The present invention provides methods for inhibiting the growth
of breast cancer cells and methods for treating breast cancers expressing
Wilms' Tumor 1 (WT1) gene product using a WT1 antisense oligonucleotide.
It further provides methods of predicting breast cancer progression
and methods for the screening of candidate substances for activity
against breast cancer.
What is claimed is:
1. A method of inhibiting the growth of a breast cancer cell expressing
a Wilms' Tumor 1 (WT1) gene product comprising contacting said cell
with an amount of a WT1 antisense molecule effective to inhibit
the growth of the breast cancer cell.
2. The method of claim 1, wherein said WT1 antisense molecule is
3. The method of claim 1, wherein said WT1 antisense molecule is
4. The method of claim 1 wherein the antisense molecule is produced
from an expression vector encoding said antisense under the control
of a promoter active in said cell.
5. The method of claim 4, wherein said promoter is a constitutive
6. The method of claim 5, wherein said constitutive promoter is
a CMV promoter, an RSV promoter, an SV40 promoter.
7. The method of claim 4, wherein said promoter is a tissue specific
8. The method of claim 7, wherein said tissue specific promoter
is leptin gene promoter, IGF binding protein-3 promoter, adenomatous
polyposis coli gene promoter.
9. The method of claim 4, wherein said promoter is an inducible
10. The method of claim 9, wherein said inducible promoter is Tet-On
system, Tet-Off system.
11. The method of claim 1, wherein said breast cancer cell is estrogen
12. The method of claim 1, wherein said breast cancer cell is estrogen
13. The method of claim 2, wherein said DNA is an oligonucleotide.
14. The method of claim 13, wherein said oligonucleotide is 6 to
about 50 bases in length.
15. The method of claim 13, wherein said oligonucleotide comprises
one or more modifed bases.
16. The method of claim 1, wherein said antisense molecule hybridizes
to a WT1 transcript.
17. The method of claim 16, wherein said antisense molecule hybridizes
to a translation initiation site or a splice site.
18. The method of claim 1, wherein said antisense molecule hybridizes
to a WT1 genomic sequence.
19. The method of claim 18, wherein said antisense molecule hybridizes
to a transcription start site, an intron, an exon, or an intron-exon
20. The method of claim 2, wherein said DNA is a double-stranded
21. The method of claim 2, wherein said DNA is a single-stranded
22. The method of claim 4, wherein said expression vector is a
23. The method of claim 4, wherein said expression vector is a
24. The method of claim 23, wherein said viral vector is selected
from the group consisting of adenovirus, retrovirus, herpesvirus,
vaccinia virus, adeno-associated virus, lentivirus and polyoma virus.
25. The method of claim 1, wherein said antisense molecule is associated
with one or more lipid.
26. The method of claim 25, wherein said antisense molecule is
encapsulated in a liposome.
27. The method of claim 25, wherein the lipid comprises at least
one neutrally charged lipid.
28. The method of claim 27, wherein said neutrally charged lipid
29. The method of claim 25, further defined as comprising more
than one lipids wherein the lipids on a whole are neutrally charged.
30. The method of claim 17, wherein said antisense molecule hybridizes
to a translation initiation site and comprises 5'-GTCGGAGCCCATTTGCTG-3'.
31. The method of claim 30, wherein said antisense molecule consists
32. The method of claim 1, wherein said cell expresses multiple
33. The method of claim 1, wherein said cell expresses one or more
adverse oncogene products.
34. A method of treating a subject having a breast cancer tumor,
cells of which express a Wilms' Tumor 1 (WT1) gene product, comprising
administering to said subject an effective amount of a WT1 antisense
35. The method of claim 34, wherein said antisense molecule is
administered to said tumor by intratumoral injection.
36. The method of claim 34, wherein said antisense moleclue is
administered to the tumor vasculature.
37. The method of claim 34, wherein said antisense molecule is
administered locally to said tumor.
38. The method of claim 34, wherein said antisense molecule is
administered regionally to said tumor.
39. The method of claim 34, wherein said antisense molecule is
administered to the lymphatic system locally or regionally to said
40. The method of claim 34, further comprising administering to
said subject a second breast cancer therapy.
41. The method of claim 40, wherein said second breast cancer therapy
is chemotherapy, radiation therapy, immunotherapy, hormonal therapy,
or gene therapy.
42. The method of claim 40, wherein said second breast cancer therapy
is provided to said subject prior to said WT1 antisense molecule.
43. The method of claim 40, wherein said second breast cancer therapy
is provided to said subject after said WT1 antisense molecule.
44. The method of claim 40, wherein said second breast cancer therapy
is provided to said subject at the same time as said WT1 antisense
45. A method of predicting breast cancer progression in a subject
having breast cancer comprising: (a) obtaining a sample from said
subject comprising breast cancer tumor cells; and (b) assessing
expression of one or more isoforms of Wilms' Tumor 1 (WT1) gene
product in said cells.
46. The method of claim 45, wherein assessing comprises measuring
WT1 protein levels.
47. The method of claim 44, wherein measuring comprises quantitative
48. The method of claim 45, wherein assessing comprises measuring
WT1 mRNA levels.
49. The method of claim 48, wherein measuring comprises quantitative
50. A method of screening a candidate substance for activity against
breast cancer comprising: (i) providing a cell that expresses one
or more isoforms of the Wilms' Tumor 1 (WT1) gene product; (ii)
contacting the cell with the candidate substance suspected of inhibiting
WT1; and (iii) measuring the effect of the candidate substance on
the cell. wherein a decrease in the amount of WT1 gene product in
said cell, as compared to a cell not treated with said candidate
substance, indicates that said candidate substance has activity
against breast cancer.
51. The method of claim 50, wherein said candidate substance is
a protein, a nucleic acid or a small molecule pharmaceutical.
52. The method of claim 50, wherein measuring comprises determining
the level of a WT1 gene product in said cell.
53. The method of claim 50, wherein said cell is a breast cancer
 The present application claims priority to provisional U.S.
Patent Application Serial No. 60/345,102 filed Jan. 3, 2002. The
entire text of the above referenced applications are incorporated
herein by reference and without disclaimer.
BACKGROUND OF THE INVENTION
 1. Field of the Invention
 The present invention relates generally to the fields of
cancer therapy, specifically treatment of breast cancer. More particularly,
these treatments involve the use of antisense oligonucleotides against
the Wilms' Tumor 1 (WT1) gene, and lipid associated and liposomal
 2. Description of Related Art
 Breast cancer is the second most common form of cancer among
women in the U.S., and the second leading cause of cancer deaths
among women. Although several forms of radiation-therapy and chemotherapy
are available for the treatment of such cancers, these therapies,
especially when used in high doses, have side effects such as killing
non-cancerous cells. When used in lower doses, they may not be enough
to eradicate the cancer completely. Gene therapy is another form
of anti-cancer therapy that has been receiving much attention. However,
for a gene therapy to be effective it is necessary to identify genes
and gene products that are involved in the disease and may be targeted
 Wilms' Tumor is a pediatric kidney cancer arising from pluripotent
embryonic renal precursors (Lee et al., 2001). WT1 is a Wilms' Tumor
gene that was isolated from chromosome 11p13 by a positional cloning
technique (Call et al., 1990; Gessler et al., 1990). Abnormalities
of the WT1 gene are found in approximately 10% of patients with
Wilms' tumor and the WT1 has been categorized to be a tumor suppressor
gene (Haber et al., 1990; Little et al., 1992).
 It has been shown that WT1 participates in leukemogenesis
and all leukemic cells express high levels of WT1 expression (Inoue
et al., 1994). It has also been shown that a WT1 antisense oligomer
suppresses and inhibits growth of leukemia cells (U.S. Pat. No.
6,034,235; Yamagami et al., 1996).
 Oji et al., (1999), have determine the role of the Wilms'
tumor gene WT1 in tumorigenesis of solid tumors, by examining the
expression of the WT1 gene in 34 solid tumor cell lines including
four gastric cancer cell lines, five colon cancer cell lines, 15
lung cancer cell lines, four breast cancer cell lines, one germ
cell tumor cell line, two ovarian cancer cell lines, one uterine
cancer cell line, one thyroid cancer cell line, and one hepatocellular
carcinoma cell line. WT1 gene expression was detected in three of
the four gastric cancer cell lines, all of the five colon cancer
cell lines, 12 of the 15 lung cancer cell lines, two of the four
breast cancer cell lines, the germ cell tumor cell line, the two
ovarian cancer cell lines, the uterine cancer cell line, the thyroid
cancer cell line, and the hepatocellular carcinoma cell line. Furthermore,
when a gastric cancer cell line AZ-521, a lung cancer cell line
OS3, and an ovarian cancer cell line TYK-nu were treated with WT1
antisense oligomers, the growth of these cells was significantly
inhibited in association with a reduction in WT1 protein levels.
Thus, there is indication that the WT1 gene plays an oncogenic role
in the growth of several types of solid tumors.
 It has been recently shown that the expression of high levels
of the WT1 mRNA is associated with invasive breast cancers with
poor patient prognosis (Miyoshi et al., 2002). However, the role
of WT1 antisense molecules as possible treatments for breast cancer
has not been investigated. As current cancer therapies have only
limited therapeutic benefits, especially with regard to breast cancers,
there exists a need for a treatment that is specific for different
types of breast tumors.
SUMMARY OF THE INVENTION
 The present invention overcomes these and other defects
in the art and demonstrates that antisense WT1 molecules are effective
in inhibiting cancer cell growth in breast cancers expressing the
Wilms' Tumor 1 (WT1) gene.
 Thus, provided are methods for treating and/or preventing
breast cancer. The invention also provides methods for diagnosing
breast cancer and methods for screening for substances with activity
against breast cancer.
 In some embodiments, methods of inhibiting the growth of
breast cancer cells expressing a WT1 gene product comprising contacting
the cell with an amount of a WT1 antisense molecule effective to
inhibit the growth of the breast cancer cell are provided.
 An "effective amount" is defined here as an amount
of a WT1 antisense molecule that will decrease, reduce, inhibit
or otherwise abrogate the growth of a cancer cell, arrest-cell growth,
induce apoptosis, inhibit metastasis, induce tumor necrosis, kill
cells or induce cytotoxicity in cancer cells.
 In some aspects of these embodiments, the cell may express
one or more WT1 isoforms and/or one or more adverse oncogenes. The
present invention contemplates that the growth of any breast cancer
cell expressing a WT1 gene product may be inhibited. Thus, the breast
cancer cell may be estrogen negative. Alternatively, the breast
cancer cell may be estrogen positive.
 In some embodiments, the WT1 antisense molecule may be a
double stranded or single stranded DNA. In some specific embodiments,
the DNA may be an oligonucleotide wherein the oligonucleotide may
be 6 to about 50 bases in length comprising one or more modified
bases. In other embodiments, the WT1 antisense molecule may be an
 The antisense molecule may be produced from an expression
vector encoding the WT1 antisense molecule under the control of
a promoter active in the cell.
 Any promoter active in a breast cancer cell may be used.
However, some non-limiting examples are provided. For example, in
some embodiments of the method, one may use a constitutive promoter,
such as, a CMV promoter, an RSV promoter, or an SV40 promoter. In
other embodiments, the promoter may be a tissue-specific promoter
such as leptin gene promoter, IGF binding protein-3 promoter, adenomatous
polyposis coli gene promoter. In yet other embodiments, the promoter
may be an inducible promoter, for example, Tet-On system, Tet-Off
 Expression vectors for the expression of antisense molecules
as set forth herein are well known to one of skill in the art. In
some embodiments, the expression vector may be a non-viral vector
and/or a viral vector. Some examples of viral vectors include adenoviral
vectors, retroviral vectors, herpesviral vectors, vaccinia viral
vectors, adeno-associated viral vectors, lentiviral vectors or polyoma
 In some embodiments of the method, the antisense molecule
may hybridize to a WT1 transcript, a translation initiation site
that may comprise 5'-GTCGGAGCCCATTTGCTG-3' (SEQ ID NO: 1), a splice
site, a genomic sequence, a transcription start site, an intron,
an exon, and/or an intron-exon junction.
 In other embodiments, the antisense molecule may be associated
with one or more lipid molecules. In some specific aspects, the
lipid may comprise at least one neutrally charged lipid. One example
of a neutrally charged lipid is dioleoylphosphatidylcholine (DOPC).
Other neutrally charged lipids known in the art may also be used.
This includes lipids such as phosphatidylcholines, phosphatidylglycerols,
 In yet other aspects, the WT1 antisense molecule may be
associated with more than one lipids wherein the lipids on a whole
are neutrally charged. For example, the lipid component can comprise
a mixture of positively and negatively charged lipids such that
the overall charge of the lipid component is neutral.
 In yet other embodiments, the antisense molecule may be
encapsulated in a liposome. In some specific embodiments, the liposome
may be comprised of at least one or more neutrally charged lipid
 Another embodiment of the invention also provides methods
of treating a subject having a breast cancer which express a Wilms'
Tumor 1 (WT1) gene product, comprising administering to the subject
an amount of an WT1 antisense molecule that is effective to treat
 The term "treat cancer" is defined as a decrease
in cancer cell growth, reduction in cancer cell growth, inhibition
or abrogation of growth of a cancer cell, cancer cell growth arrest,
induction of apoptosis, killing of cancer cells, inhibition of metastasis,
induction of tumor necrosis, and/or induction of cytotoxicity in
 In such embodiments, the antisense molecule or formulations
thereof may be administered to the tumor by intratumoral injection.
In other embodiments, it may be administered to the tumor vasculature.
In some other embodiments, it may be administered locally to the
tumor. In yet other embodiments, it may be administered regionally.
In other embodiments, it may be administered to the lymphatic system
locally or regionally to the tumor.
 In yet other embodiments, the antisense molecule or formulations
thereof may be administered to the subject having such a tumor by
systemic or parenteral methods of administration. This includes
among others intravenous, intraarterial, intramuscular, intraperitoneal
routes of administration.
 The composition may advantageously be delivered to a human
patient in a volume of 0.50-10.0 ml per dose, or in an amount of
5-100 mg antisense oligonucleotide per m.sup.2 or 5-30 mg antisense
oligonucleotide per m.sup.2. Thus, one may administer 5 mg/m.sup.2,
6 mg/m.sup.2, 7 mg/m.sup.2, 8 mg/m.sup.2, 9 mg/m.sup.2, 10 mg/m.sup.2,
11 mg/m.sup.2, 12 mg/m.sup.2, 13 mg/m.sup.2, 14 mg/m.sup.2, 15 mg/m.sup.2,
16 mg/m.sup.2, 17 mg/m.sup.2, 18 mg/m.sup.2, 19 mg/m.sup.2, 20 mg/m.sup.2,
21 mg/m.sup.2, 22 mg/m.sup.2, 23 mg/m.sup.2, 24 mg/m.sup.2, 25 mg/m.sup.2,
26 mg/m.sup.2, 27 mg/m.sup.2, 28 mg/m.sup.2, 29 mg/m.sup.2, 30 mg/m.sup.2,
35 mg/m.sup.2, 40 mg/m.sup.2, 45 mg/m.sup.2, 50 mg/m.sup.2, 55 mg/m.sup.2,
60 mg/m.sup.2, 65 mg/m.sup.2, 70 mg/m.sup.2, 75 mg/m.sup.2, 80 mg/m.sup.2,
85 mg/m.sup.2, 90 mg/m.sup.2, 95 mg/m.sup.2, or 100 mg/m.sup.2 of
a WT1 antisense oligonucleotide. Of course intermediate ranges are
also contemplated as useful and this includes ranges such as 10.5
mg/m.sup.2, 92 mg/m.sup.2, and the like. As will be appreciated
by one of skill in the art, the final dose of administration will
be determined by a skilled physician depending on the disease status
and individual suffering from the disease taking into effect factors
such as age, sex, and the like. The composition may further be administered
multiply, daily, weekly and/or monthly. As an example, it is contemplated
that one particular therapeutic regimen the composition may be administered
3 times per week for 8 weeks.
 It is also contemplated that the therapeutic methods may
further comprise administering to a subject a second breast cancer
therapy such as chemotherapy, radiation therapy, immunotherapy,
hormonal therapy and/or gene therapy. Such methods are well known
to a person of ordinary skill in the art and are also described
elsewhere in the specification.
 In some embodiments of the invention, the second breast
cancer therapy may be provided to the subject prior to the WT1 antisense
molecule. In other embodiments, the second breast cancer therapy
may be provided to the subject after the WT1 antisense molecule.
In yet other embodiments, the second breast cancer therapy may be
provided to the subject at the same time as said WT1 antisense molecule.
 The present invention also provides methods of predicting
breast cancer progression in a subject having breast cancer that
comprise obtaining a sample from the subject comprising breast cancer
tumor cells and assessing expression of one or more isoforms of
Wilms' Tumor 1 (WT1) gene product in the cells. In some embodiments,
the assessing comprises measuring WT1 protein levels. In other embodiments,
the assessing comprises measuring WT1 mRNA levels. In some embodiments,
measuring these levels may comprise quantitative immunodetection
methods and/or quantitative PCR. All these methods are known to
a person of ordinary skill in the art and are also described elsewhere
in the specification.
 The present invention also provides methods of screening
candidate substances for growth inhibitory activity against breast
cancer comprising providing a cell that expresses one or more isoforms
of the Wilms' Tumor 1 (WT1) gene product, contacting the cell with
the candidate substance suspected of inhibiting WT1 and measuring
the effect of the candidate substance on the cell wherein a decrease
in the amount of WT1 gene product in the cell, as compared to a
cell not treated with the candidate substance, indicates that the
candidate substance has activity against breast cancer. The candidate
substance may be a protein, a polypeptide, a nucleic acid and/or
a small molecule pharmaceutical. In some embodiments of this method,
the measuring may comprise determining the level of a WT1 gene product
in the cell and/or determining the level of a WT1 gene transcript
in the cell and/or determining the level of more than one WT1 gene
product and/or determining the level of more than one WT1 transcript
isoform and/or measuring the level of WT1 gene product in a cell
not treated with the candidate substance.
 "A" or "an" is defined herein to mean
one or more than one. Other objects, features and advantages of
the present invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration only,
since various changes and modifications within the spirit and scope
of the invention will become apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
 The following drawings form part of the present specification
and are included to further demonstrate certain aspects of the present
invention. The invention may be better understood by reference to
one or more of these drawings in combination with the detailed description
of specific embodiments presented herein.
 FIG. 1. Western blot analysis of WT1 expression in nuclear
extracts of breast cancer cells. Nuclear mitotic apparatus protein
(NUMA) was used as an internal control.
 FIGS. 2A, 2B, 2C, and 2D. Growth inhibition of breast cancer
cell lines by L-WT1. FIG. 2A. K562 cells--light bars: L-control;
dark bars: L-WT1. FIG. 2B. MDA-MB-453 (.quadrature.), and MCF-7
(.DELTA.) cells treated with L-control oligos; MDA-MB-453(.box-solid.)
and MCF-7 (.tangle-solidup.) cells treated with L-WT1 oligos. FIG.
2C. Effect of 12 .mu.M L-WT1 in 9 breast cancer cell lines. light
bars: L-control; dark bars: L-WT1. FIG. 2D. Western blot of WT1
protein expression in MCF-7 and MDA-MB-453 cells exposed to L-WT1
and L-control oligos.
 FIG. 3. Reduction in numbers of breast cancer cells by L-WT1.
MCF-7 and MDA-MB-453 cells were treated with 12 .mu.M L-WT1 or L-control
oligos for 3 days and observed under light microscopy.
 FIGS. 4A, 4B, and 4C. Expression of WT1 mRNA isoforms in
breast cancer cell lines. ER-positive cell lines: 1: MCF-7; 2: BT-474;
3: T-47D; 4: MDA-MB-361. ER-negative cell lines: 5: SKBr-3; 6: MDA-MB-231;
7: MDA-MB-453; 8: BT-20; 9: MDA-MB-468, and 10: K562 leukemic cells.
FIG. 4A. Results from a single round of RT-PCR analysis of total
WT1 mRNA in breast cancer cell lines. FIG. 4B. Results from nested
RT-PCR analysis of the KTS+ and the KTS- isoforms of WT1 mRNA. FIG.
4C. Results from nested RT-PCR analysis of all 4 isoforms of WT1
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
 As mentioned above, breast cancer is the second most common
form of cancer among women in the U.S., and the second leading cause
of cancer deaths among women. While many therapies exist, these
are either insufficient to eradicate the disease or are too toxic
or both. Thus, there is a need to provide improved therapies and
to better predict the progression of breast cancer.
I. THE PRESENT INVENTION
 The Wilms' Tumor 1 (WT1) gene modulates the expression of
several genes involved in mammary glands. The inventors have identified
a role for WT1 in the proliferation of breast cancer cells. The
present invention provides a therapy that makes use of antisense
oligonucleotides to reduce WT1 protein expression and induce growth
inhibition of breast cancer cells. A particular method for delivering
these antisense molecules is in association with lipids and in some
embodiments via liposomes.
 Some breast cancer cells are estrogen receptor (ER)-positive
and some are ER-negative. While WT1 is expressed in higher levels
in ER-positive cells, liposomal WT1 (L-WT1) is effective at inhibiting
proliferation of breast cancer cells irrespective of their ER status.
In addition, it is contemplated that the L-WT1 will be useful in
inhibiting even those cells that have a high level of expression
of adverse oncogenes such as EGFR, Her2/neu, and the mutant p53
protein. Thus, this technology holds great promise as a therapeutic
agent for the treatment of cancer.
 The present invention further contemplates the prediction
of breast cancer progression in an individual having breast cancer
by assessing expression of one or more isoforms of Wilms' Tumor
1 (WT1) gene product in said cells.
 It also contemplates a method of screening a substance for
its ability to suppress the WT1 protein expression in a cancer cell
thus acting as a potential inhibitor of breast cancer. The invention,
in its various embodiments, is described in greater detail below.
II. WILMS' TUMOR GENE (WT1)
 The chromosome 11p13 Wilms' Tumor susceptibility gene (WT1)
appears to play a crucial role in regulating the proliferation and
differentiation of nephroblasts and gonadal tissue. When present
in the germline, specific heterozygous dominant-negative mutations
are associated with severe abnormalities of renal and sexual differentiation,
pointing to the essential role of WT1 for normal genitourinary development.
 WT1 encodes a protein migrating around 50 kDa, which contains
two domains with apparent functional properties: a C-terminal domain
that consists of four Cys.sub.2-His.sub.2 zinc finger domains involved
in DNA binding and an N-terminal proline/glutamine-rich transactivational
domain. The zinc finger domains have a high degree of homology to
the early growth response 1 and 2 products (Sukhatme et al., 1988;
Joseph et al., 1988). The coding sequence is comprised of 10 exons,
with each zinc finger encoded by an individual exon. Each of the
four zinc finger domains is contained within a separate exon. The
genomic structure of the zinc finger domains has been analyzed in
which a small deletion has been detected (Haber et al., 1990). The
analysis demonstrated that each zinc finger is separated from the
next by a short intron.
 Two alternative pre-mRNA splicing events give rise to four
distinct transcripts or isoforms. Alternative splice I consists
of 51 nucleotides, encoding 17 amino acids, including 5 serines
and 1 threonine, potential sites of protein phosphorylation. The
proline rich amino-terminus domain is encoded by the first exon
alone, and the 51 nucleotides of alternative splice I compose exon
5. Splice I is inserted between the proline-rich amino terminus
of the predicted protein and the first zinc finger domain.
 Alternative splice II, results from the use of a variable
splice donor site between exon 9 and 10, leading to the insertion
of three amino acids, lysine-threonine-serine (commonly referred
to as KTS), between third and fourth zinc finger. This insertion
disrupts the critical spacing between these zinc fingers resulting
in the loss of DNA binding to the consensus WT1 DNA-binding sequence
(Wang et al., 1995).
 The presence of two alternative splices in the WT1 trancript
may reflect a degree of complexity in gene product function. The
molecular mechanisms resulting in alternative mRNA splicing are
poorly understood, but are thought to reflect both nucleotide sequence
information contained in the splice junction, as well as cell type-specific
regulatory factors (Breitbart et al., 1987).
 Genetic evidence suggests that WT1 mutations, deletions,
or imbalances among the different WT1 isoforms may alter the transcriptional-regulator
function of WT1 leading to developmental abnormalities and possibly
cancer (Klamt et al., 1998; Guan et al., 1998; Liu et al., 1999).
High expression of WT1 has been correlated with poor prognosis and
increased drug resistance in acute myeloid leukemia (Inoue et a.,
1994), probably because increased WT1 expression can stimulate the
proliferation and block the differentiation of leukemic cells (Yamagami
et al., 1996). Therefore, WT1 seems to act as both a tumor suppressor
gene and an oncogene in certain types of malignancies. Recently,
two groups have reported that breast cancer cells also express WT1
protein, but they did not describe the function of WT1 in breast
cancer cells (Silberstein et al., 1997; Loeb et al., 2001).
III. ANTISENSE CONSTRUCTS
 The term "antisense" is intended to refer to oligonucleotide
or polynucleotide molecules complementary to a portion of a WT1
RNA, or the DNA's corresponding thereto. "Complementary"
oligonucleotides are those which are capable of base-pairing according
to the standard Watson-Crick complementarity rules. That is, the
larger purines will base pair with the smaller pyrimidines to form
combinations of guanine paired with cytosine (G:C) and adenine paired
with either thymine (A:T) in the case of DNA, or adenine paired
with uracil (A:U) in the case of RNA. Inclusion of less common bases
such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine
and others in hybridizing sequences does not interfere with pairing.
 As used herein, the terms "complementary" or "antisense"
mean oligonucleotides that are substantially complementary over
their entire length and have very few base mismatches. For example,
sequences of seven bases in length may be termed complementary when
they have a complementary nucleotide for five or six positions out
of seven. Naturally, sequences which are "completely complementary"
will be sequences which are entirely complementary throughout their
entire length and have no base mismatches.
 Alternatively, the hybridizing segments may be shorter oligonucleotides.
While all or part of the gene sequence may be employed in the context
of antisense construction, it is important that the antisense when
constructed binds/hybridizes the target sequence and does not face
interference from other sequences that may be present in the gene
sequence. Statistically, any sequence 17 bases long should occur
only once in the human genome and, therefore, suffice to specify
a unique target sequence. Although shorter oligomers are easier
to make and increase in vivo accessibility, numerous other factors
are involved in determining the specificity of hybridization. Both
binding affinity and sequence specificity of an oligonucleotide
to its complementary target increases with increasing length. It
is contemplated that oligonucleotides of 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 base pairs
will be used. In the present invention, SEQ ID NO: 1 is the sequence
of the WT1 antisense oligos targeted against the translation initiation
site and SEQ ID NO: 2 is the sequence of the control oligos. One
can readily determine whether a given antisense nucleic acid is
effective in targeting of the corresponding host cell gene simply
by testing the constructs in vitro to determine whether the endogenous
gene's function is affected or whether the expression of related
genes having complementary sequences is affected.
 Targeting double-stranded (ds) DNA with oligonucleotides
leads to triple-helix formation; targeting RNA will lead to double-helix
formation. Antisense oligonucleotides, when introduced into a target
cell, specifically bind to their target oligonucleotide and interfere
with transcription, RNA processing, transport, translation and/or
stability. Antisense RNA constructs, or DNA encoding such antisense
RNA's, may be employed to inhibit gene transcription or translation
or both within a host cell, either in vitro or in vivo, such as
within a host animal, including a human subject.
 The intracellular concentration of monovalent cation is
approximately 160 mM (10 mM Na.sup.+; 150 mM K.sup.+). The intracellular
concentration of divalent cation is approximately 20 mM (18 mM Mg.sup.++;
2 mM Ca.sup.++). The intracellular protein concentration, which
would serve to decrease the volume of hybridization and, therefore,
increase the effective concentration of nucleic acid species, is
150 mg/ml. Constructs can be tested in vitro under conditions that
mimic these in vivo conditions.
 Antisense constructs may be designed to hybridize to a WT1
transcript, a translation initiation site, a splice site, a WT1
genomic sequence, a start site, an intron, an exon or an intron-exon
 Hybridization is a process by which two complementary nucleic
acid strands, such as DNA and DNA, RNA and DNA or RNA and RNA, recognize
and bind to each other and form a double stranded structure. Intracellular
hybridization is the basis of antisense therapy. This involves the
administration/delivery of an antisense nucleic acid to a cell where
the antisense molecule finds its complementary target-nucleic acid,
which may be either DNA or RNA, and hybridizes to it thereby preventing
further transcription or translation of the target-nucleic acid.
In a particular embodiment of the invention, it is contemplated
that the most effective antisense constructs for the present invention
will include regions complementary to portions of the mRNA start
site. One can readily test such constructs simply by testing the
constructs in vitro to determine whether levels of the target protein
are affected. Similarly, detrimental non-specific inhibition of
protein synthesis also can be measured by determining target cell
viability in vitro. It is envisioned that hybridization of the antisense
oligonucleotides of the present invention to the translation initiation
site of mRNA will be the basis of the antisense-gene therapy aimed
at WT1 mediated diseases. Intracellular hybridization will prevent
the transcription of mRNA and thereby decrease the protein content
in the cell to which the antisense oligonucleotide is administered.
 Other sequences with lower degrees of homology also are
contemplated. For example, an antisense construct which has limited
regions of high homology, but also contains a non-homologous region
(e.g., a ribozyme) could be designed. These molecules, though having
less than 50% homology, would bind to target sequences under appropriate
 As mentioned above, the oligonucleotides according to the
present invention may encode a WT1 gene or a portion of that gene
that is sufficient to effect antisense inhibition of expression
of WT1 protein. These oligonucleotides may be derived from genomic
DNA, i.e., cloned directly from the genome of a particular organism.
In other embodiments, however, the oligonucleotides may be complementary
DNA (cDNA). cDNA is DNA prepared using messenger RNA (mRNA) as template.
Thus, a cDNA does not contain any interrupted coding sequences and
usually contains almost exclusively the coding region(s) for the
corresponding protein. In other embodiments, the antisense oligonucleotide
may be produced synthetically.
 It may be advantageous to combine portions of the genomic
DNA with cDNA or synthetic sequences to generate specific constructs.
For example, where an intron is desired in the ultimate construct,
a genomic clone will need to be used. The cDNA or a synthesized
oligonucleotide may provide more convenient restriction sites for
the remaining portion of the construct and, therefore, would be
used for the rest of the sequence.
 In certain embodiments, one may wish to employ antisense
constructs which include other elements, for example, those which
include C-5 propyne pyrimidines. Oligonucleotides which contain
C-5 propyne analogues of uridine and cytidine have been shown to
bind RNA with high affinity and to be potent antisense inhibitors
of gene expression (Wagner et al., 1993).
 As an alternative to targeted antisense delivery, targeted
ribozymes may be used. The term "ribozyme" refers to an
RNA-based enzyme capable of targeting and cleaving particular base
sequences in both DNA and RNA. Ribozymes can either be targeted
directly to cells, in the form of RNA oligonucleotides incorporating
ribozyme sequences, or introduced into the cell as an expression
vector encoding the desired ribozymal RNA. Ribozymes may be used
and applied in much the same way as described for antisense oligonucleotide.
Ribozyme sequences also may be modified in much the same way as
described for antisense oligonucleotide. For example, one could
incorporate non-Watson-Crick bases, or make mixed RNA/DNA oligonucleotides,
or modify the phosphodiester backbone.
 Alternatively, the antisense oligo- or polynucleotides of
the present invention may be provided as mRNA via transcription
from expression constructs that carry nucleic acids encoding the
 A nucleic acid may be made by any technique known to one
of ordinary skill in the art, such as for example, chemical synthesis,
enzymatic production or biological production. Non-limiting examples
of a synthetic nucleic acid (e.g., a synthetic oligonucleotide),
include a nucleic acid made by in vitro chemically synthesis using
phosphotriester, phosphite or phosphoramidite chemistry and solid
phase techniques, as described in EP 266,032 incorporated herein
by reference, or via deoxynucleoside H-phosphonate intermediates
as described by Froehler et al., 1986 and U.S. Pat. No. 5,705,629,
each incorporated herein by reference. In the methods of the present
invention, one or more oligonucleotides may be used. Various different
mechanisms of oligonucleotide synthesis have been disclosed in for
example, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566,
4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which
is incorporated herein by reference.
 A non-limiting example of an enzymatically produced nucleic
acid includes one produced by enzymes in amplification reactions
such as PCR.TM. (see for example, U.S. Pat. No. 4,683,202 and U.S.
Pat. No. 4,682,195, each incorporated herein by reference), or the
synthesis of an oligonucleotide described in U.S. Pat. No. 5,645,897,
incorporated herein by reference. A non-limiting example of a biologically
produced nucleic acid includes a recombinant nucleic acid produced
(i.e., replicated) in a living cell, such as a recombinant DNA vector
replicated in bacteria (see for example, Sambrook et al. 1989, incorporated
herein by reference).
IV. GENETIC CONSTRUCTS
 The nucleic acid segments of the present invention, regardless
of the length of the coding sequence itself, may be combined with
other DNA sequences, such as promoters, enhancers and polyadenylation
signals. It will be important to employ a promoter that effectively
directs the expression of the DNA segment in the cell type, organism,
or even animal, chosen for expression. Throughout this application,
the term "expression construct" is meant to include any
type of genetic construct containing an antisense product in which
part or all of the nucleic acid sequence is capable of being transcribed.
Typical expression vectors include bacterial plasmids or phage,
such as any of the pUC or Bluescript.TM. plasmid series or, as discussed
further below, viral vectors adapted for use in eukaryotic cells.
 A. Promoters
 In particular embodiments, the antisense oligonucleotide
or polynucleotide is part of an expression construct and is under
the transcription control of a promoter. A "promoter"
is a control sequence that is a region of a nucleic acid sequence
at which initiation and rate of transcription are controlled. It
may contain genetic elements at which regulatory proteins and molecules
may bind, such as RNA polymerase and other transcription factors,
to initiate the specific transcription a nucleic acid sequence.
The phrases "operatively positioned," "operatively
linked," "under control," and "under transcriptional
control" mean that a promoter is in a correct functional location
and/or orientation in relation to a nucleic acid sequence to control
transcriptional initiation and/or expression of that sequence.
 The term promoter will be used here to refer to a group
of transcriptional control modules that are clustered around the
initiation site for RNA polymerase II. Much of the thinking about
how promoters are organized derives from analyses of several viral
promoters, including those for the HSV thymidine kinase (tk) and
SV40 early transcription units. These studies, augmented by more
recent work, have shown that promoters are composed of discrete
functional modules, each consisting of approximately 7-20 bp of
DNA, and containing one or more recognition sites for transcriptional
activator or repressor proteins.
 At least one module in each promoter functions to position
the start site for RNA synthesis. The best known example of this
is the TATA box, but in some promoters lacking a TATA box, such
as the promoter for the mammalian terminal deoxynucleotidyl transferase
gene and the promoter for the SV40 late genes, a discrete element
overlying the start site itself helps to fix the place of initiation.
 Additional promoter elements regulate the frequency of transcriptional
initiation. Typically, these are located in the region 30-110 bp
upstream of the start site, although a number of promoters have
recently been shown to contain functional elements downstream of
the start site as well. The spacing between promoter elements frequently
is flexible, so that promoter function is preserved when elements
are inverted or moved relative to one another. In the tk promoter,
the spacing between promoter elements can be increased to 50 bp
apart before activity begins to decline. Depending on the promoter,
it appears that individual elements can function either co-operatively
or independently to activate transcription.
 The particular promoter employed to control the expression
of a nucleic acid encoding the antisense oligonucleotides of this
invention is not believed to be important, so long as it is capable
of directing the expression of the antisense oligonucleotides in
the targeted cell. Thus, where a human cell is targeted, it is preferable
to position the nucleic acid coding the antisense oligonucleotide
described in the invention adjacent to and under the control of
a promoter that is capable of being expressed in a human cell. Generally
speaking, such a promoter might include either a human or viral
 In various embodiments, the human cytomegalovirus (CMV)
immediate early gene promoter, the SV40 early promoter, the Rous
sarcoma virus (RSV) long terminal repeat can be used to obtain high-level
expression of the antisense oligonucleotides described and contemplated
in the present invention. The use of other viral or mammalian cellular
or bacterial phage promoters which are well-known in the art to
achieve expression of an antisense oligonucleotide of interest is
contemplated as well, provided that the levels of expression are
sufficient for a given purpose.
 Selection of a promoter that is regulated in response to
specific physiologic or synthetic signals can permit inducible expression
of the WT1 antisense oligonucleotide. For example, in the case where
expression of a transgene or transgenes when a multicistronic vector
is utilized, is toxic to the cells in which the vector is produced,
it may be desirable to prohibit or reduce expression of one or more
of the transgenes. Examples of transgenes that may be toxic to the
producer cell line are pro-apoptotic and cytokine genes. Several
inducible promoter systems are available for production of viral
vectors where the transgene product may be toxic.
 The ecdysone system (Invitrogen, Carlsbad, Calif.) is one
such system. This system is designed to allow regulated expression
of a gene of interest in mammalian cells. It consists of a tightly
regulated expression mechanism that allows virtually no basal level
expression of the transgene, but over 200-fold inducibility. The
system is based on the heterodimeric ecdysone receptor of Drosophila,
and when ecdysone or an analog such as muristerone A binds to the
receptor, the receptor activates a promoter to turn on expression
of the downstream transgene high levels of mRNA transcripts are
attained. In this system, both monomers of the heterodimeric receptor
are constitutively expressed from one vector, whereas the ecdysone-responsive
promoter which drives expression of the gene of interest is on another
plasmid. Engineering of this type of system into the gene transfer
vector of interest would therefore be useful. Cotransfection of
plasmids containing the gene of interest and the receptor monomers
in the producer cell line would then allow for the production of
the gene transfer vector without expression of a potentially toxic
transgene. At the appropriate time, expression of the transgene
could be activated with ecdysone or muristeron A.
 Another inducible system that would be useful is the Tet-Off.TM.
or Tet-On.TM. system (Clontech, Palo Alto, Calif.) originally developed
by Gossen and Bujard (Gossen and Bujard, 1992; Gossen et al., 1995).
This system also allows high levels of gene expression to be regulated
in response to tetracycline or tetracycline derivatives such as
doxycycline. In the Tet-On.TM. system, gene expression is turned
on in the presence of doxycycline, whereas in the Tet-Off.TM. system,
gene expression is turned on in the absence of doxycycline. These
systems are based on two regulatory elements derived from the tetracycline
resistance operon of E. Coli. The tetracycline operator sequence
to which the tetracycline repressor binds, and the tetracycline
repressor protein. The gene of interest is cloned into a plasmid
behind a promoter that has tetracycline-responsive elements present
in it. A second plasmid contains a regulatory element called the
tetracycline-controlled transactivator, which is composed, in the
Tet-Off.TM. system, of the VP16 domain from the herpes simplex virus
and the wild-type tertracycline repressor. Thus in the absence of
doxycycline, transcription is constitutively on. In the Tet-On.TM.
system, the tetracycline repressor is not wild-type and in the presence
of doxycycline activates transcription. For gene therapy vector
production, the Tet-Off.TM. system would be preferable so that the
producer cells could be grown in the presence of tetracycline or
doxycycline and prevent expression of a potentially toxic transgene,
but when the vector is introduced to the patient, the gene expression
would be constitutively on.
 In some circumstances, it may be desirable to regulate expression
of a transgene in a gene therapy vector. For example, different
viral promoters with varying strengths of activity may be utilized
depending on the level of expression desired. In mammalian cells,
the CMV immediate early promoter is often used to provide strong
transcriptional activation. Modified versions of the CMV promoter
that are less potent have also been used when reduced levels of
expression of the transgene are desired. When expression of a transgene
in hematopoetic cells is desired, retroviral promoters such as the
LTRs from MLV or MMTV are often used. Other viral promoters that
may be used depending on the desired effect include SV40, RSV LTR,
HIV-1 and HIV-2 LTR, adenovirus promoters such as from the E1A,
E2A, or MLP region, AAV LTR, cauliflower mosaic virus, HSV-TK, and
avian sarcoma virus.
 Similarly tissue specific promoters may be used to effect
transcription in specific tissues or cells so as to reduce potential
toxicity or undesirable effects to non-targeted tissues. For example,
promoters such as leptin gene promoter (O'Neil et al., 2001), CDH13
(Toyooka et al., 2001), adenomatous polyposis coli (APC) gene promoter
(Jin et al., 2001), IGF binding protein-3 promoter (IGFBP-3) (Walker
et al., 2001) may be used to target gene expression in breast cancers.
 By employing a promoter with well-known properties, the
level and pattern of expression of an antisense oligonucleotide
of interest can be optimized. Further, selection of a promoter that
is regulated in response to specific physiologic signals can permit
inducible expression of an antisense oligonucleotide. For example,
a nucleic acid under control of the human PAI-1 promoter results
in expression inducible by tumor necrosis factor. Tables 1 and 2
list several elements/promoters which may be employed, in the context
of the present invention, to regulate the expression of antisense
constructs. This list is not intended to be exhaustive of all the
possible elements involved in the promotion of expression but, merely,
to be exemplary thereof.
 B. Enhancers
 Enhancers are genetic elements that increase transcription
from a promoter located at a distant position on the same molecule
of DNA. Enhancers are organized much like promoters. That is, they
are composed of many individual elements, each of which binds to
one or more transcriptional proteins. The basic distinction between
enhancers and promoters is operational. An enhancer region as a
whole must be able to stimulate transcription at a distance; this
need not be true of a promoter region or its component elements.
On the other hand, a promoter must have one or more elements that
direct initiation of RNA synthesis at a particular site and in a
particular orientation, whereas enhancers lack these specificities.
Promoters and enhancers are often overlapping and contiguous, often
seeming to have a very similar modular organization.
 Below is a list of viral promoters, cellular promoters/enhancers
and inducible promoters/enhancers that could be used in combination
with the nucleic acid encoding an antisense oligonucleotide described
in this invention in an expression construct (Table 1 and Table
2). Additionally any promoter/enhancer combination (as per the Eukaryotic
Promoter Data Base EPDB) also could be used to drive expression
of a nucleic acid according to the present invention. Use of a T3,
T7 or SP6 cytoplasmic expression system is another possible embodiment.
Eukaryotic cells can support cytoplasmic transcription from certain
bacterial promoters if the appropriate bacterial polymerase is provided,
either as part of the delivery complex or as an additional genetic
1TABLE 1 Other Promoter/Enhancer Elements Promoter/Enhancer References
Immunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al.,
1983; Grosschedl and Baltimore, 1985; Atchinson and Perry, 1986,
1987; Imler et al., 1987; Weinberger et al., 1988; Kiledjian et
al., 1988; Porton et al., 1990 Immunoglobulin Light Chain Queen
and Baltimore, 1983; Picard and Schaffner, 1984 T-Cell Receptor
Luria et al., 1987, Winoto and Baltimore, 1989; Redondo et al.,
1990 HLA DQ .alpha. and DQ .beta. Sullivan and Peterlin, 1987 .beta.-Interferon
Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn and Maniatis,
1985 Interleukin-2 Greene et al., 1989 Interleukin-2 Receptor Greene
et al., 1989; Lin et al., 1990 MHC Class II Koch et al., 1989 MHC
Class II HLA-DR.alpha. Sherman et al., 1989 .beta.-Actin Kawamoto
et al., 1988; Ng et al., 1989 Muscle Creatine Kinase Jaynes et al.,
1988; Horlick and Benfield, 1989; Johnson et al., 1989a Prealbumin
(Transthyretin) Costa et al., 1988 Elastase I Omitz et al., 1987
Metallothionein Karin et al., 1987; Culotta and Hamer, 1989 Collagenase
Pinkert et al., 1987; Angel et al., 1987 Albumin Gene Pinkert et
al., 1987, Tronche et al., 1989, 1990 .alpha.-Fetoprotein Godbout
et al., 1988; Campere and Tilghman, 1989 .gamma.-Globin Bodine and
Ley, 1987; Perez-Stable and Constantini, 1990 .beta.-Globin Trudel
and Constantini, 1987 c-fos Cohen et al., 1987 c-HA-ras Triesman,
1986; Deschamps et al., 1985 Insulin Edlund et al., 1985 Neural
Cell Adhesion Molecule Hirsch et al., 1990 (NCAM) a.sub.1-antitrypsin
Latimer et al., 1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse
or Type I Collagen Ripe et al., 1989 Glucose-Regulated Proteins
Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsen et
al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 Troponin
I (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech
et al., 1989 Duchenne Muscular Dystrophy Klamut et al., 1990 SV40
Banerji et al., 1981; Moreau et al., 1981; Sleigh and Lockett, 1985;
Firak and Subramanian, 1986; Herr and Clarke, 1986; Imbra and Karin,
1986; Kadesch and Berg, 1986; Wang and Calame, 1986; Ondek et al.,
1987; Kuhl et al., 1987 Schaffner et al., 1988 Polyoma Swartzendruber
and Lehman, 1975; Vasseur et al., 1980; Katinka et al., 1980, 1981;
Tyndell et al., 1981; Dandolo et al., 1983; deVilliers et al., 1984;
Hen et al., 1986; Satake et al., 1988; Campbell and Villarreal,
1988 Retroviruses Kriegler and Botchan, 1982, 1983; Levinson et
al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al.,
1986; Miksicek et al., 1986; Celander and Haseltine, 1987; Thiesen
et al., 1988; Celander et al., 1988; Chol et al., 1988; Reisman
and Rotter, 1989 Papilloma Virus Campo et al., 1983; Lusky et al.,
1983; Spandidos and Wilkie, 1983; Spalholz et al., 1985; Lusky and
Botchan, 1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika
et al., 1987, Stephens and Hentschel, 1987; Glu et al., 1988 Hepatitis
B Virus Bulla and Siddiqui, 1986; Jameel and Siddiqui, 1986; Shaul
and Ben-Levy, 1987; Spandau and Lee, 1988 Human Immunodeficiency
Virus Muesing et al., 1987; Hauber and Cullan, 1988; Jakobovits
et al., 1988; Feng and Holland, 1988; Takebe et al., 1988; Rowen
et al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp
and Marciniak, 1989; Braddock et al., 1989 Cytomegalovirus Weber
et al., 1984; Boshart et al., 1985; Foecking and Hofstetter, 1986
Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinn et al., 1989
2TABLE 2 Inducible Elements Element Inducer MT II Phorbol Ester
(TPA) Heavy metals MMTV (mouse mammary tumor Glucocorticoids virus)
.beta.-Interferon poly(rI)X poly(rc) Adenovirus 5 E2 Ela c-jun Phorbol
Ester (TPA), H.sub.2O.sub.2 Collagenase Phorbol Ester (TPA) Stromelysin
Phorbol Ester (TPA), IL-1 SV40 Phorbol Ester (TPA) Murine MX Gene
Interferon, Newcastle Disease Virus GRP78 Gene A23187 .alpha.-2-Macroglobulin
IL-6 Vimentin Serum MHC Class I Gene H-2kB Interferon HSP70 Ela,
SV40 Large T Antigen Proliferin Phorbol Ester-TPA Tumor Necrosis
Factor FMA Thyroid Stimulating Hormone .alpha. Thyroid Hormone Gene
Insulin E Box Glucose
 In certain embodiments of this invention, the delivery of
a nucleic acid to a cell may be identified in vitro or in vivo by
including a marker in the expression construct. The marker would
result in an identifiable change to the transfected cell permitting
easy identification of expression. Enzymes such as herpes simplex
virus thymidine kinase (tk) (eukaryotic) or chloramphenicol acetyltransferase
(CAT) (prokaryotic) may be employed.
 C. Polyadenylation Signals
 Where a cDNA insert is employed, one will typically desire
to include a polyadenylation signal to effect proper polyadenylation
of the gene transcript. The nature of the polyadenylation signal
is not believed to be crucial to the successful practice of the
invention, and any such sequence may be employed such as human or
bovine growth hormone and SV40 polyadenylation signals. Also contemplated
as an element of the expression cassette is a terminator. These
elements can serve to enhance message levels and to minimize read
through from the cassette into other sequences.
V. LIPID FORMULATIONS
 In a particular embodiment of the invention, the antisense
oligonucleotides and expression vectors may be associated with a
lipid. An oligonucleotide associated with a lipid may be encapsulated
in the aqueous interior of a liposome, interspersed within the lipid
bilayer of a liposome, attached to a liposome via a linking molecule
that is associated with both the liposome and the oligonucleotide,
entrapped in a liposome, complexed with a liposome, dispersed in
a solution containing a lipid, mixed with a lipid, combined with
a lipid, contained as a suspension in a lipid, contained or complexed
with a micelle, or otherwise associated with a lipid. The lipid
or lipid/oligonucleotide associated compositions of the present
invention are not limited to any particular structure in solution.
For example, they may be present in a bilayer structure, as micelles,
or with a "collapsed" structure. They may also simply
be interspersed in a solution, possibly forming aggregates which
are not uniform in either size or shape.
 Lipids are fatty substances which may be naturally occurring
or synthetic lipids. For example, lipids include the fatty droplets
that naturally occur in the cytoplasm as well as the class of compounds
which are well known to those of skill in the art which contain
long-chain aliphatic hydrocarbons and their derivatives, such as
fatty acids, alcohols, amines, amino alcohols, and aldehydes. An
example is the lipid dioleoylphosphatidylcholine (DOPC).
 Phospholipids may be used for preparing the liposomes according
to the present invention and can carry a net positive charge, a
net negative charge or are neutral. Diacetyl phosphate can be employed
to confer a negative charge on the liposomes, and stearylamine can
be used to confer a positive charge on the liposomes. The liposomes
can be made of one or more phospholipids.
 In a particular embodiment, the lipid material is comprised
of a neutrally charged lipid. A neutrally charged lipid can comprise
a lipid without a charge, a substantially. uncharged lipid or a
lipid mixture with equal number of positive and negative charges.
 In one aspect, the lipid component of the composition comprises
a neutral lipid. In another aspect, the lipid material consists
essentially of neutral lipids which is further defined as a lipid
composition containing at least 70% of lipids without a charge.
In other aspects, the lipid material may contain at least 80% to
90% of lipids without a charge. In yet other aspects, the lipid
material may comprise about 90%, 95%, 96%, 97%, 98%, 99% or 100%
lipids without a charge.
 In specific aspects, the neutral lipid comprises a phosphatidylcholine,
a phosphatidylglycerol, or a phosphatidylethanolamin- e. In a particular
aspect, the phosphatidylcholine comprises DOPC.
 In other aspects the lipid component comprises a substantially
uncharged lipid. A substantially uncharged lipid is described herein
as a lipid composition that is substantially free of anionic and
cationic phospholipids and cholesterol. In yet other aspects the
lipid component comprises a mixture of lipids to provide a substantially
uncharged lipid. Thus, the lipid mixture may comprise negatively
and positively charged lipids.
 Lipids suitable for use according to the present invention
can be obtained from commercial sources. For example, dimyristyl
phosphatidylcholine ("DMPC") can be obtained from Sigma
Chemical Co., dicetyl phosphate ("DCP") is obtained from
K & K Laboratories (Plainview, N.Y.); cholesterol ("Chol")
is obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol
("DMPG") and other lipids may be obtained from Avanti
Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids
in chloroform or chloroform/methanol can be stored at about -20.degree.
C. Preferably, chloroform is used as the only solvent since it is
more readily evaporated than methanol.
 Phospholipids from natural sources, such as egg or soybean
phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol,
heart cardiolipin and plant or bacterial phosphatidylethanolamine
are preferably not used as the primary phosphatide, i.e., constituting
50% or more of the total phosphatide composition, because of the
instability and leakiness of the resulting liposomes.
 "Liposome" is a generic term encompassing a variety
of single and multilamellar lipid vehicles formed by the generation
of enclosed lipid bilayers or aggregates. Liposomes may be characterized
as having vesicular structures with a phospholipid bilayer membrane
and an inner aqueous medium. Multilamellar liposomes have multiple
lipid layers separated by aqueous medium. They form spontaneously
when phospholipids are suspended in an excess of aqueous solution.
The lipid components undergo self-rearrangement before the formation
of closed structures and entrap water and dissolved solutes between
the lipid bilayers (Ghosh and Bachhawat, 1991). However, the present
invention also encompasses compositions that have different structures
in solution than the normal vesicular structure. For example, the
lipids may assume a micellar structure or merely exist as nonuniform
aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic
 Liposome-mediated oligonucleotide delivery and expression
of foreign DNA in vitro has been very successful. Wong et al. (1980)
demonstrated the feasibility of liposome-mediated delivery and expression
of foreign DNA in cultured chick embryo, HeLa and hepatoma cells.
Nicolau et al. (1987) accomplished successful liposome-mediated
gene transfer in rats after intravenous injection.
 In certain embodiments of the invention, the lipid may be
associated with a hemagglutinating virus (HVJ). This has been shown
to facilitate fusion with the cell membrane and promote cell entry
of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,
the lipid may be complexed or employed in conjunction with nuclear
non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In
yet further embodiments, the lipid may be complexed or employed
in conjunction with both HVJ and HMG-1. Such expression vectors
have been successfully employed in transfer and expression of an
oligonucleotide in vitro and in vivo and thus are applicable for
the present invention. Where a bacterial promoter is employed in
the DNA construct, it also will be desirable to include within the
liposome an appropriate bacterial polymerase.
 Liposomes used according to the present invention can be
made by different methods. The size of the liposomes varies depending
on the method of synthesis. A liposome suspended in an aqueous solution
is generally in the shape of a spherical vesicle, having one or
more concentric layers of lipid bilayer molecules. Each layer consists
of a parallel array of molecules represented by the formula XY,
wherein X is a hydrophilic moiety and Y is a hydrophobic moiety.
In aqueous suspension, the concentric layers are arranged such that
the hydrophilic moieties tend to remain in contact with an aqueous
phase and the hydrophobic regions tend to self-associate. For example,
when aqueous phases are present both within and without the liposome,
the lipid molecules may form a bilayer, known as a lamella, of the
arrangement XY--YX. Aggregates of lipids may form when the hydrophilic
and hydrophobic parts of more than one lipid molecule become associated
with each other. The size and shape of these aggregates will depend
upon many different variables, such as the nature of the solvent
and the presence of other compounds in the solution.
 Liposomes within the scope of the present invention can
be prepared in accordance with known laboratory techniques. A particular
method of the invention describes the preparation of liposomes and
is described below. Briefly, P-ethoxy oligonucleotides (also referred
to as PE oligos) are dissolved in DMSO and the phospholipids (Avanti
Polar Lipids, Alabaster, Ala.), such as for example the preferred
neutral phospholipid dioleoylphosphatidylcholine (DOPC), is dissolved
in tert-butanol. The lipid is then mixed with the antisense oligonucleotides.
In the case of DOPC, the molar ratio of the lipid to the antisense
oligos is 20:1. Tween 20 is added to the lipid:oligo mixture such
that Tween 20 is 5% of the combined weight of the lipid and oligo.
Excess tert-butanol is added to this mixture such that the volume
of tert-butanol is at least 95%. The mixture is vortexed, frozen
in a dry ice/acetone bath and lyophilized overnight. The lyophilized
preparation is stored at -20.degree. C. and can be used up to three
months. When required the lyophilized liposomes are reconstituted
in 0.9% saline. The average diameter of the particles obtained using
Tween 20 for encapsulating the lipid with the oligo is 0.7-1.0 .mu.m
 Alternatively liposomes can be prepared by mixing liposomal
lipids, in a solvent in a container, e.g., a glass, pear-shaped
flask. The container should have a volume ten-times greater than
the volume of the expected suspension of liposomes. Using a rotary
evaporator, the solvent is removed at approximately 40.degree. C.
under negative pressure. The solvent normally is removed within
about 5 min. to 2 hours, depending on the desired volume of the
liposomes. The composition can be dried further in a desiccator
under vacuum. The dried lipids generally are discarded after about
1 week because of a tendency to deteriorate with time.
 Dried lipids can be hydrated at approximately 25-50 mM phospholipid
in sterile, pyrogen-free water by shaking until all the lipid film
is resuspended. The aqueous liposomes can be then separated into
aliquots, each placed in a vial, lyophilized and sealed under vacuum.
 In other alternative methods, liposomes can be prepared
in accordance with other known laboratory procedures: the method
of Bangham et al. (1965), the contents of which are incorporated
herein by reference; the method of Gregoriadis (1979), the contents
of which are incorporated herein by reference; the method of Deamer
and Uster (1983), the contents of which are incorporated by reference;
and the reverse-phase evaporation method as described by Szoka and
Papahadjopoulos (1978). The aforementioned methods differ in their
respective abilities to entrap aqueous material and their respective
aqueous space-to-lipid ratios.
 The dried lipids or lyophilized liposomes prepared as described
above may be dehydrated and reconstituted in a solution of inhibitory
peptide and diluted to an appropriate concentration with an suitable
solvent, e.g., DPBS. The mixture is then vigorously shaken in a
vortex mixer. Unencapsulated nucleic acid is removed by centrifugation
at 29,000.times.g and the liposomal pellets washed. The washed liposomes
are resuspended at an appropriate total phospholipid concentration,
e.g., about 50-200 mM. The amount of nucleic acid encapsulated can
be determined in accordance with standard methods. After determination
of the amount of nucleic acid encapsulated in the liposome preparation,
the liposomes may be diluted to appropriate concentrations and stored
at 4.degree. C. until use.
 P-ethoxy oligonucleotides, nucleases resistant analogues
of phosphodiesters, are preferred because they are stable in serum.
Neutral lipids are also preferred and specifically the lipid dioleoylphosphatidylchoine
is preferred. However other lipids such as other phosphatidylcholines,
phosphatidylglycerols, and phosphatidylethanolamines may also be
useful. In yet another particular method described herein, the nuclease-resistant
oligonucleotides and lipids are dissolved in DMSO and t-butanol
respectively. The lipid is then mixed with the oligonucleotides
in a molar ratio of between about 5:1 to about 100:1, and preferably
in a ratio of 20:1. The preferred lipid:oligonucleotide ratio for
P-ethoxy oligonucleotides and the lipid dioleoylphosphatidylchoine
is 20:1. Tween 20 is then added to the mixture to obtain the liposomes.
Excess t-butanol is added and the mixture is vortexed, frozen in
an acetone/dry-ice bath, and then lyophilized overnight. The preparation
is stored at -20.degree. C. and may be used within one month of
preparation. When required for use the lyophilized liposomal antisense
oligonucleotides are reconstituted in 0.9% saline.
 In an alternative embodiment, nuclease-resistant oligonucleotides
are mixed with lipids in the presence of excess t-butanol. The mixture
is vortexed before being frozen in an acetone/dry ice bath. The
frozen mixture is then lyophilized and hydrated with Hepes-buffered
saline (1 mM Hepes, 10 mM NaCl, pH 7.5) overnight, and then the
liposomes are sonicated in a bath type sonicator for 10 to 15 min.
The size of the liposomal-oligonucleotides typically ranges between
200-300 nm in diameter as determined by the submicron particle sizer
autodilute model 370 (Nicomp, Santa Barbara, Calif.).
 A pharmaceutical composition comprising the liposomes will
usually include a sterile, pharmaceutically acceptable carrier or
diluent, such as water or saline solution.
VI. NON-LIPOSOMAL DELIVERY SYSTEMS
 The delivery of antisense constructs of the present invention
may also be accomplished using expression vectors which may be viral
or non-viral in nature.
 Retroviruses. The retroviruses are a group of single-stranded
RNA viruses characterized by an ability to convert their RNA to
double-stranded DNA in infected cells by a process of reverse-transcription
(Coffin, 1990). The resulting DNA then stably integrates into cellular
chromosomes as a provirus and directs synthesis of viral proteins.
The integration results in the retention of the viral gene sequences
in the recipient cell and its descendants. The retroviral genome
contains three genes--gag, pol, and env--that code for capsid proteins,
polymerase enzyme, and envelope components, respectively. A sequence
found upstream from the gag gene, termed .PSI., functions as a signal
for packaging of the genome into virions. Two long terminal repeat
(LTR) sequences are present at the 5' and 3' ends of the viral genome.
These contain strong promoter and enhancer sequences and are also
required for integration in the host cell genome (Coffin, 1990).
 In order to construct a retroviral vector, a nucleic acid
encoding a WT1 antisense construct as described in this invention
is inserted into the viral genome in the place of certain viral
sequences to produce a virus that is replication-defective. In order
to produce virions, a packaging cell line containing the gag, pol
and env genes but without the LTR and .PSI. components is constructed
(Mann et al., 1983). When a recombinant plasmid containing an inserted
DNA, together with the retroviral LTR and .PSI. sequences, is introduced
into this cell line (by calcium phosphate precipitation for example),
the .PSI. sequence allows the RNA transcript of the recombinant
plasmid to be packaged into viral particles, which are then secreted
into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986;
Mann et al., 1983). The media containing the recombinant retroviruses
is then collected, optionally concentrated, and used for gene transfer.
Retroviral vectors are able to infect a broad variety of cell types.
However, integration and stable expression require the division
of host cells (Paskind et al., 1975).
 Adenoviruses. Human adenoviruses are double-stranded DNA
tumor viruses with genome sizes of approximate 36 kB. As a model
system for eukaryotic gene expression, adenoviruses have been widely
studied and well characterized, which makes them an attractive system
for development of adenovirus as a gene transfer system. This group
of viruses is easy to grow and manipulate, and they exhibit a broad
host range in vitro and in vivo. In lytically infected cells, adenoviruses
are capable of shutting off host protein synthesis, directing cellular
machineries to synthesize large quantities of viral proteins, and
producing copious amounts of virus.
 The E1 region of the genome includes E1A and E1B which encode
proteins responsible for transcription regulation of the viral genome,
as well as a few cellular genes. E2 expression, including E2A and
E2B, allows synthesis of viral replicative functions, e.g. DNA-binding
protein, DNA polymerase, and a terminal protein that primes replication.
E3 gene products prevent cytolysis by cytotoxic T cells and tumor
necrosis factor and appear to be important for viral propagation.
Functions associated .with the E4 proteins include DNA replication,
late gene expression, and host cell shutoff. The late gene products
include most of the virion capsid proteins, and these are expressed
only after most of the processing of a single primary transcript
from the major late promoter has occurred. The major late promoter
(MLP) exhibits high efficiency during the late phase of the infection
(Stratford-Perricaudet and Perricaudet, 1991).
 A small portion of the viral genome appears to be required
in cis adenovirus-derived vectors when used in connection with cell
lines such as 293 cells. Ad5-transformed human embryonic kidney
cell lines (Graham et al., 1977) have been developed to provide
the essential viral proteins in trans.
 Particular advantages of an adenovirus system for expressing
and delivering the antisense oligonucleotides of this invention
include (i) the structural stability of recombinant adenoviruses;
(ii) the safety of adenoviral administration to humans; and (iii)
lack of any known association of adenoviral infection with cancer
or malignancies; (iv) the ability to obtain high titers of the recombinant
virus; and (v) the high infectivity of adenovirus.
 Further advantages of adenovirus vectors over retroviruses
include the higher levels of gene expression. Additionally, adenovirus
replication is independent of host gene replication, unlike retroviral
sequences. Because adenovirus transforming genes in the E1 region
can be readily deleted and still provide efficient expression vectors,
oncogenic risk from adenovirus vectors is thought to be negligible
(Grunhaus et al., 1992).
 In general, adenovirus gene transfer systems are based upon
recombinant, engineered adenovirus which is rendered replication-incompetent
by deletion of a portion of its genome, such as E1, and yet still
retains its competency for infection. Sequences encoding relatively
large foreign proteins can be expressed when additional deletions
are made in the adenovirus genome. Surprisingly persistent expression
of transgenes following adenoviral infection has also been reported.
 Other Viral Vectors as Expression Constructs. Other viral
vectors may be employed as expression constructs in the present
invention. Vectors derived from viruses such as vaccinia virus (Ridgeway,
1988; Baichwal and Sugden, 1986; Coupar et al., 1988) adeno-associated
virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat
and Muzycska, 1984) lentivirus, polyoma virus and herpes viruses
may be employed. They offer several attractive features for various
mammalian cells (Friedman et al., 1989; Ridgeway, 1988; Baichwal
and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
 With the recent recognition of defective hepatitis B viruses,
new insight was gained into the structure-function relationship
of different viral sequences. In vitro studies showed that the virus
could retain the ability for helper-dependent packaging and reverse
transcription despite the deletion of up to 80% of its genome (Horwich
et al., 1990). This suggested that large portions of the genome
could be replaced with foreign genetic material. The hepatotropism
and persistence (integration) were particularly attractive properties
for liver-directed gene transfer. Chang et al. (1991) introduced
the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis
B virus genome in the place of the polymerase, surface, and pre-surface
coding sequences. It was cotransfected with wild-type virus into
an avian hepatoma cell line. Culture media containing high titers
of the recombinant virus were used to infect primary duckling hepatocytes.
Stable CAT gene expression was detected for at least 24 days after
transfection (Chang et al., 1991).
 Non-viral Methods. Several non-viral methods for the transfer
of expression vectors into cultured mammalian cells also are contemplated
in the present invention. These include calcium phosphate precipitation
(Graham and van der Eb, 1973; Chen and Okayama, 1987; Rippe et al.,
1990); DEAE-dextran (Gopal, 1985); electroporation (Tur-Kaspa et
al., 1986; Potter et al., 1984); direct microinjection (Harland
and Weintraub, 1985); cell sonication (Fecheimer et al., 1987);
gene bombardment using high velocity microprojectiles (Yang et al.,
1990); polycations; and receptor-mediated transfection (Wu and Wu,
1987; Wu and Wu, 1988). Some of these techniques may be successfully
adapted for in vivo or ex vivo use.
 In one embodiment of the invention, the expression construct
may simply consist of naked recombinant vector. Transfer of the
construct may be performed by any of the methods mentioned above
which physically or chemically permeabilize the cell membrane. For
example, Dubensky et al. (1984) successfully injected polyomavirus
DNA in the form of CaPO.sub.4 precipitates into liver and spleen
of adult and newborn mice demonstrating active viral replication
and acute infection. Benvenisty and Neshif (1986) also demonstrated
that direct intraperitoneal injection of CaPO.sub.4 precipitated
plasmids results in expression of the transfected genes. It is envisioned
that DNA encoding an WT1 antisense oligonucleotide construct may
also be transferred in a similar manner in vivo.
 Another embodiment of the invention for transferring a naked
DNA expression vector into cells may involve particle bombardment.
This method depends on the ability to accelerate DNA coated microprojectiles
to a high velocity allowing them to pierce cell membranes and enter
cells without killing them (Klein et al., 1987). Several devices
for accelerating small particles have been developed. One such device
relies on a high voltage discharge to generate an electrical current,
which in turn provides the motive force (Yang et al., 1990). The
microprojectiles used have consisted of biologically inert substances
such as tungsten or gold beads.
 Selected organs including the liver, skin, and muscle tissue
of rats and mice have been bombarded in vivo (Yang et al., 1990;
Zelenin et al., 1991). This may require surgical exposure of the
tissue or cells, to eliminate any intervening tissue between the
gun and the target organ. DNA encoding a WT1 antisense oligonucleotide
as described in this invention may be delivered via this method.
 Where clinical application of liposomes containing antisense
oligo- or polynucleotides or expression vectors is undertaken, it
will be necessary to prepare the liposome complex as a pharmaceutical
composition appropriate for the intended application. Generally,
this will entail preparing a pharmaceutical composition that is
essentially free of pyrogens, as well as any other impurities that
could be harmful to humans or animals. One also will generally desire
to employ appropriate buffers to render the complex stable and allow
for uptake by target cells.
 Aqueous compositions of the therapeutic composition of the
present invention comprise an effective amount of the antisense
expression vector encapsulated in a liposome as discussed above,
further dispersed in pharmaceutically acceptable carrier or aqueous
medium. Such compositions also are referred to as inocula. The phrases
"pharmaceutically" or "pharmacologically acceptable"
refer to compositions that do not produce an adverse, allergic or
other untoward reaction when administered to an animal, or a human,
 As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings, antibacterial
and antifungal agents, isotonic and absorption delaying agents and
the like. The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any conventional
media or agent is incompatible with the active ingredient, its use
in the therapeutic compositions is contemplated. Supplementary active
ingredients also can be incorporated into the compositions.
 Solutions of therapeutic compositions can be prepared in
water suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions also can be prepared in glycerol, liquid polyethylene
glycols, mixtures thereof and in oils. Under ordinary conditions
of storage and use, these preparations contain a preservative to
prevent the growth of microorganisms.
 For human administration, preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biologics standards. The biological material should
be extensively dialyzed to remove undesired small molecular weight
molecules and/or lyophilized for more ready formulation into a desired
vehicle, where appropriate. The active compounds will then generally
be formulated for parenteral administration, e.g., formulated for
injection via the intravenous, intramuscular, sub-cutaneous, intralesional,
or even intraperitoneal routes. The preparation of an aqueous composition
that contains the therapeutic composition as an active component
or ingredient will be known to those of skill in the art in light
of the present disclosure. Typically, such compositions can be prepared
as injectables, either as liquid solutions or suspensions; solid
forms suitable for using to prepare solutions or suspensions upon
the addition of a liquid prior to injection can also be prepared;
and the preparations can also be emulsified.
 The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile
and must be fluid to the extent that easy syringability exists.
It must be stable under the conditions of manufacture and storage
and must be preserved against the contaminating action of microorganisms,
such as bacteria and fungi.
 A therapeutic composition can be formulated into a composition
in a neutral or salt form. Pharmaceutically acceptable salts, include
the acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids
as acetic, oxalic, tartaric, mandelic, and the like. Salts formed
with the free carboxyl groups can also be derived from inorganic
bases such as, for example, sodium, potassium, ammonium, calcium,
or ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
 The carrier can also be a solvent or dispersion medium containing,
for example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), suitable
mixtures thereof, and vegetable oils. The proper fluidity can be
maintained, for example, by the use of a coating, such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or sodium
chloride. Prolonged absorption of the injectable compositions can
be brought about by the use in the compositions of agents delaying
absorption, for example, aluminum monostearate and gelatin.
 Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate solvent
with various of the other ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are prepared
by incorporating the various sterilized active ingredients into
a sterile vehicle which contains the basic dispersion medium and
the required other ingredients from those enumerated above. In the
case of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum-drying
and freeze-drying techniques which yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
 The therapeutic compositions of the present invention are
advantageously administered in the form of injectable compositions
either as liquid solutions or suspensions; solid forms suitable
for solution in, or suspension in, liquid prior to injection may
also be prepared. These preparations also may be emulsified. A typical
composition for such purpose comprises a pharmaceutically acceptable
carrier. For instance, the composition may contain 10 mg, 25 mg,
50 mg or up to about 100 mg of human serum albumin per milliliter
of phosphate buffered saline. Other pharmaceutically acceptable
carriers include aqueous solutions, non-toxic excipients, including
salts, preservatives, buffers and the like.
 Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oil and injectable organic esters such as ethyloleate.
Aqueous carriers include water, alcoholic/aqueous solutions, saline
solutions, parenteral vehicles such as sodium chloride, Ringer's
dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers.
Preservatives include antimicrobial agents, anti-oxidants, chelating
agents and inert gases. The pH and exact concentration of the various
components the pharmaceutical composition are adjusted according
to well known parameters.
 Additional formulations are suitable for oral administration.
Oral formulations include such typical excipients as, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,
sodium saccharine, cellulose, magnesium carbonate and the like.
The compositions take the form of solutions, suspensions, tablets,
pills, capsules, sustained release formulations or powders. When
the route is topical, the form may be a cream, ointment, salve or
 The therapeutic compositions of the present invention may
include classic pharmaceutical preparations. Administration of therapeutic
compositions according to the present invention will be via any
common route so long as the target tissue is available via that
route. This includes oral, nasal, buccal, rectal, vaginal or topical.
Topical administration would be particularly advantageous for the
treatment of skin cancers, to prevent chemotherapy-induced alopecia
or other dermal hyperproliferative disorder. Alternatively, administration
may be by orthotopic, intradernal subcutaneous, intramuscular, intraperitoneal
or intravenous injection. Such compositions would normally be administered
as pharmaceutically acceptable compositions that include physiologically
acceptable carriers, buffers or other excipients. For treatment
of conditions of the lungs, the preferred route is aerosol delivery
to the lung. Volume of the aerosol is between about 0.01 ml and
0.5 ml. Similarly, a preferred method for treatment of colon-associated
disease would be via enema. Volume of the enema is between about
1 ml and 100 ml.
 An effective amount of the therapeutic composition is determined
based on the intended goal. The term "unit dose" or "dosage"
refers to physically discrete units suitable for use in a subject,
each unit containing a predetermined-quantity of the therapeutic
composition calculated to produce the desired responses, discussed
above, in association with its administration, i.e., the appropriate
route and treatment regimen. The quantity to be administered, both
according to number of treatments and unit dose, depends on the
 Precise amounts of the therapeutic composition also depend
on the judgment of the practitioner and are peculiar to each individual.
Factors affecting the dose include the physical and clinical state
of the patient, the route of administration, the intended goal of
treatment (alleviation of symptoms versus cure) and the potency,
stability and toxicity of the particular therapeutic substance.
 Administration of the therapeutic construct of the present
invention to a patient will follow general protocols for the administration
of chemotherapeutics, taking into account the toxicity, if any,
of the vector. It is expected that the treatment cycles would be
repeated as necessary. It also is contemplated that various standard
therapies, as well as surgical intervention, may be applied in combination
with the described treatments.
 Depending on the particular cancer to be, administration
of therapeutic compositions according to the present invention will
be via any common route so long as the target tissue is available
via that route. This includes oral, nasal, buccal, rectal, vaginal
or topical. Topical administration would be particularly advantageous
for treatment of skin cancers. Alternatively, administration will
be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal
or intravenous injection. Such compositions would normally be administered
as pharmaceutically acceptable compositions that include physiologically
acceptable carriers, buffers or other excipients.
 The treatments may include various "unit doses."
Unit dose is defined as containing a predetermined-quantity of the
therapeutic composition calculated to produce the desired responses
in association with its administration, i.e., the appropriate route
and treatment regimen. The quantity to be administered, and the
particular route and formulation, are within the skill of those
in the clinical arts. Also of importance is the subject to be treated,
in particular, the state of the subject and the protection desired.
A unit dose need not be administered as a single injection but may
comprise continuous infusion over a set period of time.
 According to the present invention, one may treat the cancer
by directly injecting a tumor with the therapeutic composition of
the present invention. Alternatively, the tumor may be infused or
perfused with the antisense oligonucleotides using any suitable
delivery vehicle. Local or regional administration, with respect
to the tumor, also is contemplated. Finally, systemic administration
may be performed. Continuous administration also may be applied
where appropriate, for example, where a tumor is excised and the
tumor bed is treated to eliminate residual, microscopic disease.
Delivery via syringe or catherization is preferred. Such continuous
perfusion may take place for a period from about 1-2 hours, to about
2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2
days, to about 1-2 wk or longer following the initiation of treatment.
Generally, the dose of the therapeutic composition via continuous
perfusion will be equivalent to that given by a single or multiple
injections, adjusted over a period of time during which the perfusion
 For tumors of >4 cm, the volume to be administered will
be about 4-10 ml (preferably 10 ml), while for tumors of <4 cm,
a volume of about 1-3 ml will be used (preferably 3 ml). Multiple
injections delivered as single dose comprise about 0.1 to about
0.5 ml volumes. The viral particles or protein may advantageously
be contacted by administering multiple injections to the tumor,
spaced at approximately 1 cm intervals.
 In certain embodiments, the tumor being treated may not,
at least initially, be resectable. Treatments with therapeutic compositions
may increase the resectability of the tumor due to shrinkage at
the margins or by elimination of certain particularly invasive portions.
Following treatments, resection may be possible. Additional viral
or protein treatments subsequent to resection will serve to eliminate
microscopic residual disease at the tumor site.
 A typical course of treatment, for a primary tumor or a
post-excision tumor bed, will involve multiple doses. Typical primary
tumor treatment involves a 6 dose application over a two-week period.
The two-week regimen may be repeated one, two, three, four, five,
six or more times. During a course of treatment, the need to complete
the planned dosings may be re-evaluated.
 The preparation of more, or highly, concentrated solutions
for direct injection is also contemplated, where the use of DMSO
as solvent is envisioned to result in extremely rapid penetration,
delivering high concentrations of the active agents to a small area.
 For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially suitable
for intravenous, intramuscular, subcutaneous and intraperitoneal
administration. In this connection, sterile aqueous media which
can be employed will be known to those of skill in the art in light
of the present disclosure. For example, one dosage could be dissolved
in 1 ml of isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences"
15th Edition, pages 1035-1038 and 1570-1580). Some variation in
dosage will necessarily occur depending on the condition of the
subject being treated. The person responsible for administration
will, in any event, determine the appropriate dose for the individual
VIII. COMBINATION CANCER THERAPY
 To further enhance the efficacy of the therapy provided
by the invention, combination therapies are contemplated. Thus,
a second therapeutic agent in addition to a WT1 antisense oligonucleotide
therapy may be used. The second therapeutic agent may be a chemotherapeutic
agent, a radiotherapeutic agent, a gene therapeutic agent, a protein/peptide/polypeptide
therapeutic agent, an immunotherapeutic agent, or a hormonal therapeutic
agent. Such agents are well known in the art.
 As set forth earlier an "effective amount" is
defined as an amount of the WT1 antisense composition that can decrease,
reduce, inhibit or otherwise abrogate the growth of a cancer cell,
arrest-cell growth, induce apoptosis, inhibit metastasis, induce
tumor necrosis, kill cells or induce cytotoxicity in cells.
 The administration of the second therapeutic agent may precede
or follow the therapy using an antisense construct by intervals
ranging from minutes to days to weeks. In embodiments where the
second therapeutic agent and an antisense construct encoding nucleic
acid or protein product are administered together, one would generally
ensure that a significant period of time did not expire between
the time of each delivery. In such instances, it is contemplated
that one would administer to a patient both modalities within about
12-24 hours of each other and, more preferably, within about 6-12
hours of each other, with a delay time of only about 12 hours being
most preferred. In some situations, it may be desirable to extend
the time period for treatment significantly, however, where several
days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7
or 8) lapse between the respective administrations.
 It also is conceivable that more than one administration
of either the second therapeutic agent and an antisense oligonucleotide
will be required to achieve complete cancer cure. Various combinations
may be employed, where the second therapeutic agent is "A"
and the antisense oligonucleotide is "B", as exemplified
3 A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B
A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A
A/A/B/A A/B/B/B B/A/B/B B/B/A/B
 Other combinations also are contemplated. The exact dosages
and regimens of each agent can be suitably altered by those of ordinary
skill in the art.
 Provided below is a description of some other agents effective
in the treatment of cancer.
 (i) Radiotherapeutic Agents
 In some tumor cell lines, levels of antisense oligonucleotide
were found to correlate to the sensitivity of cells to ionizing
radiation, indicating that antisense therapy restores and/or enhances
sensitivity of tumor cells to genotoxic agents. Therefore, additional
therapy with radiotherapeutic agents and factors including radiation
and waves that induce DNA damage for example, y-irradiation, X-rays,
UV-irradiation, microwaves, electronic emissions, radioisotopes,
and the like are contemplated. Therapy may be achieved by irradiating
the localized tumor site with the above described forms of radiations.
It is most likely that all of these factors effect a broad range
of damage DNA, on the precursors of DNA, the replication and repair
of DNA, and the assembly and maintenance of chromosomes.
 Dosage ranges for X-rays range from daily doses of 50 to
200 roentgens for prolonged periods of time (3 to 4 weeks), to single
doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes
vary widely, and depend on the half-life of the isotope, the strength
and type of radiation emitted, and the uptake by the neoplastic
 (ii) Surgery
 Approximately 60% of persons with cancer will undergo surgery
of some type, which includes preventative, diagnostic or staging,
curative and palliative surgery. Curative surgery is a cancer treatment
that may be used in conjunction with other therapies, such as the
treatment of the present invention, chemotherapy, radiotherapy,
hormonal therapy, gene therapy, immunotherapy and/or alternative
 Curative surgery includes resection in which all or part
of cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery includes
laser surgery, cryosurgery, clectrosurgery, and microscopically
controlled surgery (Mohs' surgery). It is further contemplated that
the present invention may be used in conjunction with removal of
superficial cancers, precancers, or incidental amounts of normal
 Upon excision of part of all of cancerous cells, tissue,
or tumor, a cavity may be formed in the body. Treatment may be accomplished
by perfusion, direct injection or local application of the area
with an additional anti-cancer therapy. Such treatment may be repeated,
for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3,
4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12
months. These treatments may be of varying dosages as well.
 (iii) Chemotherapeutic Agents
 Agents that damage DNA are chemotherapeutics. These can
be, for example, agents that directly cross-link DNA, agents that
intercalate into DNA, and agents that lead to chromosomal and mitotic
aberrations by affecting nucleic acid synthesis. Agents that directly
cross-link nucleic acids, specifically DNA, are envisaged and are
exemplified by cisplatin, and other DNA alkylating agents. Agents
that damage DNA also include compounds that interfere with DNA replication,
mitosis, and chromosomal segregation.
 Some examples of chemotherapeutic agents include antibiotic
chemotherapeutics such as, Doxorubicin, Daunorubicin, Mitomycin
(also known as mutamycin and/or mitomycin-C), Actinomycin D (Dactinomycin),
Bleomycin, Plicomycin. Plant alkaloids such as Taxol, Vincristine,
Vinblastine. Miscellaneous agents such as Cisplatin, VP16, Tumor
Necrosis Factor. Alkylating Agents such as, Carmustine, Melphalan
(also known as alkeran, L-phenylalanine mustard, phenylalanine mustard,
L-PAM, or L-sarcolysin, is a phenylalanine derivative of nitrogen
mustard), Cyclophosphamide, Chlorambucil, Busulfan (also known as
myleran), Lomustine. And other agents for example, Cisplatin (CDDP),
Carboplatin, Procarbazine, Mechlorethamine, Camptothecin, Ifosfamide,
Nitrosurea, Etoposide (VP16), Tamoxifen, Raloxifene, Estrogen Receptor
Binding Agents, Gemcitabien, Navelbine, Farnesyl-protein transferase
inhibitors, Transplatinum, 5-Fluorouracil, and Methotrexate, Temazolomide
(an aqueous form of DTIC), or any analog or derivative variant of
 (iv) Immunotherapy
 Immunotherapeutics may be used in conjunction with the therapy
contemplated in the present invention. Immunotherapeutics, generally,
rely on the use of immune effector cells and molecules to target
and destroy cancer cells. The immune effector may be, for example,
another antibody specific for some other marker on the surface of
a tumor cell. This antibody in itself may serve as an effector of
therapy or it may recruit other cells to actually effect cell killing.
This antibody also may be conjugated to a drug or toxin (chemotherapeutic,
radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.)
and serve merely as a targeting agent. Alternatively, the effector
may be a lymphocyte carrying a surface molecule that interacts,
either directly or indirectly, with a tumor cell target.
 In one aspect the immunotherapy can be used to target a
tumor cell. Many tumor markers exist and any of these may be suitable
for targeting in the context of the present invention. Common tumor
markers include carcinoembryonic antigen, prostate specific antigen,
urinary tumor associated antigen, fetal antigen, tyrosinase (p97),
gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen
receptor, laminin receptor, erb B and p155. Alternate immune stimulating
molecules also exist including: cytokines such as IL-2, IL-4, IL-12,
GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8 and growth
factors such as FLT3 ligand.
 (a) Passive Immunotherapy
 A number of different approaches for passive immunotherapy
of cancer exist. They may be broadly categorized into the following:
injection of antibodies alone; injection of antibodies coupled to
toxins or chemotherapeutic agents; injection of antibodies coupled
to radioactive isotopes; injection of anti-idiotype antibodies;
and finally, purging of tumor cells in bone marrow.
 (b) Active Immunotherapy
 In active immunotherapy, an antigenic peptide, polypeptide
or protein, or an autologous or allogenic tumor cell composition
or "vaccine" is administered, generally with a distinct
bacterial adjuvant (Ravindranath & Morton, 1991; Morton &
Ravindranath, 1996; Morton et al., 1992; Mitchell et al., 1990;
Mitchell et al., 1993).
 (c) Adoptive Immunotherapy
 In adoptive immunotherapy, the patient's circulating lymphocytes,
or tumor infiltrated lymphocytes, are isolated in vitro, activated
by lymphokines such as IL-2 or transduced with genes for tumor necrosis,
and readministered (Rosenberg et al., 1988; 1989). To achieve this,
one would administer to an animal, or human patient, an immunologically
effective amount of activated lymphocytes in combination with an
adjuvant-incorporated antigenic peptide composition as described
herein. The activated lymphocytes will most preferably be the patient's
own cells that were earlier isolated from a blood or tumor sample
and activated (or "expanded") in vitro.
 (v) Gene Therapy
 The present invention contemplates the use of a variety
of different therapeutic transgenes in combination with the antisense
therapy of the present invention. For example, genes encoding a
tumor suppressor, an inhibitor of apoptosis, a cell cycle regulatory
gene, a toxin, a cytokine, a ribosome inhibitory protein and interferons
are contemplated as suitable genes that potentiate the inhibition
of cancer cell growth according to the present invention.
 (a) Tumor Suppressors
 The tumor suppressors function to inhibit excessive cellular
proliferation. The inactivation of these genes destroys their inhibitory
activity, resulting in unregulated proliferation. It is contemplated
that the antisense oligonucleotide may be attached to antibodies
that recognize mutant tumor suppressors or wild-type tumor suppressors.
Alternatively, an antisense construct may be linked to all or part
of the tumor suppressor. Exemplary tumor suppressors are p53, p16
and C--CAM which are described below.
 High levels of mutant p53 have been found in many cells
transformed by chemical carcinogenesis, ultraviolet radiation, and
several viruses. The p53 gene is a frequent target of mutational
inactivation in a wide variety of human tumors and is already documented
to be the most frequently mutated gene in common human cancers.
It is mutated in over 50% of human NSCLC (Hollstein et al., 1991)
and in a wide spectrum of other tumors. The p53 gene encodes a 393-amino
acid phosphoprotein that can form complexes with host proteins such
as large-T antigen and E1B. The protein is found in normal tissues
and cells, but at concentrations which are minute by comparison
with transformed cells or tumor tissue.
 Wild-type p53 is recognized as an important growth regulator
in many cell types. Missense mutations are common for the p53 gene
and are essential for the transforming ability of the oncogene.
A single genetic change prompted by point mutations can create carcinogenic
p53. Unlike other oncogenes, however, p53 point mutations are known
to occur in at least 30 distinct codons, often creating dominant
alleles that produce shifts in cell phenotype without a reduction
to homozygosity. Additionally, many of these dominant negative alleles
appear to be tolerated in the organism and passed on in the germ
line. Various mutant alleles appear to range from minimally dysfunctional
to strongly penetrant, dominant negative alleles (Weinberg, 1991).
 Another inhibitor of cellular proliferation is p16. The
major transitions of the eukaryotic cell cycle are triggered by
cyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase
4 (CDK4), regulates progression through the G.sub.1. The activity
of this enzyme may be to phosphorylate Rb at late G.sub.1. The activity
of CDK4 is controlled by an activating subunit, D-type cyclin, and
by an inhibitory subunit, the p16.sup.INK4 has been biochemically
characterized as a protein that specifically binds to and inhibits
CDK4, and thus may regulate Rb phosphorylation (Serrano et al.,
1993; Serrano et al., 1995). Since the p16.sup.INK4 protein is a
CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase
the activity of CDK4, resulting in hyperphosphorylation of the Rb
protein. p16 also is known to regulate the function of CDK6.
 p16.sup.INK4 belongs to a newly described class of CDK-inhibitory
proteins that also includes p16.sup.B, p19, p21.sup.WAF1, and p27.sup.KIP1.
The p16 gene maps to 9p21, a chromosome region frequently deleted
in many tumor types. Homozygous deletions and mutations of the p16.sup.INK4
gene are frequent in human tumor cell lines. This evidence suggests
that the p16.sup.INK4 gene is a tumor suppressor gene. This interpretation
has been challenged, however, by the observation that the frequency
of the p16.sup.INK4 gene alterations is much lower in primary uncultured
tumors than in cultured cell lines (Caldas et al., 1994; Cheng et
al., 1994; Hussussian et al., 1994; Kamb et al., 1994; Okamoto et
al., 1994; Nobori et al., 1995; Orlow et al., 1994). Restoration
of wild-type p16.sup.INK4 function by transfection with a plasmid
expression vector reduced colony formation by some human cancer
cell lines (Okamoto, 1994).
 Other genes that may be employed according to the present
invention include Rb, APC, mda-7, DCC, NF-1, NF-2, WT-1, MEN-I,
MEN-II, zac1, p73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16
fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI),
PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp, hst,
abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF,
thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.
 (b) Regulators of Programmed Cell Death
 Apoptosis, or programmed cell death, is an essential process
for normal embryonic development, maintaining homeostasis in adult
tissues, and suppressing carcinogenesis (Kerr et al., 1972). The
Bc1-2 family of proteins and ICE-like proteases have been demonstrated
to be important regulators and effectors of apoptosis in other systems.
The Bc1-2 protein, discovered in association with follicular lymphoma,
plays a prominent role in controlling apoptosis and enhancing cell
survival in response to diverse apoptotic stimuli (Bakhshi et al.,
1985; Cleary and Sklar, 1985; Cleary et al., 1986; Tsujimoto et
al., 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved
Bc1-2 protein now is recognized to be a member of a family of related
proteins, which can be categorized as death agonists or death antagonists.
 Apo2 ligand (Apo2L, also called TRAIL) is a member of the
tumor necrosis factor (TNF) cytokine family. TRAIL activates rapid
apoptosis in many types of cancer cells, yet is not toxic to normal
cells. TRAIL mRNA occurs in a wide variety of tissues. Most normal
cells appear to be resistant to TRAIL's cytotoxic action, suggesting
the existence of mechanisms that can protect against apoptosis induction
by TRAIL. The first receptor described for TRAIL, called death receptor
4 (DR4), contains a cytoplasmic "death domain"; DR4 transmits
the apoptosis signal carried by TRAIL. Additional receptors have
been identified that bind to TRAIL. One receptor, called DR5, contains
a cytoplasmic death domain and signals apoptosis much like DR4.
The DR4 and DR5 mRNAs are expressed in many normal tissues and tumor
cell lines. Recently, decoy receptors such as DcR1 and DcR2 have
been identified that prevent TRAIL from inducing apoptosis through
DR4 and DR5. These decoy receptors thus represent a novel mechanism
for regulating sensitivity to a pro-apoptotic cytokine directly
at the cell's surface. The preferential expression of these inhibitory
receptors in normal tissues suggests that TRAIL may be useful as
an anticancer agent that induces apoptosis in cancer cells while
sparing normal cells (Marsters et al., 1999).
 Subsequent to its discovery, it was shown that Bc1-2 acts
to suppress cell death triggered by a variety of stimuli. Also,
it now is apparent that there is a family of Bc1-2 cell death regulatory
proteins which share in common structural and sequence homologies.
These different family members have been shown to either possess
similar functions to Bc1-2 (e.g., Bc1.sub.XL, Bc1.sub.W, Bc1.sub.S,
Mc1-1, A1, Bf1-1) or counteract Bc1-2 function and promote cell
death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri). It is contemplated
that any of these polypeptides, including TRAIL, or any other polypeptides
that induce or promote of apoptosis, may be operatively linked to
an antisense construct, or that an antibody recognizing any of these
polypeptides may also be attached to an antisense construct.
 It will be appreciated by those of skill in the art that
monoclonal or polyclonal antibodies specific for proteins that are
preferentially expressed in metastatic or nonmetastatic cancer will
have utilities in several types of applications. These may include
the production of diagnostic kits for use in detecting or diagnosing
human cancer. An alternative use would be to link such antibodies
to therapeutic agents, such as chemotherapeutic agents, followed
by administration to individuals with cancer, thereby selectively
targeting the cancer cells for destruction. The skilled practitioner
will realize that such uses are within the scope of the present
 (c) Interferons
 Other classes of genes that are contemplated to be inserted
into the vectors of the present invention include interferons, interleukins
and cytokines. Inteferon-.alpha., interferon-.beta., interferon-.gamma.,
interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, angiostatin, thrombospondin,
endostatin, METH-1, METH-2, Flk2/Flt3 ligand, GM-CSF, G-CSF, M-CSF,
and tumor necrosis factor (TNF).
 (d) Cell Cycle Regulatory Genes
 In another embodiment, the present invention utilizes an
isolated nucleic acid segment comprising a cell cycle regulatory
gene operatively linked to an antisense oligonucleotide of the present
invention; transferring the nucleic acid segment into a cancer cell
to obtain a transfected cell; and maintaining the cancer cell under
conditions effective to express the cell cycle regulatory gene;
wherein expression of the cell cycle regulatory gene inhibits proliferation
of the cancer cell. In the practice of the method, the cell cycle
regulatory gene operatively linked to an antisense oligonucleotide
may comprise a liposomal or a non-liposomal vector. In the present
invention, it comprises a liposomal vector. Further, the cell cycle
regulatory gene may preferably encode Rb, p53, cell cycle dependent
kinase, CDK kinase, cyclin or a constitutively active Rb gene product,
or an antisense RNA.
 (e) Toxin Encoding Genes
 In another embodiment, the present invention may be described
as a method of inhibiting tumor cell growth comprising the steps
of: obtaining an isolated nucleic acid segment comprising a toxin
encoding gene. The genes may encode TNF.alpha., gelonin, ricin A
Chain, Pseudomonas exotoxin, diphtheria toxin, mitogillin, saporin,
ribosome inhibitory protein.
 (f) Oncogenes
 Oncogenes are considered to be genes that, when mutated
or activated, sponsor the development of cancer. Therapeutic intervention
involves the inhibition of these gene products. For example, one
may provide antisense or ribozymes which inhibit the transcription,
processing or translation of an oncogene. Alternatively, single
chain antibodies that encode products bind to and inhibit the oncogene
can be utilized. Table 3 provides a list of suitable oncogene targets.
4TABLE 3 Gene Source Human Disease Function Growth Factors HST/KS
Transfection FGF family member INT-2 MMTV promoter FGF family member
Insertion INTI/WNTI MMTV promoter Factor-like Insertion SIS Simian
sarcoma PDGF B virus Receptor Tyrosine Kinases ERBB/HER Avian Amplified,
deleted EGF/TGF-.alpha./ erythroblastosis squamous cell Amphiregulin/
virus; ALV cancer; Hetacellulin promoter glioblastoma receptor insertion;
amplified human tumors ERBB-2/NEU/HER- Transfected from rat Amplified
breast, Regulated by NDF/ 2 Glioblastomas ovarian, gastric Heregulin
and cancers EGF-Related factors FMS SM feline sarcoma CSF-1 receptor
virus KIT HZ feline sarcoma MGF/Steel receptor virus Hematopoieis
TRK Transfection from NGF (nerve growth human colon Factor) receptor
cancer MET Transfection from Scatter factor/HGF human Receptor osteosarcoma
RET Translocations and Sporadic thyroid Orphan receptor Tyr point
mutations cancer; Kinase familial medullary thyroid cancer; multiple
endocrine neoplasias 2A and 2B ROS URII avian sarcoma Orphan receptor
Tyr Virus Kinase PDGF receptor Translocation Chronic TEL(ETS-like
Myelomonocytic transcription Leukemia factor)/ PDGF receptor gene
Fusion TGF-.beta. receptor Colon carcinoma mismatch mutation target
NONRECEPTOR TYROSINE KINASES ABI. Abelson Mul. V Chronic Interact
with RB, myelogenous RNA leukemia polymerase, CRK, translocation
CBL with BCR FPS/FES Avian Fujinami SV; GA FeSV LCK Mul. V (murine
Src family; T-cell leukemia signaling; interacts virus) promoter
CD4/CD8 T-cells insertion SRC Avian Rous Membrane- sarcoma associated
Tyr Virus kinase with signaling function; activated by receptor
kinases YES Avian Y73 virus Src family; signaling SER/THR PROTEIN
KINASES AKT AKT8 murine Regulated by retrovirus PJ(3)K?; regulate
70-kd S6 k? MOS Moloney murine SV GVBD; cystostatic factor; MAP
kinase kinase PIM-1 Promoter insertion Mouse RAF/MIL 3611 murine
SV; Signaling in RAS MH2 Pathway avian SV MISCELLANEOUS CELL SURFACE
APC Tumor suppressor Colon cancer Interacts with catenins DCC Tumor
suppressor Colon cancer CAM domains E-cadherin Candidate tumor Breast
cancer Extracellular Suppressor homotypic binding; intracellular
interacts with catenins PTC/NBCCS Tumor suppressor Nevoid basal
cell 12 transmembrane and cancer domain; signals Drosophilia syndrome
(Gorline through Gli homology syndrome) homogue CI to antagonize
hedgehog pathway TAN-1 Notch Translocation T-ALI. Signaling? homologue
MISCELLANEOUS SIGNALING BCL-2 Translocation B-cell lymphoma Apoptosis
CBL Mu Cas NS-1 V Tyrosine- Phosphorylated RING finger interact
Abl CRK CT1010 ASV Adapted SH2/SH3 interact Abl DPC4 Tumor suppressor
Pancreatic cancer TGF-.beta.-related signaling Pathway MAS Transfection
and Possible angiotensin Tumorigenicity Receptor NCK Adaptor SH2/SH3
GUANINE NUCLEOTIDE EXCHANGERS AND BINDING PROTEINS BCR Translocated
with Exchanger; protein ABL Kinase in CML DBL Transfection Exchanger
GSP NF-1 Hereditary tumor Tumor suppressor RAS GAP Suppressor neurofibromatosis
OST Transfection Exchanger Harvey-Kirsten, N- HaRat SV; Ki Point
mutations in Signal cascade RAS RaSV; many Balb-MoMuSV; human tumors
Transfection VAV Transfection S112/S113; exchanger NUCLEAR PROTEINS
AND TRANSCRIPTION FACTORS BRCA1 Heritable suppressor Mammary Localization
cancer/ovarian unsettled cancer BRCA2 Heritable suppressor Mammary
cancer Function unknown ERBA Avian thyroid hormone erythroblastosis
receptor Virus (transcription) ETS Avian E26 virus DNA binding EVII
MuLV promotor AML Transcription factor Insertion FOS FBI/FBR murine
1 transcription osteosarcoma factor viruses with c-JUN GLI Amplified
glioma Glioma Zinc finger; cubitus interrupts homologue is in hedgehog
signaling pathway; inhibitory link PTC and hedgehog HMGI/LIM Translocation
Lipoma Gene fusions high t(3:12) mobility group t(12:15) HMGI-C
(XT hook) and transcription factor LIM or acidic domain JUN ASV-17
Transcription factor AP-1 with FOS MLL/VHRX + Translocation/fusion
Acute myeloid Gene fusion of ELI/MEN ELL with MLL leukemia DNA-
Trithorax-like gene binding and methyl transferase MLL with ELI
RNA pol II elongation factor MYB Avian DNA binding myeloblastosis
Virus MYC Avian MC29; Burkitt's lymphoma DNA binding with Translocation
B- MAX partner; cell cyclin Lymphomas; regulation; interact promoter
RB?; regulate Insertion avian apoptosis? leukosis Virus N-MYC Amplified
Neuroblastoma L-MYC Lung cancer REL Avian NF-.kappa.B family transcription
factor Retriculoendotheliosis Virus SKI Avian SKV770 Transcription
factor Retrovirus VHL Heritable suppressor Von Hippel-Landau Negative
regulator syndrome or elongin; transcriptional elongation complex
WT-1 Wilms' tumor Transcription factor CELL CYCLE/DNA DAMAGE RESPONSE
ATM Hereditary disorder Ataxia- Protein/lipid kinase telangiectasia
homology; DNA damage response upstream in P53 pathway BCL-2 Translocation
Follicular Apoptosis lymphoma FACC Point mutation Fanconi's anemia
C (predisposition leukemia MDA-7 Fragile site 3p14.2 Lung carcinoma
Histidine triad- related diadenosine 5', 3''''- tetraphosphate asymmetric
hydrolase hMLI/MutL HNPCC Mismatch repair; MutL Homologue hMSH2/MutS
HNPCC Mismatch repair; MutS Homologue hPMS1 HNPCC Mismatch repair;
MutL Homologue hPMS2 HNPCC Mismatch repair; MutL Homologue INK4/MTS1
Adjacent INK-4B at Candidate MTS1 p16 CDK inhibitor 9p21; CDK suppressor
and complexes MLM melanoma gene INK4B/MTS2 Candidate p15 CDK inhibitor
suppressor MDM-2 Amplified Sarcoma Negative regulator p53 p53 Association
with Mutated >50% Transcription factor; SV40 human checkpoint
control; T antigen tumors, including apoptosis hereditary Li- Fraumeni
syndrome PRAD1/BCL1 Translocation with Parathyroid Cyclin D Parathyroid
adenoma; hormone B-CLL or IgG RB Hereditary Retinoblastoma; Interact
cyclin/cdk; Retinoblastoma; osteosarcoma; regulate E2F Association
with breast transcription factor many cancer; other DNA virus tumor
sporadic Antigens cancers XPA xeroderma Excision repair; pigmentosum;
skin photo- cancer product predisposition recognition; zinc finger
 (g) Other Agents
 It is contemplated that other agents may be used in combination
with the present invention to improve the therapeutic efficacy of
treatment. One form of therapy for use in conjunction with chemotherapy
includes hyperthermia, which is a procedure in which a patient's
tissue is exposed to high temperatures (up to 106.degree. F.). External
or internal heating devices may be involved in the application of
local, regional, or whole-body hyperthermia. Local hyperthermia
involves the application of heat to a small area, such as a tumor.
Heat may be generated externally with high-frequency waves targeting
a tumor from a device outside the body. Internal heat may involve
a sterile probe, including thin, heated wires or hollow tubes filled
with warm water, implanted microwave antennae, or radio frequency
 A patient's organ or a limb is heated for regional therapy,
which is accomplished using devices that produce high energy, such
as magnets. Alternatively, some of the patient's blood may be removed
and heated before being perfused into an area that will be internally
heated. Whole-body heating may also be implemented in cases where
cancer has spread throughout the body. Warm-water blankets, hot
wax, inductive coils, and thermal chambers may be used for this
 Hormonal therapy may also be used in conjunction with the
present invention. The use of hormones may be employed in the treatment
of certain cancers such as breast, prostate, ovarian, or cervical
cancer to lower the level or block the effects of certain hormones
such as testosterone or estrogen and this often reduces the risk
 The terms "contacted" and "exposed,"
when applied to a cell, are used herein to describe the process
by which a therapeutic construct or protein and a chemotherapeutic
or radiotherapeutic agent are delivered to a target cell or are
placed in direct juxtaposition with the target cell. To achieve
cell killing or stasis, both agents are delivered to a cell in a
combined amount effective to kill the cell or prevent it from dividing.
IX. PROGNOSTIC APPLICATIONS
 As described earlier, the WT1 mRNA can be spliced in two
different ways leading to the expression of at least four predominant
isoforms (Haber et al., 1991). One splicing inserts or removes 17
amino acids in exon 5; the other splicing inserts or removes the
3-amino-acid Lys-Thr-Ser (KTS) in exon 9, located between zinc fingers
3 and 4 (Lee et al., 2001; Wang et al., 1995). All of the resulting
WT1 isoforms can positively or negatively regulate gene expression
(Klamt et al., 1998). Throughout the application, reference to WT1
will encompass its various isoforms.
 The WT1 splicing isoforms have different biological activities
(Lee et al., 2001). Of the two major splicing products encoded by
WT1, the -KTS isoforms have transactivational properties in some
genes that are involved in cell growth and differentiation, whereas
the +KTS isoforms have a potential role in RNA processing (Lee et
al., 2001). Exon 5 may function as a repressor domain or as an activator
domain, depending on which proteins are interacting with WT1 (Richard
et al., 2001).
 WT1 mRNA is readily detected by Northern (RNA) blot in most
Wilms tumor, as well as normal fet al kidney tissue (Haber et al.,
1990). With the more sensitive RNA PCR technique, alternatively
spliced WT1 transcripts can be easily demonstrated in all tissues.
However, PCR analysis can only provide an approximate ratio of the
various RNA species and due to the positions and sizes of two alternative
splices, it cannot be used to distinguish various splicing combinations.
 To determine the existence and relative abundance of various
forms of the WT1 transcript, an RNase protection assay has been
developed which is capable of differentiating each form based on
a protected fragment of distinctive length (Haber et al., 1991).
The functional role of each WT1 isoform in breast cancer cells such
as cell proliferation, sensitivity to estrogens and anti-estrogens,
sensitivity to apoptotic and chemotherapeutic stimuli may enable
one to determine whether a patient's breast tumor has high expression
of a certain WT1 isoform, and may potentially be able to predict
what kind of therapy the breast tumor will respond to. Techniques
such as RT-PCR or RNase protection assay may be used to determine
the level of expression of a certain WT1 isoform.
 The present invention further contemplates that the evaluation
of the expression level of one or more isoforms of WT1 gene product
in a cancer cell will be useful to effectively predict the efficacy
of a cancer therapeutic regimen, to determine whether the patient's
cancer will be responsive to a particular cancer therapeutic regimen
by analyzing the cancer tissues or cancer cells of a patient and
to monitor the progression of breast cancer in a patient. The method
of the present invention will involve obtaining a sample from said
subject comprising breast cancer cells and assessing expression
of one or more isoforms of Wilms' Tumor 1 (WT1) gene product in
 The present invention's prognostic method therefore allows
the determination of the need for specific cancer therapeutic regimens
based on the expression of WT1 in an individual patient.
 The expression levels of WT1 protein will also be useful
in monitoring the effectiveness of a treatment regimen, such as
that of the present invention, alone or in conjunction with other
cancer therapies as described above. Again, in such a situation
the level of expression of WT1 protein will be used to effectively
determine and adjust the dosage of a radiation and/or chemotherapeutic
combination regimen. In any event, the methods of the present invention
will assist physicians in determining optimal treatment courses
and doses for individuals with different tumors of varying malignancy
based on the levels of expression of WT1 proteins in such tumors.
 As described herein, the amount of a WT1 polypeptide/protein
and/or mRNA present within a biological sample or specimen, such
as a tissue, a cell(s), blood or serum or plasma sample, any other
biological fluid, a biopsy, needle biopsy cores, surgical resection
samples, lymph node tissue, or any other clinical sample may be
determined by means of a molecular biological assay to determine
the level of a nucleic acid that encodes such a polypeptide, or
by means of a polypeptide/protein detection assay such as a western
blot, northern blot (to quantitate RNA), RNA-PCR or even by means
of an immunoassay may be detected and measured or quantified. Such
detection and measuring/quantification methods may be used to measure
WT1 protein levels or WT1 mRNA levels and such methods are known
to one of skill in the art.
 In certain embodiments, nucleic acids or polypeptides would
be extracted from these samples. Some embodiments would utilize
kits containing pre-selected primer pairs or hybridization probes,
such as an antisense construct of the present invention. Antibodies
may also be used for this purpose. The amplified nucleic acids or
polypeptide would be tested for the presence of a WT1 polypeptide/protein
and/or mRNA by any of the detection methods described later in the
description or other suitable methods known in the art.
 In other embodiments, sample/specimen extracts containing
a WT1 polypeptide/protein and/or mRNA would be extracted from a
sample and subjected to an immunoassay. Immunoassays of tissue sections
are also possible. Immunoassays that are contemplated useful are
well known to one of skill in the art. Kits containing the antibodies
to WT1 polypeptides would be useful.
 In terms of analyzing tissue samples, irrespective of the
manner in which the level of a WT1 polypeptide/protein and/or mRNA
is determined, the prognostic evaluation may generally require the
amount of the WT1 product in the tissue sample to be compared to
the amount in normal cells, in other patients and/or amounts at
an earlier stage of treatment of the same patient. Comparing the
varying levels will allow the characteristics of the particular
cancer to be more precisely defined and therefore allow for prescribing
a tailor made cancer treatment regimen to a patient.
 Thus, it is contemplated that the levels of a WT1 polypeptide/protein
and/or mRNA detected would be compared with statistically valid
groups of metastatic, non-metastatic malignant, benign or normal
tissue samples; and/or with earlier WT1 levels in the same patient.
The diagnosis and prognosis of the individual patient would be determined
by comparison with such groups.
 If desired, the cancer prognostic methods of the present
invention may be readily combined with other methods in order to
provide an even more reliable indication of prognosis. Various markers
of cancer have been proposed to be correlated with metastasis and
malignancy. They are generally classified as cytological, protein
or nucleic acid markers. Any one or more of such methods may thus
be combined with those of this invention in order to provide a multi-marker
prognostic test. Some examples of tumor markers specific to breast
include p27, cyclin E, carcinoembryonic antigen (CEA), mucin associated
antigen, tumor polypeptide antigen and breast cancer specific antigen.
 Combination of the present techniques with one or more other
diagnostic or prognostic techniques or markers is certainly contemplated.
In that many cancers are multifactorial, the use of more than one
method or marker is often highly desirable.
 A. Prognostic Kits
 The materials and reagents required for detecting the levels
of expression of a WT1 polypeptide/protein and/or mRNA in a cancer
cell in a biological sample may be assembled together in a kit.
 One set of kits are designed to detect the levels of expression
of a WT1 nucleic acid. Such kits of the invention will generally
comprise one or more preselected primers or probes specific for
WT1. The antisense constructs of the present invention may be used
as hybridization probes or primers. Preferably, the kits will comprise,
in suitable container means, one or more nucleic acid probes or
primers and means for detecting nucleic acids. In certain embodiments,
such as in kits for use in Northern blotting, the means for detecting
the nucleic acids may be a label, such as a radiolabel, that is
linked to a nucleic acid probe itself.
 Preferred kits are those suitable for use in PCR.TM. which
is described later in the specification. In PCR.TM. kits, two primers
will preferably be provided that have sequences from, and that hybridize
to, spatially distinct regions of the WT1 gene. Preferred pairs
of primers for amplifying nucleic acids are selected to amplify
the sequences specified herein. Also included in PCRTM kits may
be enzymes suitable for amplifying nucleic acids, including various
polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide
the necessary reaction mixture for amplification.
 In each case, the kits will preferably comprise distinct
containers for each individual reagent and enzyme, as well as for
each cancer probe or primer pair. Each biological agent will generally
be suitable aliquoted in their respective containers.
 The container means of the kits will generally include at
least one vial or test tube. Flasks, bottles and other container
means into which the reagents are placed and aliquoted are also
possible. The individual containers of the kit will preferably be
maintained in close confinement for commercial sale. Suitable larger
containers may include injection or blow-molded plastic containers
into which the desired vials are retained. Instructions may be provided
with the kit.
 In further embodiments, the invention provides immunological
kits for use in detecting the levels of expression of WT1 in biological
samples, e.g., in cancer cells. Such kits will generally comprise
one or more antibodies that have immunospecificity for WT1 proteins
 As the anti-WT1 antibodies may be employed to detect WT1
proteins or peptides and their levels, both of such components may
be provided in the kit. The immunodetection kits will thus comprise,
in suitable container means, a WT1 protein or peptide, or a first
antibody that binds to such a protein or peptide, and an immunodetection
 In other embodiments, it is contemplated that the antibodies
will be those that bind to the WT1 antigenic epitopes. MAbs are
readily prepared and will often be preferred. Where proteins or
peptides are provided, it is generally preferred that they be highly
 In certain embodiments, the WT1 protein or peptide, or the
first antibody that binds to the WT1 protein or peptide may be bound
to a solid support, such as a column matrix or well of a microtitre
 The immunodetection reagents of the kit may take any one
of a variety of forms, including those detectable labels that are
associated with, or linked to, the given antibody or antigen itself.
Detectable labels that are associated with or attached to a secondary
binding ligand are also contemplated. Exemplary secondary ligands
are those secondary antibodies that have binding affinity for the
first antibody or antigen.
 Further suitable immunodetection reagents for use in the
present kits include the two-component reagent that comprises a
secondary antibody that has binding affinity for the first antibody
or antigen (generally anti-WT1) along with a third antibody that
has binding affinity for the second antibody, wherein the third
antibody is linked to a detectable label.
 As noted above in the discussion of antibody conjugates,
a number of exemplary labels are known in the art and all such labels
may be employed in connection with the present invention. Radiolabels,
nuclear magnetic spin-resonance isotopes, fluorescent labels and
enzyme tags capable of generating a colored product upon contact
with an appropriate substrate are suitable examples.
 The kits may contain antibody-label conjugates either in
fully conjugated form, in the form of intermediates, or as separate
moieties to be conjugated by the user of the kit.
 The kits may further comprise a suitably aliquoted composition
of a WT1 antigen whether labeled or unlabeled, as may be used to
prepare a standard curve for a detection assay.
 The kits of the invention, regardless of type, will generally
comprise one or more containers into which the biological agents
are placed and, preferably, suitable aliquoted. The components of
the kits may be packaged either in aqueous media or in lyophilized
 The immunodetection kits of the invention, may additionally
contain one or more of a variety of other cancer marker antibodies
or antigens, if so desired. Such kits could thus provide a panel
of cancer markers, as may be better used in testing a variety of
patients. By way of example, such additional markers could include,
other tumor markers such as breast cancer antigen CA15-3, p53, BR
27.29, HER-2/neu, BRCA-1, and BRCA-2. The container means of the
kits will generally include at least one vial, test tube, flask,
bottle, or even syringe or other container means, into which the
antibody or antigen may be placed, and preferably, suitably aliquoted.
Where a second or third binding ligand or additional component is
provided, the kit will also generally contain a second, third or
other additional container into which this ligand or component may
 The kits of the present invention will also typically include
a means for containing the antisense composition, and any other
reagent containers in close confinement for commercial sale. Such
containers may include injection or blow-molded plastic containers
into which the desired vials are retained.
X. SCREENING ASSAYS
 The present invention contemplates the screening of compounds
for abilities to affect expression or function of isoforms of WT1.
Particularly preferred compounds will be those useful in inhibiting
the expression of WT1, thus inhibiting the growth of breast cancer
cells. In the screening assays of the present invention, the candidate
substance may first be screened for basic activity--e.g., binding
to a target molecule--and then tested for its ability to inhibit
expression, at the cellular, tissue or whole animal level.
 The present invention provides methods of screening for
candidate substances that show activity against breast cancer. In
one embodiment, the present invention is directed to a method of:
 (i) providing a cell that expresses one or more isoforms
of the Wilms' Tumor 1 (WT1) gene product;
 (ii) contacting the cell with the candidate substance suspected
of inhibiting WT1; and
 (iii) measuring the effect of the candidate substance on
 The candidate substance may be a protein, a nucleic acid
or a small molecule pharmaceutical. As a result of measurement,
a decrease in the amount of one or more WT1 isoform gene products
or gene transcripts in said cell, as compared to a cell not treated
with said candidate substance, indicates that said candidate substance
has activity against breast cancer.
 In still yet other embodiments, one would look at the effect
of a candidate substance on the expression of WT1. This can be done
by examining mRNA expression, although the clinical results could
be insufficient. A more direct way of assessing expression is by
directly examining protein levels, for example, through Western
blot or ELISA. An inhibitor according to the present invention may
be one which exerts an inhibitory effect on the expression or function
 As used herein, the term "candidate substance"
refers to any molecule that may inhibit growth of cancer cells.
The candidate substance may be a protein or fragment thereof, a
small molecule inhibitor, or even a nucleic acid molecule. It may
prove to be the case that the most useful pharmacological compounds
will be compounds that are structurally related to compounds which
interact naturally with WT1. Creating and examining the action of
such molecules is known as "rational drug design," and
include making predictions relating to the structure of target molecules.
 The goal of rational drug design is to produce structural
analogs of biologically active polypeptides or target compounds.
By creating such analogs, it is possible to fashion drugs which
are more active or stable than the natural molecules, which have
different susceptibility to alteration or which may affect the function
of various other molecules. In one approach, one would generate
a three-dimensional structure for a molecule like a WT1, and then
design a molecule for its ability to interact with WT1. Alternatively,
one could design a partially functional fragment of a WT1 (binding
but no activity), thereby creating a competitive inhibitor. This
could be accomplished by x-ray crystallography, computer modeling
or by a combination of both approaches.
 It also is possible to use antibodies to ascertain the structure
of a target compound or inhibitor. In principle, this approach yields
a pharmacore upon which subsequent drug design can be based. It
is possible to bypass protein crystallography altogether by generating
anti-idiotypic antibodies to a functional, pharmacologically active
antibody. As a mirror image of a mirror image, the binding site
of anti-idiotype would be expected to be an analog of the original
antigen. The anti-idiotype could then be used to identify and isolate
peptides from banks of chemically- or biologically-produced peptides.
Selected peptides would then serve as the pharmacore. Anti-idiotypes
may be generated using the methods described herein for producing
antibodies, using an antibody as the antigen.
 On the other hand, one may simply acquire, from various
commercial sources, small molecule libraries that are believed to
meet the basic criteria for useful drugs in an effort to "brute
force" the identification of useful compounds. Screening of
such libraries, including combinatorially generated libraries (e.g.,
peptide libraries), is a rapid and efficient way to screen large
number of related (and unrelated) compounds for activity. Combinatorial
approaches also lend themselves to rapid evolution of potential
drugs by the creation of second, third and fourth generation compounds
modeled of active, but otherwise undesirable compounds.
 Candidate compounds may include fragments or parts of naturally-occurring
compounds or may be found as active combinations of known compounds
which are otherwise inactive. It is proposed that compounds isolated
from natural sources, such as animals, bacteria, fungi, plant sources,
including leaves and bark, and marine samples may be assayed as
candidates for the presence of potentially useful pharmaceutical
agents. It will be understood that the pharmaceutical agents to
be screened could also be derived or synthesized from chemical compositions
or man-made compounds. Thus, it is understood that the candidate
substance identified by the present invention may be polypeptide,
polynucleotide, small molecule inhibitors or any other compounds
that may be designed through rational drug design starting from
known inhibitors of hypertrophic response.
 The candidate substance suspected of inhibiting WT1 expression
may be an antisense molecule. In an assay that comprises the screening
of such molecules, the cell that expresses one or more isoforms
of Wilms' Tumor gene product is contacted with the antisense molecule
suspected of inhibiting WT1 expressing cells. The ability of the
antisense construct to inhibit the expression of WT1 may be assayed
by methods such as measuring the levels of expression of the WT1
gene or measuring the levels of the WT1 gene product in the cell.
Other suitable inhibitors include ribozymes, and antibodies (including
single chain antibodies).
 It will, of course, be understood that all the screening
methods of the present invention are useful in themselves notwithstanding
the fact that effective candidates may not be found. The invention
provides methods for screening for such candidates, not solely methods
of finding them.
 A. In vitro Assays
 A quick, inexpensive and easy assay to run is a binding
assay. Binding of a molecule to a target may, in and of itself,
be inhibitory, due to steric, allosteric or charge-charge interactions.
This can be performed in solution or on a solid phase and can be
utilized as a first round screen to rapidly eliminate certain compounds
before moving into more sophisticated screening assays.
 The target may be either free in solution, fixed to a support,
expressed in or on the surface of a cell. Either the target or the
compound may be labeled, thereby permitting determining of binding.
In another embodiment, the assay may measure the inhibition of binding
of a target to a natural or artificial substrate or binding partner
(such as a WT1).
 Competitive binding assays can be performed in which one
of the agents (WT1 for example) is labeled. Usually, the target
will be the labeled species, decreasing the chance that the labeling
will interfere with the binding moiety's function. One may measure
the amount of free label versus bound label to determine binding
or inhibition of binding.
 A technique for high throughput screening of compounds is
described in WO 84/03564. Large numbers of small peptide test compounds
are synthesized on a solid substrate, such as plastic pins or some
other surface. The peptide test compounds are reacted with, for
example, a WT1 and washed. Bound polypeptide is detected by various
 Purified target, such as a WT1, can be coated directly onto
plates for use in the aforementioned drug screening techniques.
However, non-neutralizing antibodies to the polypeptide can be used
to immobilize the polypeptide to a solid phase. Also, fusion proteins
containing a reactive region (preferably a terminal region) may
be used to link an active region (e.g., the C-terminus of a WT1)
to a solid phase.
 B. In cyto Assays
 Various cell lines that express isoforms of WT1 can be utilized
for screening of candidate substances. For example, cells containing
a WT1 with an engineered indicator can be used to study various
functional attributes of candidate compounds. In such assays, the
compound would be formulated appropriately, given its biochemical
nature, and contacted with a target cell.
 Molecular analysis may be performed in which the function
of a WT 1 and related genes may be explored. This involves assays
such as those for protein expression, enzyme function, substrate
utilization, mRNA expression (including differential display of
whole cell or polyA RNA) and others.
XI. QUANTITATING LEVELS OF EXPRESSION OF WT1
 The levels of expression of WT1 polypeptide/protein and/or
mRNA is a function of the proliferation of breast cancer cells and
is thus useful for various purposes such as a prognostic method
for determining the breast cancer progression, as a screening method
to know whether a candidate substance is able to inhibit cancer
by inhibiting the WT1 gene or gene product and also in determining
the type of treatment/combination treatment that may be used on
a patient depending on the efficacy of the treatment. It may also
be used to determine the progress of a patient when treated with
an antisense oligonucleotide therapy or to determine what type or
dose of the therapeutic regimen are suitable. Therefore, some embodiments
of the invention concern measuring and/or quantitation and/or estimation
of levels of WT1 expression.
 A. Quantitative PCR
 For quantitation of a nucleic acid, reverse transcription
(RT) of RNA to cDNA followed by relative quantitative or semi-quantitative
PCR.TM. (RT-PCR.TM.) can be used to determine the relative concentrations
of specific mRNA species in a series of total cell RNAs isolated
from the cancer cells.
 By determining that the concentration of a specific mRNA
species varies, it is shown that the gene encoding the specific
mRNA species is expressed at different levels in different types
 In PCR.TM., the number of molecules of the amplified target
DNA increase by a factor approaching two with every cycle of the
reaction until some reagent becomes limiting. Thereafter, the rate
of amplification becomes increasingly diminished until there is
not an increase in the amplified target between cycles. If one plots
a graph on which the cycle number is on the X axis and the log of
the concentration of the amplified target DNA is on the Y axis,
one observes that a curved line of characteristic shape is formed
by connecting the plotted points.
 Beginning with the first cycle, the slope of the line is
positive and constant. This is said to be the linear portion of
the curve. After some reagent becomes limiting, the slope of the
line begins to decrease and eventually becomes zero. At this point
the concentration of the amplified target DNA becomes asymptotic
to some fixed value. This is said to be the plateau portion of the
 The concentration of the target DNA in the linear portion
of the PCR.TM. is directly proportional to the starting concentration
of the target before the PCR.TM. was begun. By determining the concentration
of the PCR.TM. products of the target DNA in PCR.TM. reactions that
have completed the same number of cycles and are in their linear
ranges, it is possible to determine the relative concentrations
of the specific target sequence in the original DNA mixture.
 If the DNA mixtures are cDNAs synthesized from RNAs isolated
from different cells, the relative abundances of the specific mRNA
from which the target sequence was derived can be determined for
the respective tissues or cells. This direct proportionality between
the concentration of the PCR.TM. products and the relative mRNA
abundances is only true in the linear range portion of the PCR.TM.
 The final concentration of the target DNA in the plateau
portion of the curve is determined by the availability of reagents
in the reaction mix and is independent the original concentration
of target DNA. Therefore, the first condition that must be met before
the relative abundances of a mRNA species can be determined by RT-PCR.TM.
for a collection of RNA populations is that the concentrations of
the amplified PCR.TM. products must be sampled when the PCR.TM.
reactions are in the linear portion of their curves.
 The second condition that must be met for an RT-PCR.TM.
study to successfully determine the relative abundances of a particular
mRNA species is that relative concentrations of the amplifiable
cDNAs must be normalized to some independent standard. The goal
of an RT-PCR.TM. study is to determine the abundance of a particular
mRNA species relative to the average abundance of all mRNA species
in the sample. In such studies, mRNAs for .beta.-actin, asparagine
synthetase and lipocortin II may be used as external and internal
standards to which the relative abundance of other mRNAs are compared.
 Most protocols for competitive PCR.TM. utilize internal
PCR.TM. internal standards that are approximately as abundant as
the target. Other studies are available that use a more conventional
relative quantitative RT-PCR.TM. with an external standard protocol.
 B. Immunodetection Methods
 In still further embodiments, the present invention concerns
immunodetection methods for binding, purifying, removing, quantifying
or otherwise generally detecting WT1 gene product. The steps of
various useful immunodetection methods have been described in the
scientific literature, such as, e.g., Nakamura et al. (1987; incorporated
herein by reference). Immunoassays, in their most simple and direct
sense, are binding assays. Certain preferred immunoassays are the
various types of enzyme linked immunosorbent assays (ELISAs), radioimmunoassays
(RIA) and immunobead capture assay. Immunohistochemical detection
using tissue sections also is particularly useful. However, it will
be readily appreciated that detection is not limited to such techniques,
and Western blotting, dot blotting, FACS analyses, and the like
also may be used in connection with the present invention.
 1. Immunohistochemistry
 Fresh-frozen and formalin-fixed, paraffin-embedded tissue
blocks may be prepared from study by immunohistochemistry (IHC).
For example, each tissue block consists of 50 mg of residual "pulverized"
tumor. The method of preparing tissue blocks from these particulate
specimens has been successfully used in previous IHC studies of
various prognostic factors, e.g., in breast, and is well known to
those of skill in the art (Brown et al., 1990; Abbondanzo et al.,
1990; Allred et al., 1990).
 Briefly, frozen-sections may be prepared by rehydrating
50 ng of frozen "pulverized" tumor at room temperature
in phosphate buffered saline (PBS) in small plastic capsules; pelleting
the particles by centrifugation; resuspending them in a viscous
embedding medium (OCT); inverting the capsule and pelleting again
by centrifugation; snap-freezing in -70.degree. C. isopentane; cutting
the plastic capsule and removing the frozen cylinder of tissue;
securing the tissue cylinder on a cryostat microtome chuck; and
cutting 25-50 serial sections containing an average of about 500
remarkably intact tumor cells.
 Permanent-sections may be prepared by a similar method involving
rehydration of the 50 mg sample in a plastic microfuge tube; pelleting;
resuspending in 10% formalin for 4 h fixation; washing/pelleting;
resuspending in warm 2.5% agar; pelleting; cooling in ice water
to harden the agar; removing the tissue/agar block from the tube;
infiltrating and embedding the block in paraffin; and cutting up
to 50 serial permanent sections.
 2. FACS
 Fluorescent activated cell sorting, flow cytometry or flow
microfluorometry provides the means of scanning individual cells
for the presence of an antigen, such as WT1. The method employs
instrumentation that is capable of activating, and detecting the
excitation emissions of labeled cells in a liquid medium.
 FACS is unique in its ability to provide a rapid, reliable,
quantitative, and multiparameter analysis on either living or fixed
cells. Cells would generally be obtained by biopsy, single cell
suspension in blood or culture. FACS analyses would probably be
most useful when desiring to analyze a number of cancer antigens
at a given time, e.g., to follow an antigen profile during disease
 3. Western Blots
 Western blotting may be used to detect inhibition of proliferation
of breast cancer cell lines due to specific inhibition of WT1 protein
expression. The antisense construct of the present invention may
be used as high-affinity primary reagents for the identification
of WT1 gene product immobilized onto a solid support matrix, such
as nitrocellulose, nylon or combinations thereof. The technique
of western blots is well known to a person of ordinary skill in
 The following examples are included to demonstrate particular
embodiments of the invention. It should be appreciated by those
of skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific embodiments
which are disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention.
 Materials And Methods
 Cell Culture
 The ER.alpha.-positive breast cancer cell lines MCF-7, BT-474,
T-47D, and MDA-MB-361 (Sutherland et al., 1988; Fitzgerald et al.,
1997), and the ER.alpha.-negative breast cancer cell lines SKBr-3,
MDA-MB-231, MDA-MB-453, BT-20, and MDA-MB-468 (Fitzgerald et al.,
1997; Love-Schimenti et al., 1996) were obtained from the American
Type Culture Collection (Manassas, Va.). They were propagated in
DMEM/F12 medium supplemented with 10% FCS. The human leukemia cell
line K562, chosen as a positive control cell line because of its
high expression of WT1 protein (Yamagami et al., 1996), was also
obtained from ATCC and propagated in RPMI 1640 medium supplemented
with 10% FCS. All cell lines were incubated in 95% air and 5% CO.sub.2
at 37.degree. C.
 Western Blotting
 Western blotting was used to determine the expression levels
of WT1 proteins in nuclear extracts from breast cancer and leukemia
cell lines since these proteins are known to localize within the
nucleus (Dobashi et al., 1997). Protein concentration was determined
by using the Bio-Rad DC protein concentration assay. Briefly 50
.mu.g of proteins were subjected to electrophoresis on 12% SDS-polyacrylamide
gels and transferred to nitrocellulose membranes. Immunodetection
was done using rabbit antibodies specific for WT1 (C-19) from Santa
Cruz Biotechnology (Santa Cruz, Calif.) and anti-rabbit secondary
antibodies conjugated with horseradish peroxidase (Amersham Life
Science Inc.). Protein bands were visualized by enhanced chemiluminescence
(Kirkegaard & Perry Laboratories, Gaithersburg, Md.).
 Preparation of Liposome--Incorporated Oligonucleotides
 The following is a brief description of how oligonucleotides
may be incorporated in a liposome. For details one may refer to
Tari et al. (2000).
 The oligonucleotides were radiolabelled with .sup.32p radioisotope
and incorporated in DOPC lipids purchased from Avanti Polar Lipids
(Alabaster, Ala.). DOPC was dissolved in tert-butanol at 20 mg/ml.
Oligonucleotides are dissolved in water or DMSO at .about.8 mg/ml.
Oligonucleotides are aliquoted and mixed well before adding excess
tert-butanol. Because DMSO is present, tert-butanol should be added
for at least 95% (v/v) so that the mixture can be well frozen in
an acetone/dry ice bath before being lyophilized overnight. The
lyophilized preparation is hydrated with 0.9% normal saline at a
final oligonucleotide concentration of 0.1 mM.
 WT1 Antisense Oligos and Cell Treatment
 P-ethoxy oligos, purchased from Oligos Etc., Inc. (Wilsonville,
Oreg.), were incorporated into DOPC liposomes (Tari et al., 2000).
The sequence of the WT1 antisense oligos targeted against the translation
initiation site is 5'-GTCGGAGCCCATTTGCTG-3' (SEQ ID NO: 1), and
the sequence of the control oligos is 5'-GGGCTTTTGAACTCTGCT-3' (SEQ
ID NO: 2) (Yamagami et al., 1996). Breast cancer and leukemia cells
were plated in 96-well plates (2.times.10.sup.3 cells per well)
in DMEM/F12 supplemented with 10% FCS and allowed to adhere overnight.
Then various concentrations (0, 3, 6, or 12 .mu.M) of liposomal
WT1 antisense (L-WT1) and liposomal control (L-control) oligos were
added to the cells and incubated for 72 h. Cell growth was determined
by using the CellTiter 96 Aqueous nonradioactive proliferation assay
(Promega, Madison, Wis.).
 Light Microscopic Evaluation of Cell Growth
 MCF-7 and MDA-MB-453 cells were seeded in 6-well plates
(1.0.times.10.sup.5 cells per well) in DMEM/F12 medium supplemented
with 10% FCS. After 24 h, the cells were treated with 12 .mu.M L-WT1
or L-control oligos for 3 days, examined under light microscopy
at 100.times. magnification, and photographed with Kodak gold 400
 RNA Purification and RT-PCR
 Total RNA was prepared from the cell lines by using 1 ml
of TRIzol Reagent (Life Technologies, Gaithersburg, Md.) according
to the manufacturer's protocol. The pellet of RNA was dissolved
in DEPC-treated-water and quantified by spectrophotometry at 260
nm. cDNA was created with Superscript II according to the manufacturer's
protocol (Gibco BRL). All PCR reactions were carried out with 5
.mu.l of cDNA, 0.2 mM dNTPs, 100 ng of each primer, 10 mM Tris-HCl
(pH 8.4, 50 mM KCl, 0.01% gelatin, 1.5 mM MgCl.sub.2), and 2.5 U
of Taq DNA polymerase. PCR to detect the different WT1 isoforms
was performed with primers as described by Brenner et al (Brenner
et al., 1992). The thermal profile involved 35 cycles of denaturation
at 94.degree. C. for 40 s, primer annealing at 64.degree. C. for
30 s, and extension at 72.degree. C. for 30 s. PCR products were
subjected to electrophoresis on 2% agarose gels and the reaction
products were visualized with ethidium bromide and photographed
under UV transillumination.
 Expression of WT1 Protein in Breast Cancer Cell Lines
 The endogenous expression of the 52-54 kDa WT1 protein in
breast cancer cell lines was assessed and K562 leukemic cells were
used as positive control. WT1 protein was detected in the nuclear
extracts of both ER-positive and ER-negative cell lines (FIG. 1).
 The results obtained indicate that WT1 protein is vital
for the proliferation of breast cancer cells, regardless of whether
the cells are ER-positive or ER-negative. The inventors found no
correlation between the basal expression of WT1 protein and the
inhibitory response to L-WT 1. Nor was any correlation evident between
inhibition by L-WT1 and the status of p53 protein, as MCF-7 is the
only cell line that expresses the wild-type 53 protein (Casey et
 Reduction of WT1 Protein Expression Leads to Growth Inhibition
of Breast Cancer. It was first verified that L-WT1 oligos could
inhibit the growth of K562 leukemia cells (Yamagami et al., 1996;
Algar et al., 1996) in a dose dependent manner (FIG. 2A). Next,
the effect of L-WT1 on MCF-7 cells, an ER-positive cell line with
high endogenous WT1 expression, and on MDA-MB-453 cells, an ER-negative
cell line with low endogenous WT1 expression, was studied. L-WT1
induced dose-dependent growth inhibition in both cell lines (FIG.
2B). Maximal growth inhibition (>90%) was observed at 12 .mu.M
L-WT1; therefore, this concentration was used for the subsequent
experiments. These findings were further expanded to 7 more breast
cancer cell lines, the ER-positive BT-474, T-47D, and MDA-MB-361
and the ER-negative cell lines SKBr-3, MDA-MB-231, BT-20, and MDA-MB-468.
L-WT1 inhibited the growth of 8 of the 9 breast cancer cell lines,
with greater than 50% effects in MCF-7, T-47D, and MDA-MB-453 cells
(FIG. 2C). Under the same conditions, approximately 50% growth inhibition
was observed in BT-474 and MDA-MB-468 cells while less than 50%
growth inhibition was observed in MDA-MB-361, SKBr-3, and BT-20
cells. No growth inhibition was observed in MDA-MB-231 cells.
 Western blotting confirmed that the inhibition of proliferation
in MCF-7 and MDA-MB-453 cells was due to specific inhibition of
WT1 protein expression (FIG. 2D).
 The inventors have detected WT1 mRNA in all cell lines and
this contradicts a previous report by Loeb et al. (2001) who found
WT1 mRNA in T-47D and MDA-MB-468 cells but not in MCF-7, MDA-MB-231
or SKBr-3 cells. However these investigators used one round of PCR,
whereas the inventors subjected cells to reamplification by using
nested PCR. The inventors' data indicated low but detectable WT1
levels in breast cancer cells, and the reamplification allowed the
different WT1 isoforms to be detected as well.
 Light Microscopy. Using light microscopy, the inventors
observed that L-WT1 reduced the number of MCF-7 and MDA-MB-453 cells
as compared with untreated cells (FIG. 3). But L-control did not
decrease the number of MCF-7 and MDA-MB-453 cells.
 Expression of WT1 mRNA Isoforms. RT-PCR was used to determine
whether expression of the total WT1 mRNA and its isoforms was associated
with the growth inhibition of breast cancer cells. The highest total
mRNA expression was detected in T-47D and MDA-MB-468 cells, since
the PCR products of WT1 (857 bp) in these cell lines were detected
in the first round of PCR (FIG. 4A). However in the other cell lines,
the PCR products of WT1 were not detected until the second round
 To identify the various WT1 mRNA isoforms, the inevntors
first amplified the KTS region with specific primers to exon 7 and
primers to the KTS- or KTS+ areas in exon 9. All cell lines produce
two products, but the KTS- isoform was more abundant than the KTS+
isoform (FIG. 4B). To detect exon 5 isoforms, primers to exon 1
and primers to KTS- or KTS+ isoforms were used (FIG. 4C), all four
WT1 isoforms were detected in the control K562 cells and in the
ER-positive cells. Among the four ER-positive cells, MDA-MB-361
cells had the lowest expression of these isoforms. All four isoforms
were also detected in two of the five ER-negative cell lines MDA-MB-453
and MDA-MB-468. However, only the exon 5+/KTS+ and exon 5-/KTS-
isoforms were detected in the SKBr-3 cells, and only the exon 5+/KTS+
and exon 5+/KTS- isoforms were detected in the BT-20 cells. No PCR
products were observed in the MDA-MB-231 cells.
 As described earlier in the description, the WT1 splicing
isoforms have different biological activities (Lee et al., 2001).
The KTS- isoforms have transactivational properties in some genes
that are involved in cell growth and differentiation, whereas the
KTS+ isoforms have a potential role in RNA processing (Lee et al.,
2001). Exon 5 may function as a repressor domain or as an activator
domain, depending on which proteins are interacting with WT1 (Richard
et al., 2001). In the inventors' study, all five cell lines in which
L-WT1 led to .gtoreq.50% growth inhibition contain all four WT1
isoforms. But the two cell lines that were little affected by L-WT1
expressed only two WT1 isoforms, and the one cell line that was
not affected by L-WT1 expressed no WT1 isoforms. These data show
that the regulation of breast cancer cell growth by WT1 protein
may depend on the expression of all four WT1 isoforms.
 All of the methods and compositions disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred embodiments,
it will be apparent to those of skill in the art that variations
may be applied to the methods and compositions and in the steps
or in the sequence of steps of the method described herein without
departing from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents which are
both chemically and physiologically related may be substituted for
the agents described herein while the same or similar results would
be achieved. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended claims.
 The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
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Sequence CWU 1
2 1 18 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 1 gtcggagccc atttgctg 18 2 18 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Primer 2 gggcttttga