U.S. patent application number 16/683205 was filed with the patent office on 2021-07-08 for targeting gene amplification in cancer using triplex formation as a therapeutic strategy.
This patent application is currently assigned to Yale University. The applicant listed for this patent is Yale University. Invention is credited to Faye A. Rogers.
Application Number | 20210206874 16/683205 |
Document ID | / |
Family ID | 1000005666493 |
Filed Date | 2021-07-08 |
United States Patent
Application |
20210206874 |
Kind Code |
A9 |
Rogers; Faye A. |
July 8, 2021 |
TARGETING GENE AMPLIFICATION IN CANCER USING TRIPLEX FORMATION AS A
THERAPEUTIC STRATEGY
Abstract
Disclosed herein are methods and agents for the treatment of
cancer using p53-independent apoptosis to reduce the number of
p53-depleted or p53-mutated cancer cells that have amplified HER2
gene. Also disclosed herein are methods and agents for the
treatment of HER2-positive cancer in individuals with Li-Fraumeni
Syndrome.
Inventors: |
Rogers; Faye A.; (Norwalk,
CT) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Yale University |
New Haven |
CT |
US |
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Assignee: |
Yale University
New Haven
CT
|
Prior
Publication: |
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Document Identifier |
Publication Date |
|
US 20200190211 A1 |
June 18, 2020 |
|
|
Family ID: |
1000005666493 |
Appl. No.: |
16/683205 |
Filed: |
November 13, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62767279 |
Nov 14, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6807 20170801;
A61K 47/543 20170801; C07K 2317/76 20130101; C07K 16/3015 20130101;
C07K 2317/73 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61K 47/68 20060101 A61K047/68; A61K 47/54 20060101
A61K047/54 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under
R21CA185192 and R01GM126211 awarded by the National Institutes of
Health. The government has certain rights in the invention.
Claims
1. A method of reducing, in a population of cells, the number of
p53-depleted cancer cells in which a HER2 gene is amplified, the
method comprising contacting p53-depleted cancer cells with triplex
forming oligonucleotides (TFOs) targeted to a polypurine target
site in the amplified-HER2 gene, under conditions under which the
TFOs enter the p53-depleted cancer cells in sufficient quantity to
induce apoptosis.
2. The method of claim 1, wherein the p53-depleted cells are
mammalian cells.
3. (canceled)
4. The method of claim 1, wherein the polypurine target site
is/comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO:
6.
5. The method of claim 1, wherein the TFOs are at least 13
nucleotides in length.
6. (canceled)
7. (canceled)
8. The method of claim 1, wherein the TFOs comprise at least one of
the following: a nucleotide sequence at least 90% identical to SEQ
ID NO: 3 and/or a nucleotide sequence at least 90% identical to SEQ
ID NO: 4 and/or a nucleotide sequence at least 90% identical to SEQ
ID NO: 7 and/or a nucleotide at least 90% identical to SEQ ID NO:
8.
9-13. (canceled)
14. A method of reducing, in a population of cells, the number of
p53-mutated cancer cells in which a HER2 gene is amplified, the
method comprising contacting p53-mutated cancer cells with triplex
forming oligonucleotides (TFOs) targeted to a polypurine site in
the amplified-HER2 gene, under conditions under which the TFOs
enter the p53-mutated cancer cells in sufficient quantity to induce
apoptosis.
15. The method of claim 14, wherein the p53-mutated cells are
mammalian cells.
16. (canceled)
17. (canceled)
18. The method of claim 14, wherein the TFOs are at least 13
nucleotides in length.
19. (canceled)
20. (canceled)
21. The method of claim 14, wherein the TFOs comprise at least one
of the following: a nucleotide sequence at least 90% identical to
SEQ ID NO: 3, and/or a nucleotide sequence at least 90% identical
to SEQ ID NO: 4, and/or a nucleotide sequence at least 90%
identical to SEQ ID NO: 7 and/or a nucleotide at least 90%
identical to SEQ ID NO: 8.
22-26. (canceled)
27. A method of treating cancer in an individual with Li-Fraumeni
syndrome, the method comprising administering to the individual
TFOs targeted to a polypurine target site in an amplified-HER2
gene, under conditions under which the TFOs enter p53-depleted
cancer cells in sufficient quantity to induce apoptosis.
28. The method of claim 27, wherein the polypurine target site is
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 6.
29. The method of claim 27, wherein the TFOs are at least 13
nucleotides in length.
30. (canceled)
31. (canceled)
32. The method of claim 27, wherein the TFOs comprise at least one
of the following: a nucleotide sequence at least 90% identical to
SEQ ID NO: 3, and/or a nucleotide sequence at least 90% identical
to SEQ ID NO: 4, and/or a nucleotide sequence at least 90%
identical to SEQ ID NO: 7, and/or a nucleotide at least 90%
identical to SEQ ID NO: 8.
33. The method of claim 27, wherein the TFOs are in a delivery
vehicle or are conjugated to a delivery vehicle.
34-40. (canceled)
41. A method of administering TFOs for the treatment of cancer, the
method comprising: (i) preparing a mixture of TFOs targeted to a
polypurine target site in an amplified-HER2 gene; and (ii)
administering the mixture of TFOs to an individual, in sufficient
quantity to induce p53-independent apoptosis.
42-58. (canceled)
59. A composition, comprising: (i) TFOs targeted to a polypurine
target site in an amplified-HER2 gene in sufficient quantity to
induce p53-independent apoptosis in a p53-depleted cancer cell or a
p53-mutated cancer cell; and (ii) a pharmaceutically acceptable
carrier.
60. The composition of claim 59, further comprising: (iii) lipid
nanoparticles, wherein the TFOs are encapsulated in the lipid
nanoparticles or wherein the TFOs are conjugated to the lipid
nanoparticles.
61. The composition of claim 59, wherein the polypurine target site
is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 6.
62. The composition of claim 59, wherein the TFOs are at least 13
nucleotides in length.
63. (canceled)
64. (canceled)
65. The composition of claim 59, wherein the TFOs comprise at least
one of the following: a nucleotide sequence at least 90% identical
to SEQ ID NO: 3, and/or a nucleotide sequence at least 90%
identical to SEQ ID NO: 4, and/or a nucleotide sequence at least
90% identical to SEQ ID NO: 7 and/or a nucleotide at least 90%
identical to SEQ ID NO: 8.
66-68. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. provisional application Ser. No. 62/767,279, filed
Nov. 14, 2018, the content of which is incorporated by reference
herein in its entirety.
BACKGROUND
[0003] Gene amplification often leads to higher expression of genes
involved in normal cell growth and survival pathways.sup.1,2. As
such, gene amplification is a major mechanism driving oncogenesis
in a broad spectrum of cancers, ultimately affecting tumor
progression and clinical outcome.sup.3,4. Several drugs have been
developed to inhibit the oncogenic activity of amplified driver
genes.sup.5. The majority of these cancer therapeutics target the
overexpressed protein products and their clinical efficacy is often
hampered by drug resistance.sup.6,7.
SUMMARY
[0004] Described herein is a novel therapeutic method for the
treatment of cancers that are characterized by gene amplification,
and, in one embodiment specifically, the treatment of cancers that
are characterized by HER2 gene amplification. In the method,
manipulation of the DNA damage response with triplex-forming
oligonucleotides (TFOs) drives p53-independent tumor-specific
induction of apoptosis. The method described is particularly
applicable to p53-independent cancers, which are often aggressive
and resistant to traditional chemotherapeutic drugs. This provides
a new and specific approach in targeted cancer therapy, which can
have enormous impact on the field of precision medicine.
[0005] Accordingly, one aspect of the present disclosure provides a
method of reducing, in a population of cells, the number of
p53-depleted cancer cells in which a HER2 gene is amplified, the
method comprising contacting p53-depleted cancer cells with triplex
forming oligonucleotides (TFOs) targeted to a polypurine target
site in the amplified-HER2 gene, under conditions under which the
TFOs enter the p53-depleted cancer cells in sufficient quantity to
induce apoptosis. In some embodiments, the p53-depleted cells are
mammalian cells. In some embodiments, the p53-depleted cells are
human cells. In some embodiments, the polypurine target site
is/comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:5 or SEQ ID
NO:6. In some embodiments, the TFOs are at least 13 nucleotides in
length. In some embodiments, the TFOs are at least 22 nucleotides
in length. In some embodiments, at least 13 of the nucleotides
hybridize to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:5 or SEQ ID
NO:6.
[0006] TFOs can be administered individually (e.g., all TFOs
administered have the same sequence) or a combination of two or
more TFOs can be administered (e.g., TFOs administered comprise
different nucleotide sequences). In some embodiments, the TFOs
comprise a nucleotide sequence at least 90% identical to SEQ ID NO:
3; a nucleotide sequence at least 90% identical to SEQ ID NO: 4; a
nucleotide sequence at least 90% identical to SEQ ID NO: 7; a
nucleotide at least 90% identical to SEQ ID NO: 8; or a combination
of two, three or four of the foregoing. For example, TFOs
administered can comprise a nucleotide sequence at least 90%
identical to SEQ ID NO: 3 and a nucleotide sequence at least 90%
identical to SEQ ID NO: 4; a nucleotide sequence at least 90%
identical to SEQ ID NO: 3 and a nucleotide sequence at least 90%
identical to SEQ ID NO: 7; a nucleotide sequence at least 90%
identical to SEQ ID NO: 3 and a nucleotide at least 90% identical
to SEQ ID NO: 8; a nucleotide sequence at least 90% identical to
SEQ ID NO: 4 and a nucleotide sequence at least 90% identical to
SEQ ID NO: 7; a nucleotide sequence at least 90% identical to SEQ
ID NO: 4 and a nucleotide sequence at least 90% identical to SEQ ID
NO:8; a nucleotide sequence at least 90% identical to SEQ ID NO: 7
and a nucleotide sequence at least 90% identical to SEQ ID NO:
8.
[0007] In some embodiments, three different TFOs are administered.
For example, the following can be administered: [0008] a TFO that
comprises a nucleotide sequence at least 90% identical to SEQ ID
NO: 3, a TFO that comprises a nucleotide sequence at least 90%
identical to SEQ ID NO: 4 and a TFO that comprises a nucleotide
sequence at least 90% identical to SEQ ID NO: 7; [0009] a TFO that
comprises a nucleotide sequence at least 90% identical to SEQ ID
NO: 3, a TFO that comprises a nucleotide sequence at least 90%
identical to SEQ ID NO: 4 and a TFO that comprises a nucleotide
sequence at least 90% identical to SEQ ID NO: 8; [0010] a TFO that
comprises a nucleotide sequence at least 90% identical to SEQ ID
NO: 3, a TFO that comprises a nucleotide sequence at least 90%
identical to SEQ ID NO: 7 and a TFO that comprises a nucleotide
sequence at least 90% identical to SEQ ID NO: 8; or [0011] a TFO
that comprises a nucleotide sequence at least 90% identical to SEQ
ID NO: 4, a TFO that comprises a nucleotide sequence at least 90%
identical to SEQ ID NO: 7 and a TFO that comprises a nucleotide
sequence at least 90% identical to SEQ ID NO: 8.
[0012] In further embodiments, four different TFOs are
administered: a (at least one) TFO that comprises a nucleotide
sequence at least 90% identical to SEQ ID NO: 3; a (at least one)
TFO that comprises a nucleotide sequence at least 90% identical to
SEQ ID NO: 4; a (at least one) TFO that comprises a nucleotide
sequence at least 90% identical to SEQ ID NO: 7; and a (at least
one) TFO that comprises a nucleotide at least 90% identical to SEQ
ID NO: 8.
[0013] Alternatively, TFOs that comprise a nucleotide sequence
identical to SEQ ID NO: 3; TFOs that comprise a nucleotide sequence
identical to SEQ ID NO: 4; TFOs that comprise a nucleotide sequence
identical to SEQ ID NO: 7; and TFOs that comprise a nucleotide
sequence identical to SEQ ID NO: 8 can be administered individually
(e.g., all TFOs administered have the same sequence) or a
combination of two or more TFOs can be administered (e.g., TFOs
administered comprise different nucleotide sequences).
[0014] For example, TFOs administered can comprise a nucleotide
sequence identical to SEQ ID NO: 3 and a nucleotide sequence
identical to SEQ ID NO: 4; a nucleotide sequence identical to SEQ
ID NO: 3 and a nucleotide sequence identical to SEQ ID NO: 7; a
nucleotide sequence identical to SEQ ID NO: 3 and a nucleotide
sequence identical to SEQ ID NO: 8; a nucleotide sequence identical
to SEQ ID NO: 4 and a nucleotide sequence identical to SEQ ID NO:
7; a nucleotide sequence identical to SEQ ID NO: 4 and a nucleotide
sequence identical to SEQ ID NO:8; a nucleotide sequence identical
to SEQ ID NO: 7 and a nucleotide sequence identical to SEQ ID NO:
8.
[0015] In some embodiments, three different TFOs are administered.
For example, the following can be administered: [0016] a TFO that
comprises a nucleotide sequence identical to SEQ ID NO: 3, a TFO
that comprises a nucleotide sequence identical to SEQ ID NO: 4 and
a TFO that comprises a nucleotide sequence identical to SEQ ID NO:
7; [0017] a TFO that comprises a nucleotide sequence identical to
SEQ ID NO: 3, a TFO that comprises a nucleotide sequence identical
to SEQ ID NO: 4 and a TFO that comprises a nucleotide sequence
identical to SEQ ID NO: 8; [0018] a TFO that comprises a nucleotide
sequence identical to SEQ ID NO: 3, a TFO that comprises a
nucleotide sequence identical to SEQ ID NO: 7 and a TFO that
comprises a nucleotide sequence identical to SEQ ID NO: 8; or
[0019] a TFO that comprises a nucleotide sequence identical to SEQ
ID NO: 4, a TFO that comprises a nucleotide sequence identical to
SEQ ID NO: 7 and a TFO that comprises a nucleotide sequence
identical to SEQ ID NO: 8.
[0020] In further embodiments, four different TFOs are
administered: a (at least one) TFO that comprises a nucleotide
sequence identical to SEQ ID NO: 3; a (at least one) TFO that
comprises a nucleotide sequence identical to SEQ ID NO: 4; a (at
least one) TFO that comprises a nucleotide sequence identical to
SEQ ID NO: 7; and a (at least one) TFO that comprises a nucleotide
identical to SEQ ID NO: 8.
[0021] In some embodiments, the triplex forming oligonucleotides
(TFOs) are in a delivery vehicle or are conjugated to a delivery
vehicle. In some embodiments, the delivery vehicle is lipid
nanoparticles. In some embodiments, the TFOs have backbone
modifications. In some embodiments, the backbone modifications
include phosphorothioates, phosphorodithioates, methylphosphonates,
phosphoramidates, boranophosphate oligos, polyamides,
methylene(methylimino) linkages, morpholino oligos, or some
combination thereof. In some embodiments, the p53-depleted cancer
cells are renal cell carcinoma cells, lung cancer cells, colon
cancer cells, colon carcinoma cells, ovarian cancer cells, breast
cancer cells, colorectal cancer cells, gastric cancer cells, and/or
endometrial cancer cells.
[0022] Another aspect of the present disclosure provides a method
of reducing, in a population of cells, the number of p53-mutated
cancer cells in which a HER2 gene is amplified, the method
comprising contacting p53-mutated cancer cells with triplex forming
oligonucleotides (TFOs) targeted to a polypurine site in the
amplified-HER2 gene, under conditions under which the TFOs enter
the p53-mutated cancer cells in sufficient quantity to induce
apoptosis. In some embodiments, the p53-mutated cells are mammalian
cells. In some embodiments, the p53-mutated cells are human cells.
In some embodiments, the polypurine target site is SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the
TFOs are at least 13 nucleotides in length. In some embodiments,
the TFOs are at least 22 nucleotides in length. In some
embodiments, at least 13 of the nucleotides hybridize to SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO:5 or SEQ ID NO:6. In some embodiments,
the TFOs comprise a nucleotide sequence at least 90% identical to
SEQ ID NO: 3, and/or a nucleotide sequence at least 90% identical
to SEQ ID NO: 4, and/or a nucleotide sequence at least 90%
identical to SEQ ID NO: 7 and/or a nucleotide at least 90%
identical to SEQ ID NO: 8. In some embodiments, the TFOs are in a
delivery vehicle or are conjugated to a delivery vehicle. In some
embodiments, the delivery vehicle is lipid nanoparticles. In some
embodiments, the TFOs have backbone modifications. In some
embodiments, the backbone modifications include phosphorothioates,
phosphorodithioates, methylphosphonates, phosphoramidates,
boranophosphate oligos, polyamides, methylene(methylimino)
linkages, morpholino oligos, or some combination thereof. In some
embodiments, the p53-mutated cancer cells are renal cell carcinoma
cells, lung cancer cells, colon cancer cells, colon carcinoma
cells, ovarian cancer cells, breast cancer cells, colorectal cancer
cells, gastric cancer cells, and/or endometrial cancer cells.
[0023] Another aspect of the present disclosure provides a method
of treating cancer in an individual with Li-Fraumeni syndrome, the
method comprising administering to the individual TFOs targeted to
a polypurine target site in an amplified-HER2 gene, under
conditions under which the TFOs enter p53-depleted cancer cells in
sufficient quantity to induce apoptosis. In some embodiments, the
polypurine target site is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:5
or SEQ ID NO:6. In some embodiments, the TFOs are at least 13
nucleotides in length. In some embodiments, the TFOs are at least
22 nucleotides in length. In some embodiments, at least 13 of the
nucleotides hybridize to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:5 or
SEQ ID NO:6. In some embodiments, the TFOs comprise a nucleotide
sequence at least 90% identical to SEQ ID NO: 3, and/or a
nucleotide sequence at least 90% identical to SEQ ID NO: 4, and/or
a nucleotide sequence at least 90% identical to SEQ ID NO: 7 and/or
a nucleotide at least 90% identical to SEQ ID NO: 8. In some
embodiments, the TFOs are in a delivery vehicle or are conjugated
to a delivery vehicle. In some embodiments, the delivery vehicle is
lipid nanoparticles. In some embodiments, the TFOs are encapsulated
in the lipid nanoparticles. In some embodiments, the TFOs have
backbone modifications. In some embodiments, the backbone
modifications include phosphorothioates, phosphorodithioates,
methylphosphonates, phosphoramidates, boranophosphate oligos,
polyamides, methylene (methylimino) linkages, morpholino oligos, or
some combination thereof. In some embodiments, the TFOs are
administered by injection. In some embodiments, the TFOs are
administered intratumorally or intraperitoneally. In some
embodiments, an anticancer agent that is not a TFO is administered
with the TFOs.
[0024] In some embodiments, the anticancer agent is a protein, a
nucleic acid, a small molecule, or a drug. In some embodiments, the
anticancer agent is a protein, a nucleic acid, a small molecule, or
a drug.
[0025] Another aspect of the present disclosure provides a method
of administering TFOs for the treatment of cancer, the method
comprising preparing a mixture of TFOs targeted to a polypurine
target site in an amplified-HER2 gene and administering the mixture
of TFOs to an individual, in sufficient quantity to induce
p53-independent apoptosis. In some embodiments, the mixture of TFOs
is encapsulated in lipid nanoparticles. In some embodiments, the
polypurine target site is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:5
or SEQ ID NO:6. In some embodiments, the TFOs are at least 13
nucleotides in length. In some embodiments, the TFOs are at least
22 nucleotides in length. In some embodiments, at least 13 of the
nucleotides hybridize to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:5 or
SEQ ID NO:6. In some embodiments, the mixture of TFOs comprises a
nucleotide sequence at least 90% identical to SEQ ID NO: 3, and/or
a nucleotide sequence at least 90% identical to SEQ ID NO: 4,
and/or a nucleotide sequence at least 90% identical to SEQ ID NO: 7
and/or a nucleotide at least 90% identical to SEQ ID NO: 8. In some
embodiments, the TFOs have backbone modifications. In some
embodiments, the backbone modifications include phosphorothioates,
phosphorodithioates, methylphosphonates, phosphoramidates,
boranophosphate oligos, polyamides, methylene(methylimino)
linkages, morpholino oligos, or some combination thereof. In some
embodiments, the mixture of TFOs is administered by injection. In
some embodiments, the mixture of TFOs is administered
intratumorally or intraperitoneally. In some embodiments, an
anticancer agent that is not a TFO is administered with the mixture
of TFOs. In some embodiments, the anticancer agent is a protein, a
nucleic acid, a small molecule, or a drug. In some embodiments, the
individual is a mammal. In some embodiments, the individual is a
human. In some embodiments, the individual is a model of cancer. In
some embodiments, the cancer is a carcinoma, a sarcoma or a
melanoma with HER-2 gene amplification. In some embodiments, the
model of cancer is selected from a group including a p53-knockout
mouse, a Li-Fraumeni Syndrome mouse, a mouse with MDA-MB-453 cells,
SKBR3 cells, BT474 cells, PEO1 cells, SKOV3 cells, and p53-knockout
mouse.
[0026] Another aspect of the present disclosure provides a
composition, comprising TFOs targeted to a polypurine target site
in an amplified-HER2 gene in sufficient quantity to induce
p53-independent apoptosis in a p53-depleted cancer cell or a
p53-mutated cancer cell, a pharmaceutically acceptable carrier, and
optionally lipid nanoparticles, wherein the TFOs are encapsulated
in the lipid nanoparticles or are conjugated to the lipid
nanoparticles. In some embodiments, the polypurine target site is
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:5 or SEQ ID NO:6. In some
embodiments, the TFOs are at least 13 nucleotides in length. In
some embodiments, the TFOs are at least 22 nucleotides in length.
In some embodiments, at least 13 of the nucleotides hybridize to
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:5 or SEQ ID NO:6. In some
embodiments, the TFOs comprise a nucleotide sequence at least 90%
identical to SEQ ID NO: 3, and/or a nucleotide sequence at least
90% identical to SEQ ID NO: 4, and/or a nucleotide sequence at
least 90% identical to SEQ ID NO: 7 and/or a nucleotide at least
90% identical to SEQ ID NO: 8. In some embodiments, the TFOs have
backbone modifications. In some embodiments, the backbone
modifications include phosphorothioates, phosphorodithioates,
methylphosphonates, phosphoramidates, boranophosphate oligos,
polyamides, methylene(methylimino) linkages, morpholino oligos, or
some combination thereof. In some embodiments, the pharmaceutically
acceptable carrier comprises water or saline.
[0027] The details of one or more embodiments of the invention are
set forth in the description below. Other features or advantages of
the present invention will be apparent from the drawings and
detailed description of several embodiments, and also from the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present disclosure, which can be better understood
by reference to one or more of these drawings in combination with
the detailed description of specific embodiments presented herein.
It is to be understood that the data illustrated in the drawings in
no way limit the scope of the disclosure. In the drawings:
[0029] FIGS. 1A-1E include diagrams showing targeting of gene
amplification in cancer via triplex formation. FIG. 1A: a schematic
illustration showing a drug design scheme. Targeting the HER2 gene
on a genomic level using DNA-binding molecules provides a novel
therapeutic option to directly manipulate the DNA damage response
pathways to specifically attack the HER2-amplified tumor.
Triplex-induced DNA damage will only provoke apoptosis when
multiple triplex structures are formed, while nucleotide excision
repair (NER)-dependent repair prevails in the presence of one or
two structures. FIG. 1B: a table showing the gene copy number
characteristics of breast cancer cells lines .sup.18. FIG. 1C: a
photo showing the western blot analysis of HER2 protein levels in
breast cancer cell lines with varying gene copy number. FIG. 1D: a
schematic illustration showing that TFOs bind as third strands in a
sequence-specific manner within the major groove of duplex DNA at
polypurine sites. The specificity of these molecules arises from
the formation of base triplets via reverse Hoogsteen hydrogen bonds
between the third strand and the polypurine strand of the duplex
DNA. Results shown are from the use of TFOs HER2-1 and HER2-205,
designed to bind to a polypurine sequence located either in the
promoter or the coding region of the HER2 gene. FIG. 1E: photos of
non-denatured metaphase chromosome spreads of MCF7 and BT474 breast
cancer cells demonstrate chromosomal binding of TAMRA-HER2-205
(red) to its target site located on chromosome (chr.) 17
(green).
[0030] FIGS. 2A-2H include diagrams showing that triplex induced
DNA damage and apoptosis correlate with gene copy number.
"UT"=untreated cells. "Mock"=cells with transfection reagent only.
"MIX24"=cells treated with control mixed sequence oligonucleotide,
MIX24. "HER2-1"=HER2-1-treated cells. "HER2-205"=HER2-205-treated
cells. FIG. 2A: Representative images of neutral comet assays
performed 24 h after HER2-205 treatment in MCF7 and BT474 cells.
FIG. 2B: a chart showing the quantification of triplex-induced DNA
double strand breaks using the neutral comet assay as measured by
tail moment in multiple breast cancer cell lines. FIG. 2C: a chart
showing that triplex-induced DNA damage increases in cell lines
containing multiple copies of the HER2 gene. FIG. 2D: a chart
showing the frequency of cells with more than 5 .gamma.H2AX foci
per nuclei following 24 h HER2-205 treatment. FIG. 2E:
Representative images of HER2-205 induced 53BP1 (green) and
.gamma.H2AX (red) foci in nuclei (blue) compared to MIX24 24 h
post-treatment in BT474 cells. FIG. 2F: a chart showing the
analysis of triplex-induced apoptosis as measured by Annexin-V
staining in breast cancer cell lines 24 h post TFO-treatment. FIG.
2G: a chart showing that the level of triplex-induced apoptosis
increases with gene copy number. FIG. 2H: a chart showing the
analysis of triplex-induced apoptosis in HER2-positive ovarian
cancer cells as measured by Annexin V staining 48 h post-treatment.
**** denotes p<0.0001, *** denotes p<0.001, ** denotes
p<0.01, and * denotes p<0.05
[0031] FIGS. 3A-3I include diagrams showing triplex-induced DNA
damage and activation of apoptosis in several HER2-positive breast
and ovarian cancer cell lines. "UT"=untreated cells. "Mock"=cells
with transfection reagent only. "MIX24"=cells treated with control
mixed sequence oligonucleotide, MIX24. "HER2-1"=HER2-1-treated
cells. "HER2-205"=HER2-205-treated cells. FIG. 3A: a chart showing
the quantification of cells with more than 5 .gamma.H2AX and/or
53BP1 foci per nuclei in BT474 cells treated with HER2-205 or
MIX24. FIG. 3B: images showing that triplex formation induces
apoptosis in HER2-positive breast cancer cell lines as measured by
Western blot analysis of cleaved PARP. FIG. 3C: images showing the
detection of HER2 copies in interphase nuclei by dual color FISH
with HER2 probe (red) and chromosome 17 probe (green). FIG. 3D:
images showing the immunofluorescence of .gamma.H2AX in PE01
ovarian cancer cells 24 h post-treatment with HER2-205 or MIX24.
FIG. 3E: representative immunofluorescence images of .gamma.H2AX
foci in SKOV3 ovarian cancer cells 24 h following treatment with
HER2-205 or MIX24. FIG. 3F: a chart showing the frequency of PEO1
and SKOV3 cells positive for .gamma.H2AX following 24 h treatment.
FIG. 3G: a chart showing the quantification of triplex-induced DNA
double strand breaks using the neutral comet assay as measured by
tail moment. FIG. 3H: images showing a monolayer growth assay that
demonstrates a decrease in cell survival in PEO1 and SKOV3 cells
treated with HER2-205 72 h after treatment. FIG. 3I: images showing
a western blot analysis of activation of apoptosis as measured by
cleaved PARP in ovarian cancer cells following TFO treatment.
[0032] FIGS. 4A-4G include diagrams showing the in vivo effect of
HER2-205 on human HER2-positive cancer xenografts. Tumor growth
delay curves of BT474 xenografts generated by subcutaneous
injection of female athymic nude mice. Twenty-eight days after
implantation mice were treated by intraperitoneal (IP) injection
with three doses of (FIG. 4A) HER2-205, (FIG. 4B) trastuzumab and
(FIG. 4C) MIX24 at a concentration of 20 mg/kg. Arrow indicates
administration of first dose. Tumor growth measurements .+-.SEM are
shown. FIG. 4D: a Kaplan-Meier plot of the percentage of tumors
smaller than three times baseline size. Baseline size was defined
as tumor size on the first day of treatment [Day 28 in (FIG. 4A)
and Day 21 in (FIG. 4B) and (FIG. 4C)]. FIG. 4E: images showing a
histopathologic analysis of BT474 tumor sections from mice 24 h
after treatment with a single dose of HER2-205 (20 mg/kg body
weight) or vehicle. Haematoxylin and eosin (H&E), caspase 3,
HER2, Ki67 stain at 4.times. magnification. Scale bar=10 .mu.m.
FIG. 4F: an image showing a higher magnification of H&E tumor
section from HER2-205 treatment specimen. FIG. 4G: a Kaplan-Meier
plot of the percentage of SKOV3 ovarian cancer tumors smaller than
three times baseline size. Mice were treated with 3 doses of
HER2-205 at a concentration of 20 mg/kg or cisplatin at a
concentration of 10 mg/kg.
[0033] FIGS. 5A-5F include diagrams showing the molecular mechanism
of anticancer activity. "UT"=untreated cells. "Mock"=cells with
transfection reagent only. "MIX24"=cells treated with control mixed
sequence oligonucleotide, MIX24. "HER2-205"=HER2-205-treated cells.
FIG. 5A: images showing a western blot analysis of the
phosphorylation status of the DNA damage response proteins Chk1 and
Chk2 following TFO treatment. FIG. 5B: images showing that the
knockdown of the NER factor, XPD, in BT474 cells results in a
decrease in the induction of apoptosis as measured by cleaved PARP
and pH2AX Y142. pH2AX Y142 is an essential post-translational
modification for the recruitment of pro-apoptotic factors to the
tail of .gamma.H2AX. FIG. 5C: images showing that HER2-205
activates p53-independent apoptosis in HER2-positive BT474 cells.
FIG. 5D: a chart showing the analysis of HER2 gene expression by
RT-PCR and FIG. 5E: images showing that determination of HER2
protein levels and phosphorylation status using Western blot
analysis provide evidence that HER2-205 achieves therapeutic
activity using a mechanism that is independent of HER2 cellular
function. FIG. 5F: a schematic illustration of molecular mechanism
of gene-targeted apoptosis. TFO binding in the major groove of
duplex DNA causes a distortion of the double helix, which can
induce DNA replication fork collapse and induction of DNA double
strand breaks (DSBs). DNA damage response activates an
XPD-dependent but p53-independent apoptotic pathway.
[0034] FIGS. 6A-6D include diagrams that support a molecular
mechanism that is independent of HER2 signaling pathways.
"UT"=untreated cells. "Mock"=cells with transfection reagent only.
"MIX24"=cells treated with control mixed sequence oligonucleotide,
MIX24. "HER2-205"=HER2-205-treated cells. FIG. 6A: a chart showing
the quantification of phosphorylated ATM by flow cytometry
following treatment with HER2-205. Western blot analysis of the
phosphorylation status of HER family receptors (FIG. 6B) HER3,
(FIG. 6C) HER4, and (FIG. 6D) EGFR (HER1) in multiple breast cancer
cell lines following HER2-205 treatment.
[0035] FIG. 7 includes a schematic illustration showing TFOs that
are designed to bind to polypurine sites in non-coding regions of
the HER2 gene, which is located on chromosome 17. Two TFOs,
HER2-5922-2 and HER2-40118, were designed to target the introns of
the HER2 gene.
[0036] FIGS. 8A-8B include charts showing that TFOs targeting
non-coding regions can also induce DSBs and apoptosis. FIG. 8A
includes a chart showing TFOs targeting non-coding regions of the
HER2 gene can induce DNA DSBs. The chart shows quantification of
triplex-induced DSBs using the neutral comet assay as measured by
tail moment. Triplex-induced DNA damage was assessed 24 hours post
treatment. FIG. 8B includes a chart showing that TFOs targeting
non-coding regions of the HER2 gene can induce apoptosis. The chart
shows analysis of triplex-induced apoptosis as measured by
Annexin-V staining 24 hours post-treatment in BT474 cells. ****
denotes p<0.0001, *** denotes p<0.001, ** denotes p<0.01,
and * denotes p<0.05.
DETAILED DESCRIPTION
[0037] Described herein is a method of reducing, in a population of
cells, the number of p53-depleted cancer cells in which a HER2 gene
is amplified and agents useful to reduce the number of p53-depleted
cancer cells comprising an amplified HER2 gene. In specific
embodiments, the agents are triplex-forming oligonucleotides (TFOs)
that are targeted to a polypurine site in an amplified HER2 gene in
p53-depleted cancer cells.
[0038] In one embodiment, the method comprises contacting a
population of cells, such as tissue, comprising p53-depleted cancer
cells in which a HER2 gene is amplified with triplex forming
oligonucleotides (TFOs) targeted to a polypurine target site in the
HER2 gene(s), under conditions under which the TFOs enter the
p53-depleted cancer cells in sufficient quantity to induce
apoptosis.
[0039] Advancements in DNA sequencing technology have not only
revealed commonly mutated and deleted genes across cancer types,
but also enabled identification of amplified cancer-promoting
genes.sup.8. These amplified genes include epigenetic regulators,
cell cycle-associated genes, and genes linked to signaling
pathways, such as the EGFR and HER2 genes.sup.9. Described herein
is an approach for targeted therapeutics that can be used in the
treatment of p53-depleted cancers characterized by gene
amplification and that has limited toxicity to normal tissue. The
limited toxicity is at least due to the localized effect and
targeting of the TFOs to cells that have HER2-amplified genes and
thus are likely to be the cancerous cells in the tissue. Described
herein are agents and methods demonstrating that manipulation of
DNA damage response is as effective in its anticancer activity as
targeting the individual overexpressed protein product.
[0040] Provided herein are triplex-forming oligonucleotides (TFOs,
also referred to as triplex-inducing oligonucleotides) for the
induction of p53-independent apoptosis. TFOs are molecules that
function as sequence-specific gene targeting/modification tools.
Without wishing to be bound by theory, it has been shown that the
TFOs bind to the major groove of duplex DNA and are restricted to
sites with purines (also referred to as polypurine sites) on one
strand and pyrimidines on the other.
HER2 Gene Amplification and NER-Dependent Repair
[0041] Gene amplification is observed in a broad spectrum of
cancers, contributing not only to incipient cancer development, but
also to the development of drug resistance. HER2 gene amplification
(amplification of human epidermal growth factor receptor 2-encoding
gene) is observed in a vast majority of cancers. Cancers with HER2
gene amplification or over-expression of the HER2 protein are
sometimes referred to as HER2-positive cancers. Non-limiting
examples of such cancers include breast cancer, ovarian cancer,
colorectal cancer, gastric cancer, lung cancer, and endometrial
cancer. HER2 gene amplification has been identified in about 25% of
breast cancers.
[0042] Disclosed herein are TFOs targeted to specific regions of
the HER2 gene. These TFOs can be utilized for a p53-independent
cancer therapy. There are several polypurine sites in the HER2 gene
that are susceptible to triplex formation. Binding of TFOs to the
HER2 gene (e.g., major groove regions on the HER2 gene) causes DNA
perturbation that can impede replication fork progression,
resulting in fork collapse and helix distorting structures (e.g.,
lesions or, more specifically, DNA double strand breaks
(DSBs)).sup.14. Under normal circumstances (e.g., low HER2 gene
copy levels), these helix distorting structures trigger the
nucleotide excision repair (NER) pathway, which repairs the helix
distorting structures. This ability of the NER pathway to resolve
low levels of triplex-induced DNA damage allows normal cells to
tolerate the formation of a few triplexes.sup.16,17. In contrast,
if there is HER2 gene amplification and consequently high levels of
triplex formation, NER-dependent DNA repair is ineffective and
instead apoptosis is triggered.sup.15. HER2 gene amplification in
cancers, such as breast cancers, provides an opportunity to test
the efficacy of TFOs as an apoptosis-inducing agent in cancer
cells, but not in healthy cells, which lack HER2
amplification.sup.18 (FIGS. 1A-1C).
[0043] XPD, a transcription factor II H (TFIIH) subunit, plays a
key role in this NER pathway by operating as a 5'-3' helicase to
unzip the DNA. In instances of high DNA damage (or high triplex
formation), XPD is required for p53-mediated apoptosis (see U.S.
Pat. No. 9,587,238, the relevant disclosures of which are herein
incorporated as reference). Previous studies established that in
cases of excess DNA damage, an apoptotic pathway is initiated that
is dependent on the presence of both XPD and p53. This, in part,
explains the chemotherapeutic drug resistance and the difficulty in
treating p53-defective conditions.
P53 Tumor Supressor
[0044] Disclosed herein are methods and compositions for inducing
apoptosis in p53-depleted cells comprising an amplified HER2 gene.
P53 (also referred to as TP53 or p53 tumor suppressor) is a gene on
the 17.sup.th chromosome (17p13.1) that encodes p53 protein (also
referred to as TP53 or tumor protein). The protein is a regulator
in the cell cycle and plays the role of a tumor suppressor. The p53
tumor suppressor regulates pro-apoptotic pathways in response to
severe DNA damage. Under normal, non-pathological conditions, p53
expression is low. DNA damage and related signals upregulate its
expression to initiate growth arrest, DNA repair, and, in extreme
cases, apoptosis. Typically, growth arrest inhibits replication of
damaged DNA; however, in cancerous cells this is bypassed. As
explained herein, gene amplification manifests in cancerous cells
and can result in ineffective DNA repair (for example, ineffective
NER-mediated DNA repair). In such cases, p53 is relied on for
apoptosis of the damaged cells.
[0045] Mutations in p53 are correlated with a broad spectrum of
aggressive cancers and have been implicated in as many as 50% of
all human tumors, highlighting the importance of this gene and the
impact of a p53-independent chemotherapeutic approach. Over 50% of
human cancers exhibit chemotherapeutic resistant phenotypes due to
loss of function p53 mutations, which lead to an inability to
trigger apoptosis. Previous studies attempted to address cancer
treatment in p53-defective conditions by upregulating wild-type p53
or augmenting the activity of wild-type p53 (Smith and Seo,
Mutagenesis 17(2), 149-156, 2002). Additional strategies to
overcome this challenge include attempts to modify the p53 gene
through gene editing, reactivate p53 genes with chemotherapeutic
drugs, or suppress p53 mutant aggregation. These strategies have
had limited success.
[0046] The term "p53-depleted" refers to cells, such as cancer
cells, in which p53 is reduced, lacking or mutated. In some
embodiments, a p53-depleted cancer cell is a cell that does not
express p53. It can also refer to significantly decreased p53
expression under conditions under which p53 is typically
upregulated (e.g., in response to a DNA lesion). Mutations in p53
have been shown to give rise to different isoforms, some of which
give rise to tissue-specific cancers. The term "p53-depleted" also
includes cells, such as cancer cells, in which the p53 gene has a
mutation (e.g., loss of function mutation, gain of function
mutation, etc.) and produces a protein that is dysfunctional (e.g.,
displays no or reduced function). This may also occur through the
production of a truncated p53 protein that is dysfunctional. Most
p53 mutations are missense mutations. In some embodiments, the
p53-mutated cell is a homozygote mutant. In some embodiments, the
p53-mutant cell is a heterozygote, carrying a wild-type p53 allele
and a mutant p53 allele. Previous studies have shown that in some
heterozygote cases, the mutant allele functions in a dominant
negative manner, suppressing the expression of the wild-type
allele. Loss of wild-type p53 and p53 mutations have been shown to
occur in both early and late tumorigenesis. Some p53 mutations,
referred to as gain-of-function p53 mutations, result in p53 mutant
proteins that have additional oncogenic properties and promote
cancer progression (Rivlin et al., Genes & Cancer 2(4):466-474,
2011).
Triplex Forming Oligonucleotides (TFOs)
[0047] Triplex-forming oligonucleotides (TFOs) form triplexes,
which are DNA structures comprised of an additional RNA or DNA
binding sequence. Without wishing to be bound by theory, they are
believed to bind in the major groove of duplex DNA. Purine motif
TFOs (comprised of G and A) form G*G:C and A*A:T triplets and bind
in antiparallel orientation, via reverse Hoogsteen base pairing,
with regard to the purine strand of the duplex. In contrast,
pyrimidine motif TFOs (C/T) form triplexes in parallel orientation,
via forward Hoogsteen alignment, and form C.sup.+*G:C and T*A:T
triplets. Mixed purine and pyrimidine TFOs bind in either parallel
or antiparallel orientation and form G*G:C and T*A:T triplets.
(Maldonado, R., et al. RNA 24(3): 371-380, 2018 and Basye, J., et
al. Nucleic acids research 29(23): 4873-4880, 2001).
Applications of HER2 Targeted TFOs
[0048] As described, HER2-targeted TFOs trigger an alternative
pathway, a p53-independent apoptotic pathway. HER2-targeted TFOs
induce copy number dependent DNA double strand breaks (DSBs) and
activate apoptosis in HER2 gene amplified cancer cells and human
tumor xenografts via a mechanism that is independent of HER2
cellular function as well as independent of p53. In specific
embodiments, HER2-targeted TFOs, HER2-1 (SEQ ID NO:3), HER2-205
(SEQ ID NO:4), HER2-5922-2 (SEQ ID NO:7), and HER2-40118 (SEQ ID
NO: 8), trigger p53-independent apoptosis in cancer cells
comprising amplified HER2 gene.
[0049] In some embodiments, the HER2-targeted TFOs target
polypurine target sites in the promoter region of the HER2 gene
(e.g., the HER2-1 TFO (SEQ ID NO: 3)). In some embodiments, the
HER2-targeted TFOs target polypurine target sites in the coding
region of the HER2 gene (e.g., the HER2-205 TFO (SEQ ID NO: 4)). In
some embodiments, the HER2-targeted TFOs target introns or
non-coding regions of the HER2 gene. For example, the HER2-5922-2
TFO (SEQ ID NO: 7) targets a site within intron 2 of the HER 2
gene, and the HER2-40118 (SEQ ID NO: 8) TFO targets intron 19 of
the HER2 gene.
[0050] Disclosed herein is a method of reducing, in a population of
cells, the number of p53-depleted cancer cells comprising an
amplified HER2 gene. As described, this method can be carried out
in a population of cells, such as in a tissue or organ. As used
herein, the term "reducing" refers to decreasing the number of
living cells by inducing apoptosis in the cells. The reduction
could decelerate rapid cell growth or decelerate hyperplasia, which
are two common characteristics of cancerous cells.
[0051] The methods disclosed herein includes contacting
p53-depleted cancer cells comprising amplified HER2 gene with
triplex-forming oligonucleotides (TFOs) targeted to a polypurine
target site in the amplified-HER2 gene.
[0052] In some embodiments, the TFOs are polypurine TFOs.
Polypurine TFOs are rich in adenine and/or guanine bases, and,
without wishing to be bound by theory, are believed to bind to the
major groove of their polypurine target sites in an antiparallel
fashion. As used, the term "polypurine TFO" refers to purine motif
TFOs or TFOs rich in purines (adenine and/or guanine bases). As
used, the term "polypurine target site" refers to a DNA duplex
having a strand rich in purines (adenine and/or guanine bases). The
terms "polypurine target strand" and "polypurine strand" refer to
the strand in the polypurine target site that is rich in purines
(adenine. guanine or both adenine and guanine bases). In some
embodiments, a sequence is referred to as "rich in purines" when
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more
of its nucleotides have adenine and/or guanine bases.
[0053] An example of a polypurine target site identified in the
promoter region of the HER2 gene is a DNA duplex having the
sequence 5'-AGGAGAAGGAGGAGGTGGAGGAGGAGGG-3' (SEQ ID NO:1) bound to
5'-CCCTCCTCCTCCACCTCCTCCTTCTCCT-3' (SEQ ID NO:10). Another example
of a polypurine target site identified in the coding region of the
HER2 gene is a DNA duplex having the sequence
3'-CCCCGAGGAGGAGCGGGAGAACGGGGGG-5' (SEQ ID NO:2) bound to
5'-GGGGCTCCTCCTCGCCCTCTTGCCCCCC-3' (SEQ ID NO:11). (FIG. 1B).
[0054] Another example of a polypurine target site identified in
the non-coding region of the HER2 gene is a DNA duplex having the
sequence 3'-GGGAAAGAGGAGGGGGTGAGAGGAGTGGGG -5' (SEQ ID NO: 5) bound
to 5'-CCCTTTCTCCTCCCCCACTCTCCTCACCCC-3' (SEQ ID NO: 12). Another
example of a polypurine target site identified in the non-coding
region of the HER2 gene is a DNA duplex having the sequence
3'-GGGGGAAACAGGGAGGGTGGGG-5' (SEQ ID NO: 6) bound to
5'-CCCCCTTTGTCCCTCCCACCCC-3' (SEQ ID NO: 13). (FIG. 7).
[0055] Formation of the triplex after introduction of a TFO occurs
via reverse Hoogsteen hydrogen bonds between the third strand (TFO)
and the polypurine strand of the duplex
[0056] In some embodiments, a TFO has a nucleotide sequence that is
complementary to SEQ ID NOs: 10, 11, 12 and 13 and/or binds to at
least 13 nucleotides in a polypurine target strand, such as at
least 13 nucleotides in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 5 or
SEQ ID NO: 6. In some embodiments, a TFO binds to 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30
nucleotides in a polypurine target strand. TFOs can bind to
contiguous or non-contiguous nucleotides in a polypurine target
site. The TFOs described herein can be any TFO sequence that is
targeted to a polypurine target site in a HER2 gene, for example,
polypurine target sites SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 5 or
SEQ ID NO: 6. In some embodiments, the TFOs are at least 13
nucleotides in length. In some embodiments, the TFOs range from 13
to 30 nucleotides in length. For example, the TFOs can be 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
nucleotides in length. In further embodiments, the TFOs are shorter
(e.g., 8, 9, 10, 11, or 12 nucleotides).
[0057] Examples of HER2-targeted TFOs are HER2-1
(5'-GGGAGGAGGAGGTGGAGGAGGAAGAGGA-3'; SEQ ID NO:3), HER2-205
(5'-GAGGAGGAGTGGGAGAATGGGGGG-3'; SEQ ID NO:4), HER2-5922-2
(5'-GGGAAAGAGGAGGGGGTGAGAGGAGTGGGG-3'; SEQ ID NO: 7), and
HER2-40118 (5'-GGGGGAAATAGGGAGGGTGGGG-3'; SEQ ID NO: 8). HER2-1
hybridizes to SEQ ID NO:1, under physiological conditions. HER2-205
hybridizes to SEQ ID NO:2, under physiological conditions.
HER2-5922-2 hybridizes to SEQ ID NO: 5, under physiological
conditions. HER2-40118 hybridizes to SEQ ID NO: 6, under
physiological conditions (FIGS. 1D and 7).
[0058] As used, the term "complementary" refers to the capacity for
precise pairing (also referred to as hybridization) between two
nucleotides. For example, if a nucleotide at a certain position of
an oligonucleotide is capable of hydrogen bonding with a nucleotide
at a corresponding position of a target RNA, then the nucleotide of
the oligonucleotide and the nucleotide of the target RNA are
complementary to each other at that position. As understood by one
of ordinary skill in the art, for complementary base pairings,
adenosine-type bases (A) are complementary to thymidine-type bases
(T) or uracil-type bases (U), cytosine-type bases (C) are
complementary to guanosine-type bases (G), and universal bases such
as 3-nitropyrrole or 5-nitroindole can hybridize to and are
considered complementary to any A, C, U, or T. In some embodiments,
the methods and agents of the present disclosure can include
Inosine (I). Inosine has also been considered in the art to be a
universal base and is considered complementary to A, C, U or T.
[0059] In some embodiments, the TFO is a nucleotide sequence that
is at least 90% identical to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7
or SEQ ID NO: 8. As used herein, the term "identity" or "identical"
refers to sequence identity, which refers to two nucleotides being
identical. In some embodiments, the TFO is a nucleotide at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical
to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7 or SEQ ID NO: 8.
[0060] The sequence on a HER2 gene to which a TFO is targeted is
referred to as a "target sequence." For example, a TFO that is
targeted to a polypurine site is one that hybridizes to that
polypurine site under physiological conditions, such as in the case
of in vivo administration or treatment. "Targeted" can also refer
to a TFO that specifically hybridizes to a polypurine site
(partially or completely). For example, the TFO hybridizes to a
sequence in the target sequence or target sequence, but does not
hybridize to any other (off-target) nucleotide sequence within the
cell and would not hybridize to a sequence within a cell that lacks
the polypurine site, under physiological conditions.
[0061] In the methods of the present disclosure, TFOs are contacted
with p53-depleted cells that comprise amplified HER2 gene by a
variety of approaches, such as by administering TFOs to an
individual in need of a reduction in a population of p53-deficient
cells that comprise amplified HER2 gene. For example, TFOs in an
appropriate delivery vehicle can be administered to an individual
with cancer in which cancer cells are p53 depleted and comprise
amplified HER2 gene. The TFOs are contacted with such cells by any
manner and under conditions that result in entry into cells in the
individual. For example, TFOs can be introduced into an individual
by injection, infusion, or any delivery method, such as those
described further below.
[0062] In some embodiments of the present disclosure, the TFOs are
administered to an individual who has been screened for a p53
mutation, has HER2-positive cancer cells, and thus has been
identified as a candidate for this p53-independent TFO-treatment.
Non-limiting examples of methods for identifying an individual with
p53 mutations include genetic testing of the DNA found in sera or
other body fluids (see Rivlin et al., Genes & Cancer
2(4):466-474, 2011, the relevant disclosures of which are herein
incorporated as reference).
[0063] In some embodiments, the methods of the present disclosure
are for the treatment of p53-mutated cancers. Non-limiting examples
of such p53-mutated cancers include breast cancer, ovarian cancer,
renal carcinoma, lung cancer, colon carcinoma, hepatocellular
carcinoma, prostate cancer, bladder cancer, and pancreatic
neoplasia. The methods of the present disclosure can include
administering the TFOs herein to the cells of the aforementioned
cancers (e.g., breast cancer cells, ovarian cancer cells, renal
cell carcinoma cells, lung cancer cells, colon cancer cells,
colorectal cancer cells, gastric cancer cells, and endometrial
cancer cells).
Li-Fraumeni Syndrome Application
[0064] In some embodiments, the present disclosure includes methods
and compositions for the treatment of an individual with
Li-Fraumeni Syndrome (LFS) or treatment of a cancer in an
individual with LFS. LFS is a cancer predisposition syndrome
characterized by germline mutations of p53 (Smith and Seo,
Mutagenesis 17(2), 149-156, 2002). Individuals with LFS are
susceptible to a broad spectrum of cancers and are susceptible to
early onset of these cancers. Of the spectrum of
Li-Fraumeni-associated tumors, breast cancer, sarcomas of the soft
tissues and bone, acute leukemias, and brain tumors are among the
most common (Nichols et al., Cancer Epidemiology and Prevention
Biomarkers 10(2): 83-87, 2001, the relevant disclosures of which
are herein incorporated by reference). The lifetime risk of an LFS
patient to develop cancer has been estimated to be as high as 90%.
Non-limiting examples of types of cancer commonly found in families
with LFS include osteosarcoma (bone cancer), soft-tissue sarcoma,
acute leukemia, breast cancer, brain cancer, adrenal cortical
tumors, and acute leukemia.
[0065] In some embodiments, an individual is screened for HER2 gene
amplification before administration of the TFO. Methods for
detection of gene amplification are known in the art. Non limiting
examples of these methods include conventional cytogenetics,
Southern blotting, quantitative PCR, fluorescence in situ
hybridization (FISH), comparative genomic hybridization (CGH), and
microarray technology.
[0066] Due to the limited toxicity associated with the described
TFOs, in alternative embodiments, a TFO targeted to at least one
sequence in HER2 gene can be administered to an individual
diagnosed with cancer to induce p53 independent apoptosis in
p53-depleted cells comprising an amplified HER2 gene prior to or
without screening for HER2 gene amplification.
Chemical Modifications to TFOs
[0067] In some embodiments, the TFOs have backbone modifications.
Unmodified purine TFOs bind well under physiologic conditions, but
binding efficiency can sometimes be inhibited at physiologic
K.sup.+ conditions. Backbone modifications can augment the binding
efficiency of such TFOs. Various modifications for purine TFOs are
disclosed in Knauert and Glazer, Human Molecular Genetics 10(20):
2243-2251, 2001, the relevant disclosures of which are herein
incorporated by reference. Non-limiting examples of backbone
modifications to the TFOs include phosphorothioates,
phosphorodithioates, methylphosphonates, phosphoramidates,
boranophosphate oligos, polyamides, methylene (methylimino)
linkages, morpholino oligos, and combinations thereof. TFOs with
polyamide backbone modifications bind to the minor groove of the
DNA duplex, rather than the major groove.
Combination Therapies
[0068] Disclosed herein are methods of administering TFOs in
sufficient quantity to induce p53-independent apoptosis in
p53-depleted cells comprising an amplified HER2 gene. In some
embodiments, one type of TFO (e.g., either HER2-1 or HER2-205) is
administered. In some embodiments, TFOs of more than one type are
administered (e.g., a mixture of TFOs, a mixture of HER2-1 and
HER2-205, etc.). Herein, reference to "administering TFOs" can also
refer to the administration of TFOs of more than one type.
[0069] In some embodiments, the one type of TFO is administered in
combination with at least one non-TFO (e.g., a non-TFO anticancer
agent). In some embodiments, more than one type of TFO is
administered with at least one non-TFO (e.g., a non-TFO anticancer
agent). An anticancer agent that is not a TFO can be, for example,
a protein, a nucleic acid, a small molecule, or a drug for the
treatment of cancer. This anticancer agent can have any anti-cancer
effect on the population of cells that it is administered to
including, but not limited to, a cytotoxic, apoptotic, anti-mitotic
anti-angiogenesis or inhibition of metastasis effect. This
anticancer agent can also affect DNA damage response (e.g., a DNA
repair inhibitor). In some embodiments, the second anticancer agent
is a drug directed against overexpressed protein products.
[0070] Anticancer agents include, for example, antimetabolites,
inhibitors of topoisomerase I and II, alkylating agents and
microtubule inhibitors (e.g., taxol). Non-limiting examples of
anticancer agents include adriamycin aldesleukin; alemtuzumab;
alitretinoin; allopurinol; altretamine; amifostine; anastrozole;
arsenic trioxide; Asparaginase; BCG Live; bexarotene capsules;
bexarotene gel; bleomycin; busulfan intravenous; busulfan oral;
calusterone; capecitabine; carboplatin; carmustine; carmustine with
Polifeprosan 20 Implant; celecoxib; chlorambucil; cisplatin;
cladribine; cyclophosphamide; cytarabine; cytarabine liposomal;
dacarbazine; dactinomycin; actinomycin D; Darbepoetin alfa;
daunorubicin liposomal; daunorubicin, daunomycin; Denileukin
diftitox, dexrazoxane; docetaxel; doxorubicin; doxorubicin
liposomal; Dromostanolone propionate; Elliott's B Solution;
epirubicin; Epoetin alfa estramustine; etoposide phosphate;
etoposide (VP-16); exemestane; Filgrastim; floxuridine
(intraarterial); fludarabine; fluorouracil (5-FU); fulvestrant;
gemcitabine, gemtuzumab ozogamicin; goserelin acetate; hydroxyurea;
Ibritumomab Tiuxetan; idarubicin; ifosfamide; imatinib mesylate;
Interferon alfa-2a; Interferon alfa-2b; irinotecan; letrozole;
leucovorin; levamisole; lomustine (CCNU); meclorethamine (nitrogen
mustard); megestrol acetate; melphalan (L-PAM); mercaptopurine
(6-MP); mesna; methotrexate; methoxsalen; mitomycin C; mitotane;
mitoxantrone; nandrolone phenpropionate; Nofetumomab; LOddC;
Oprelvekin; oxaliplatin; paclitaxel; pamidronate; pegademase;
Pegaspargase; Pegfilgrastim; pentostatin; pipobroman; plicamycin;
mithramycin; porfimer sodium; procarbazine; quinacrine;
Rasburicase; Rituximab; Sargramostim; streptozocin; talbuvidine
(LDT); talc; tamoxifen; temozolomide; teniposide (VM-26);
testolactone; thioguanine (6-TG); thiotepa; topotecan; toremifene;
Tositumomab; Trastuzumab; tretinoin (ATRA); uracil mustard;
valrubicin; valtorcitabine (monoval LDC); vinblastine; vinorelbine;
zoledronate; and mixtures thereof, among others (see U.S. Pat. No.
9,643,922, the relevant disclosures of which are herein
incorporated by reference).
[0071] Non-limiting examples of anticancer agents include oestrogen
receptor modulators, androgen receptor modulators, retinoid
receptor modulators, cytotoxic agents, antiproliferative agents,
prenyl-protein transferase inhibitors, HMG-CoA reductase
inhibitors, reverse transcriptase inhibitors and further
angiogenesis inhibitors.
[0072] Non-limiting examples of retinoid receptor modulators
include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic
acid, .alpha.-difluoromethylornithine, ILX23-7553,
trans-N-(4'-hydroxyphenyl)retinamide and
N-4-carboxyphenylretinamide (see U.S. Pat. No. 10,093,623, the
relevant disclosures of which are herein incorporated by
reference).
[0073] Non-limiting examples of cytotoxic agents include
tirapazimine, sertenef, cachectin, ifosfamide, tasonermin,
lonidamine, carboplatin, altretamine, prednimustine,
dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin,
temozolomide, heptaplatin, estramustine, improsulfan tosylate,
trofosfamide, nimustine, dibrospidium chloride, pumitepa,
lobaplatin, satraplatin, profiromycin, cisplatin, irofulven,
dexifosfamide, cis-aminedichloro(2-methylpyridine)platinum,
benzylguanine, glufosfamide, GPX100,
(trans,trans,trans)bis-mu-(hexane-1,6-diamine)-mu-[diamineplatinum(II)]bi-
-s[diamine(chloro)platinum(II)]tetrachloride, diarisidinylspermine,
arsenic trioxide,
1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine,
zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone,
pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston,
3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin,
annamycin, galarubicin, elinafide, MEN10755 and
4-demethoxy-3-deamino-3-aziridinyl-4-methylsulfonyldaunorubicin
(see WO 00/50032, the relevant disclosures of which are herein
incorporated by reference).
[0074] Non-limiting examples of antiproliferative agents include
antisense RNA and DNA oligonucleotides such as G3139, ODN698,
RVASKRAS, GEM231 and INX3001 and antimetabolites such as
enocitabine, carmofur, tegafur, pentostatin, doxifluridine,
trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine
ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid,
emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,
nelzarabine, 2'-deoxy-2'-methylidenecytidine,
2'-fluoromethylene-2'-deoxycytidine,
N-[5-(2,3-dihydrobenzofuryl)sulfonyl]-N'-(3,4-dichlorophenyl)urea,
N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]-glycylamino]-L-glycero-B-L-
-mannoheptopyranosyl]adenine, aplidine, ecteinascidin,
troxacitabine,
4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b]-1,4-thiazin-6-yl-
-(S)ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin,
5-fluorouracil, alanosine,
11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetr-
-acyclo(7.4.1.0.0)tetradeca-2,4,6-trien-9-ylacetic acid ester,
swainsonine, lometrexol, dexrazoxane, methioninase,
2'-cyano-2'-deoxy-N4-palmitoyl-1-B-D-arabinofuranosyl cytosine and
3-aminopyridine-2-carboxaldehyde thiosemicarbazone.
"Antiproliferative agents" also include monoclonal antibodies to
growth factors other than those listed under "angiogenesis
inhibitors", such as trastuzumab (for examples, see U.S. Pat. No.
6,069,134, the relevant disclosures of which are herein
incorporated by reference).
[0075] The first drugs directed against overexpressed protein
products were major breakthroughs in cancer therapeutics. For
example, trastuzumab (HERCEPTIN.RTM.) targets the HER2 receptor
tyrosine kinase, which is overexpressed in about 25% of breast
tumors due to gene amplification.sup.10. Trastuzumab works, at
least in part, by disrupting HER2 signaling, which results in cell
cycle arrest and suppression of cell growth and
proliferation.sup.11. While trastuzumab has proven to be effective
in prolonging the survival of HER2-positive breast cancer patients,
primary and acquired drug resistance limits overall success rates.
Similar problems hamper the long-term efficacy of other cancer
drugs, including the tyrosine kinase inhibitors gefitinib
(IRESSA.RTM.) and erlotinib (TARCEVA.RTM.), which target EGFR gene
amplification in breast, colorectal, and lung cancer.sup.12,13.
Methods of Administering TFOs
[0076] The TFOs of the present disclosure may be administered to an
individual by any route or in any delivery vehicle.
[0077] In some embodiments, the TFOs are administered in a delivery
vehicle (e.g., lipid-based nanoparticles). The TFOs can be
conjugated to the lipid-based nanoparticles. Alternatively, the
TFOs can be encapsulated in the lipid-based nanoparticles. One
example of lipid-based nanoparticles is lipid nanoparticles that
contain a solid lipid core matrix with the ability to solubilize
lipophilic molecules. The term "solid" refers to a nanoparticle
that is solid at room temperature and atmospheric pressure. The
lipid nanoparticles can have a nanostructure core (solid or hollow)
and a lipid layer. The diameter of the core can be less than or
equal to about 500 nm, less than or equal to about 250 nm, less
than or equal to about 100 nm, less than or equal to about 75 nm,
less than or equal to about 50 nm, less than or equal to about 40
nm, less than or equal to about 35 nm, less than or equal to about
30 nm, less than or equal to about 25 nm, less than or equal to
about 20 nm, less than or equal to about 15 nm, or less than or
equal to about 5 nm. In some embodiments, the core is less than
1000 nm. In some embodiments, the core is 1 nm, 2 nm, 3 nm, 4 nm, 5
nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm 50 nm, 60
nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm,
200 nm, 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, 320 nm, 340 nm, 360
nm, 380 nm, 400 nm, 420 nm, 440 nm, 460 nm, 480 nm, or 500 nm in
diameter.
[0078] The lipid nanoparticle can be a solid lipid nanoparticle or
a polymeric nanoparticle. Methods for making solid liquid
nanoparticles are well-established in the art (see, for example,
Gasco, M. R., Nanoparticelle Lipidiche Solide Quali Sistemi
Terapeutici Colloidali, NCF nr. 7: 71-73, 1996; Kozariara et al.,
Pharmaceutical Research, 20(11): 1772, 2003; and Lockman et al.,
Journal of Controlled Release, 93:271-282, 2003, the relevant
disclosures of which are herein incorporated by reference).
[0079] In a polymeric lipid nanoparticle the polymer can be any
ionic or ionizable polymer or copolymer known to those of skill in
the art including polymers and copolymers of, for example,
polyglycine, polyethylene glycol, heparin,
hydroxypropylmethylcellulose, hydroxyethylcellulose,
hydroxypropylcellulose, polyvinylpyrrolidone, polyvinyl alcohol,
poly-beta amino esters (PBAEs), methacrylic acid copolymers, ethyl
acrylate-methyl methacrylate copolymers, and mixtures thereof. In
some embodiments, the first functionalized polymer can be
poly(glycolic acid), poly(lactic acid) (PLA), or copolymers
thereof, such as poly(D,L-lactide-co-glycolide), or mixtures
thereof.
[0080] In some embodiments, the TFOs are delivered using
poly(lactic-co-glycolic acid) (PLGA) nanoparticles or PLA
nanoparticles. In some embodiments, the PLGA nanoparticles or PLA
nanoparticles are loaded with the TFOs of the present disclosure.
In some embodiments the nanoparticles include an agent conjugated
to their surface, such as polyethylene glycol (PEG) and
hyperbranched polyglycerols (HPG).
[0081] In some embodiments, the lipid nanoparticles include
cationic lipids or anionic lipids. Alternatively, the lipid
nanoparticles can include neutral lipids. Non-limiting examples of
cationic lipids include
3.beta.-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
hydrochloride (DC-Chol); 1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP);
dimethyldioctadecylammonium bromide salt (DDAB);
1,2-dilauroyl-sn-glycero-3-ethylphosphocholine chloride (DL-EPC);
N-(1-(2,3-dioleyloyx)propyl)-N-N-N-trimethyl ammonium chloride
(DOTMA); N-(1-(2,3-dioleyloyx)propyl)-N-N-N-dimethyl ammonium
chloride (DODMA); N,N-dioctadecyl-N,N-dimethylammonium chloride
(DODAC);
N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
ammonium trifluoracetate (DOSPA);
1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide
(DMRIE); dioctadecylamidoglycylspermine (DOGS); neutral lipids
conjugated to cationic modifying groups; and combinations thereof.
Non-limiting examples of anionic lipids include fatty acids such as
oleic, linoleic, and linolenic acids; cholesteryl hemisuccinate;
1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1'-rac-glycerol) (Diether
PG); 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium
salt); 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt);
1-hexadecanoyl,2-(9Z,12Z)-octadecadienoyl-sn-glycero-3-phosphate;
1,2-dioleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (DOPG);
dioleoylphosphatidic acid (DOPA); and
1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); anionic
modifying groups conjugated to neutral lipids; and combinations
thereof. Non-limiting examples of neutral lipids include
phosphatidylcholine (PC), phosphatidylethanolamine, ceramide,
cerebrosides, sphingomyelin, cephalin, cholesterol,
diacylglycerols, glycosylated diacylglycerols, prenols, lysosomal
PLA2 substrates, and N-acylglycines. Additional examples of lipids
and lipid components can be found in U.S. Pat. No. 9,833,416.
[0082] The lipid nanoparticles can comprise surfactants and/or
emulsifiers. Non-limiting examples of surfactants include
phospholipids, phosphatidylcholines, TWEENs, Soy lecithin, egg
lecithin (Lipoid E 80), phosphatidylcholine, poloxamer 188, 182,
and 407, poloxamine 908, Tyloxapol, polysorbate 20, 60, and 80,
sodium cholate, sodium glycocholate, taurocholic acid sodium salt,
taurodeoxycholic acid sodium salt, butanol, butyric acid, dioctyl
sodium sulfosuccinate, and monooctylphosphoric acid sodium.
Non-limiting examples of emulsifiers include cationic phospholipid
or non-ionic surfactant. Examples of cationic surfactant include,
but are not limited to, 1,2-dimyristoyl-3-trimethylammonium-propane
(DMTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),
1,2-distearoyl-3-trimethylammonium-propane (DSTAP),
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),
1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP),
1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP),
1,2-dilauroyl-3-dimethylammonium-propane (DLDAP),
1,2-distearoyl-3-dimethylammonium-propane (DSDAP),
dimethyldioctadecylammonium chloride (DDAB),
N-[1-(1,2-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA) and 1,2-dioleoyl-3-ethylphosphocholine (DOEPC). Examples of
non-ionic surfactants include, but are not limited to poloxamers,
sorbitan esters (Span), polyoxyethylene-sorbitan fatty acid esters
(Tween) and polyoxyethylene ethers (Brij).
[0083] In certain embodiments, the lipid comprises one or more of:
a) cationic or anionic lipids or surfactants; b) neutral lipids or
surfactants; c) cholesterol; and d) PEGylated lipids or
surfactants.
[0084] Other non-limiting examples of lipid-based nanoparticles
include liposomes, bolaamphiphiles, nanostructured lipid carriers
(NLC), and monolayer membrane structures (e.g., archaeosomes and
micelles). Methods of encapsulating agents in lipid nanoparticles
are disclosed in Puri et al. Critical Reviews in Therapeutic Drug
Carrier Systems 26(6):523-580, 2009, the relevant disclosures of
which are herein incorporated by reference.
[0085] In some embodiments, the TFOs are conjugated to cholesterol
to enhance delivery into cells. In some embodiments, the TFO are
administered absent of a transport peptide or cell-penetrating
peptide (CPP). In some embodiments, the TFOs are administered with
a peptide, e.g., cell-penetrating peptides (CPPs), primary
amphipathic peptides, such as MPG or Pep-1.
[0086] The administration of the TFOs can be directly to tissue in
an individual. In some embodiments, the TFOs are delivered
systemically. In some embodiments, the TFOs are delivered locally
or intratumorally. In some embodiments, the TFOs are administered
as an injection. As used, an injection can use different delivery
routes. In some embodiments, the TFOs are administered
intravenously or intraperitoneally. In some embodiments, the TFOs
are administered intravenously, intradermally, intraarterially,
intralesionally, intratumorally, intracranially, intraarticularly,
intraprostaticaly, intrapleurally, intratracheally, intranasally,
intravitreally, intravaginally, intrarectally, topically,
intramuscularly, intraperitoneally, subcutaneously,
subconjunctival, intravesicularlly, mucosally, intrapericardially,
intraumbilically, intraocularally, orally, locally, inhalation
(e.g., aerosol inhalation), injection, infusion, continuous
infusion, localized perfusion bathing target cells directly, via a
catheter, via a lavage, in creams, in lipid compositions (e.g.,
liposomes, lipid nanoparticles, etc.), or by other method or any
combination of the forgoing as would be known to one of ordinary
skill in the art (see, for example, Remington's Pharmaceutical
Sciences (1990), incorporated herein by reference).
[0087] In some embodiments, the TFOs are administered as a
composition having a pharmaceutically acceptable carrier. In some
embodiments, the pharmaceutically acceptable carrier comprises
water or saline.
[0088] The term "pharmaceutically-acceptable carrier" refers to one
or more compatible solid or liquid filler, diluents or
encapsulating substances which are suitable for administration to a
human or other subject contemplated by the disclosure.
"Pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, surfactants, antioxidants,
preservatives (e.g., antibacterial agents, antifungal agents),
isotonic agents, absorption delaying agents, salts, preservatives,
drugs, drug stabilizers (e.g., antioxidants), gels, binders,
excipients, disintegration agents, lubricants, sweetening agents,
flavoring agents, dyes, such like materials and combinations
thereof, as would be known to one of ordinary skill in the art
(see, for example, Remington's Pharmaceutical Sciences (1990),
incorporated herein by reference). Except insofar as any
conventional carrier is incompatible with the active ingredient,
its use in the therapeutic or pharmaceutical compositions is
contemplated.
[0089] The TFOs can be administered once, or alternatively they may
be administered in a plurality of administrations. If administered
multiple times, the compounds may be administered via different
routes. For example, the first (or the first few) administrations
may be made directly into the affected tissue while later
administrations may be systemic.
[0090] In some aspects of the present disclosure, the term
"individual" refers to a mammal. In some embodiments, individual
refers to a human. Alternatively, individual can refer to a mammal,
wherein the mammal is selected from a group including but not
limited to non-human primates, cows, horses, pigs, sheep, goats,
dogs, cats, rabbits, ferrets, and rodents. In some embodiments, the
term "individual" is used to refer to a model of cancer.
Non-limiting examples of models of cancer include p53-knockout
mice, BALB/c mice injected with MDA-MB-453 cells, SKBR3 cells,
BT474 cells, PEO1 cells, or SKOV3 cells, and LFS mouse models
(engineered to express mutant p53).
[0091] As used herein, the term "sufficient quantity" refers to a
"therapeutically effective amount" or "effective amount" that
elicits a biological or medicinal response in a tissue, system,
animal, individual or human that is being sought by a researcher,
veterinarian, medical doctor or other clinician. In this case, the
response would be a reduction (partial or total/complete) in the
number of p53-depleted cancer cells comprising a HER2 gene by
apoptosis. The appropriate response can be determined in vitro by
trypsinization and cell counting (using methods established in the
art). In vivo, the appropriate response from a therapeutically
effective amount can be determined by measuring or visualizing
(e.g., imaging) tumor size.
[0092] In some embodiments, the TFOs are administered to an
individual in a dose of approximately 20 mg/kg. In some
embodiments, the TFOs are administered to an individual in a dose
of 5 mg/kg, 7 mg/kg, 9 mg/kg, 10 mg/kg, 12 mg/kg, 14 mg/kg, 16
mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg,
25 mg/kg, or 30 mg/kg. These disclosed amounts can be increased or
decreased by one of ordinary skill in order to personalize a
chemotherapeutic treatment plan based on an individual's stage of
cancer, the size of tumor, the presence or absence of an adjuvant
chemotherapeutic agent, etc.
TFO Use for Assays
[0093] In alternate embodiments, the method is an assay in which
TFOs are contacted in vitro with p53-depleted cancer cells
comprising a HER2 gene. As used, the term "contacting" refers to
the use of TFOs in in vitro assays. The contacting can be through
the transfection of cells with the TFOs in vitro. In some
embodiments, the cells are cancer cells. In some embodiments, the
cancer cells are contacted with TFOs under conditions under which
the TFOs enter the p53-depleted cancer cells in sufficient quantity
to induce apoptosis under the conditions of the in vitro assay.
[0094] Methods of transfection are well established in the arts and
include chemical, biological, and physical methods. Chemical
methods include, but are not limited to, calcium phosphate
transfection, cationic polymer transfection (e.g.,
polyethylenimine), lipofection, Oligofectamine.TM.,
DharmaFECT-1.TM., FUGENE.RTM., and DEAE-Dextran-mediated
transfection. Other methods of transfection include, but are not
limited to, electroporation, microinjection, sonoporation, cell
squeezing, impalefection, optical transfection, protoplast fusion,
magnetofection.TM., and particle bombardment.
[0095] Non-limiting examples of cells that can be contacted by any
of the TFOs described herein for in vitro assays, include carcinoma
cells, lung cancer cells, colon cancer cells, human colon carcinoma
cells, ovarian cancer cells, breast cancer cells, colorectal
cancer, gastric cancer cells, and endometrial cancer cells.
Additional non-limiting examples of cells that can be contacted by
any of the TFOs described herein for in vitro assays, include
MDA-MB-453 cells, SKBR3 cells, BT474 cells, PEO1 cells, or SKOV3
cells.
[0096] Without further elaboration, it is believed that one skilled
in the art can, based on the above description, utilize the present
invention to its fullest extent. The following specific embodiments
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever. All publications cited herein are incorporated by
reference.
EXAMPLES
Materials and Methods
[0097] Oligonucleotides. Oligonucleotides were synthesized by IDT
with a 3'-amino modifier and purified by reverse-phase HPLC. The
TFO, HER2-1 was designed to bind to the HER2 promoter and had the
sequence 5'-GGGAGGAGGAGGTGGAGGAGGAAGAGGA-3' (SEQ ID NO: 3).
HER2-205 was synthesized with the sequence 5'-GAG GAG GAG TGG GAG
AAT GGG GGG-3' (SEQ ID NO: 4) and has been designed to bind to a
polypurine sequence in the coding region of the HER2 gene. The
control mixed-sequence oligonucleotide MIX24 has the following
sequence: 5'-AGT CAG TCA GTC AGT CAG TCA GTC-3' (SEQ ID NO:9).
Labeled oligonucleotide was synthesized with 5'-TAMRA
modifications. HER2-5922-2 was synthesized with the sequence
5'-GGGAAAGAGGAGGGGGTGAGAGGAGTGGGG-3' (SEQ ID NO: 7). HER2-40118 was
synthesized with the sequence 5'-GGGGGAAATAGGGAGGGTGGGG-3' (SEQ ID
NO: 8).
[0098] Cell Lines and Transfections. Human breast cancer cell lines
were obtained from ATCC and routinely tested for mycoplasma. The
human cell lines, MDA-MB-453, SKBR3, and BT474 are HER2-amplified
breast cancer cell lines. BT20 and MCF7 cells are non-amplified
breast cancer cell lines. MCF-10A is a non-tumorigenic breast
epithelial cell line. PEO1 and SKOV3 are human ovarian cancer cell
lines with HER2 gene amplification.
[0099] Cells were seeded in six-well plates at a density of
2-4.times.10.sup.5 cells per well the day before transfection.
Cells were transfected with 2 .mu.g of HER2-targeted TFO or MIX24
using Oligofectamine (Invitrogen) or Dharmafect-1 (Dharmacon)
transfection reagent. Transfection was performed as per
manufacturer's instructions. siRNA directed against p53, XPD and
non-target controls (ON-Target plus SMARTpool reagents; Dharmacon)
were transfected into BT474 cells using Dharmafect-1 transfection
reagent (Dharmacon) according to the manufacturer's instructions.
Western blot analysis was used to confirm knockdown of protein.
[0100] Metaphase Chromosome Spreads. Cells were transfected with 2
.mu.g of TAMRA labeled HER2-205. Twenty-four hours
post-transfection, cells were treated for 5 h with Colcemid (0.1
.mu.g/.mu.l). Cells were then collected and washed once with PBS.
To the cell pellet a 75 mM KC1 solution was added for 20 minutes at
37.degree. C. Cell pellets were then resuspended in Carnoy's
fixative solution (75% methanol, 25% acetic acid). Following 10
minutes incubation at room temperature, the cells were pelleted and
resuspended in an additional 500 .mu.l of Carnoy's fixative
solution (3:1 methanol:acetic acid). Cells were dropped from a
height onto glass slides and mounting medium with DAPI (Prolong
Gold antifade reagent, Invitrogen) was added to each slide. A FITC
labeled satellite probe specific for human chromosome 17 (Cytocell)
was used to detect gene-specific triplex formation. Pictures were
taken of 50-60 metaphase spreads using an Axiovert 200 microscope
(Carl Zeiss Micro Imaging, Inc.).
[0101] Western blotting. Whole cell lysates were prepared from
floating and adherent cells using RIPA buffer according to standard
protocols. Total protein (30-50 .mu.g per sample) was resolved by
SDS-PAGE. Proteins were detected by a standard immunoblot protocol
using the following primary antibodies: cleaved PARP, cleaved
caspase 3, .gamma.H2AX, XPD, p53, HER2, pHER2 (Y11221/1222), HER3,
pHER3 (Y1289), HER4, pHER4 (Y1284), EGFR, pEGFR (Y1068), Chk1,
pChk1(S345), pChk2 (T68), and Chk2 (Cell Signaling Technology);
pH2AX (tyrosine 142; EMD Millipore); tubulin (clone B-512; Sigma),
and GAPDH-HRP (Proteintech). Each experiment was repeated with
independent sample preparation a minimum of three times, and
representative western blots are shown.
[0102] Apoptosis analysis. Cells (2-4.times.10.sup.5) were seeded
in six-well plates 24 h prior to treatment with MIX24, HER2-1 or
HER2-205 (2 .mu.g). Post-treatment analysis was performed using the
Annexin V-FITC/PI apoptosis detection kit (BD Pharmingen) according
to the manufacturer's protocol. Apoptotic frequency was calculated
as the combined percentage of early and late apoptotic cells. Data
analysis was performed using FlowJo software.
[0103] Immunofluorescence. Cells were seeded onto UV-irradiated
coverslips and were treated for 24 h with HER2-205, MIX24, or a
mock transfection. Cells were processed 24 h post-transfection,
fixed with 4% formaldehyde and then incubated with ice-cold 100%
methanol for 20 minutes followed by a methanol and acetone solution
(1:1) for 20 minutes each at -20.degree. C. After washing with PBS,
cells were blocked with blocking buffer (4% BSA, 0.2% Triton X-100
in PBS) for 30 minutes and then incubated overnight with the
following primary antibodies: .gamma.H2AX (1:500; Cell Signaling or
Millipore) and 53BP1 (1:100; Santa Cruz) in blocking buffer at
4.degree. C. After three washes, cells were incubated with
secondary antibodies Alexa 488 F(ab')2 fragment goat anti-rabbit
IgG or Alexa 568 F(ab')2 fragment goat anti-mouse IgG (1:1000;
Molecular Probes) for 1 h at room temperature. Cells were then
mounted on microscope glass slides with anti-fade mounting media
containing DAPI (Life Technologies), and pictures were taken with a
Leica SP5 microscope. Immunofluorescence experiments were repeated
for validation.
[0104] Comet Assay. Neutral comet assays were performed 24 h post
TFO-transfection as per the manufacturer's instructions (Trevigen)
with the adjustment of 3.5.times.10.sup.5 cells/ml for each single
cell suspension and 30 minutes electrophoresis. Comets were
visualized using an Axiovert 200 microscope and analyzed with
Autocomet software. Approximately 100-200 comets were analyzed per
experiment. Results were expressed as mean tail moment.
[0105] Mouse Models. All mice were maintained at Yale School of
Medicine in accordance with guidelines of the Animal Care and Use
Committee of Yale University and conformed to the recommendations
in the Guide for the Care and Use of Laboratory Animals (Institute
of Laboratory Animal Resources, National Research Council, National
Academy of Sciences).
[0106] Six to seven-week old female BALB/c athymic, ovariectomized
nude mice (Harlan Sprague-Dawley) were implanted with 0.72 mg,
60-day release 17.beta.-estradiol pellets (Innovative Research).
The following day 2.5.times.10.sup.7 BT474 cells suspended in 100
.mu.l equal volume of media and Matrigel Basement Membrane Matrix
(BD Bioscience) were injected subcutaneously in the right flank of
each mouse. Mice bearing a tumor of about 100 mm.sup.3 in volume
were randomly divided into four treatment groups: vehicle (PBS);
mixed-sequence oligonucleotide, MIX24; HER2-targeted TFO, HER2-205;
and trastuzumab (HERCEPTIN.RTM.). Mice were treated with 20 mg/kg
body weight of MIX24, HER2-205 or trastuzumab in PBS by
intraperitoneal (IP) injection (3 doses evenly administered over 7
days). Tumor volumes in each group were then monitored and mice
were sacrificed when tumor volumes reached 1000 mm.sup.3. Error
bars represent standard error of the mean. Tumor tripling time was
calculated as the time required for tumors to increase in volume
three-fold over baseline (defined as tumor volume before
administration of dose on first day of treatment). Harvested tumors
were fixed in 10% neutral buffered formalin and processed by Yale
Pathology Tissue Services for H&E, Caspase 3, HER2, and Ki67.
Images were taken at 4.times. magnification.
[0107] To establish an ovarian cancer model, female BALB/c athymic
nude mice were injected subcutaneously in the flank with
5.times.10.sup.6 SKOV3 cells suspended in 100 .mu.l equal volume of
media and Matrigel. Mice bearing a tumor of about 100 mm.sup.3 in
volume were randomly divided into three treatment groups: vehicle
(PBS), n=5; HER2-targeted TFO, HER2-205, n=5; and cisplatin, n=7.
HER2-205 (20 mg/kg) and cisplatin (10 mg/kg) were administered by
intraperitoneal injection (3 doses/once per week for three weeks).
Tumor volumes were monitored and tumor tripling times were
calculated as described above.
[0108] Gene Expression. RNA was extracted from snap-frozen cells
using the RNeasy Kit (Qiagen) per the manufacturer's protocol. cDNA
synthesis was carried out with 10 .mu.g of RNA via reverse
transcription reactions and the High-Capacity cDNA Reverse
Transcription Kit (ThermoFisher Scientific). cDNA (10 ng) was then
combined with TaqMan Universal PCR master mix (20 .mu.l) (Applied
Biosystem) and primers specific to HER2 (HER2, Hs01001580_ml,
ThermoFisher Scientific) or the internal control, .beta.-actin
(Hs99999903_ml, ThermoFisher Scientific). qRT-PCR was performed in
96-well optical plates in triplicate for each sample. Briefly,
reactions were performed at 50.degree. C. for 2 minutes, followed
by 95.degree. C. for 10 minutes. Amplification of the target or
control gene was carried out with 40 cycles of the two-step
reaction, which included 95.degree. C. for 15 seconds and 1 minute
at 60.degree. C. .beta.-actin expression levels were used to
normalize the difference between cDNA levels in different samples.
Relative expression levels were calculated using the 2(-Delta Delta
C(T)) method.
[0109] Flow Cytometry. BT474 cells were collected 24 h following
treatment with either MIX24 or HER2-205. After washing with PBS,
cells were incubated with 1% paraformaldehyde for 15 minutes on
ice. Cells were then fixed with cold 70% ethanol at -20 .degree. C.
for 2 h or kept for up to 2 weeks until further analysis. Cells
were centrifuged and rinsed with PBS, blocked with PBST buffer (1%
w/v bovine serum albumin and 0.2% v/v Triton X-100 in PBS) for 15
minutes on ice, followed by another PBS rinse. Cells were first
incubated with anti-phospho-ATM (S1981, EMD Millipore) in PBST at
1:100 dilution for 1 h at room temperature. Cells were rinsed with
PBST and incubated with anti-rabbit IgG Fab2 Alexa 488 (Molecular
Probes) at 1:100 dilution at room temperature for 1 h, and then
rinsed with PBST. Acquisition of labeled cells and analysis of data
was completed using a flow cytometer (FACS Calibur) and FlowJo
software respectively.
[0110] Survival Assay. Cell survival was assayed by visualization
of monolayer growth. Briefly, cells were plated at a defined
density in 6 or 12-well dishes and treated with either transfection
reagent alone (mock), MIX24, or HER2-205 as previously described.
Monolayers were visualized by staining cells with crystal violet 72
h post-treatment.
[0111] Fluorescence in situ Hybridization (FISH). HER2 and
chromosome 17 probes were obtained from Cytocell. The HER2 gene
(17q12) probe was labeled with fluorescent Texas Red spectrum and
the CEP17 (17p11.1-q11.1) probe was tagged with FITC. PEO1 and
SKOV3 cells were treated with colcemid (0.1 .mu.g/ml) for three
hours and collected by trypsinizing the monolayer. After washing
the cells with PBS, cells were treated with a hypotonic solution
(0.075 M KC1) at 37.degree. C. for 20 minutes. Cells were then
washed and fixed with Carnoy's fixative solution (methanol and
acetic acid in 3:1 ratio). Cells were dropped on slides and
fluorescent in situ hybridization was performed on the spreads as
per the manufacturer's instructions. Images were obtained using a
Zeiss microscope with Metafer software. A minimum of 50 cells were
scored to quantify HER2 and chromosome 17 positive foci.
[0112] Statistical analysis. Statistical analysis was performed by
one-way or two-way ANOVA with the Tukey's test as post hoc. All
analysis was completed using GraphPad Prism software. Herein, ****
denotes p<0.0001, *** denotes p<0.001, ** denotes p<0.01,
and * denotes p<0.05.
Results
(A) Targeting Gene Amplification in Cancer via Triplex
Formation
[0113] The present disclosure relates to a promising drug platform
that directly converts the amplified oncogenic driver genes into
DNA damage to trigger cell death (FIG. 1A). This approach employs
TFOs that recognize unique polypurine sites within the amplified
chromosomal region. First, a TFO, HER2-1, was designed to target
the polypurine sequence in the promoter region of the HER2 gene at
positions -218 to -245 relative to the transcription start site
(FIG. 1D). Another polypurine site favorable for high affinity
triplex formation is located within the coding region beginning at
position 205 and was targeted by another TFO, HER2-205 (FIG. 1D).
Chromosomal TFO binding was confirmed by preparing non-denatured
metaphase spreads from MCF7 and BT474 breast cancer cells that had
been treated with TAMRA-labeled HER2-205. The generation of
chromosomal HER2-205 foci represent third strand binding to fixed
chromosomes with intact DNA double helix.sup.15. Gene-specific
triplex formation was verified using a FITC labeled satellite probe
specific for human chromosome 17 (FIG. 1E). TAMRA-HER2-205
chromosomal foci were only generated on chromosome 17, the location
of the HER2 gene, thus validating target site specificity (FIG.
1E).
(B) The Level of Triplex-Induced DNA Damage Correlates with Higher
Gene Copy Numbers
[0114] A neutral comet assay showed that HER2-205 induced
significantly more DSBs in cell lines containing multiple copies of
the HER2 gene as indicated by an increase in DNA tail moment (FIGS.
2A-2B). Importantly, the level of triplex-induced DNA damage was
directly proportional to gene copy number (FIG. 2C). There was also
a markedly increased number of .gamma.H2AX positive cells,
indicative of DSBs, upon treatment of breast cancer cells with high
HER2 gene copy numbers (FIG. 2D). Then 53BP1 foci, which colocalize
with .gamma.H2AX at damage sites, were further assessed. HER2-205
treated BT474 cells exhibited substantially increased .gamma.H2AX
and 53BP1 foci compared to cells treated with the control
oligonucleotide MIX24 (FIG. 2E). Furthermore, colocalization of
.gamma.H2AX and 53BP1 was observed in 49% of cells following
HER2-205 treatment (FIG. 3A).
An experiment was conducted to determine whether HER2-targeting
TFOs would be capable of inducing apoptosis specifically in
amplified breast cancer cells. The results revealed TFO-induced
apoptosis specifically in the HER2-positive cell lines and that
HER2-205 treatment resulted in a higher percentage of apoptotic
cells than that with HER2-1 (FIGS. 2F-2G and FIG. 3B). Together,
the results demonstrate that the intensity of triplex-induced DNA
damage and apoptosis is dependent on gene copy number (FIG. 2C and
2G). Furthermore, these findings indicate that triplex-induced
apoptosis provides the basis to develop novel therapeutics that
specifically target amplified cancers, while sparing normal
non-amplified tissues.
(C) HER2-205 Treatment Effectively Targets HER2 Positive Ovarian
Cancers
[0115] The therapeutic efficacy in HER2-positive ovarian cancers
was evaluated. When administered to PEO1 and SKOV3 cells, both of
which have HER2 copy number gains (FIG. 3C), HER2-205 treatment
induced increased .gamma.H2AX foci and DNA tail moments (FIGS.
3D-G). There were also elevated levels of unrepaired DSBs in the
untreated PEO1 cells, which harbor a deficiency in BRCA2, a key
factor involved in DSB repair by homologous recombination (FIGS. 3F
and 3G). Importantly, TFO treatment significantly increased the
level of DSBs above baseline (FIG. 3G). In addition, HER2-205
reduced cell viability (FIG. 3H) and activated apoptosis in both
cancer cell lines (FIGS. 2H and 3I).
(D)Active HER2-Targeted TFO Could Potentially be used Clinically to
Treat HER2-Positive Cancers
[0116] To test whether active HER2-targeted TFO could be used
clinically to treat HER2-positive cancers in a preclinical model,
two independent subcutaneous xenograft tumor models were developed.
Treatment of BT474 human breast cancer tumors in athymic nude mice
with HER2-205 suppressed tumor growth to a significantly greater
degree than the controls, vehicle, and MIX24 (FIGS. 4A and 4C). IP
administration of HER2-205 resulted in a notable reduction in tumor
growth that was comparable to the currently used targeted therapy
trastuzumab, thus demonstrating the potential utility of this
gene-targeted cancer therapy (FIGS. 4A-4B). A tumor tripling time
of 29.+-.5.7 days post-initial dose was observed in tumors treated
with HER2-205 compared to 24.+-.2.1 days in tumors treated with
trastuzumab (FIG. 4D). In contrast, the control oligonucleotide,
MIX24 had no impact on BT474 tumor growth relative to the control
buffer alone, with a tumor tripling time for control tumors of
15.7.+-.4.9 days versus 16.3.+-.6.6 days in tumors treated with
MIX24 (ANOVA, p=0.99; FIG. 4D). Histological and
immunohistochemical analyses were performed on paraffin-embedded
tumor tissue sections. Tumor cell apoptosis (evidenced by the
presence of cleaved caspase 3), decreased proliferation as measured
by Ki67 staining, and a confluent area of tumor necrosis were
observed in the HER2-205 treated specimen (FIG. 4E). Magnification
of the HER2-205 treated tumor revealed that areas of tumor cell
apoptosis are accompanied by a brisk infiltrate of inflammatory
cells consisting predominantly of neutrophils and macrophages (FIG.
4F).
[0117] The standard of care for epithelial ovarian cancers consists
of platinum-based chemotherapy and surgical cytoreduction .sup.20.
However, as in the case of the SKOV3 cell line, many human ovarian
cancers are resistant to platinum-based drugs. Using SKOV3 ovarian
cancer xenografts, we find that HER2-205 treatment showed a
substantial survival advantage compared with cisplatin (FIG. 4G).
HER2-205 demonstrated significant tumor growth inhibitory activity
with the average tumor volume being 49% smaller than those in
cisplatin-treated mice (ANOVA, p=0.006). These data demonstrate
that triplex-induced apoptosis may provide a feasible therapeutic
alternative for drug resistant cancers with copy number gains.
(E) Apoptosis Corresponds with the Phosphorylation of Specific DNA
Damage Response Proteins and XPD is Required for the Induction of
Apoptosis
[0118] Given that the novelty of the approach herein is based upon
the development of agents with a unique mechanism of action, the
status of DNA damage response proteins, including ATM, Chk1/Chk2,
and the NER factor, XPD in HER2 positive cells was determined
following HER2-205 treatment. As shown in FIG. 5A, Chk1
phosphorylation at serine 345 was observed after HER2-205 treatment
in the HER2-amplified cells and not in the cells with normal HER2
gene copy numbers. Chk1 activation in BT474 cells corresponds to
induction of DSBs and apoptosis as determined by Western blot
analysis of pH2AX 5139 and cleaved PARP, respectively. In addition,
phosphorylation of Chk2 at threonine 68 was observed in response to
triplex-induced DSBs in the BT474 cells (FIG. 5A). These
phosphorylation events correspond to an increase in pATM positive
cells following HER2-205 treatment (FIG. 6A).
(F) HER2-205 Treatment Activates p53-Independent Apoptosis
[0119] To test whether triplex-induced DNA damage could activate
p53-independent apoptosis, p53-depleted BT474 cells were treated
with HER2-205. The results showed that TFO-treatment of
p53-depleted cells results in a similar level of PARP cleavage
compared to treatment of control cells, confirming that triplex
formation can activate apoptosis irrespective of p53 status (FIG.
5C). Unlike XPD-depleted cells, which displayed a decrease in
TFO-induced apoptosis we also demonstrate that triplex-induced DSBs
trigger robust H2AX Y142 phosphorylation in the absence of p53
(FIG. 5C).
[0120] Regulation of the phosphorylation status of H2AX at tyrosine
142 (Y142) is crucial for determining the recruitment of either DNA
repair or pro-apoptotic factors to the DSBs site.sup.21. H2AX Y142
was found to phosphorylate in response to HER2-205 induced DSBs to
trigger apoptosis as indicated by Western blot analysis of cleaved
PARP (FIG. 5B). XPD occupies a central role in the mechanism that
modulates survival/death decisions in response to triplex-induced
DNA damage.sup.15. Accordingly, a requirement for XPD in the
phosphorylation of Y142 in H2AX and activation of apoptosis
following HER2-205 treatment was seen (FIG. 5B). These results
suggest that the absence of XPD disrupts the signaling pathway used
to activate apoptosis following TFO treatment and support a
mechanism of action that is dependent upon DNA damage response.
(G) HER2-Targeted TFO Treatment is Independent of HER 2 Gene
Expression and Cellular Function
[0121] Trastuzumab's anticancer activity has been attributed in
part to changes in HER2 tyrosine phosphorylation and downregulation
of total HER2.sup.22,23. To further demonstrate that HER2-205
activity is based on a mechanism independent of the receptor's
cellular function, HER2 gene expression was analyzed by RT-PCR
(FIG. 5D) and total HER2 protein and phosphorylation levels were
monitored by Western blot following treatment in several breast
cancer cell lines (FIG. 5E). The results showed that HER2 gene
expression is not significantly affected by HER2-205 treatment in
either the non-amplified or amplified breast cancer cell lines
(FIG. 5D) and that total and activated HER2 levels remain the same
following triplex-induced apoptosis in the HER2-positive cells
compared to the control samples (FIG. 5E). In general, no changes
were noted in the levels of HERS, HER4 and EGFR following drug
treatment compared to the untreated or MIX24 treated cells (FIGS.
6B-6D). When combined, these studies revealed no consistent
evidence of an alteration of the expression or phosphorylation of
HER2 or the HER2 family receptors due to drug treatment, thus
supporting a mechanism of action that is independent of HER2
cellular function and dependent on DNA damage response (FIG.
5F).
(H) TFOs Designed to Target Non-Coding Regions of HER2 Gene Induce
DNA Double Strand Breaks
[0122] A neutral comet assay was conducted on BT474 cells 24 hours
post-treatment and showed that the TFOs targeting the non-coding
regions of HER2 (HER2-5922-2 and HER2-40118) and the TFO targeting
the coding region of HER2 (HER2-205) induced significantly more
DSBs in cell lines containing multiple copies of the HER2 gene as
indicated by an increase in DNA tail moment (FIG. 8A).
(I) TFOs Designed to Target Non-Coding Regions of the HER2 Gene
Activate Apoptosis
[0123] Triplex-induced apoptosis of BT474 cells was measured by
Annexin-V staining. Cells were stained 24 hours post-treatment and
the results revealed that TFOs targeting non-coding regions of the
HER2 gene can induce. HER2-40118 and HER2-5922-2 TFOs had more than
2-fold the percentage of apoptotic cells 24 hours post-treatment
(FIG. 8B).
Discussion
[0124] Herein, HER2-205 treatment of HER2-positive breast cancer
xenografts resulted in a 52% reduction in tumor volumes compared to
controls, which is comparable to the 58% reduction observed with a
current HER2-associated chemotherapeutic drug, trastuzumab. TFOs
targeting the coding and non-coding regions can induce DNA double
strand breaks and apoptosis. Notably, it was confirmed that triplex
formation can activate p53-independent apoptosis, which is
especially important since p53 mutations are associated with
therapeutically challenging cancers. The compositions and methods
disclosed herein can be used as drug design platform and treatment
option for several cancers with gene amplification and resistance
to currently used targeted-therapies.
OTHER EMBODIMENTS
[0125] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
From the above description, one skilled in the art can easily
ascertain the essential characteristics of the present invention,
and without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions. Thus, other embodiments are also
within the claims.
Equivalents
[0126] While several inventive embodiments have been described and
illustrated herein, those of ordinary skill in the art will readily
envision a variety of other means and/or structures for performing
the function and/or obtaining the results and/or one or more of the
advantages described herein, and each of such variations and/or
modifications is deemed to be within the scope of the inventive
embodiments described herein. More generally, those skilled in the
art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the inventive teachings is/are used. Those
skilled in the art will recognize, or be able to ascertain using no
more than routine experimentation, many equivalents to the specific
inventive embodiments described herein. It is, therefore, to be
understood that the foregoing embodiments are presented by way of
example only and that, within the scope of the appended claims and
equivalents thereto, inventive embodiments may be practiced
otherwise than as specifically described and claimed. Inventive
embodiments of the present disclosure are directed to each
individual feature, system, article, material, kit, and/or method
described herein. In addition, any combination of two or more such
features, systems, articles, materials, kits, and/or methods, if
such features, systems, articles, materials, kits, and/or methods
are not mutually inconsistent, is included within the inventive
scope of the present disclosure.
[0127] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0128] All references, patents and patent applications disclosed
herein are incorporated by reference with respect to the subject
matter for which each is cited, which in some cases may encompass
the entirety of the document.
[0129] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0130] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0131] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0132] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0133] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
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Sequence CWU 1
1
13128DNAArtificial SequenceSynthetic Polynucleotide 1aggagaagga
ggaggtggag gaggaggg 28228DNAArtificial SequenceSynthetic
Polynucleotide 2ggggggcaag agggcgagga ggagcccc 28328DNAArtificial
SequenceSynthetic Polynucleotide 3gggaggagga ggtggaggag gaagagga
28424DNAArtificial SequenceSynthetic Polynucleotide 4gaggaggagt
gggagaatgg gggg 24530DNAArtificial SequenceSynthetic Polynucleotide
5ggggtgagga gagtggggga ggagaaaggg 30622DNAArtificial
SequenceSynthetic Polynucleotide 6ggggtgggag ggacaaaggg gg
22730DNAArtificial SequenceSynthetic Polynucleotide 7gggaaagagg
agggggtgag aggagtgggg 30822DNAArtificial SequenceSynthetic
Polynucleotide 8gggggaaata gggagggtgg gg 22924DNAArtificial
SequenceSynthetic Polynucleotide 9agtcagtcag tcagtcagtc agtc
241028DNAArtificial SequenceSynthetic Polynucleotide 10ccctcctcct
ccacctcctc cttctcct 281128DNAArtificial SequenceSynthetic
Polynucleotide 11ggggctcctc ctcgccctct tgcccccc 281230DNAArtificial
SequenceSynthetic Polynucleotide 12ccctttctcc tcccccactc tcctcacccc
301322DNAArtificial SequenceSynthetic Polynucleotide 13ccccctttgt
ccctcccacc cc 22
* * * * *