi oligomers listed in SEQ ID NOs
2-281, 283-461, 463-935, 937-1080, 1082-1248, 1250-1253 and
1267-1447 and the complements thereof. In another aspect, the DNAi
oligonucleotides are SEQ ID NOs 2-22, 283-301, 463-503, 937-958,
1082-1109, 1250-1254 and 1270-1447 and the complements thereof. In
an embodiment of these aspects, the DNAi oligonucleotides are from
15-35 base pairs in length.
[0339] For the bcl-2 gene, the DNAi oligomers can include any DNAi
oligomer that hybridizes to SEQ ID NOs: 1249 or 1254. In another
aspect, the DNAi oligomer can be any oligomer that hybridizes to
nucleotides 500-2026, nucleotides 500-1525, nucleotides 800-1225,
nucleotides 900-1125, nucleotides 950-1075 or nucleotides 970-1045
of SEQ ID NO:1249 or the complement thereof. In another aspect, the
DNAi oligonucleotides can be any DNAi oligomer that hybridizes
under physiological conditions to exemplary hot zones in SEQ ID
NO:1249. Examples of DNAi oligomers include, without limitation,
those DNai oligomers listed in SEQ ID NOs 1250-1253 and 1267-1447
and the complements thereof. In an embodiment of these aspects, the
DNAi oligonucleotides are from 15-35 base pairs in length.
[0340] In another embodiment, the DNAi oligomer can be SEQ ID
NO:1250, 1251, 1252, 1253, 1267-1447 or the complement thereof. In
yet another embodiment, the DNAi oligomer can be SEQ ID NO:1250,
1251, 1267, 1268, 1276, 1277, 1285, 1286 or the complement thereof.
In still another embodiment, the DNAi oligomer can be SEQ ID NOs
1250, 1251, 1289-1358 or the complements thereof. In an additional
embodiment the DNAi oligomer can be SEQ ID NO:1250 or 1251.
[0341] In a further embodiment of these aspects, the DNAi oligomer
has the sequence of the positive strand of the bcl-2 sequence, and
thus, binds to the negative strand of the sequence.
[0342] In other aspects, the DNAi oligomers can include mixtures of
DNAi oligonucleotides. For instance, the DNAi oligomer can include
multiple DNAi oligonucleotides, each of which hybridizes to
different parts of SEQ ID NOs 1249 and 1254. DNAi oligomers can
hybridize to overlapping regions on those sequences or the DNAi
oligomers may hybridize to non-overlapping regions. In other
embodiments, DNAi oligomers can be SEQ ID NOs 1250, 1251, 1252,
1253, 1267-1447 or the complement thereof, wherein the mixture of
DNAi oligomers comprises DNAi oligomers of at least 2 different
sequences.
[0343] In other embodiments, the DNAi oligomer can include a
mixture of DNAi oligomers, each of which hybridizes to a regulatory
region of different genes. For instance, the DNAi oligomer can
include a first DNAi oligomer that hybridizes to SEQ ID NO:1249 or
1254 and a second DNAi oligomer that hybridizes to a regulatory
region of a second gene. In some embodiments, the DNAi oligomer
includes a DNAi oligomer of SEQ ID NOs 1250-1254 or 1267-1447 or
the complements thereof, and a DNAi oligomer that hybridizes to SEQ
ID NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ ID NO:936, or SEQ ID
NO:1081 or the complement thereof. In other embodiments, the DNAi
oligomer includes SEQ ID NO 1250 or 1251 or the complement thereof
and a DNAi oligomer that hybridizes to SEQ ID NO:1, SEQ ID NO:282,
SEQ ID NO:462, SEQ ID NO:936, or SEQ ID NO:1081 or the complement
thereof. In yet other embodiments, the DNAi oligomer includes SEQ
ID NO:1250 or 1251 or the complement thereof and any of SEQ ID NOs
2-281, 283-461, 463-935, 937-1080 and 1082-1248, or the complement
thereof.
[0344] In some embodiments, the present invention provides DNAi
oligonucleotide therapeutics that are methylated at specific sites.
The present invention is not limited to a particular mechanism.
Indeed, an understanding of the mechanism is not necessary to
practice the present invention. Nonetheless, it is contemplated
that one mechanism for the regulation of gene activity is
methylation of cytosine residues in DNA. 5-methylcytosine (5-MeC)
is the only naturally occurring modified base detected in DNA
(Ehrlick et al., Science 212:1350-1357 (1981)). Although not all
genes are regulated by methylation, hypomethylation at specific
sites or in specific regions in a number of genes is correlated
with active transcription (Doerfler, Ann. Rev. Biochem. 52:93-124
[1984]; Christman, Curr. Top. Microbiol. Immunol. 108:49-78 [1988];
Cedar, Cell 34:5503-5513 [1988]). DNA methylation in vitro can
prevent efficient transcription of genes in a cell-free system or
transient expression of transfected genes. Methylation of C
residues in some specific cis-regulatory regions can also block or
enhance binding of transcriptional factors or repressors (Doerfler,
supra; Christman, supra; Cedar, Cell 34:5503-5513 (1988); Tate et
al., Curr. Opin. Genet. Dev. 3:225-231 [1993]; Christman et al.,
Virus Strategies, eds. Doerfler, W. & Bohm, P. (VCH, Weinheim,
N.Y.) pp. 319-333 [1993]).
[0345] Disruption of normal patterns of DNA methylation has been
linked to the development of cancer (Christman et al., Proc. Natl.
Acad. Sci. USA 92:7347-7351 [1995]). The 5-MeC content of DNA from
tumors and tumor derived cell lines is generally lower than normal
tissues (Jones et al., Adv. Cancer Res 40:1-30 [1983]).
Hypomethylation of specific oncogenes such as c-myc, c-Ki-ras and
c-Ha-ras has been detected in a variety of human and animal tumors
(Nambu et al., Jpn. J. Cancer (Gann) 78:696-704 [1987]; Feinberg et
al., Biochem. Biophys. Res. Commun. 111:47-54 [1983]; Cheah et al.,
JNCI73:1057-1063 [1984]; Bhave et al., Carcinogenesis (Lond)
9:343-348 [1988]. In one of the best studied examples of human
tumor progression, it has been shown that hypomethylation of DNA is
an early event in development of colon cancer (Goetz et al.,
Science 228:187-290 [1985]). Interference with methylation in vivo
can lead to tumor formation. Feeding of methylation inhibitors such
as L-methionine or 5-azacytodine or severe deficiency of
5-adenosine methionine through feeding of a diet depleted of
lipotropes has been reported to induce formation of liver tumors in
rats (Wainfan et al., Cancer Res. 52:2071s-2077s [1992]). Studies
show that extreme lipotrope deficient diets can cause loss of
methyl groups at specific sites in genes such as c-myc, ras and
c-fos (Dizik et al., Carcinogenesis 12:1307-1312 [1991]).
Hypomethylation occurs despite the presence of elevated levels of
DNA MTase activity (Wainfan et al., Cancer Res. 49:4094-4097
[1989]). Genes required for sustained active proliferation become
inactive as methylated during differentiation and tissue specific
genes become hypomethylated and are active. Hypomethylation can
then shift the balance between the two states. In some embodiments,
taking advantage of this naturally occurring phenomena, the mixture
of the present invention may be adapted for site specific
methylation of specific gene promoters, thereby preventing
transcription and hence translation of certain genes. In other
embodiments, the mixture of the present invention may be adapted
for upregulating the expression of a gene of interest (e.g., a
tumor suppressor gene) by altering the gene's methylation
patterns.
[0346] The present invention is not limited to the use of
methylated DNAi oligonucleotides. Indeed, the use of non-methylated
DNAi oligonucleotides for the inhibition of gene expression is
specifically contemplated by the present invention.
[0347] The DNAi oligonucleotides can be in a naturally occurring
state, and can also contain modifications or substitutions in the
nucleobases, the sugar moiety and/or in the internucleoside
linkage.
[0348] Nucleobases comprise naturally occurring nucleobases as well
as non-naturally occurring nucleobases. Illustrative examples of
such nucleobases include without limitation adenine, cytosine,
5-methylcytosine, isocytosine, pseudoisocytosine, guanine, thymine,
uracil, 5-bromouracil, 5-propynyluracil, 5-propynylcytosine,
5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine,
2-aminopurine, inosine, diaminopurine, 7-deazaguanine,
7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 8-azaguanine,
8-azaadenine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine,
2-chloro-6-aminopurine, 4-acetylcytosine, 5-hydroxymethylcytosine,
8-hydroxy-N6-methyladenosine, aziridinylcytosine,
5-(carboxyhydroxyl-methyl) uracil, 5-fluorouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
N6-methyladenine, 7-methylguanine and other alkyl derivatives of
adenine and guanine, 2-propyl adenine and other alkyl derivatives
of adenine and guanine, 2-aminoadenine, 5-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5'-methoxycarbonylmethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid
methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,
queosine, 2-thiocytosine, 2-thiothymine, 5-halouracil,
5-halocytosine, 6-azo uracil, cytosine and thymine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
8-halo, 8-amino, 8-thiol, 8-hydroxyl and other 8-substituted
adenines and guanines, 5-trifluoromethyl uracil and cytosine,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
queosine, xanthine, hypoxanthine, 2-thiocytosine,
2,6-diaminopurine, 5-substituted pyrimidines, 6-azapyrimidines and
N-2, N-6 and 0-6 substituted purines, including
2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
[0349] The DNAi oligonucleotides can also have sugars other than
ribose and deoxy ribose, including arabinofuranose (described in
International Publication number WO 99/67378, which is herein
incorporated by reference), xyloarabinofuranose (described in U.S.
Pat. Nos. 6,316,612 and 6,489,465, which are herein incorporated by
reference), .alpha.-threofuranose (Schoning, et al. (2000) Science,
290, 1347-51, which is herein incorporated by reference) and
L-ribofuranose. Sugar mimetics can replace the sugar in the
nucleotides. They include cyclohexene (Wang et al. (2000) J. Am.
Chem. Soc. 122, 8595-8602; Vebeure et al. Nucl. Acids Res. (2001)
29, 4941-4947, which are herein incorporated by reference), a
tricyclo group (Steffens, et al. J. Am. Chem. Soc. (1997) 119,
11548-11549, which is herein incorporated by reference), a
cyclobutyl group, a hexitol group (Maurinsh, et al. (1997) J. Org.
Chem, 62, 2861-71; J. Am. Chem. Soc. (1998) 120, 5381-94, which are
herein incorporated by reference), an altritol group (Allart, et
al., Tetrahedron (1999) 6527-46, which is herein incorporated by
reference), a pyrrolidine group (Scharer, et al., J. Am. Chem.
Soc., 117, 6623-24, which is herein incorporated by reference),
carbocyclic groups obtained by replacing the oxygen of the furnaose
ring with a methylene group (Froehler and Ricca, J. Am. Chem. Soc.
114, 8230-32, which is herein incorporated by reference) or with an
S to obtain 4'-thiofuranose (Hancock, et al., Nucl. Acids Res. 21,
3485-91, which is herein incorporated by reference), and/or
morpholino group (Heasman, (2002) Dev. Biol., 243, 209-214, which
is herein incorporated by reference) in place of the pentofuranosyl
sugar. Morpholino oligonucleotides are commercially available from
Gene Tools, LLC (Corvallis Oreg., USA).
[0350] The DNAi oligonucleotides can also include "locked nucleic
acids" or LNAs. The LNAs can be bicyclic, tricyclic or polycyclic.
LNAs include a number of different monomers, one of which is
depicted in Formula I.
##STR00005## [0351] wherein [0352] B constitutes a nucleobase;
[0353] Z* is selected from an internucleoside linkage and a
terminal group; [0354] Z is selected from a bond to the
internucleoside linkage of a preceding nucleotide/nucleoside and a
terminal group, provided that only one of Z and Z* can be a
terminal group; [0355] X and Y are independently selected from
--O--, --S--, --N(H)--, --N(R)--, --CH.sub.2-- or --C(H).dbd.,
CH.sub.2--O--, --CH.sub.2--S--, --CH.sub.2--N(H)--,
--CH.sub.2--N(R)--, --CH.sub.2--CH.sub.2-- or --CH.sub.2--C(H)=,
--CH.dbd.CH--; [0356] provided that X and Y are not both O.
[0357] In addition to the LNA
[2'-Y,4'-C-methylene-.beta.-D-ribofuranosyl]monomers depicted in
formula XVIII (a[2,2,1]bicyclo nucleoside), an LNA or LNA*
nucleotide can also include "locked nucleic acids" with other
furanose or other 5 or 6-membered rings and/or with a different
monomer formulation, including 2'-Y,3' linked and 3'-Y,4' linked,
1'-Y,3 linked, 1'-Y,4' linked, 3'-Y,5' linked, 2'-Y, 5' linked,
1'-Y,2' linked bicyclonucleosides and others. All the above
mentioned LNAs can be obtained with different chiral centers,
resulting, for example, in LNA [3'-Y-4'-C-methylene (or
ethylene)-.beta. (or .alpha.)-arabino-, xylo- or
L-ribo-furanosyl]monomers. LNA oligonucleotides and LNA nucleotides
are generally described in International Publication No. WO
99/14226 and subsequent applications; International Publication
Nos. WO 00/56746, WO 00/56748, WO 00/66604, WO 01/25248, WO
02/28875, WO 02/094250, WO 03/006475; U.S. Pat. Nos. 6,043,060,
6,268,490, 6,770,748, 6,639,051, and U.S. Publication Nos.
2002/0125241, 2003/0105309, 2003/0125241, 2002/0147332,
2004/0244840 and 2005/0203042, all of which are incorporated herein
by reference. LNA oligonucleotides and LNA analogue
oligonucleotides are commercially available from, for example,
Proligo LLC 6200 Lookout Road, Boulder, Colo. 80301 USA.
[0358] The nucleotide derivatives of the DNAi oligonucleotides can
include nucleotides containing one of the following at the 2' sugar
position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-
or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and
alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl,
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10, C.sub.1 to C.sub.10 lower alkyl,
substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,
SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl,
2'-methoxyethoxy(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta
78:486 [1995]) i.e., an alkoxyalkoxy group,
2'-dimethylaminooxyethoxy (i.e., an
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group), also known as 2'-DMAOE,
and 2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2,
2'-methoxy(2'-O--CH.sub.3),
2'-aminopropoxy(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro
(2'-F). Similar modifications may also be made at other positions
on the DNAi oligonucleotide, particularly the 3' position of the
sugar on the 3' terminal nucleotide or in 2'-5' linked DNAi
oligonucleotides and the 5' position of 5' terminal nucleotide.
[0359] In some embodiments, the DNAi oligonucleotides have
non-natural internucleoside linkages. As defined in this
specification, oligonucleotides having modified backbones include
those that retain a phosphorus atom in the backbone and those that
do not have a phosphorus atom in the backbone. For the purposes of
this specification, modified oligonucleotides that do not have a
phosphorus atom in their internucleoside backbone can also be
considered to be oligonucleosides.
[0360] Some modified DNAi oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphoroselenates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free acid forms are also
included.
[0361] Other modified DNAi oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N,
O, S and CH.sub.2 component parts.
[0362] In yet other DNAi oligonucleotide mimetics, both the sugar
and the internucleoside linkage (i.e., the backbone) of the
nucleotide units are replaced with novel groups. The base units are
maintained for hybridization with an appropriate nucleic acid
target compound. One such oligomeric compound, an oligonucleotide
mimetic that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone. Representative patents that teach the
preparation of PNA compounds include, but are not limited to, U.S.
Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is
herein incorporated by reference. Further teaching of PNA compounds
can be found in Nielsen et al., Science 254:1497 (1991).
[0363] In some embodiments, DMAi oligonucleotides of the invention
are oligonucleotides with phosphorothioate backbones and
oligonucleosides with heteroatom backbones, and in particular
--CH.sub.2--, --NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2--, and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2-] of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240.
Oligonucleotides can also have a morpholino backbone structure of
the above-referenced U.S. Pat. No. 5,034,506.
[0364] In some embodiments the DNAi oligonucleotides have a
phosphorothioate backbone having the following general
structure.
##STR00006##
[0365] Another modification of the DNAi oligonucleotides of the
present invention involves adding additional nucleotides to the 3'
and/or 5' ends of the DNAi oligonucleotides. The 3' and 5' tails
can comprise any nucleotide and can be as short as one nucleotide
and as long as 20 nucleotides.
[0366] Yet another modification of the DNAi oligonucleotides of the
present invention involves chemically linking to the
oligonucleotide one or more moieties or conjugates that enhance the
activity, cellular distribution or cellular uptake of the
oligonucleotide. Such moieties include but are not limited to lipid
moieties such as a cholesterol moiety, cholic acid, a thioether,
(e.g., hexyl-S-tritylthiol), a thiocholesterol, an aliphatic chain,
(e.g., dodecandiol or undecyl residues), a phospholipid, (e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or a
polyethylene glycol chain or adamantane acetic acid, a palmityl
moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol
moiety.
[0367] One skilled in the relevant art knows well how to generate
oligonucleotides containing the above-described modifications. The
present invention is not limited to the DNAi oligonucleotides
described above. Any suitable modification or substitution may be
utilized, provided that the DNAi oligonucleotide is a single
stranded nucleic acid oligonucleotide or derivative thereof, whose
sequence is complementary, in part, to a portion of the longest
non-transcribed region of a gene in which the oligonucleotide
affects indirectly or directly the expression, regulation or
production of the same or different gene, wherein the longest
non-transcribed region includes any portion of the gene that is not
transcribed when the transcriptional start site is the site closest
to the translation start site. DNAi oligonucleotides do not include
RNAi and antisense oligonucleotides that base pair only with mRNAs
or pre-mRNAs and interfere with RNA processing and/or message
translation.
[0368] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within a DNAi
oligonucleotide. The present invention also includes pharmaceutical
compositions and formulations that include the DNAi oligonucleotide
compounds of the present invention as described below.
[0369] 5. Preparation and Formulation of DNAi Oligonucleotides
[0370] Any of the known methods of oligonucleotide synthesis can be
used to prepare the modified DNAi oligomers of the present
invention. In some embodiments utilizing methylated DNAi
oligonucleotides the nucleotide, dC is replaced by 5-methyl-dC
where appropriate, as taught by the present invention. The modified
or unmodified DNAi oligonucleotides of the present invention are
most conveniently prepared by using any of the commercially
available automated nucleic acid synthesizers. They can also be
obtained from commercial sources that synthesize custom
oligonucleotides pursuant to customer specifications.
[0371] In additional embodiments, chemotherapeutic agents,
including docetaxel and others can be combined with DNAi oligomers
before or while sequestering in liposomes.
[0372] C. Amphoteric Liposome Formulations
[0373] 1. Description
[0374] Advantageously, the amphoteric liposome formulations of the
mixture of the present invention (1) exhibit low toxicity; (2) can
sequester high concentrations of DNAi oligomers e.g., the
efficiency of sequestering the DNAi oligonucleotides associated
with the amphoteric liposomes is at least about 35%; (3) are stable
in the bloodstream, such as when administered systemically, such
that the oligonucleotide and/or other agents are stably sequestered
in the liposomes until eventual uptake in the target tissue or
cells; (4) can be optimized for delivery to animals, such as by
adjusting the concentration of sequestered DNAi oligonucleotide to
between about 1 to 4 mg/ml (such as about 2 mg/ml) for a lipid
concentration of about 10 to 100 mM or less which provides dosing
at 10 mg/kg in 200 .mu.l of injection volume; and (5) can be
produced with an average amphoteric liposome size that is smaller
than 200 .eta.m, such as about 100 .eta.m, which maximizes tumor
penetration.
[0375] As described above, the amphoteric liposomes include one or
more DNAi oligonucleotides, one or more amphoteric lipids or a
mixture of anionic and cationic lipid components with amphoteric
properties and one or more neutral lipids.
[0376] In general, cationic lipids or positive charges on the
amphoteric lipids act to bind DNAi oligonucleotides. Anionic
lipids, such as CHEMS, or anionic charges on amphoteric lipids and
neutral lipids, such as phosphatidylethanolamines allow for the
fusogenic properties of the amphoteric liposomes.
[0377] In some embodiments of the present invention, the amphoteric
liposomes can be formed from a lipid phase comprising an amphoteric
lipid. The lipid phase can comprise 5 to 30 mole % or 10 to 25 mole
% of the amphoteric lipid. Alternatively, the amphoteric liposomes
can be formed from a lipid phase comprising a mixture of lipid
components with amphoteric properties. The total amount of charged
lipids may vary from 5 to 95 mole %, from 20 to 80 mole % or from
30 to 70 mole % of the lipid mixture.
[0378] The ratio of the percent of cationic lipids to anionic
lipids can be between about 3 and 0.5 or between about 2 to 0.5. In
some embodiments, the ratio of cationic lipids to anionic lipids is
about 2. In other embodiments, the ratio of cationic lipids to
anionic lipids is about 1. In other embodiments, the ratio of
cationic lipids to anionic lipids is about 0.5.
[0379] Specific pairs of cationic and anionic lipids include,
without limitation, MoChol and CHEMS, DOTAP and CHEMS, MoChol and
Cet-P, and MoChol and DMGSucc. Examples of charged lipid pairs
further include, without limitation, between about 10 to 60 mole %
of MoChol and between about 10 to 30 mole % of CHEMS; between about
5 to 30 mole % of DOTAP and between about 10 to 30 mole % of CHEMS;
between about 10 to 40 mole % MoChol and between about 5 to 30 mole
% Cet-P; and between about 20 to 60 mole % MoChol and between about
20 to 60 mole % DMGSucc.
[0380] The amphoteric liposomes also contain neutral lipids, which
can be either sterols or phospholipids, and mixtures thereof. The
amphoteric liposomes include neutral lipids in an amount between
about 5 to 95 mole % of the lipid mixture, between about 20 to 80
mole %, or between 30 and 70 mole %.
[0381] A number of neutral lipid combinations are useful in forming
the amphoteric liposomes, such as POPC and DOPE; and POPC and
cholesterol. In contrast, a combination of the neutral lipids DOPE
and cholesterol is not preferred. In some embodiments, the mixture
of neutral lipids includes 5 to 40 mole % POPC and 20 to 50 mole %
DOPE; or 10 to 50 mole % of POPC and 30 to 50 mole % of
cholesterol. The ratio of the percentage of charged lipids to
neutral lipids can be between about 3 and 0.2. In some embodiments,
the ratio of the percentage of charged lipids to neutral lipids is
about 2. In other embodiments, the ratio of the percentage of
charged lipids to neutral lipids is about 0.5.
[0382] Examples of specific combinations of charged and neutral
lipids for sequestering an DNAi oligomer, such as PNT-100 (SEQ ID
NO:1251), include POPC, DOPE, MoChol and CHEMS; POPC, DOPE, DMGSucc
and MoChol; POPC, DOTAP, CHEMS and cholesterol; and POPC, MoChol,
Cet-P and cholesterol. In some embodiments, the amphoteric liposome
for sequestering a DNAi oligomer, such as SEQ ID NO:1251, includes
3-20 mole % of POPC, 10 to 60 mole % of DOPE, 10 to 60 mole % of
MoChol and 10 to 60 mole % of CHEMS. The amphoteric liposome may
include POPC/DOPE/MoChol/CHEMS in molar ratios of about 6/24/47/23
and about 15/45/20/20. In another embodiment, the amphoteric
liposomes include 3-20 mole % of POPC, 10 to 40 mole % of DOPE, 15
to 60 mole % of MoChol and 15 to 60 mole % of DMGSucc. The
amphoteric liposome can include POPC/DOPE/DMGSucc/MoChol in molar
ratios of about 6/24/23/47 and about 6/24/47/23. In still another
embodiment, the amphoteric liposome includes 10 to 50 mole % of
POPC, 20 to 60 mole % of Chol, 10 to 40 mole % of CHEMS and 5 to 20
mole % of DOTAP. The amphoteric liposome can include
POPC/Chol/CHEMS/DOTAP in a molar ratio of about 30/40/20/10. In
still another embodiment, the amphoteric liposome includes 10 to 40
mole % of POPC, 20 to 50 mole % of Chol, 5 to 30 mole % of Cet-P
and 10 to 40 mole % of MoChol. The amphoteric liposome can include
POPC/Chol/Cet-P/MoChol in a molar ratio of about 35/35/10/20.
[0383] In general, any Amphoter I, II, or III lipid pair of
cationic and anionic lipids together with neutral lipids can be
used to form liposomes provided that the resulting liposome is
amphoteric, exhibits serum stability, has low toxicity, sequesters
an ample quantity of the DNAi oligonucleotides, e.g., at an
efficiency of about 35%, (about 5%, 10%, 15%, 20%, 25%, 30%, 35% or
higher) and provides for an adjustment of the DNAi oligonucleotide
concentration to at least 2 mg/ml for a lipid concentration of 100
mM or less.
[0384] 2. Preparation of the Amphoteric Liposomes
[0385] DNAi-amphoteric liposomes of the invention can be prepared
by standard methods for preparing and sizing liposomes known to
those skilled in the art. These include hydration of lipid films
and powders, solvent injection and reverse-phase evaporation. Often
multilamellar vesicles will form spontaneously when amphiphilic
lipids are hydrated, whereas the formation of small unilamellar
vesicles usually requires a process involving substantial energy
input, such as ultrasonication, high pressure homogenization,
injection of lipid solutions in ethanol into a water phase
containing the DNAi oligonucleotides to be sequestered and/or
extrusion through filters or membranes of defined pore size.
Methods for preparing and characterizing liposomes have been
described, for example, by S. Vemuri et al. (Preparation and
characterization of liposomes as therapeutic delivery systems: a
review. Pharm Acta Helv. 1995, 70(2):95-111).
[0386] A solution of the DNAi oligonucleotide may be contacted with
an excipient at a neutral pH, thereby resulting in a passive
loading procedure of a certain percentage of the solution. The use
of high concentrations of the excipient, ranging from about 50 mM
to about 150 mM, is one method to achieve substantial encapsulation
of the active agent. Excipients include substances that can
initiate or facilitate loading of DNAi oligonucleotides. Examples
of excipients include, without limitation, acid, sodium or ammonium
forms of monovalent aniond such as chloride, acetate, lactobionate
and formate; divalent anions such as aspartate, succinate and
sulfate; and trivalent ions such as citrate and phosphate.
[0387] Amphoteric liposomes used with the present invention offer
the distinct advantage of binding oligonucleotides at or below
their isoelectric point, thereby concentrating the active agent at
the liposome surface. The advanced loading procedure is described
in more detail in PCT International Publication Number
WO02/066012.
[0388] To form unilammellar liposomes, a shearing force is applied
to the aqueous dispersion of the DNAi-oligonucleotide lipid
mixture. The shearing force can be applied by sonication, using a
microfluidizing apparatus such as a homogenizer or French press,
injection, freezing and thawing, dialyzing away a detergent
solution from lipids, ultrafiltration, extrusion through filters,
or other known methods used to prepare liposomes. The size of the
liposomes can be controlled using a variety of known techniques,
including the duration of shearing force.
[0389] Unentrapped DNAi oligomers can be removed from the
amphoteric liposome dispersion by buffer exchange using dialysis,
size exclusion chromatography (e.g., Sephadex G-50 resin),
ultrafiltration (100,000-300,000 molecular weight cutoff), or
centrifugation.
[0390] In one embodiment, DNAi oligonucleotide loaded amphoteric
liposomes may be manufactured by a machine extrusion. Once the
lipids are mixed with the oligonucleotides, they may be extruded
using machine extrusion, where the machine is described in U.S.
Pat. No. 6,843,942 and US Patent Application No. 2004/0032037. The
liposomes are loaded and filtered so that the diameter of the
liposome is between 50 .eta.m and 200 .eta.m, the encapsulation
efficiency of the oligonucleotide is at least about 35% and the
resulting liposomes have a DNAi oligonucleotide concentration of at
least 2 mg/ml at a lipid concentration of 10 to 100 mM or less.
VII. Treating Animals or Cells with Amphoteric Liposomes
Sequestering DNAi Oligomers
[0391] The compositions of the invention are useful for treating
animals, including humans, or cells to treat cancer, such as by
inhibiting or reducing tumor growth. The animal can be a non-human
animal, including mice, horses, cats, dogs, or other animals or it
can be a human. In one embodiment, the mixture is introduced to the
animal at a dosage of between 1 mg to 100 mg/kg of body weight. In
another embodiment, the amphoteric liposomes can be introduced to
the animal one or more times per day or continuously.
[0392] The mixture can be administered to the animal via different
routes. Administration can be topical (including ophthalmic and to
mucous membranes including vaginal and rectal delivery), pulmonary
(e.g., by inhalation or insufflation of powders or aerosols,
including by nebulizer; intratracheal, intranasal, epidermal and
transdermal), oral or parenteral. Parenteral administration
includes intravenous, intraarterial, subcutaneous, intraperitoneal
or intramuscular injection or infusion; or intracranial, e.g.,
intrathecal or intraventricular, administration. Administration can
also be via a medical device.
[0393] The liposomes can be administered to cultured cells derived
from various cancers, including pancreatic cancer, colon cancer,
breast cancer, bladder cancer, lung cancer, leukemia, prostate
cancer, lymphoma, ovarian cancer or melanoma.
[0394] The liposomes can be used to target DNAi oligonucleotides to
selected tissues using several techniques. The procedures involve
manipulating the size of the liposomes, their net surface charge as
well as the route of administration. More specific manipulations
include labeling the liposomes with receptor ligands, including
membrane and nuclear receptor ligands or antibodies for specific
tissues or cells. Antibodies or ligands can be bound to the surface
of the liposomes.
[0395] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Materials
[0396] The synthesis of MoChol and HisChol were described in US
Patent Application No. 2004/0131666 (WO 02/066490). The other
lipids are available from commercial sources. For example, DOTAP
and Cholesterol are available from Merck, DMG-Succ is available
from Chiroblock GmbH, CHEMS can be obtained from Sigma Chemical
Company, DPPG, DOPE and POPC are available from Genzyme or Lipoid
GMBH and Egg phosphatidylcholine is available from Lipoid GMBH.
Example 2
Production of Amphoteric Liposomes Charged with DNAi
Oligonucleotides
[0397] The lipid composition of the liposomes as well as the
methods of preparing them are chosen so that the encapsulation
efficiency is about 35% or higher and the liposome size is smaller
than 200 .eta.m, and optimally near 100-120 .eta.m to maximize
tumor penetration. For administering to animals, the DNAi
oligonucleotide concentration is preferably at least 2 mg/ml at a
lipid concentration of 100 mM or less. This allows dosing at 10
mg/kg in 200 .mu.l of injection volume.
[0398] Liposomes are produced by a modified lipid
film/hydration/extrusion method. Lipids are dissolved in chloroform
or chloroform/methanol and dried completely in a rotary evaporator.
The lipid films are next hydrated with various amounts of the DNAi
oligonucleotides SEQ ID NO:1251 (PNT100) or the complement of
PNT-100 (PNT-100R) hydrated in buffer.
A. Advanced Loading Procedure
[0399] 2400 .mu.mole of lipid is hydrated with 10 mM NaOAc, 150 mM
NaCl (pH adjusted using citrate) containing 48.8 to 95.2 mg of DNAi
oligonucleotides for 30 min. at 40.degree. C. After three
freeze-thaw steps, the resulting multilamellar vesicles is passed
several times through a polycarbonate membrane (100 .eta.m pore
size) using high pressure pumps. Immediately after the extrusion
step, the pH of the liposome suspension is shifted to pH 7.5. The
resulting suspension is sedimented at 25s using T865 (Sorvall Ultra
Pro80) or TLA 100.4 rotors (Beckman Optima-MAX) to remove
unsequestered DNAi oligonucleotide and to exchange the buffer with
phosphate buffered saline (PBS).
B. Passive Loading Procedure
[0400] The lipid film is hydrated with PBS containing 405 mg of
DNAi oligonucleotides for 1 hr at 40.degree. C. at a final lipid
concentration of 100 mM. After three freeze-thaw steps, the
resulting vesicles are extruded through a polycarbonate membrane
stack containing different pore sizes between 100 and 800 nm. The
resulting suspension is sedimented three times at 25 s. (PBS is
Phosphate Buffered Saline, which has the formula: 10.1 mM
Na.sub.2PO.sub.4, 1.76 mM KH.sub.2PO.sub.4, 137 mM NaCl, 2.68 mM
KCl, pH 7.5.)
C. Machine Extrusion
[0401] The vesicles are prepared by either the passive or advanced
loading procedure and extruded using a device for producing lipid
vesicle.
[0402] Particle properties were measured using a Zetasizer 3000 HAS
(Malvern). Liposomes were diluted in appropriate buffer to a final
lipid concentration of 0.2-0.6 mM. Size values are recorded as Z
average and size distribution was calculated in the Multimodal
mode. For Zeta potential measurement, liposomes were also diluted
to 0.2-0.6 mM concentration.
TABLE-US-00002 TABLE 2 Liposome Formulations Formulation
Composition Molar Ratios A POPC/DOPE/MoChol/CHEMS 15/45/20/20 B
POPC/DOTAP/CHEMS/Chol 30/10/20/40 C POPC/MoChol/Cet-P/Chol
35/20/10/35 D POPC/DOPE/MoChol/CHEMS 6/24/47/23 E
POPC/DOPE/MoChol/DMG-Succ 6/24/47/23 F POPC/DOPE/MoChol/DMG-Succ
6/24/23/47
[0403] For all formulations in Table 2, active loading using either
manual or machine extrusion in general gave better results. The
encapsulation efficiencies ranged from 37-77%, liposome size ranged
from 124-201 .eta.m and the DNAi oligonucleotide concentration at a
lipid concentration of 100 mM ranged from 1.1 to 3.5 mg/ml. Machine
extrusion gave similar results as manual extrusion with the
possible exception that machine extrusion resulted in more uniform
liposome size, ranging from 135-179 .eta.m. Machine extrusion is
preferred for larger volumes.
[0404] The passive loading procedure resulted in lower
encapsulation efficiencies, ranging from 11-21%. However, liposome
size ranged from 122-182 nm and the oligonucleotide concentration
at a lipid concentration of 100 mM ranged from 2.0-3.7 mg/ml. All
formulations that were passively loaded were manually extruded
because attempts at machine extrusion created a high back
pressure.
[0405] The advanced loading procedure could not be used for all
formulations because of the low loading capacity of formulations
that contain less than 20% cationic lipid. Consequently,
formulation B with DOTAP at 10%, could not be loaded efficiently by
the advanced loading procedure, and the passive loading procedure
was used.
[0406] A ratio of cationic lipid charge to anionic nucleotide
charge at low pH (N/P) of 3.3 was found to be the best compromise
to produce small particles, high encapsulation efficiency and DNAi
oligonucleotide concentration to lipid concentration of at least
2.0 mg/ml of DNAi oligonucleotide at 10 to 100 mM lipid
concentration.
D. Preparation of PNT 2254 and PNT 2253
[0407] Liposomes are produced with a modified ethanol injection
method. Briefly, 3 volumes of ethanol, containing the lipid mixture
D (POPC/DOPE/MoChol/Chems 6:24:47:23) (133 mM, heated to 55.degree.
C.) and 8 volumes of 20 mM NaAc/300 mM Sucrose/pH 4, containing
2.71 mg/ml PNT100 (SEQ ID NO:1251) or PNT100-R (SEQ ID NO: 1288) in
case of PNT2254 or PNT2254R production, or containing 1.36 mg/ml
PNT100 in case of PNT2253 production, were continuously mixed using
an injection device as disclosed in U.S. Pat. No. 6,843,942 and US
patent application No. 2004/0032037. The acidic mixture was shifted
to pH 7.5 by an additional continuous mixing step with 32 volumes
of 100 mM NaCl/136 mM Phosphate/pH 9. The resulting liposomal
suspension was concentrated 10 fold and dialyzed against PBS, pH
7.4 to wash out non encapsulated PNT100 or PNT100-R and excess
ethanol.
Example 3
Serum Resistance of and Leakage of DNAi Oligonucleotides from
Amphoteric Liposomes
[0408] The lipid ratios can be optimized for both stability of the
liposomes in serum and minimal leakage of the DNAi
oligonucleotides. The above formulations are stable in serum and
can exhibit minimal leakage of oligonucleotide.
Example 4
Response of WSU-DLCL2 Tumors to PNT-100
[0409] Three formulations which met the specifications of at least
2 mg/ml of encapsulated PNT-100 (SEQ ID NO:1251), greater than 40%
encapsulation efficiency and less than 200 .eta.m particle size
(formulations B, D, and F, see example 2) were tested in a human
lymphoma model. Lymphoma cells (WSU-DLCL.sub.2--Wayne State
University Diffuse Large Cell Lymphoma) were obtained from Dr.
Ramzi Mohammad, Karmanos Cancer Institute, Wayne State University.
Xenografts were transplanted subcutaneously into C17/SCID mice.
Seven days after transplantation, mice were injected intravenously
with 10 mg/kg of the PNT-100 (SEQ ID NO:1251) formulations and 10
mg/kg of PNT-100R (SEQ ID NO:1288) formulations. The injections
were performed daily for 8 days in six mice. The size of the tumors
were measured up to 30 days after implantation. All animals
survived with no gross toxic pathology.
[0410] Results in FIG. 1 show that PNT-100 slows tumor growth.
340.9 and 340.8 are formulations with PNT-100 and PNT-100R,
respectively. Formulation D with PNT-100 slowed tumor growth better
and was less toxic than formulations B and F. (Data not shown.)
[0411] Experiments done with other lots of PNT-100-liposome
formulation D gave similar results, as shown in FIG. 2. Mice were
administered 10 mg/kg PNT2253 daily for eight days, an i.v. bolus
injection and tumor volume response was caliper measured (left
panel). Data show 57% tumor growth inhibition at day 28 post
xenograft transplantation or 14 days post drug treatment (n=6;
p=0.004). Mice were administered 10 mg/kg PNT2253 daily for five
days an i.v. bolus injection and tumor response was caliper
measured. Data shows 46% tumor growth inhibition at day 26 post
xenograft transplantation or 19 days post drug treatment (n=8;
p=0.007). Studies were concluded when control animal xenografts
reached >2000 mm.sup.3.
[0412] The tumor burden was calculated from the size measurements
of the tumors. FIG. 3 shows that the tumor burden in mice treated
with PNT2253, which is PNT-100 in formulation D, was dramatically
less than the tumor burden in mice treated with PNT2253R (PNT-100R
in formulation D) or PBS.
[0413] A dose response experiment was performed in WSU-DLCL2
xenograft bearing mice with PNT-100 in formulation D, with a
PNT-100 concentration of 4 mg/ml (PNT2254) and 2 mg/ml (PNT2253).
C.B.-17 ACID mice between 6-8 weeks old were supplied by Taconic
(Hudson, N.Y.). When the tumors reached approximately 100 mm3
volume, treatment with PNT2253 or PNT2254 was initiated. The mice
received 0, 0.3, 3, 10, or 20 mg/kg of PNT2254 daily for five days,
30 mg/kg of PNT2254 daily for 2 days, 60 mg/kg of PNT2254 once,
0.3, 3, or 10 mg/kg of PNT2253 daily for 5 days, 20 mg/kg of
PNT2253 daily for 2 days, or 30 mg/kg of PNT2253 once via an iv
bolus injection. (n=7 (PNT2254) or 8 (PNT2255). The animals were
checked at least three times weekly for tumor growth by caliper
measurements, and the animals were weighed at least three times
weekly. Tumor volumes of all treatment groups were analyzed using
GraphPad.TM. statistical software.
[0414] A maximum tolerated dose of 20 mg/kg/day of PNT2254 and 10
mg/kg/day of PNT2253 was established. (FIGS. 4 and 5.) Toxicity was
achieved at 30 mg/kg/day for PNT2254 and at 20 mg/kg/day for
PNT2253, and dosing was stopped after two days due to animal
efficacy. A steep dose response was seen with strong anti-tumor
efficacy for an extended time period after one dosing cycle. The
effect of the two formulations at various dosages on body weight of
the mice was determined and is shown in FIG. 6. For both
formulations, a dose of 10 mg/kg/day was efficacious while causing
minimal weight loss.
[0415] A mathematical measure of each dose was calculated that
determined the drug response in delaying tumor growth rate to 750
mg size in PNT2254 and PNT2253 drugged vs. control non-drugged
tumors (Tables 3 and 4).
TABLE-US-00003 TABLE 3 Antitumor Activity of PNT2254 in
WSU-DLCL.sub.2-Bearing SCID Mice Log.sub.10 No. of T/C kill Agent
Animals (%) T - C gross PBS control daily for 5 days 7 100 0.0 0.0
0.3 mg/kg PNT2254 daily for 5 days 7 100 0.0 0.0 3 mg/kg PNT2254
daily for 5 days 7 75 3 0.45 10 mg/kg PNT2254 daily for 5 days 7 34
10 1.5 20 mg/kg PNT2254 daily for 5 days 7 32 10 1.5 30 mg/kg
PNT2254 daily for 2 days 5 27 11 1.65 (5/7 mice survived)
TABLE-US-00004 TABLE 4 Antitumor Activity of PNT2253 in
WSU-DLCL.sub.2-Bearing SCID Mice Log.sub.10 No. of T/C kill Agent
Animals (%) T - C gross PBS control daily for 5 days 8 100 0.0 0.0
0.3 mg/kg PNT2253 daily for 5 days 8 92 0.0 0.0 3 mg/kg PNT2253
daily for 5 days 8 90 2 0.3 10 mg/kg PNT2253 daily for 5 days 8 38
9 1.4 20 mg/kg PNT2253 daily for 2 days 6 28 12 1.8 (6/8 mice
survived 30 mg/kg PNT2253 daily for 1 day 8 -- -- -- (8/8 dead)
[0416] T and C are the median times in days for the treatment group
(T) and the control group (C) tumors to reach a predetermined
weight (750 mg). T-C is a measure of tumor growth delay and is the
difference in the median days to 750 mg between the treated (T) and
the control (C) group. Log.sub.10 kill Gross=T-C value in
days/3.32.times.T.sub.d. T.sub.d is the mean tumor doubling time
(days) estimated from a log-linear growth plot of the control
tumors growing in exponential phase. The higher the Log.sub.10 kill
Gross value, the more efficacious the drug, and a value over 2.8 is
considered highly efficacious (Corbett, T. H. et al.,
"Transplantable Syngeneic Rodent Tumors". Tumor Models in Cancer
Research. Ed. Teicher B. A. Totowa, N.J.: Humana Press Inc., 2002.
41-71). Volume and weight were calculated according to the formula
described by Cammisuli, S., et al., Int. J. Cancer, 65, 351-9,
1996.
[0417] PNT2253 treatment resulted in increased toxicity compared to
PNT2254. The most efficacious dose was 10 mg/kg/day for both
PNT2253 and PNT2254, and the maximum tolerated dose is 20 mg/kg/day
for PNT2254 and 10 mg/kg/day of PNT2253.
Example 5
Response of PC-3 Tumors to PNT-100
[0418] The different formulations were tested in a PC-3 human
prostate carcinoma model. Xenografts were generated by
sub-cutaneous injection of 2.times.10.sup.6 PC-3 cells (ATCC CRL
1435) into nude mice. Mice bearing 50-200 mm.sup.3 xenografts were
injected intravenously with 10 mg/kg of PNT-100 (SEQ ID NO:1251) or
PNT-100R (SEQ ID NO:1288) in one of the formulations B, D or F on
days 1, 2, and 5 and with 7.5 mg/kg on days 3 and 4. Results show a
decrease in tumor growth with PNT-100, but not with PNT-100R (FIGS.
7 and 8). N=5.
Example 6
Toxicity in Monkeys
[0419] Toxicity of PNT-100 in formula D was explored in Cynomolgus
monkeys. Two primates were treated via two hour i.v. infusion with
PBS control, 5 mg/kg PNT2254, 25 mg/kg PNT2254, and one primate was
treated with 67 mg/kg PNT2254. There was a one week "washout"
period between each dosing. Liver enzymes toxicology analysis,
complement activation, and gross behavior and physiology
measurements were collected before and after each treatment. The
purpose of the study was to establish a maximum tolerated dose
threshold, and to ensure that there was not a CARPA toxic response
to the PNT2254 lipids. CARPA is a toxic response that is
historically known to result from a non-classical complement
pathway activation toxic response that can cause extreme
hypertension and death. The primates tolerated and survived all
doses and only a classical complement activation and not
non-classical (innate) complement activation was detected. The
liver enzyme toxicology analysis demonstrated modest increases in
liver enzyme response to PNT2254.
Other Embodiments
[0420] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages and modifications are
within the scope of the following claims.
[0421] All references cited herein are incorporated herein by
reference in their entirety.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150118291A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150118291A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References