Note: Descriptions are shown in the official language in which they were submitted.
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REGULATION OF HER2/neu ONCOGENE (EXPRESSION BY SYNTHETIC
POLYAMIDENS
The U.S. Government has certain rights in this invention pursuant to Grant
Nos.
GM 26453, 27681 and AI 29182 awarded by the National Institutes of Health.
RELATED APPLICATIONS
'This application claims benefit of priority from U.S. Provisional Application
60/099,906, filed September 1 l; 1998, which is hereby incorporated by
reference as if fully
set forth.
FIELD OF THE INVENTION
This invention is directed generally to methods and compositions for the
t 5 modulation, or regulation, of gene expression by the use of polyamides
that bind DNA.
The methods and compositions result in inhibition, crr down-regulation, of
gene
expression or overexpression by interactions between the polyamides and the
minor
groove of double-stranded DNA (dsDNA). The polyamides of these methods and
compositions bind predetermined target nucleic acid sequences located within
the
promoter region of genes to be down-regulated or inhibited. Inhibition or down-
regulation of target oncogenes that are expressed, or overexpressed, at
undesirable levels
is one application of the invention. In particular, the invention is directed
to reducing the
expression or overexpression of target endogenous cellular oncogenes.
BACKGROUND OF THE INVENTION
The tyrosine kinase membrane growth factor receptor HER2/neu, also known as
p185HE~, is encoded by a cellular oncogene of i:he same name that is
overexpressed
and ampiified in 20 to 30% of human breast cancers, among others, including
other
human gynecologic adenocarcinomas, such as those of the ovary, endornetrium,
fallopian
3o tube, and cervix.. See Baert, J.-L. et al., Int. J. Cancer 70, 590-597
(1997); Benz, C., et
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2
al., Orecogene 15, 1513-1525 (1997); Chang, C.-13., et al., Oncogene 14, 1617-
1622
(1997); Scott, G. K., et al., J. Biol. Chem. 269, 19848-19858 (1994); Pasleau,
F.; et al.,
Oncogene 8, 849-854 (1993); Tal, M. et al., Molecular and Cellular Biology 7,
2597-
2601 (1987); (Cirisano, F.D.; & Karlan, B.Y., J. Soc, Gynecol. Investig: 3 99-
105 (1996)}.
The neu oncogene gene product was originally described in chemically-induced
(ethylnitosourea) tumors in rodents. Subsequently, the human counterpart, c-
erbB-2 or
Her-2/neu, was found to be homologous to the EGF~ receptor, a i $5 kDa
transmembrane
protein with protein tyrosine kinase activity. Overexpression of HER2lneu is
also
associated with the likelihood that tumors will metastasize, with a resulting
poor
~ o prognosis for the patient. Mutation, amplification, and overexpression of
the Her-2/neu
oncogene has been reported to be associated with breast tumor progression,
early
metastasis and poor prognosis. Her-2/neu gene amplification directly
correlates with
lymph node metastasis. Additionally, in an animali model, activating mutations
lead to
rapid tumor progression. As a result, it is believed that the Her-2/neu
protein likely plays
a role in cell motility and hence in metastasis. Thus, inhibition of Her-2/neu
gene
expression by direct interference at the DNA Ievel may be a potent therapeutic
approach
for metastatic disease.
Several transcription factors - such as ESX, .AP-2, and the TATA binding
protein
("TBP") - play an important role in the regulation ~of the expression of the
gene for the
2o HER2lneu growth factor receptor. See Baert, J.-L. et al., Benz, C., et al;
supra; Chang, C.
H., et aL, supra; Bosher, J. M., et al., Proc. Natl. Acad. Sci. USA 92, 744-
747 (1995).
These transcription factors activate the expression of p185HE~ upon binding to
sites
within the HER2lneu promoter. The nucleotide sequence of the HER.2/neu
promoter and
a schematic representation are shown in Figure 1.
TBP is ubiquitous transcription factor that its involved in the activation of
most
protein-encoding genes. TBP is a DNA-binding protein that interacts to the
minor groove
of DNA. It should be noted that, apart from ESX, AP-2, and TBP, there are
other
potential transcription factor binding sites within the HER2lneu promoter.
Considerable effort has been expended in the, art to devise methods to
interfere
with gene expression in living cells in the hope that therapeutic strategies
will come from
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these studies. These approaches include interference. with the translation of
messenger
RNA into protein by the introduction of antisense oliigonucleotides into cells
(natural or
peptide nucleic acid based) or by ribozyme-mediatedl destruction of specific
RNAs.
Several approaches for direct inhibition of gene trap;>cription have also been
attempted;
these include triple helix forming oligonucleotides, designed or selected zinc
f nger
peptides that recognize pre-determined sequences,' and DNA-binding
calicheamicin
oligosaccharides.
For any therapeutic approach based in interference with gene expressionto be
successful, several criteria must be met by the therapeutic agent: first, the
agent must not
possess any general cell toxicity; second, the agent must be cell-permeable
and, in the
case of the DNA-binding agents, the compounds must transit to the nucleus and
bind their
target sequence with high affinity and specificity in the context of cellular
chromatin;
and, third, binding of the agent to its DNA target sequence must interfere
with gene
transcription. Each of the potential approaches listed above has its own
peculiar
limitations. For example, while triple helix-forming oligonucleotides have the
potential
for sequence selectivity and can effectively inhibit transcription irx vitro,
these molecules
suffer from poor cell permeability and permeabilizedl cells need to be used
for effective
gene inhibition. Similarly, zinc finger peptides must be introduced via a gene
therapy
approach with an appropriate viral or non-viral expression vector since these
peptides
cannot directly enter cells. In contrast, the calicheamicin oligosaccharides
are sufficiently
hydrophobic to pass through cell membranes, but these molecules possess
severely
limited sequence specificity (4 bp) and bind DNA wiith very Iow affinities
(100 p.M or
higher required for inhibition of protein-DNA interac;tions). Thus, new
classes of cell-
permeable molecules that possess higher degrees of IDNA sequence specificity
and
affinity are needed for any human gene therapeutic approach to be feasible.
Another approach utilizes cell-permeable small molecules that taxget
particular
DNA sequences. These molecules would be useful :for the regulation of gene
expression.
The design of small synthetic DNA-binding ligands that recognize specific
sequences in
the DNA double helix has been a long standing goal of chemistry:
3o Oligodeoxynucieotides that recognize the major groove of double-helical DNA
via triple-
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helix formation bind to a broad range of sequences with high affinity and
specificity.
Although oligonucleotides and their analogs have been shown to interfere with
gene
expression, the triple helix approach is limited to purine tracks and suffers
from poor
cellular uptake.
Other small molecules have also been of interest as DNA-binding ligands. Wade,
et al. reported the design of peptides that bind in the minor groove of DNA at
5'-
(A,T)G(A,T)C(A,T)-3' sequences by a dimeric side-by-side motif (J. Am. Chem.
Soc.
114, 8783-8794 (1992)). Mrksich, et al. reported antiparallel side-by-side
motif for
sequence specific-recognition in the minor groove of DNA by the designed
peptide I-
o methylimidazole-2-carboxamidenetropsin (Proc. Natl. Acad. Sci. USA 89, 7586-
7590
(1992)). Pelton, J.G. & Wemmer, D.E. reported th.e structural characterization
of a 2-1
distamycin A-d(CGCAAATTTGGC) complex by two-dimensional NMR (Proc. Natl.
Acad. Sci. USA 86, 5723-5727 (1989)}.
Dervan and colleagues have shown that synthetic pyrrole-imidizole polyamides
i 5 bind DNA with excellent specificity and very high a;Ffinities, even
exceeding the affinities
of many sequence-specific transcription factors (T'rauger, et al., Nature 382,
559-561
( 1996)). They further describe the recognition of DNA by designed ligands at
subnanomolar concentrations. DNA recognition depends on side-by-side amino
acid
pairing of imidizole-pyrrole or pyrrole-pyrrole pairs in the minor groove.
White, S., et
2o al., (1996) reported the effects of the A~T/T~A degeneracy of pyrrole-
imidazole
polyamide recognition in the minor groove of IDNA (Biochemistry 35, 6147-6152
( 1996}). White, et al. ( I 997) reported pairing rules for recognition in the
minor groove of
DNA by pyrrole-imidazole polyamides CChem. & Biol. 4, 569-578 (1997)), and
demonstrated the 5'-3' N-C orientation preference for polyamide binding in the
minor
25 groove. Thus, polyamide molecules thus have the potential to act as
inhibitors of protein-
DNA interactions in the minor groove.
The development of pairing rules for minor groove binding polyamides derived
from N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids provides
another
means to confer sequence specificity. An Im/Py pair distinguishes G~C frorn
C~G, and
3o both of these from A~T or T~A base pairs, while Py/lm targets a C-G
basepair. A Py/Py
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pair specifies A~T from G~C but does not distinguish A~T from T~A. The
generality of
this approach to the rational design of sequence-spec;ific DNA ligands is
supported by
direct NMR structural studies (Geierstanger, et al., Science 266,646-
650(1994)) and the
recent success in synthesis of an eight ring hairpin polyamide which targets a
six base
5 pair sequence with an apparent dissociation constant of 0.03 nM (Trauger, et
al., above}.
Moreover, two eight-ring pyrrole-imidizole polyamides differing in sequence by
a single
amino acid residue bind specifically to respective sips; base-pair target
sites that differ in
sequence by a single base pair. The replacement of a single nitrogen atom with
a C-H
can regulate specificity and affinity by two orders of magnitude.
l0 Since a six base-pair sequence would be highly redundant in the human
genome
{occurring at random once every 4 kiiobases, or 500,000 times in the human
genome),
polyamides have been synthesized to recognize much longer sequences. For
example, a
twelve-ring double hairpin polyamide has been designed to target a 12 by site
and
binding is again observed with nanomoiar affinity. Such a sequence would be
predicted
to occur at random only once every 16 million base pairs, or only 125 times in
the human
genome. Such molecules thus have the potential to acct as specific inhibitors
of gene
transcription in vivo and as human therapeutic agents. if the conditions
outlined above can
be met.
2o SUMMARY OF THE INVENTION
The present invention relates to and includes methods and compositions for the
modulation, or regulation, of gene expression or overexpression by reducing
the
transcription of genes. Preferably, the transcription of specific individual
target genes is
reduced. Such reductions result from the application of polyamides that bind
or interact
with the minor groove of double-stranded DNA (dsD~NA) within the promoter
regions of
target genes. Preferably, the binding or interaction is. with a predetermined
target nucleic
acid sequence within the promoter regions to inhibit ~or down-regulate
transcription.
The present invention reduces gene expression and overexpression by use of
sequence-specific DNA-binding small molecules that are cell-permeable and
capable of
3o inhibiting gene transcription. Appropriate application of such molecules
may inhibit
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overexpression of endogenous oncogenes to provide a fundamentally new
therapeutic
strategy for the treatment of various diseases, including cancer. The small
molecules of
the invention are polyamides that bind to or inter2~ct with nucleic acid
sequences within
the promoter region of target genes. Preferably, these sequences are
recognized, or
proximal to those that are recognized, by one or more transcription factors.
Preferably, the polyamides bind to the minor groove of double-stranded DNA in
a
promoter region that controls the transcription and expression of a gene.
Preferably the
transcription of the gene is inhibited by modulating; the binding of a protein
transcription
factor to dsDNA. In preferred embodiments, the transcription factors are ESX,
ETS, and
TBP.
Previous studies directed toward inhibition of the transcriptional activity of
the
HIV-1 promoter have demonstrated that polyarnides can block binding of TBP as
well as
an Ets family transcription factor (see PCT published application
PCTlUS98/02444, now
WO 98/35702, the teachings of which are incorporated by reference as if fully
set forth).
In principle, both classes of transcription factors can be inhibited by
polyamides that
contact or bind the minor groove of dsDNA. DNA compiexation of proteins
contacting
the minor groove may be inhibited by direct steric hinderance, repulsion, or
exclusion or,
alternatively, byallosteric efforts. For example, t:he binding of major groove
binding
proteins may be suppressed by a polyamide-induced change of the DNA
conformation.
2o Of course, inhibition can also be achieved other ways, for example, by
conjugating a
DNA cleavage agent to a polyamide targeted t:o a desired site, or by
chemically
modifying DNA.
In one preferred aspect of the invention, the expression or overexpression of
oncogenes is targeted. Preferably, the oncogenes are endogenous cellular
oncogenes
involved in cancer, particularly human breast cancer. One oncogene target of
the
invention is the HER-2/neu gene, which may be down-regulated or inhibited by
the use of
polyamides that bind to target sequences within the HER-2/neu promoter region.
Preferably, these sequences are, or are proximal to, transcription factor
binding sites
within the HER2/neu promoter. Interactions or binding between the polyamide
and the
3o target sequence can inhibit the transcription of t:he HER2/neu gene. The
degree of
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inhibition of HER2/neu expression may be extensive and includes the inhibition
of
HER2/neu overexpression. The invention further encompasses application of
polyamides
for the treatment of various tumors or cancers, including breast cancer.
Suitable polyamides have a binding affinity at the dsDNA target sequence of at
least 109 M'' and a selectivity of at least about two. Selectivity is defined
as the ratio of
the binding amity for the identified dsDNA target: sequence to the binding
affinity for a
single base-pair mismatch dsDNA sequence. In preferred embodiments,
selectivity
against at least 90% of single base mismatch sequences is greater than about
10.
In a related aspect of the present invention, compositions are provided that
to comprise a pharmaceutically acceptable excipient and a transcription-
inhibiting amount
of at least one polyamide of the invention. EaEch polyamide contains at Least
four
complementary pairs of aromatic carboxamide residues, which pairs are selected
to
correspond to an identified nucleotide sequence of a dsDNA target. Preferably,
the
polyamides additionaly comprise at least two aliphatic amino acid residues
chosen from
t5 the group consisting of glycine, ~i-alanine, y-aminobutyric acid, R-2,4-
diaminobutyric
acid, and 5-arninovaleric acid, and at least one terminal alkylamino residue,
the
polyamide having a binding affinity at the target ds:DNA sequence of at least
109 M'' and
a selectivity of at least about two, selectivity being defined as the ratio of
the binding
affinity for the identified target dsDNA sequence to the binding affinity for
a single base
2o pair mismatch dsDNA sequence.
The invention further provides methods su.itabie for treating a subject having
a
condition associated with abnormal expression of a cellular oncogene. The
subject is
preferably a human patient and, more particularly, one afflicted with breast
cancer or
other diseases or conditions associated with aberrant Her-2/neu oncogene
expression.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the HER2/neu promoter, showing the nucleotide sequence in A,
including binding sites of Ets, AP-2, and TBP ("TATA") transcription factors
and the
"CCAAT box", and in B, a schematic diagram, not ~to scale.
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Figure 2A is a graphical representation of thc~ results of a DNase I footprint
titration of polyamide HER2-1 {left} and the mismatch polyarnide ImPy-(3-PyIm-
y-
PyPyPyPyPy-~i-Dp (right) and 2B, the schematic structures and association
constants of
the polyamides, where the polyamides are represented by closed circles for
imidazole
rings, open circles for pyrrole rings, curved Iines for y-aminobutyric acid,
diamonds for (3-
alanine, and a half circle with a positive charge for
diimethylaminopropylamide.
Figure 3 compares the sequence of the HEIt1/neu promoter and polyamide
IO structures and binding sites; the binding site for the 'FATA binding
protein (TBP) is
indicated along with the structures of the polyamide;s HER2-A, HER2-1, 70, and
the
mismatch polyamide 86.
Figure 4 is a graphical representation of the results of experiments showing
the
15 effects of polyamides Her2-1 (A) and 70 on TBP binding.
Figure 5 is a graphical representation of the results of experiments showing
the
effects of the polyamide HER2-1 on HER2/neu transcription in vitro in a cell
free
system.
Figure 6 is a graphical representation of the results of experiments showing
the
effects of the polyamides HER2-I and 70 on HER2/neu mRNA production in the
human
breast cancer cell line SK-BR-2.
DETAILED DESCRIPTION
The present invention is directed to methods and compositions for modulating
or
regulating gene expression or overexpression by reducing gene transcription.
The methods
and compositions are preferably directed toward the: inhibition of oncogene
transcription,
especially of oncogenes involved in cancer, particula~~ly human cancer and
especially breast
cancer.
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The reductions in gene transcription resuht from binding or other interactions
between polyamides and the minor groove of dsDNA within the promoter regions
of target
genes. Preferably, the polyamides bind or interact with specific target
nucleic acid
sequences within the promoter regions to inhibit or down-regulate
transcription. Preferably,
the sequences are recognized, or proximal to those that are recognized, by one
or more
transcription factors.
The polyamides are preferably cell-permeable and capable of inhibiting gene
transcription in vivo, in vitro, or in cell free systems. Appropriate
application of such
polyamide molecules may be used to inhibit expression or overexpression of
endogenous
1 o oncogenes as a treatment of various diseases, including cancer.
In preferred embodiments, the polyamides bind to the minor groove of double
stranded DNA in a promoter region that controls ahe transcription and
expression of a
target gene. Preferred target genes are endogenous oncogenes involved in
cancer
formation or progression. Preferably the transcription of the gene is
inhibited by
t 5 modulating the binding of a protein, such a transcription factor, to the
same promoter
region with which the polyamide binds or interacts. In especially preferred
embodiments,
the transcription factors are one or more of the follo~,wing: ESX; ETS; and
TBP.
Inhibition of transcriptional activity at the: HIV-1 promoter demonstrates
that
polyamides can block binding of TBP as well as a~n Ets family transcription
factor See
2o W4 98/35702, which includes a discussion of polyamide synthesis. The
present
invention includes the use of polyamides that inhibit: or modulate the
activity of both TBP
and Ets transcription factors. The invention may affect transcription factor
activity by use
of one or more polyamides that contact or bind the minor groove of dsDNA. Such
contact or binding may inhibit formation of DNA-transcription factor complexes
in the
25 minor groove by direct steric repulsion, allosteric effects, or other
mechanisms (e.g.,
cleavage or chemical modification of the dsDNA). This is possibly in contrast
to major
groove DNA binding proteins, such as TBP, which may be inhibited by a
polyamide-
induced change in DNA conformation.
In a preferred aspect of the invention, tlhe expression or overexpression of
3o oncogenes; especially endogenous cellular oncol;enes, is targeted.
Preferably, the
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oncogenes are those implicated in human breast cancer, and their expression or
overexpression is inhibited by polyamides that contact or bind the minor
groove in the
region of the oncogene promoter. Preferably, the; contacted or bound portions
of the
promoter region are, or are proximal to, transcription factor binding sites.
The degree of
5 inhibition is preferably large and more preferably enough to inhibit even
overexpression
of the oncogene, in when the copy number of the gene increases.
One oncogene target of the invention is the HER-2/neu gene, which may be
down-regulated or inhibited by the use of polyamides that bind to target
sequences within
the HER-2/neu promoter region. Preferably, these sequences are, or are
proximal to,
1 o transcription factor binding sites within the HERZ/neu promoter. These
transcription
factors include TBP, ESX and AP-2. Interactions or binding between the
polyamide and
the target sequence result in inhibition of the HER2/neu gene transcription.
In a preferred embodiment, a polyamide was designed to bind immediately
downstream of the TATA element found in the human Her-2/neu breast cancer
oncogene
promoter. This polyamide, Her2-1, of composition ImPy-(3-PyIm-y-PyPy-(3-PyPy-
(3-Dp,
binds the sequence 5'-AGAATGA-3' (where the 5' A of this sequence is the 3' A
of the
TATA element) with an apparent dissociation constant of 200 pM. Her2-1 is an
effective
inhibitor of TBP binding and transcription.
The present invention includes compositiions comprising a pharmaceutically
acceptable excipient and a transcription-inhibiting .amount of at least one
polyamide for
the inhibition of gene expression or overexpression. These compositions may
also be
used far the treatment of various tumors or carucers, including breast cancer.
The
invention further provides methods of administering such compositions to
result in
inhibition of gene expression or overexpression. The methods and compositions
are
preferably suited for treating a subject having a condition associated with
abnormal
expression of a cellular oncogene. The subject is preferably a human patient
particularly
one afflicted with cancer, especially breast cancer.
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Polyamides of the invention
The polyamides used in the present invention comprise N-methylimidazole and
N-methylpyrrole carboxamides. These polyamides generally have a crescent-
shaped
structure that permits interaction and complexation t;he minor groove of
double-stranded
s DNA. NMR studies have confirmed that these compounds can bind to DNA in a
2:1
ratio by a motif in which two polyamide ligands are arranged in an
antiparallel way, side-
by-side to each other (Pelton, J., et al., Proc. Natl. A~:ad. Sci. USA 86,
5723-5727 (1986);
Mrksich, M., et al., Proc. Natl. Acad. Sci. USA, 89, 7586-7590 {1992); Wade,
W. S., et
al.,. J. Am. Chem. Soc. 114, 8783 (1992)).
t o One means to increase the binding affinity of two polyamides is to
covalentiy
linked them with a turn-unit such as y-aminobutyric acid (see Mrksich, M., et
al., J. Am.
Chem. Soc. 116, 7983 (1994)). Such polyamides are; called "hairpin
polyamides", as they
adopt a hairpin-like conformation in the DNA complex. The sequence of the
imidazole
and the pyrrole carboxamides in the polyamide determines the DNA sequence
specificity
t 5 of the ligand, according to the scheme of carboxamide pairs that recognize
nucleotide
pairs described above. In some cases it has been usE;ful to replace one or
several pyrrole
carboxamide units with (3-alanine moieties in order to adjust the curvature of
the
polyamide to that of the DNA. It has recently been shown that polyamides
comprising
N-methylimidazole and N-methylpyrrole carboxam.ides can inhibit gene
expression in
2o eukaryotic cells (Gottesfeid, J.M., et al. Nature 387, 203-205 {1997)).
It has been found that polyamides containing a new aromatic amino acid, 3-
hydroxy-N-methylpyrrole (Hp), paired opposite Py, have the ability to
discriminate A~T
nucleotide pairs from T~A nucleotide pairs in DNA sequences. The replacement
of a
single hydrogen atom on the pyrrole with a hydroxyll group in a Hp/Py pairing
affects the
25 affinity and specificity of a polyamide by an order of magnitude. By using
Hp together
with Py and Im in four pairs of aromatic amino acid residue combinations
(Im/Py; PylIm,
Hp/Py, and Py/Hp), polyamides can selectively distinguish all four Watson-
Crick base
pairs in the minor groove of double stranded DNA. White, et al., Nature 391,
468-471
( 1998).
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The invention encompasses the use of improved polyamides for binding to the
minor groove of DNA in methods and compositiions far reducing gene expression
or
overexpression. The preparation and use of polyaniides for binding in the
minor groove
of DNA are described in the art. Included in the invention is an improvement
of the
existing technology which utilizes 3-hydroxy-N-methylpyrrole to provide
carboxamide
binding pairs for DNA binding polyamides. The imlorovement relates to the
inclusion of a
binding pair of Hp/Py carboxamides in the polyamide to bind to a T~A base pair
in the
minor groove of DNA or Py/Hp carboxamide binding pair in the polyamide to bind
to an
A~T base pair in the minor groove of DNA. The polyamides used in the invention
have
1 o at least four carboxamide binding pairs that will distinguish A~T, T~A,
C~G, and G~C
base pairs in the minor groove. The polyamides may also have y-aminobutyric
acid or
another tum unit to form a hairpin-loop with a member of each carboxamide
pairing on
each side of it.
The invention also includes polyamides containing a (3-alanine substituted for
a
Py residue that would ordinarily be used in a caboxamide binding pair to match
a
particular nucleotide pair. The (3-alanine is referred to in formulas of this
invention as ji.
The ~3-alanine becomes a member of a carboxamide; binding pair, and serves to
optimize
hydrogen bonding of neighboring amino acid moieties to nucleotide base pairs.
The
invention further includes the substitution of a (3~~3 binding pair for a non-
Hp containing
2o binding pair. Thus. binding pairs in addition to the; Hp/Py and Py/Hp are
Py/Py, Im/Py,
PylIm, Iml(3, (3/Im, Pyl~i, (3/Py, and ~il(3.
In general, the polyamides of the invention are suitable far inhibiting the
transcription of a gene, preferably an oncogene. The polyamides consist of at
least four
complementary pairs of aromatic carboxamide residues, which pairs are selected
to
correspond to the nucleotide sequence of a dsDNA target. These polyamides
contain at
least two aliphatic amino acid residues chosen from the group consisting of
glycine, (3-
alanine, y-aminobutyric acid, and 5-aminovaleric acid, and at least one
terminal
alkylamino residue. The complementary pairs of aromatic carboxamide residues
selected
to correspond to the nucleotide sequence of an identified dsDNA target are
chosen from
3o the group consisting of Im/Py to correspond to the nucleotide pair G/C,
Py/Im to
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correspond to the nucleotide pair C/G, Py/Py to correspond to the nucleotide
pair A/T,
Py/Py to correspond to the nucleotide pair T/A, Hfp/Py to~ correspond to the
nucleotide
pair T/A, and Py/Hp to correspond to the nucleotide pair A/T, where Im is N-
methyl
imidazole, Py is N-methyl pyrrole and Hp is 3-hydroxy N-methyl pyrrole.
Application of
the above principles permits the design of specific polyamides that bind or
interact with
specific target nucleic acid sequences for usf~ in reducing gene expression or
overexpression.
Preferred polyamides contain at least one (3-alanine as an aliphatic amino
acid
residue. In preferred embodiments the terminal alkylamino residue is a N,N-
t o dimethylaminopropyl residue. Suitable polyamides containing at least two
j3-alanine
residues aligned to form complementary paired residues corresponding to a
nucleotide
pair chosen from the group AIT and T/A. Alternatively, corresponding pairs can
be
formed between aliphatic amino acids and aromatiic carboxamides, such as
Im/~i, (3/Im,
Py/~i and ~3/Py. In preferred polyamides, a hairpin molecule is formed by
inclusion an
aliphatic amino acid residue such as y-aminobutyric acid. Additionally, in
some
polyamides of the invention, at least one Py of a carboxamide pair is replaced
by a (3-
alanine.
Suitable polyamides have a binding affinity at the dsDNA target sequence of at
least 109 M'' and a selectivity of at least about two. Selectivity is defined
as the ratio of
2o the binding affinity for the identified dsDNA target sequence to the
binding affinity for a
single base-pair mismatch dsDNA sequence. In preferred embodiments,
selectivity
against at least 90% of single base mismatch sequences is greater than about
10.
Each polyamide used in the compositions o~f the invention preferably contains
at
least four complementary pairs of aromatic carl~oxamide residues, which pairs
are
selected to correspond to an identified nucleotide sequence of a dsDNA target.
The
polyarnides also preferably contain at least two aliphatic amino acid residues
chosen from
the group consisting of glycine, ~i-alanine, y-amin:~obutyric acid, R-2,4-
diaminobutyric
acid, and 5-aminovaleric acid, and at least one terminal alkylamino residue.
The
polyamides also preferably have a binding affinity at the target dsDNA
sequence of at
3o least 109 M'' and a selectivity of at least about two,, selectivity being
defined as the ratio
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of the binding affinity for the identified target dsDl\fA sequence to the
binding affinity far
a single base-pair mismatch dsDNA sequence.
Poiyamide Her2-1 was designed to bind to the DNA sequence 5'-AGAATGA-3',
which, as discussed above, is immediately adjacent to the TATA box of the
HEf2lneu
promoter. DNAse i footprint analysis confirms thavt this polyamide binds to
the desired
sequence with a dissociation constant (Kd) of about 0.2 nM. Polyamide 70 also
binds
adjacent to, and partially overlaps, the HER2/neu T.~1TA box. These polyamides
targeted
to the DNA sequences flanking or overlapping the Her-2lneu TATA element were
synthesized by solid phase methods.
t o Polyamide Her2-A, of sequence composition ImIm-(3-PyIm-y-PyPy-~i-PyPy-(3-
Dp
(where Im represents imidazole, Py represents pyrrole, y represents y-
aminobutyric acid,
(3 represents ~3-alanine, and Dp represents dimethyla.minopropylamide), binds
the
sequence 5'-AGGAAGT-3' at the 5' boundary of th.e Her-2/neu TATA element while
polyamide Her2-1, of sequence composition ImPy-[3-PyIm-y-PyPy-(3-PyPy-(i-Dp,
binds
the sequence 5'-AGAATGA-3' at the 3' boundary of the TATA element (see Figure
1 ).
A mismatch polyamide of sequence composition ImIm-(3-ImIm-y-PyPy-[3-PyPy-
~i-Dp (termed HIV-1) was also used in these studies. Polyamide 70, of sequence
composition ImPyPyPy-y-PyPyPyPy-(3-Dp, binds the sequence 5'-AGTATA-3'
overlapping the TATA box, while polyamide 86, of'sequence composition ImPyImPy-
y-
2o PyPyPyPy-(3-Dp, is a mismatch polyamide, with a single atom substitution
fiom
polyamide 70.
Figure 1 shaves the sequence of the Her-2/ne:u promoter region and the binding
sites of several transcription factors. The hairpin polyamide ImPy-J3-PyIm-'y-
PyPy-j3
PyPy-(3-Dp was synthesized to bind immediately downstream of the TBP binding
site
(Figure 2B, left).
Quantitative DNase I footprinting experiments {Figure 2A) conducted on a 3zP-
end-labeled restriction fragment isolated from a HER2/neu gene-containing
plasmid
(Ebbinghaus, et al. "Triplex formation inhibits HEf: 2/neu transcription in
vitro." J. Clin
Invest. 92:2433-2439 (1993)) revealed that this polyamide (Her2-I) binds its
target
sequence with an equilibrium association constant of 5 x 109 M-'. The mismatch
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polyamide ImPy-ø-PyIm-y-PyPyPyPyPy-ø-Dp {polyamide 70) bound the same sequence
with an equilibrium association constant of 2 x 108 M-~ (Figure 2B, right).
The
footprinting experiments indicated that this polyami~de also binds the S'-
AGGAAGT-3'
single-base pair mismatch sequence proximal to the ESX binding site with
comparable
off nity.
The TATA box region and binding models for each of these polyamides are
shown in Figure 3. In this f gore, polyamides are represented schematically
between the
two DNA strands at their respective binding sites. Shaded and unshaded circles
represent
imidazole {Im) and pyrrole {Py) rings, respectively; curved lines represent y-
aminobutyric
~ o acid (y); diamonds represent ø-alanine (ø); and Dp represents
dimethylaminopropylamide. The apparent binding affinities for each of these
polyamides
was determined by quantitative DNase I footprint titrations. Polyamide Her2-A
binds its
match site with a Ka of <108 M-' while polyamide Her2-1 binds with a Ka of S x
109 M''.
Given the higher affinity of Her2-1 for its target site, most of the examples
described
15 below utilized this compound. The binding constama for polyamide 70 has
been reported
previously (polyamide 2 in Trauger,et aL, Nature 38.2, SS9-561, 1996) and
corresponds to
3.S x 109 M-'. The mismatch polyamides bind with ;greatly reduced affnity.
Pharmaceutical and therapeutic compositions
2o The polyamides of the invention, as well as the pharmaceutically acceptable
salts
thereof, may be formulated into pharmaceutical or therapeutic compositions,
formulations, or preparations. Pharmaceutically acceptable salts of the
polyamide
compounds of the invention are formed where appropriate with strong or
moderately
strong, non-toxic, organic, or inorganic acids or bases by methods known in
the art.
25 Exemplary of the salts that are included in this invention are maleate,
fumarate, lactate,
oxalate, methanesulfonate, ethanesuifonate, benzeneaulfonate, tartrate,
citrate,
hydrochloride, hydrobromide, sulfate, phosphate, and nitrate salts.
As stated above, the polyaxnide compounds of the invention possess the ability
to
inhibit gene expression or overexpression, properties that are exploited in
the treatment of
3o any of a number of diseases or conditions, most notably cancer and
especially breast
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cancer. A composition of this invention may be active per se, or may act as a
"pro-drug"
that is converted in vivo to an active form.
The compounds of the invention, as well as the pharmaceutically acceptable
salts
thereof, may be incorporated into convenient dosage. forms, such as capsules,
impregnated wafers, tablets, or injectable preparatio3zs. Solid or liquid
pharmaceutically
acceptable carriers may be employed. Pharmaceutical compositions designed for
timed
release may also be formulated.
Preferably, the compounds of the invention acre administered systemically,
e.g., by
injection. When used, injection may be by any known route, preferably
intravenous,
to subcutaneous, intramuscular, intracranial, or intraperitoneai. Injectables
can be prepared
in conventional forms, either as solutions or suspensions, solid forms
suitable for solution
or suspension in liquid prior to injection, or as emulsions.
Solid Garners include starch, lactose, calciurr~ sulfate dihydrate, terra
alba, sucrose,
talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.
Liquid carriers
i5 include syrup, peanut oil, olive oil, saline, water, dextrose, glycerol and
the like.
Similarly, the carrier or diluent may include any prolonged release material,
such as
glyceryl monostearate or glyceryl distearate, alone or with a wax. When a
liquid carrier
is used, the preparation may be in the form of a syrup, elixir, emulsion, soft
gelatin
capsule, liquid containing capsule, sterile injectabie liquid (e.g., a
solution), such as an
2o ampoule, or an aqueous or nonaqueous liquid suspension. A summary of such
pharmaceutical compositions may be found, for example, in Rer~ingto~a's
Pharmaceutical
Sciences, Mack Publishing Company, Easton Pennsylvania (Gennaro 18th ed.
1990):
'The pharmaceutical preparations are made following conventional techniques of
pharmaceutical chemistry involving such steps as mixing, granulating and
compressing,
25 when necessary for tablet forms, or mixing, filling aand dissolving the
ingredients, as
appropriate, to give the desired products for oral or parenteral, including
topical,
transdermal, intravaginal, intranasal, intrabronchial, intracranial,
intraocular, intraaural
and rectal administration. The pharmaceutical compasitions may also contain
minor
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amounts of nontoxic auxiliary substances such as wetting or emulsifying
agents, pH
buffering agents and so forth.
Although the preferred routes of administration are systemic, the
pharmaceutical
composition may be administered topically or transdermally, e.g., as an
ointment, cream
or gel, orally, rectally, e.g., as a suppository, parente;rally, by injection
or continuously by
infusion, intravaginally, intranasally, intrabronchially, intracranially intra-
aurally, or
intraocularly.
For topical application, the composition may be incorporated into topically
applied vehicles such as a salve or ointment. The carrier for the active
ingredient may be
to either in sprayable or nonsprayable form. Non-spra;yable forms can be semi-
solid or solid
forms comprising a carrier indigenous to topical application and having a
dynamic
viscosity preferably greater than that of water. Suitable formulations
include, but are not
limited to, solution, suspensions, emulsions, creams, ointments, powders,
liniments,
salves, and the like. If desired, these may be sterilized or mixed with
auxiliary agents,
15 e.g., preservatives, stabilizers, wetting agents, buffers, or salts for
influencing osmotic
pressure and the like. Preferred vehicles for non-sprayable topical
preparations include
ointment bases, e.g., polyethylene glycol-1000 (PECi-1000}, conventional
creams such as
HEB cream, gels, as well as petroleum jelly and the Like.
Also suitable for topical application are spra;yable aerosol preparations
wherein
2o the compound, preferably in combination with a solid or liquid inert
carrier material, is
packaged in a squeeze bottle or in admixture with a pressurized volatile,
normally'
gaseous propellant. The aerosol preparations can contain solvents, buffers,
surfactants,
perfumes, and/or antioxidants in addition to the compounds of the invention:
For the preferred topical applications, especially for humans, it is preferred
to
2s administer an effective amount of the compound to a target area, e.g., skin
surface,
mucous membrane, eyes, etc. This amount will generally range from about 0.001
mg to
about 1 g per application, depending upon the area t~o be treated, the
severity of the
symptoms or disease, and the nature of the topical vehicle employed.
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The compositions of the invention can also be administered in combination with
one or more additional compounds that are used to great the disease or
condition. For
treating cancer, the polyamides and derivatives are given in combination with
anti-tumor
agents, such as mitotic inhibitors, e.g., vinblastine; alkylating agents,
e.g.,
cyclophosphamide; folate inhibitors, e.g., methotre~;ate, pritrexim or
trimetrexate,
antimetabolites, e.g., 5-fluorouracil and cytosine ara~binoside, intercalating
antibiotics,
e.g., adriamycin and bleomycirr, enzymes or enzyme inhibitors, e.g.,
aspaxaginase,
topoisomerase inhibitors, e.g., etoposide, or biological response modifiers,
e,.g.,
interferon. In fact, pharmaceutical compositions comprising any known cancer
to therapeutic in combination with the polyamine analogues and derivatives
disclosed herein
are within the scope of this invention.
Typical single dosages of the compounds of this invention are between about 1
ng
and about 10 glkg body weight. The dose is preferably between about O.OImg and
about
1g/kg body wt. and, most preferably, between about: 0.lmg and about lOOmg/kg
body
wt. For topical administration, dosages in the range; of about 0.01-20%
concentration of
the compound, preferably 1-5%, are suggested. A total daily dosage in the
range of about
I-500 mg is preferred for oral administration. The i:oregoing ranges are,
however,
suggestive, as the number of variables in regard to au~ individual treatment
regime is
large, and considerable excursions from these recommended values are expected
and may
2o be routinely made by those skilled in the art.
Effective amounts or doses of the compound for treating a disease or condition
can be determined using recognized in vitro systems or in vivo animal models
for the
particular disease or condition. In the case of cancer, many art-recognized
models are
known and are representative of a broad spectrum of human tumors. The
compositions
may be tested for inhibition of tumor cell growth in culture using standard
assays with
any of a multitude of tumor cell Iines of human or n.onhuman animal origin.
Many of
these approaches, including animal models, are des<~ribed in detail in Geran,
R.I. et al.,
"Protocols for Screening Chemical Agents and Natwal Products Against Animal
Tumors
and Other Biological Systems (Third Edition)", Canc. Chemother. Reports, Part
3, 3:1-
112.
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Administration methods
As noted above, the treatment methods of the invention are directed to the
administration of polyamide-containing compositions. The polyamide-containing
preparations of the invention may be administered systemically or locally and
may be
used alone or as components of mixtures. The route. of administration may be
topical,
intravenous, oral, or by use of an implant. For example, polyamides may be
administered
by means including, but not limited to, topical prep~u~ations, intravenous
injection or
infusion, oral intake, or local administration in the firm of intradermal
injection or an
Io implant. Additional routes of administration are sut>cutaneous,
intramuscular, or
intraperitoneal injections of the polyamides in convf;ntional ar convenient
forms.
Liposomal or lipophilic formulations may also be used when desired. Far
topical
administration, the polyamides may be in standard topical formulations and
compositions
including lotions, suspensions or pastes. Oral administration of suitable
formulations
t5 may also be appropriate in those instances where the; polyarnides may be
readily
administered to the target cells or tissues via this route.
The dose of polyamides may be optimized b;y the skilled artisan depending on
factors such as, but not limited to, the polyamides chosen, the physical
delivery system in
which it is carried, the individual subject, and the judgment of the skilled
practitioner.
2o Having now generally described the invention, the same will be more readily
understood through reference to the following examples which are provided by
way of
illustration, and are not intended to be limiting of the present invention,
unless specified.
EXAMPLE I
25 EIectrophoretic Mobility Shift Assays
Electrophoretic Mobility Shift Assays were performed to determine whether the
addition of various concentrations of polyamides specific for the sequences
flanking the
TATA box of the HER2/neu promoter could interfere with the DNA binding
activity of
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the TATA binding protein (TBP). Oligonucleotides corresponding to the HER2/neu
TATA box and the adjacent sequences were synthesized. The first
oligonucleotide,
HERTATA I , has the sequence:
S'-GCTGCTTGAGGAAGTATAAGAATG.AAGTTGTGAAG-3' (the TATA
5 box is in bold). The complementary oligonucleotide;, HERTATA2, has the
sequence:
5'-CTTCACAACTTCATTCTTATACTTC(:TCAAGCAGC-3'. These
complementary 35 base oligonucleotides were 5' end-labeled with y-3'-P-ATP and
T4
polynucleotide kinase and then annealed to give a double-stranded 35 base pair
oligonucleotide. This oligonucleotide was then used in electrophoretic
mobility shift
~ o assays employing 5% nondenaturing polyacrylamide gels (29:1 acrylamide to
bisacrylamide) containing 4 mM MgCI, and 0.02% (v/v) NP-40 nonionic detergent
along
with 44 mM Tris-borate, pH 8.3, 1 mM EDTA: The: labeled oligo, at a
concentration of
0. I nM, was reacted with 1 nM final concentration of TBP (Promega) in a
reaction
volume of 20 ~.1, containing 10% glycerol (v/v), 20 ~mM HEPES-OH, pH 7.9, 25
rnM
15 KCI, 0.025% NP-40 (v/v), I00 ~.g/ml bovine serum albumin, 0.5 mM
dithiothreitol, 0.8
mM spermidine, 0.1 mM EDTA, 2 mM MgCh.
Various concentrations of the different polyaunides, ranging from 0:I nM to 30
nM, were added to this binding reaction. The reactiyns were subjected to
polyacrylamide
gel electrophoresis and the dried gels were subsequently imaged and quantified
using a
2o Molecular Dynamics phosphorimager equipped with ImageQuant software. The
results
are shown in (Figure 4). It is clear that the HER2/ne:u-specific polyamides
(polyamides
HER2-I (Figure 4A), 70 (Figure 4B, squares), and RPR70, of composition
ImPyPyPy-y-
PyPyPyPy-(3-RPR, (Figure 4B, x's) significantly decrease TBP binding to the
HER2/neu
TATA box in vitro. A control polyamide 86 (FigurE: 4B, circles), which is not
specific
for the HER2/neu TATA box, has little effect on the; binding of TBP to the
HER2/neu
TATA box.
"RPR" indicates the presence of a charged aarginine-proiine-arginine tail on
the
polyamide.
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EXAMPLE II
Inhibition of Transcription ih vitro lln a Cell-Free System
The restriction endonuclease Dra I was used t:o linearize the plasmid
pGEM/HNP,
containing the HER2/neu promoter (Ebbinghaus, et al. ( 1993)), to produce a
template for
transcription. This template contained 270 base pairs of the HER2/neu promoter
as well
as 400 base pairs of downstream sequence. Transcription reactions were
performed in
20p,1 reactions containing 1 OOng of template DNA and 2 p.l of a HeLa cell
nuclear extract
in a reaction volume of 25 wl as recommended by thc: supplier (Promega). These
reactions were incubated at 30° C for 1 hour in the presence of I O
p.Ci a-'ZP-GTP along
with unlabeled nucleoside triphosphates at 0.6 mM and 20 p.M GTP. The labeled
transcripts were purified using RNAzoI as recommended by the supplier
(Teltest) and
subjected to denaturing polyacrylamide gel electrophoresis on 8%
polyacrylamide gels
containing 8.3 M urea and 88 mM Tris-borate, pH 8.3, 2 mM EDTA.
The dried gels were imaged and quantitated using a phosphorirnager (Molecular
Dynamics). The in vitro transcription from this HER2/neu promoter template was
inhibited by the addition of increasing amounts of pc~lyamide HER2-1 (Figure
5). The
addition of 10 nM HER2-1 had no effect, relative to the reaction with no added
polyamide, while the addition of 1 OOnM repressed transcription two-fold. As a
control,
in ultra transcription was also performed using the wnrelated cytomegalovirus
(CMV)
promoter as a transcription template. Addition of 10 nM or 100nM polyarnide
HER2-1 to
this template did not significantly decrease the level of transcription.
EXAMPLE IIIi
Inhibition of HER2Ineu Expressiion in Cell Culture
A number of breast cancer cell lines were obl;ained from the American Type
Culture Collection and maintained in cell culture. Tlhese cell lines included
breast cancer
cells in which the HER2/neu gene is amplified and greatly overexpressed (SIB-
BR-3 and
ZR 75-1 ) and breast cancer cells that have normal copy numbers of the
HER2/neu gene
and express HER2/neu at normal low levels {Hs 578'T). Various concentrations
of
polyamides were added directly into the appropriate cell culture medium
supplemented
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with I O% (v/v) fetal bovine serum during the growth phase of these cells at
37° C in a 5%
CO,lair mixture. For the initial experiments, polyam.ide HER2-1 was added to
the
medium at various concentrations. A control polyamide (HIV-1) was added in
various
concentrations to different flasks of cells. PoIyamide; HIV-i is similar in
structure to
s HER2-1, but does not specifically recognize the HER2lneu TATA box or its
adjacent
sequences.
In subsequent experiments, the cell line SK-BR-3 was treated with polyamides
for
6 days. In these experiments, polyamide HER2-1 and polyamide 70 in separate
experiments were added to the cell culture media for a final concentration of
O.Sp,M.
t o After 3 days of incubation, fresh media and fresh polyamide were added to
the cells.
These cells were incubated for an additional 3 days and then harvested for RNA
extraction.
Once the cells had grown to confluence in 75 cm2 culture flasks, the polyamide-
treated breast cancer cells were harvested by treating the adherent cells with
2 ml of
15 0.05% trypsin-0.53 mM EDTA (Gibco BRL) to detach the cells from the culture
flask.
These cells were collected and pelleted in a clinical c:entrifiige at 5000 rpm
(IEC ). The
cells were rinsed with cold lx phosphate buffered saJline (PBS) and pelieted
in the clinical
centrifuge. Total RNA was extracted from the cells Busing RNAzoI (Teltest). To
a packed
cell volume of approximately 100 p.l, lml of RNAzoI and 100p.1 of chloroform
were
20 added. This mixture was vortexed for 10 seconds and placed on ice for i 0
minutes. The
mixture was then spun for 15 minutes in a microfuge: at 4° C at 14,000
g. The top layer
was removed and total RNA precipitated with one volume of isopropanol. The RNA
pellet was washed with 70% ethanol, dried under vacuum, and resuspended in 50-
100 p,l
of RNAse-free (DEPC-treated) water containing 1 pl of RNasin (40 units).
25 The effects of polyamide addition were subsequently analyzed using reverse
transcriptase (RT)-polymerase chain reaction (PCR) as an assay for the
relative level of
HER2/neu mRNA. These HER2/neu mRNA levels ahould correlate with the amount of
transcription from the HER2/neu promoter, allowing; the determination of
whether
polyamide HER2-1 has any effect on transcription ire vivo. Using PCR primers
specific
30 for the HER2/neu oncogene, the PCR product will correspond to HER2/neu
cDNA,
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reflecting the relative levels of HER2/neu mRNA. T'he PCR primers were:
(Her2A) 5'-
GCTGGCCCGATGTATTTGATGGT-3' and (Her2,B) 5'-
GTTCTCTGCCGTAGGTGTCCCTTT-3', and 50 ng of each were used in PCR reactions
as described below.
The relative amounts of HER2/neu mRNA from the various cells can be
determined using reverse transcriptase (RT)-polymerase chain reaction (PCR).
After total
RNA has been extracted from the polyamide-treated! breast cancer cells, as
described
above, the concentration of total RNA is determined by spectrophotometry
(using the
optical density at 260 nM) for each different cell type and polyamide
concentration. An
t o equal amount of total RNA ( 10 ng) is used for each RT-PCR. RT-PCR was
carried out
using the Reverse Transcription System kit (Prome~;a). Using an oligo dT
primer,
cDNAs are synthesized from the mRNA templates by the enzyme reverse
transcriptase at
42° C for 25 min, as recommended. These cDNAs ~~re then used as
templates for PCR.
Using the buffers and Taq polymerase provided in the kit, PCR was carried out
at 26
15 cycles of denaturation at 94° C for 45 seconds, annealing at
60° C for 45 seconds, and
extension at 72° C for 2 minutes. Five p.Ci of the radioactive
nucleotide a 3zP-dATP is
included in the PCR step to produce a radiolabeled 1PCR product which can be
analyzed
on an acrylamide gel and visualized by autoradiography. The relative amount of
PCR
product can be quantitated using a Phosphorimager (Molecular Dynamics). The
level of
2o HER2/neu mRNA from cells which have not been treated with polyamide are the
positive
control and are given a value of 1.0 and the HER2/neu mRNA levels for the
polyamide-
treated samples are given a value relative to the value for untreated cells.
T'he results of these RT-PCR assays are sho~,vn in Figure 6. Treatment of the
cell
lines SK-BR-3 and Hs 578-T with polyamide HER:?-1 for 1-2 days resulted in
slightly
25 less than two-fold reduction in the relative levels of HER2/neu mRNA. The
control
polyamide HIV-1 had no apparent effect on the relative levels of HER2/neu
mRNA.
When SK-BR-3 cells were treated for 6 days with either polyamide HER2-1 or 70,
the
relative levels of mRNA decreased more significantly than for the 1-2 day
treated cells.
SK-BR-3 cells showed a 4-fold and 3-fold decrease in the relative levels of
HER2/neu
30 mRNA when treated with polyamide HER2-1 or 70, respectively. These results
suggest
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that the polyamides can enter the cells and bind to their target nucleotide
sequence,
thereby interfering with the expression of that gene.
All references cited herein are hereby incorporated by reference in their
entireties,
whether previously specifically incorporated or not.
Having now fully described this invention, it will be appreciated by those
skilled
in the art that the same can be performed within a wide range of equivalent
parameters,
concentrations, and conditions without departing from the spirit and scope of
the
~ o invention and without undue experimentation.
While this invention has been described in connection with specific
embodiments
thereof, it will be understood that it is capable of further modifications.
This application
is intended to cover any variations, uses, or adaptations of the invention
following, in
general, the principles of the invention and including; such departures from
the present
15 disclosure as come within known or customary practice within the art to
which the
invention pertains and as may be applied to the essential features herein
before set forth
as follows in the scope of the appended claims.
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