Title: hKIS composition and
methods of use
United States Patent: 7,414,035
Issued: August 19, 2008
Inventors: Nabel; Gary J
(Washington, DC), Nabel; Elizabeth G. (Washington, DC), Boehm; Manfred
Assignee: The Regents of
the University of Michigan (Ann Arbor, MI)
Appl. No.: 11/128,063
Filed: May 12, 2005
Disclosed herein are novel composition
and methods for altering the proliferation of a cell. Included are
wild-type and mutant hKIS polypeptides along with cyclin kinase inhibitors
containing mutations that prevent their inhibition with serine/threonine
Description of the
BRIEF SUMMARY OF THE INVENTION
The inventors have discovered a novel mechanism of regulation of CKIs.
Specifically, disclosed herein are serine/threonine kinases that inhibit the
ability of CKIs to arrest cells in G1. In light of this discovery, the
inventors were able to construct a transdominant mutant of a serine/threonine
kinase that interferes with the respective endogenous serine/threonine
kinase when introduced into a cell transgenically. Furthermore, the
inventors were able to construct a CKI unable to be inhibited by a serine/threonine
kinase. Such constructs may be used alone, together, or in conjunction with
other therapies for inhibiting or reducing cell proliferation.
Thus, the present invention provides isolated nucleic acid segments. Such
isolated nucleic acid segments may encode wild-type or mutant hKIS
polypeptides. In preferred embodiments, the isolated nucleic acid segments
encode a transdominant mutant hKIS. A transdominant mutant hKIS is a
polypeptide that is capable of interfering with the ability of endogenous
hKIS to phosphorylate p27. Thus, a transdominant mutant hKIS would lead to
or enhance cell cycle arrest in a cell containing the mutant. An example of
a transdominant mutant hKIS is an hKIS that contains a mutation altering its
serine/threonine kinase activity, such as that encoded by SEQ ID NO:3).
In other embodiments of the present invention, the isolated nucleic acid
encodes a cyclin kinase inhibitor containing a mutation at a serine or
threonine amino acid. It is preferred that the cyclin kinase inhibitor
retains its ability to arrest the cell cycle. Examples of mutated cyclin
dependent kinases include mutated of p16, p21, p27, and p57.
The isolated nucleic acids of the present invention may be contained in an
expression vector. The expression vector may be a plasmid or a viral vector.
The viral vector may be replication deficient and includes a retroviral
vector, an adenoviral vector, an adenovirus associated viral vector, or a
Furthermore, the isolated nucleic acids of the present invention may be
contained in or associated with a medical device, such as a catheter.
The isolated nucleic acids or polypeptides of the present invention may be
included in a kit, such kits may also include one or more medical devices
for administering the nucleic acid or polypeptide to a patient or one or
more cells of a patient.
DETAILED DESCRIPTION OF THE INVENTION
In order to identify proteins that bind to p27, the inventors first utilized
the yeast two-hybrid system to identify proteins that associate with p27 in
vivo (Fields and Song, 1989; Chien et al., 1991; Durfee et al., 1993; Harper
et al., 1993). Such analyses led to the discovery a novel gene that encodes
a polypeptide that binds to p27 in the yeast assay. This gene is included
herein as hKIS (human KIS) DNA and protein sequences (SEQ ID NO:1 and SEQ ID
Although the sequence of the hKIS gene is 92% identical to a rat KIS
sequence that has been reported (Maucuer et al., 1997; GenBank Acc. No.
X98374)), its potential role in p27 binding and/or as part of the cell cycle
pathway(s) has not previously been suggested.
It is well established that certain dominant genes promote tumorigenesis or
cell proliferation by binding and reducing the activity of tumor suppressor
proteins. Prominent examples include MDM2, which binds and inhibits the
tumor suppressor function of p53, and the transforming proteins encoded by
certain DNA viruses (e.g., the SV40 large T antigen), that also bind and
inactivate tumor suppressors such as p53 and Rb. The inventors have
determined that the interaction between hKIS and p27 reduces the ability of
p27 to arrest cells in G1.
The gene encoding hKIS serves as a dominant gene controlling cell
proliferation. Thus, inhibiting hKIS is a therapeutic approach. hKIS
inhibition could be achieved by providing to a hyperproliferative cell, or
administering to a patient, any compound that inhibits the hKIS gene, mRNA,
Thus, embodiments of the present invention include assays to find compounds
capable of reducing the level of transcription of the hKIS gene. Such assays
include contacting a cell with a compound and comparing the amount of hKIS
RNA in the cell as compared to a control. This comparison may be done
through any of a number of techniques including, Northern blotting,
semi-quantitative PCR, or RNase protection assays. Preferred compounds are
hKIS antisense oligonucleotides.
Other embodiments of the present invention include assays to find compounds
that inhibit the ability of serine/threonine kinases, such as hKIS, to
phosphorylate CKIs, such as p27. Such methods may be in vitro or in vivo.
For example, the assay may include contacting a cell or protein composition
comprising a hKIS and p27 protein with a compound and determine the ability
of the two proteins to interact with each other. The ability of the two
proteins to interact may be determined by a number of methods including
immunoprecipitation, plasmon resonance techniques, or fluorescence energy
transfer techniques. In some instances the substance may permit binding of
the two proteins but inhibit phosphorylation. Such instances may be
determined by detecting phosphorylation of the CKI.
In some embodiments, cell proliferation may be inhibited by providing to a
hyperproliferative cell, or administering to a patient, a CKI comprising one
or more mutations that prevent or reduce phosphorylation of the CKI's
serine/threonine residues by a serine/kinase, such as hKIS. A such mutated
CKI would maintain the ability to arrest cells in G1 phase, as shown in
example 1, yet not be inhibited by the expression of a serine/threonine
kinase. The mutation may prevent interaction of the CKI with the respective
serine/threonine or prevent phosphoryation of one or more serine/threonine
subsequent to interaction of the two proteins. In a preferred embodiment,
the mutated CKI is p27 comprising a serine to alanine mutation at amino acid
Other methods of inhibiting cell proliferation are described herein and
include, but are not limited to, compounds that reduce transcription of the
endogenous hKIS, compounds that prevent translation of hKIS mRNA, compounds
that prevent the interaction of hKIS and p27, and compounds that prevent
phosphorylation of p27 by hKIS.
Genes and DNA Segments
Important aspects of the present invention concern isolated DNA segments and
recombinant vectors encoding wild-type, polymorphic or mutant hKIS, and the
creation and use of recombinant host cells that express wild-type,
polymorphic or mutant hKIS, using the sequence of SEQ ID NO:1.
The present invention concerns DNA segments, isolatable from mammalian and
human cells, that are free from total genomic DNA and that are capable of
expressing a protein or polypeptide that has p27-binding activity. In
preferred embodiments, the DNA segments encode hKIS.
As used herein, the term "DNA segment" refers to a DNA molecule that has
been isolated free of total genomic DNA of a particular species. Therefore,
a DNA segment encoding hKIS refers to a DNA segment that contains wild-type,
polymorphic or mutant hKIS coding sequences yet is isolated away from, or
purified free from, total mammalian or human genomic DNA. Included within
the term "DNA segment", are DNA segments and smaller fragments of such
segments, and also recombinant vectors, including, for example, plasmids,
cosmids, phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified wild-type,
polymorphic or mutant hKIS gene refers to a DNA segment including wild-type,
polymorphic or mutant hKIS coding sequences and, in certain aspects,
regulatory sequences, isolated substantially away from other naturally
occurring genes or protein encoding sequences. In this respect, the term
"gene" is used for simplicity to refer to a functional protein, polypeptide,
or peptide encoding unit. As will be understood by those in the art, this
functional term includes both genomic sequences, cDNA sequences and smaller
engineered gene segments that express, or may be adapted to express,
proteins, polypeptides, domains, peptides, fusion proteins and mutants.
"Isolated substantially away form other coding sequences" means that the
gene of interest, in this case the wild-type, polymorphic or mutant hKIS
gene forms the significant part of the coding region of the DNA segment, and
that the DNA segment does not contain large portions of naturally-occurring
coding DNA, such as large chromosomal fragments or other functional genes or
cDNA coding regions. Of course, this refers to the DNA segment as originally
isolated, and does not exclude genes or coding regions later added to the
segment by the hand of man.
In particular embodiments, the invention concerns isolated DNA segments and
recombinant vectors incorporating DNA sequences that encode a wild-type,
polymorphic or mutant hKIS protein or peptide that includes within its amino
acid sequence a contiguous amino acid sequence in accordance with, or
essentially as set forth in, SEQ ID NO:2 corresponding to wild-type,
polymorphic or mutant human KIS. Moreover, in other particular embodiments,
the invention concerns isolated DNA segments and recombinant vectors that
encode a hKIS protein or peptide that includes within its amino acid
sequence the substantially full length protein sequence of SEQ ID NO:2.
The term "a sequence essentially as set forth in SEQ ID NO:2" means that the
sequence substantially corresponds to a portion of SEQ ID NO:2 and has
relatively few amino acids that are not identical to, or a biologically
functional equivalent of, the amino acids of SEQ ID NO:2.
The term "biologically functional equivalent" is well understood in the art
and is further defined in detail herein. Accordingly, sequences that have
between about 70% and about 80%; or more preferably, between about 81% and
about 90%; or even more preferably, between about 91% and about 99%; of
amino acids that are identical or functionally equivalent to the amino acids
of SEQ ID NO:2 will be sequences that are "essentially as set forth in SEQ
ID NO:2", provided the biological activity of the protein is maintained.
In certain other embodiments, the invention concerns isolated DNA segments
and recombinant vectors that include within their sequence a nucleic acid
sequence essentially as set forth in SEQ ID NO:1. The term "essentially as
set forth in SEQ ID NO:1, is used in the same sense as described above and
means that the nucleic acid sequence substantially corresponds to a portion
of SEQ ID NO:1 and has relatively few codons that are not identical, or
functionally equivalent, to the codons of SEQ ID NO:1. DNA segments that
encode proteins exhibiting p27-binding activity will be most preferred.
The term "functionally equivalent codon" is used herein to refer to codons
that encode the same amino acid, such as the six codons for arginine or
serine, and also refers to codons that encode biologically equivalent amino
acids (see Table 1, see Original Patent).
It will also be understood that amino acid and nucleic acid sequences may
include additional residues, such as additional N- or C-terminal amino acids
or 5' or 3' sequences, and yet still be essentially as set forth in one of
the sequences disclosed herein, so long as the sequence meets the criteria
set forth above, including the maintenance of biological protein activity
where protein expression is concerned. The addition of terminal sequences
particularly applies to nucleic acid sequences that may, for example,
include various non-coding sequences flanking either of the 5' or 3'
portions of the coding region or may include various internal sequences,
i.e., introns, which are known to occur within genes.
Excepting intronic or flanking regions, and allowing for the degeneracy of
the genetic code, sequences that have between about 70% and about 79%; or
more preferably, between about 80% and about 89%; or even more preferably,
between about 90%, 92%, 93%, 94% and about 99%; of nucleotides that are
identical to the nucleotides of SEQ ID NO:1 will be sequences that are
"essentially set forth in SEQ ID NO:1."
Sequences that are essentially the same as those set forth in SEQ ID NO:1
may also be functionally defined as sequences that are capable of
hybridizing to a nucleic acid segment containing the complement of SEQ ID
NO:1 under relatively stringent conditions. Suitable relatively stringent
hybridization conditions will be well known to those of skill in the art, as
Naturally, the present invention also encompasses DNA segments that are
complementary, or essentially complementary, to the sequence set forth in
SEQ ID NO:1. Nucleic acid sequences that are "complementary" are those that
are capable of base-pairing according to the standard Watson-Crick
complementarily rules. As used herein, the term "complementary sequences"
means nucleic acid sequences that are substantially complementary, as may be
assessed by the same nucleotide comparison set forth above, or as defined as
being capable of hybridizing to the nucleic acid segment of SEQ ID NO:1
under relatively stringent conditions such as those described herein.
The nuclear acid segments of the present invention, regardless of the length
of the coding sequence itself, may be combined with other DNA sequences,
such as promoters, polyadenylation signals, additional restriction enzyme
sites, multiple cloning sites, other coding segments, and the like, such
that their overall length may very considerably. It is therefore
contemplated that a nucleic acid fragment of almost any length may be
employed, with the total length preferably being limited by the ease of
preparation and use in the intended recombinant DNA protocol.
For example, nucleic acid fragments may be prepared that include a short
contiguous stretch identical to or complementary to SEQ ID NO:1, such as
about 8, about 10 to about 14, or about 15 to about 20 nucleotides. DNA
segments with total lengths of about 1,000, about 500, about 200, about 100
and about 50 base pairs in length (including all intermediate lengths) are
also contemplated to be useful.
It will be readily understood that "intermediate lengths", in these
contexts, means any length between the quoted ranges, such as 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.;
50, 51, 52, 53, etc.; 60, 61, 62, 63, 64, etc.; 100, 101, 102, 103, etc.;
150, 151, 152, 153, etc.; including all integers through the 200-500;
500-1000; 1,000-2,000 ranges.
The various probes and primers designed around the disclosed nucleotide
sequences of the present invention may be of any length. By assigning
numeric values to a sequence, for example, the first residue is 1, the
second residue is 2, etc., an algorithm defining all primers can be
n to n+y
where n is an integer from 1 to the last number of the sequence and y is the
length of the primer minus one, where n+y does not exceed the last number of
the sequence. Thus, for a 10-mer, the probes correspond to bases 1 to 10, 2
to 11, 3 to 12 . . . and so on. For a 15-mer, the probes correspond to bases
1 to 15, 2 to 16, 3 to 17 . . . and so on. For a 20-mer, the probes
correspond to bases 1 to 20, 2 to 21, 3 to 22 . . . and so on.
It will also be understood that this invention is not limited to the
particular nucleic acid and amino acid sequences of SEQ ID NO:1. Recombinant
vectors and isolated DNA segments may therefore variously include these
coding regions themselves, coding regions bearing selected alterations or
modifications in the basic coding region, or they may encode larger
polypeptides that nevertheless include such coding regions or may encode
biologically functional equivalent proteins or peptides that have variant
amino acids sequences.
The DNA segments of the present invention encompass biologically functional
equivalent hKIS proteins and peptides. Such sequences may arise as a
consequence of codon redundancy and functional equivalency that are known to
occur naturally with nucleic acid sequences and the proteins thus encoded.
Alternatively, functionally proteins or peptides may be created via the
application of recombinant DNA technology, in which changes in the protein
structure may be engineered, based on considerations of the properties of
the amino acids being exchanged. Changes designed by man may be introduced
through the application of site-directed mutagenesis techniques, e.g., to
disrupt the kinase properties of hKIS or to alter the domain of hKIS
responsible for interaction with p27.
One may also prepare fusion proteins and peptides, e.g. where the hKIS
coding regions are aligned within the same expression unit with other
proteins or peptides having desired functions, such as for purification or
immunodetection purposes (e.g., proteins that may be purified by affinity
chromatography and enzyme label coding regions, respectively).
Encompassed by the invention are DNA segments encoding relatively small
peptides, such as, for example, peptides of from about 15 to about 50 amino
acids in lengths, and more preferably, of from about 15 to about 30 amino
acids in length; and also larger polypeptides up to and including proteins
corresponding to the full-length sequences set forth in SEQ ID NO:2. It is
contemplated that such DNA segments encoding polypeptides or mutants thereof
may be useful in methods of interring with the biological function of the
endogenous hKIS protein. In a preferred embodiment, the DNA segment is that
of SEQ ID NO: 3.
In other aspects, the present invention concerns isolated DNA segments and
recombinant vectors encoding mutant CKIs and the creation and use of
recombinant host cells that express mutant CKIs. CKIs have the ability to
arrest the cell cycle and are useful in therapies designed to inhibit or
otherwise limit cell proliferation. Examples of cell proliferation disorders
that can be affected by the modified CKIs described herein include
restenosis, atherosclerosis, cancer, smooth muscle cell proliferative
diseases or disorders of any vessel in the body, and the like. Examples of
CKI include p16, p21, p27, p57. The present invention includes a DNA segment
encoding a CKI modified such that it containing a mutation of one or more
serine or threonine residues. Such mutants maintain the ability to arrest
cells under going proliferation and are no longer able to be phosphorylated/inhibited
by a serine/threonine kinase. In preferred embodiments, the serine or
threonine residue is changed to alanine. However, it is contemplated that
the serine or threonine may be changed to essentially any other amino acid
as long as the change allows the CKI protein to maintain its functional
activity and is no longer phosporylatable/inhibited by a serine/threonine
kinase. Alanine generally is preferred because it tends to have the least
amount of effect on the conformation or function of the protein possessing
the mutation. Other amino acids, such as glycine, also can be used to
The serine and threonine residues available for modification to a non-phophorylatable
residue, such as alanine, are provided in Table 2 (see Original Patent). In
light of the present disclosure, one of ordinary skill in the art readily
could construct a nucleic acid construct with a mutation at one or more
serine or threonine codons, express the nucleic acid, and test the activity
of the encoded protein. One or more nucleotides of a serine or threonine
codon can be modified to create an alanine codon. The codons for alanine are
listed in table 1. One of ordinary skill in the art would be able to create
the mutation and test for a decrease in the ability of the CKI to be
phosphorylated/inhibited by a serine/threonine kinase and maintain its
ability to arrest the cell cycle. Methods of testing the ability of a CKI to
inhibit the cell cycle is described herein (Example 1).
In one embodiment, the serine or threonine residue/codon to be modified
occurs within residues 1-20 of the encoded protein. In another embodiment
the serine/threonine residue to modified occurs within residues 1-15 or 1-10
of the encoded protein. In other embodiments, p16 is modified at S4 or T10;
p21 is modified at S2 or S15; p27 is modified at S2, S7, S10, or S12; p57 is
modified at S2, S5, S8, T9, S10 or T11.
Recombinant Vectors, Host Cells and Expression
Recombinant vectors form important further aspects of the present invention.
The term "expression vector or construct" means any type of genetic
construct containing a nucleic acid coding for a gene product in which part
or all of the nucleic acid encoding sequence is capable of being
transcribed. The transcript may be translated into a protein, but it need
not be. Thus, in certain embodiments, expression includes both transcription
of a gene and translation of a RNA into a gene product. In other
embodiments, expression only includes transcription of the nucleic acid, for
example, to generate antisense constructs.
Particularly useful vectors are contemplated to be those vectors in which
the coding portion of the DNA segment, whether encoding a full length
protein or smaller peptide, is positioned under the transcriptional control
of a promoter. A "promoter" refers to a DNA sequence recognized by the
synthetic machinery of the cell, or introduced synthetic machinery, required
to initiate the specific transcription of a gene. The phrases "operatively
positioned", "under control" or "under transcriptional control" means that
the promoter is in the correct location and orientation in relation to the
nucleic acid to control RNA polymerase initiation and expression of the
The promoter may be in the form of the promoter that is naturally associated
with the expressed gene, as may be obtained by isolating the 5' non-coding
sequences located upstream of the coding segment or exon, for example, using
recombinant cloning and/or PCR technology, in connection with the
compositions disclosed herein (PCR technology is disclosed in U.S. Pat. No.
4,683,202 and U.S. Pat. No. 4,682,195, each incorporated herein by
In other embodiments, it is contemplated that certain advantages will be
gained by positioning the coding DNA segment under the control of a
recombinant, or heterologous, promoter. As used herein, a recombinant or
heterologous promoter is intended to refer to a promoter that is not
normally associated with the gene in its natural environment. Such promoters
may include promoters normally associated with other genes, and/or promoters
isolated from any other bacterial, viral, eukaryotic, or mammalian cell.
Naturally, it will be important to employ a promoter that effectively
directs the expression of the DNA segment in the cell type, organism, or
even animal, chosen for expression. The use of promoter and cell type
combinations for protein expression is generally known to those of skill in
the art of molecular biology, for example, see Sambrook et al. (1989),
incorporated herein by reference. The promoters employed may be
constitutive, or inducible, and can be used under the appropriate conditions
to direct high level expression of the introduced DNA segment, such as is
advantageous in the large-scale production of recombinant proteins or
The particular promoter that is employed to control the expression of a
nucleic acid is not believed to be critical, so long as it is capable of
expressing the nucleic acid in the targeted cell. Thus, where a human cell
is targeted, it is preferable to position the nucleic acid coding region
adjacent to and under the control of a promoter that is capable of being
expressed in a human cell. Generally speaking, such a promoter might include
a human or viral promoter.
In various other embodiments, the human cytomegalovirus (CMV) immediate
early gene promoter, the SV40 early promoter and the Rous sarcoma virus long
terminal repeat can be used to obtain high-level expression of transgenes.
The use of other viral or mammalian cellular or bacterial phage promoters
which are well-known in the art to achieve expression of a transgene is
contemplated as well, provided that the levels of expression are sufficient
for a given purpose. Tables 3 and 4 (see Original Patent) list several
elements/promoters which may be employed, in the context of the present
invention, to regulate the expression of a heterologous gene. This list is
not intended to be exhaustive of all the possible elements involved in the
promotion of transgene expression but, merely, to be exemplary thereof.
Any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base
EPDB) could also be used to drive expression of a transgene. Use of a T3, T7
or SP6 cytoplasmic expression system is another possible embodiment.
Eukaryotic cells can support cytoplasmic transcription from certain
bacterial and viral promoters if the appropriate bacterial or viral
polymerase is provided, either as part of the delivery complex or as an
additional genetic expression construct.
It may also be desirable to modify the identified regulatory unit by adding
additional sequences to the unit. The added sequences may include additional
enhancers, promoters or even other genes. Thus one may, for example prepare
a DNA fragment that contains the native regulatory elements positioned to
regulate one or more copies of the native gene and/or another gene or
prepare a DNA fragment which contains not one but multiple copies of the
promoter region such that transcription levels of the desired gene are
Turning to the expression of a transgene to produce a protein, once a
suitable clone or clones have been obtained, whether they be cDNA based or
genomic, one may proceed to prepare an expression system. Both cDNA and
genomic sequences are suitable for eukaryotic expression, as the host cell
will generally process the genomic transcripts to yield functional mRNA for
translation into protein. Generally speaking, it may be more convenient to
employ as the recombinant gene a cDNA version of the gene. It is believed
that the use of cDNA version will provide advantages in that the size of the
gene will generally be much smaller and more readily employed to transfect
the targeted cell than will be a genomic gene, which will typically be up to
an order of magnitude larger than the cDNA gene. However, the inventors do
not exclude the possibility of employing a genomic version of a particular
gene where desired.
In expression, one will typically include a polyadenylation signal to effect
proper polyadenylation of the transcript. The nature of the polyadenylation
signal is not believed to be crucial to the successful practice of the
invention, and any such sequence may be employed. Preferred embodiments
include the SV40 polyadenylation signal and the bovine growth hormone
polyadenylation signal, convenient and known to function well in various
target cells. Also contemplated as an element of the expression cassette is
a terminator. These elements can serve to enhance message levels and to
minimize read through from the cassette into other sequences.
A specific initiation signal also may be required for efficient translation
of coding sequences. These signals include the ATG initiation codon and
adjacent sequences. Exogenous translational control signals, including the
ATG initiation codon, may need to be provided. One of ordinary skill in the
art would readily be capable of determining this and providing the necessary
signals. It is well known that the initiation codon must be "in-frame" with
the reading frame of the desired coding sequence to ensure translation of
the entire insert. The exogenous translational control signals and
initiation codons can be either natural or synthetic. The efficiency of
expression may be enhanced by the inclusion of appropriate transcription
It is proposed that a wild-type, polymorphic or mutant hKIS gene may be
co-expressed with wild-type or mutant p27, wherein the proteins may be
co-expressed in the same cell or wherein wild-type, polymorphic or mutant
hKIS genes may be provided to a cell that already has wild-type or mutant
p27. Co-expression may be achieved by co-transfecting the cell with two
distinct recombinant vectors, each bearing a copy of either the respective
DNA. Alternatively, a single recombinant vector may be constructed to
include the coding regions for both of the proteins, which could then be
expressed in cells transfected with the single vector. In either event, the
term "co-expression" herein refers to the expression of both the wild-type,
polymorphic or mutant hKIS and wild-type or mutant p27 proteins in the same
In addition to co-expression with p27, it is proposed that the wild-type,
polymorphic or mutant hKIS gene may be co-expressed with genes encoding
other CKI or tumor suppressor proteins or polypeptides. Tumor suppressor
proteins contemplated for use include, but are not limited to, the
retinoblastoma, p53, Wilms tumor (WT-1), DCC, neurofibromatosis type 1
(NF-1), von Hippel-Lindau (VHL) disease tumor suppressor, Maspin, Brush-1,
BRCA-2, and the multiple tumor suppressor (MTS). Further particularly,
contemplated is co-expression with a selected wild-type version of a
selected oncogene. Wild-type oncogenes contemplated for use include, but are
not limited to, tyrosine kinases, both membrane-associated and cytoplasmic
forms, such as members of the Src family, serine/threonine kinases, such as
Mos, growth factor and receptors, such as platelet derived growth factor (PDDG),
SMALL GTPases (G proteins) including the ras family, cyclin-dependent
protein kinases (cdk), members of the myc family members including c-myc, N-myc,
and L-myc and bcl-2 and family members.
As used herein, the terms "engineered" and "recombinant" cells are intended
to refer to a cell into which an exogenous DNA segment or gene, such as a
cDNA or gene encoding a hKIS has been introduced. Therefore, engineered
cells are distinguishable from naturally occurring cells which do not
contain a recombinantly introduced exogenous DNA segment or gene. Engineered
cells are thus cells having a gene or genes introduced through the hand of
man. Recombinant cells include those having an introduced cDNA or genomic
gene, and also include genes positioned adjacent to a promoter not naturally
associated with the particular introduced gene.
Expression vectors for use in mammalian cells may include an origin of
replication (as necessary), a promoter located in front of the gene to be
expressed, along with any necessary ribosome binding sites, RNA splice
sites, polyadenylation site, and transcriptional terminator sequences. The
origin of replication may be provided either by construction of the vector
to include exogenous origin, such as may be derived from SV40 or other viral
(e.g., polyoma, adenovirus, VSV, or BPV) source, or may be provided by the
host cell chromosomal replication mechanism. If the vector is integrated
into the host cell chromosome, the later is often is sufficient.
The promoters may be from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., CMV immediate
early, the adenovirus late promoter; the vaccinia virus 7.5K promoter).
Further, it is also possible, and may be desirable, to utilize promoter or
control sequences normally associated with the desired gene sequence,
provided such control sequences are compatible with the host cell systems.
A number of viral based expression systems may be utilized, for example,
commonly used promoters are derived from polyoma, Adenovirus 2, and most
frequently Simian Virus 40 (SV40). The early and late promoters of SV40
virus are particularly useful because both are obtained easily from the
virus as a fragment which also contains the SV40 origin of replication.
Smaller or larger SV40 fragments may also be used, providing there is
included the approximately 250 bp sequence extending from the HindIII site
toward the BgIl site located in the viral origin of replication.
In cases where an adenovirus is used as an expression vector, the coding
sequence may be ligated to an adenovirus transcription/translation control
complex, e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in vitro or
in vivo recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus that is
viable and capable of expressing the transgene in infected hosts.
In eukaryotic expression, one will also typically desire to incorporate into
the transcriptional unit an appropriate polyadenylation site (e.g.,
5'-AATAAA-3') if one was not contained within the original cloned segment.
Typically, the poly A addition site is placed about 30 to 2000 nucleotides
"downstream" of the termination site of the protein at a position prior to
Also, a number of selection systems may be used, including, but not limited,
to the herpes simplex virus thymidine kinase, hypoxanthine-guanine
phosphoribosyltransferase and adenine phosphoribosyltransferase genes. Also,
antimetabolite resistance can be used as the basis of selection for dhfr,
that confers resistance to methotrexate; gpt, that confers resistance to
mycophenolic acid; neo, that confers resistance to the amonoglycoside G-418;
and hygro, that confers resistance to hygromycin.
It is contemplated that a gene of the present invention may be "overexpressed",
i.e., expressed in increasing levels of relative to its natural expression
in cells. Such overexpression may be assessed by a variety of methods,
including semi-quanitative PCR, Northern blotting, RNase protection assays,
radio- and Immuno-assays. However, simple and direct methods are preferred,
for example, those involving SDS/PAGE and protein staining or western
blotting, followed by quantitative analyses, such as densitometric scanning
of the resultant gel or blot. A specific increase in the level of the mRNA,
recombinant protein or peptide in comparison to the level in natural cells
is indicative of overexpression.
Certain embodiments of the present invention may require the introduction of
mutations into hKIS or a CKI gene. Site-specific mutagenesis provides a
ready ability to prepare and test sequence variants, incorporating one or
more of the foregoing considerations, by introducing one or more nucleotide
sequence changes into the DNA. Site-specific mutagenesis allows the
production of mutants through the use of specific oligonucleotide sequences
which encode the DNA sequence of the desired mutation, as well as a
sufficient number of adjacent nucleotides, to provide a primer sequence of
sufficient size and sequence complexity to form a stable duplex on both
sides of the deletion junction being traversed. Typically, a primer of about
17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on
both sides of the junction of the sequence being altered.
In general, the technique of site-specific mutagenesis is well known in the
art and commercial kits are available (ALTERED SITES.RTM. ii in vitro
Mutagenesis Systems; Promega Corp., Madison, Wis.). In some cases, the
technique employs a bacteriophage vector that exists in both a single
stranded and double stranded form. Typical vectors useful in site-directed
mutagenesis include vectors such as the M13 phage. These phage vectors are
commercially available and their use is generally well known to those
skilled in the art. Double stranded plasmids are also routinely employed in
site directed mutagenesis, which eliminates the step of transferring the
gene of interest from a phage to a plasmid.
In general, site-directed mutagenesis is performed by first obtaining a
single-stranded vector, or melting of two strands of a double stranded
vector which includes within its sequence a DNA sequence encoding the
desired protein. An oligonucleotide primer bearing the desired mutated
sequence is synthetically prepared. This primer is then annealed with the
single-stranded DNA preparation, and subjected to DNA polymerizing enzymes
such as E. coli polymerase I Klenow fragment, in order to complete the
synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed
wherein one strand encodes the original non-mutated sequence and the second
strand bears the desired mutation. This heteroduplex vector is then used to
transform appropriate cells, such as E. coli cells, and clones are selected
that include recombinant vectors bearing the mutated sequence arrangement.
The preparation of sequence variants of the selected gene using
site-directed mutagenesis is provided as a means of producing potentially
useful species and is not meant to be limiting, as there are other ways in
which sequence variants of genes may be obtained. For example, recombinant
vectors encoding the desired gene may be treated with mutagenic agents, such
s hydroxylamine, to obtain sequence variants.
Proteins and Peptides
The present invention provides purified, and in preferred embodiments,
substantially purified proteins and peptides. The term "purified protein or
peptide" as used herein, is intended to refer to an aqueous composition,
isolatable from mammalian cells or recombinant host cells, wherein the
protein or peptide is purified to any degree relative to its
naturally-obtainable state, i.e., relative to its purity within a cellular
extract. A purified protein or peptide therefore also refers to a protein or
peptide free from the environment in which it naturally occurs.
The proteins or polypeptides may be full length proteins or may also be less
then full length proteins, such as individual domains, regions or even
epitopic peptides. Where less than full length proteins are concerned the
most preferred will be those containing the functional domains.
Generally, "purified" will refer to protein or peptide composition that has
been subjected to fractionation to remove various other protein or peptide
components, and which composition substantially retains the biological
activity of the desired protein. For example, a purified wild-type hKIS
protein would still maintain biological activity, as may be assessed by
binding to p27, forming complexes with p27, or phosphorylating p27.
Where the term "substantially purified" is used, this will refer to a
composition in which the protein or peptide forms the major component of the
composition, such as constituting about 50% of the proteins in the
composition or more. In preferred embodiments, a substantially purified
protein will constitute more than 60%, 70%, 80%, 90%, 95%, 99% or even more
of the proteins in the composition.
A polypeptide or protein that is "purified to homogeneity," as applied to
the present invention, means that he polypeptide or protein has a level or
purity where the polypeptide or protein is substantially free from other
proteins and biological components. For example, a purified polypeptide or
protein will often be sufficiently free of other protein components so that
degradative sequencing may be performed successfully.
Various methods for quantifying the degree of purification of proteins or
peptides will be known to those of skill in the art in light of the present
disclosure. These include, for example, determining the specific biological
activity of a faction, or assessing the number of polypeptides within a
fraction by gel electrophoresis. Assessing the number of polypeptides within
a fraction by SDS/PAGE analysis will often be preferred in the context of
the present invention as this is straightforward.
To purify a protein or peptide of interest, a natural or recombinant
composition comprising at least some proteins or peptides of interest will
be subjected to fractionation to remove various other polypeptide or protein
components from the composition. Various techniques suitable for use in
protein purification will be well known to those of skill in the art. These
include, for example, precipitation with ammonium sulfate, PEG, antibodies
and the like or by heat denaturation, followed by centrifugation;
chromatography steps such as ion exchange, gel filtration, reverse phase,
hydroxylapatite, lectin affinity and other affinity chromatography steps;
isoelectric focusing; gel electrophoresis; and combinations of such and
Although preferred for use in certain embodiments, there is no general
requirement that the protein or peptide always be provided in their most
purified state. Indeed, it is contemplated that less substantially purified
proteins or peptides, which are nonetheless enriched the protein of
interest, relative to the natural state, will have utility in certain
Methods exhibiting a lower degree of relative purification may have
advantages in total recovery of protein product, or in maintaining the
activity of an expressed protein. Inactive products also have utility in
certain embodiments, such as, e.g., in antibody generation.
The general approach to the cell proliferation suppression aspect of the
present invention is to provide a cell with a transdominant hKIS protein, a
CKI mutated such that it is no longer inhibited by a serine/threonine kinase,
or both. By "transdominant hKIS", it is meant that the mutated hKIS or hKIS
polypeptide fragment interferes the ability of endogenous hKIS protein to
inhibit p27 mediated G1 arrest. While it is conceivable that a protein may
be delivered directly to a cell, a preferred embodiment involves providing a
nucleic acid encoding a protein to the cell. Following this provision, the
polypeptide is synthesized by the transcriptional and translational
machinery of the cell, as well as any that may be provided by the expression
construct. In providing antisense, ribozymes and other inhibitors, the
preferred mode is also to provide a nucleic acid encoding the construct to
the cell. All such approaches are herein encompassed within the term "gene
The delivery and entry of recombinant material into target cells is
facilitated by use of vectors. DNA can be directly transferred to somatic
target cells by viral vectors, such as retroviruses and adenoviruses, and
non-viral methods, such as cationic liposomes, liposome viral conjugates,
Viruses naturally infect mammalian cells and introduce their viral DNA to
convert the host biosynthetic pathway to produce viral DNA, RNA, and
protein. Molecular biologists have been able to modify these viruses so that
they deliver foreign DNA to the target cell but cannot replicate in the host
cell nor express viral proteins necessary for encapsulation. In general,
early response viral sequences, involved in viral transcription,
translocation or capsid synthesis, have been removed from the viral genome
and are replaced by the foreign gene of interest.
Therefore, these recombinant viruses can only propagate in specific
packaging cell lines which express the deleted viral proteins.
Replication-deficient retroviruses, adenoviruses, adeno-associated viruses
and adenoviral conjugates are now used in gene transfer techniques.
Retroviruses are RNA viruses that require vector integration into the host
genome for expression of the transgene thus limiting their use to dividing
cells. As most of the vascular and myocardial cellular components are
non-replicating cells, retroviruses are of limited use in cardiovascular
gene transfer. In addition, integration at random locations may lead to
insertional mutagenesis and transformation. However, there have been no
reported short- or long-term toxicity associated with their use in human
gene therapy trials. Retrovirus-mediated gene transfer has been used for
cell-mediated gene transfer using endothelial cells and for direct gene
transfer into porcine arteries. The long-term, high-level expression renders
retroviral vectors in particular ideal for ex vivo, cell-mediated gene
In cell-mediated gene transfer, endothelial cells or vascular smooth muscle
cells may be isolated, expanded and transduced in the laboratory and
reseeded on to an artery in vivo. The technique of ex vivo gene transfer is
however fairly cumbersome since it requires cell expansion. However, ex vivo
gene transfer of endothelial cells and smooth muscle cells may be useful in
seeding stents, grafts or injured arteries during vascular procedures to
treat thrombotic disorders or graft hyperplasia.
Recombinant gutted lentiviruses may represent an attractive alternative to
retroviruses. Lentiviruses have not been directly implicated in any
malignancies and, in contrast to retroviral based vector systems, human,
simian and bovine immunodeficiency viral (HIV, BIV, SIV) vector systems have
been shown to mediate stable gene transfer in terminally differentiated
neurons and macrophages in culture. In vivo, transgene expression is
detected for up to 6 months in liver, muscle, retinal tissue, and brain of
immune-competent rats in vivo and does not appear to evoke an immune
response or local inflammation, permitting repeated viral challenge.
Recombinant adenoviruses efficiently transfect proliferating and
non-proliferating cells, but lack mutagenicity since the transgenic genome
is not integrated into the host chromosome but remains episomal. Deletions
of EI A, EI B, E2 and E3 regions of the viral genome prevent viral
replication in transfected cells, reduce expression of early response viral
proteins, and hence, limit cellular inflammation. Recombinant adenoviruses
have been successfully used for in vivo gene transfer in carotid and jugular
veins rat and rabbit myocardium and rabbit peripheral arteries. In vivo
adenovirus-mediated gene transfer using biological active gene products have
also been shown to exert effects in vascular diseases. Since immunogenicity
remains limiting in adenoviral vectors, adenoviral vectors gutted of almost
the entire adenoviral genome may prove to be beneficial in circumventing the
deleterious immune response.
Recombinant adeno-associated viruses (rAAV) are promising vectors given the
ability to integrate into the host genome, resulting in stable transgenic
expression, and lack of immunogenicity due to a lack of viral genes in the
vector that express surface proteins. rAAV vectors are described in U.S.
Pat. No. 5,139,941. rAAV has not been associated with disease in any host
and has not been associated with malignancies despite integration of the
transgene into the host genome. rAAV integrates viral and transgenic DNA
preferentially but not exclusively at chromosome 19q locus. Adeno-associated
viruses are incapable of replication and depend on co-infection with
adenovirus or a herpes virus for replication. In vivo, long-term expression
of .beta.-galactosidase and tyrosine hydroxylase have been achieved in
non-dividing neurons in the rat CNS by rAAV, and intravenous delivery of
rAAV encoding human clotting factor IX resulted intransduction of 3% of all
hepatocytes over a 5 month observation period. Also, intraluminal and
periadventitial vascular delivery of rAAV in atherosclerotic carotid
arteries of cynomolgus monkeys results in efficient transgenic expression.
However, in contrast to retroviruses and adenoviruses, transgenic expression
is predominantly found in adventitial endothelial cells of microvessels.
Other viral vectors that may be used for gene therapy include herpes simplex
virus (U.S. Pat. No. 5,288,641) and cytomegalovirus (Miller, 1992).
Because of safety concerns regarding viral vectors, an interest arose in
developing synthetic delivery system avoiding the infectious complications
presented by the first generation viral vectors. Non-viral gene transfer can
be performed by microinjection, DEAE-dextran transfection, calcium phosphate
precipitation, electroporation, liposomes, and particle-mediated gene
transfer (i.e. introducing DNA-coated particles).
The most common non-viral gene transfer vectors are DNA-liposomes. Cationic
liposomes condense and entrap the DNA through electrostatic interaction.
They are prepared by sonification and remain stable in aqueous solution for
months. The positively charged liposome complex fuses with the negatively
charged cell surface to release the DNA into the cytoplasm of target cells,
bypassing the lysosomal compartment and degradation by serum. It is
postulated that plasmid DNA is subsequently incorporated in the nucleus as
an episome. The relatively safe profile of liposomes, the lack of vector
size or target cell constraints, as well as the relative ease of
liposome-DNA complex preparation favors this gene transfer technique.
Preclinical studies using different forms of these lipids (DOTMA, DC-ChoI,
DMRIE, and DLRIE) have shown promise for efficient in vivo transfection.
Lipofection-mediated gene transfer, using either catheter-based delivery or
direct injection, results in site-specific expression of foreign recombinant
genes in vascular endothelial and smooth muscle cells and alters the biology
of the vessel wall. Cationic liposomes are well tolerated in vivo and do not
induce any biochemical, hemodynamic or cardiac intoxications.
Additional advances in lipid chemistry are developing newer generations of
cationic liposomes, which permit higher transfection with minimal toxicity.
The transfection efficacy and specificity of lipofection may be further
augmented by coupling of ligands or viral particles (Ad, HVJ, VSVG) to the
liposomes. In particular, HVJ-coated liposomes have been successfully
utilized to transduce venous bypass grafts ex vivo and in vivo.
In certain embodiments, plasmid DNA or RNA may be injected directly into
tissue such as skeletal muscle or myocardium. In other embodiments,
anti-sense oligonucleotides are used for gene therapy (Morishita et al.,
1993). Anti-sense oligonucleotides do not require a vector for cell
transduction and can be directly injected in the target tissue. Anti-sense
oligonucleotides are short DNA sequences complementary to the RNA message of
interest, which are chemically modified to resist nuclease degradation. The
oligonucleotide may be modified at the 5; end to prevent nuclease
degradation or may made up of ribonucleotide bases attached to a peptide
backbone (protein nucleic acid).
Various animal and cell culture studies have shown that anti-sense
oligonucleotides are able to efficiently modify intracellular expression of
factors involved in smooth muscle cell and endothelial cell migration and
proliferation, including by use of anti-sense oligonucleotides against c-myc,
c-myb, cdc2, and PCNA. The nucleotide sequence hybridizes to target RNA,
which prevents translation of RNA, targets the message for degradation by
ribonuclease H, and interferes with cytosolic translocation.
Gene Transfer in the Cardiovascular System
In certain embodiments of the present invention, gene therapy is used to
treat or prevent cell proliferation. In preferred embodiments, vascular cell
proliferation such as that associated with restenosis or atherosclerosis is
prevented using gene therapy. It is contemplated that a gene therapy vector
or composition of the present invention may be tested in an animal model.
Studies in animal models of cardiovascular disease have demonstrated that
transgenes can be expressed at high levels at local sites in the vasculature.
Local Delivery to the Vasculature
An attractive feature of cardiovascular gene transfer is that recombinant
genes may be delivered to local sites in the vasculature by a medical
device. Medical devices that are suitable for use in the present invention
include known devices for the localized delivery of therapeutic agents. Such
devices include, for example, catheters such as injection catheters, balloon
catheters, double balloon catheters, microporous balloon catheters, channel
balloon catheters, infusion catheters, perfusion catheters, etc., which are,
for example, coated with the therapeutic agents or through which the agents
are administered; needle injection devices such as hypodermic needles and
needle injection catheters; needleless injection devices such as jet
injectors; coated stents, bifurcated stents, vascular grafts, stent grafts,
etc.; and coated vaso-occlusive devices such as wire coils.
Exemplary devices are described in U.S. Pat. Nos. 5,935,114; 5,908,413;
5,792,105; 5,693,014; 5,674,192; 5,876,445; 5,913,894; 5,868,719; 5,851,228;
5,843,089; 5,800,519; 5,800,508; 5,800,391; 5,354,308; 5,755,722; 5,733,303;
5,866,561; 5,857,998; 5,843,003; and 5,933,145; the entire contents of which
are incorporated herein by reference. Exemplary stents that are commercially
available and may be used in the present application include the RADIUS.TM.
(Scimed Life Systems, Inc.), the SYMPHONY.RTM. (Boston Scientific
Corporation), the Wallstent (Schneider Inc.), the Precedent II.TM. (Boston
Scientific Corporation) and the NIR.TM. (Medinol Inc.). Such devices are
delivered to and/or implanted at target locations within the body by known
The double balloon catheter was an initial catheter employed in animal model
studies and was useful to demonstrate the basic principles of gene transfer.
The catheter consists of two balloons placed about 1.5 cm apart with an
inner protected space. The genetic vector is instilled into the isolated
arterial segment between the balloons. Adenoviral-mediated recombinant gene
expression is detected in endothelial cells, vascular smooth muscle cells
and adventitial cells for several weeks following infection and is not found
downstream to the arterial segment or in other tissues by PCR.
Retroviral-mediated gene expression can be detected for up to 6 months. A
disadvantage to this catheter is the possibility of distal ischemia due to
occlusion of blood flow. Alternate delivery devices permit flow distal to
the isolated segment allowing a prolonged instillation time period without
compromising distal perfusion.
Porous and microporous balloons infuse the vector directly into the
juxtapositioned arterial wall through small pores in the catheter. The depth
of delivery is directly related to the perfusion pressure. Channel balloon
catheters combine two separate inflatable compartments for balloon
angioplasty and drug infusion, allowing separate control of balloon
inflation pressure for positioning and drug infusion pressure. A hydrogel
coated balloon catheter has a hydrophilic polyacrylic acid polymer coating
of the balloon. This polymer absorbs the DNA suspension and when the balloon
is inflated, the DNA coating is pressed against the vessel wall. The
iontophoretic balloon uses a local current between the balloon and the skin
of the subject to drive the negatively charged DNA into the arterial wall.
Other delivery devices include stents coated with a DNA-impregnated polymer
or cells comprising a nucleic acid of the present invention (ex vivo gene
transfer) into arterial and venous grafts. Furthermore, tissue may be
selectively targeted for gene therapy by use of tissue specific promoters
Expression Constructs in Combination with Other Therapies
The method of the present invention can be combined with other methods for
treating cell proliferation. For example, other genes such as thymidine
kinase, cytosine deaminase, wild-type or mutated p21, p27, and p53 and
combinations thereof can be concomitantly transformed into cells and
expressed. For example, the herpes simplex-thymidine kinase (HS-tK) gene,
when delivered to blood vessel walls by a adenoviral vector system,
successfully resulted in the decrease in neointimal proliferation associated
with restenosis (Chang et al., Mol. Med., 1995, 172-181). In the context of
the present invention, it is contemplated that mutant hKIS or CKI gene
expression could be used similarly in conjunction with other gene therapy
approaches. The genes may be encoded on a single nucleic acid but separately
transcribed. Alternatively, the genes may be operably linked such that they
are contranscribed. In preferred embodiments, the genes are operably linked
to encode a fusion protein. In other embodiments the co-transcribed genes
are separated by an internal ribosome binding site allowing the proteins to
be translated separately. Such combination therapies are described in WO
99/03508 (incorporated herein by reference in its entirety).
In certain embodiments of the present invention, nucleic acid or protein
compositions of the present invention may be introduced into the myocardium.
Myocardial gene transfer requires tranfection of terminally differentiated
myocytes. Adenoviral gene transfer by intracoronary or intramyocardial
delivery results in transient gene expression for several weeks in a limited
number of cells. Adeno-associated viral vectors have been shown to induce
stable transgene expression in up to 50% of murine, rat and porcine
cardiomyocytes after ex vivo intracoronary infusion and myocardial
injections for at least 6 months. These vectors may be useful for gene
delivery to treat human myocardial diseases.
Many vascular diseases are characterized by abnormalities of cell
proliferation. One approach to therapies is to express genes that inhibit
cell proliferation within vascular lesions, for example, after angioplasty
or in a by-pass graft. Most approaches regulate the cell cycle in vascular
smooth muscle, endothelial or macrophage cells.
Progression through the cell cycle is regulated by the assembly and
phosphorylation of cyclin/cyclin-dependent kinase complexes (CDKs).
Endogenous inhibitors of the cyclin-CDKs, termed the cyclin-dependent kinase
inhibitors (CKIs) result in cell cycle arrest and cessation of cell
Genetic strategies to abrogate vascular lesion formation have focused on
regulatory gene products that interfere with DNA synthesis, cell cycle
progression, and cell viability. Gene products interfering with DNA and RNA
replication have been evaluated for their capacity to block smooth muscle
cell proliferation and reduce vascular lesion formation. Prodrug-enzyme
therapies, using thymidine kinase or cytosine deaminase, constitute a form
of local therapy in which an enzyme is expressed locally that converts a
prodrug into an active form. Gene transfer of DNA encoding these converting
enzymes to the injured arterial wall combined with systemic prodrugs
administration produces high levels of growth inhibitory drugs in the target
tissue. The therapeutic effect of transgene expression can be regulated by
administration of the prodrug and can be initiated independently of the gene
Herpes simplex virus thymidine kinase (HSV-tk) converts an inert nucleoside
analog, ganciclovir into a phosphorylated, toxic form in transduced cells.
Its subsequent incorporation into the host DNA induces chain termination and
cell death in dividing cells, while non-dividing cells remain unaffected.
Local delivery of recombinant adenovirus encoding for HSV-tk at the time of
the balloon injury and systemic administration to ganciclovir inhibited
smooth muscle cell proliferation in vivo, and decreased intimal formation in
balloon-injured porcine and rat arteries and atherosclerotic rabbit
arteries. A similar reduction of neointimal hyperplasia was observed in
arterial interposition grafts which overexpress HSV-tk in the rabbit.
Cytosine deaminase (CD) catalyzes the hydrolytic deamination of non-toxic
cytosine and 5-fluorocytosine (5-FC) into uracil and 5-fluorouracil, which
inhibits thymidilate synthase and hence DNA and RNA synthesis. In human and
rabbit primary smooth muscle cells, CD/5-FC does not induce significant
necrosis or apoptosis but results in cytostatic effects on vascular smooth
muscle cells. CD gene transfer in the rabbit femoral injury model followed
by systemic 5-FC treatment resulted in a decrease of the intima to media
area ratio, comparable to the efficacy of HSV-tk/ganciclovir in a rat and
pig model of vascular injury.
The Fas/FasL death-signaling pathway mediates cellular immunocytotoxicity in
activated lymphocytes. Binding of the Fas receptor to FasL activates the
caspase pathway leading to apoptosis. FasL is expressed in intimal smooth
muscle cells and immune competent cells in atherosclerotic plaques. Studies
using adenoviral-mediated gene transfer of FasL to balloon-injured rat
carotid arteries demonstrated an attenuation of T cell extravasation in FasL
expressing arteries as opposed to sham virus treated arteries, accompanied
with a 60% reduction of neointima formation (intima/media area ratio). FasL
may function to protect the vessel from leukocyte extravasation to the
subendothelial space during arterial repair by inducing T lymphocyte
Targeting of cell cycle regulatory proteins promotes inhibition of cell
proliferation, and cell differentiation. Cell cycle arrest prevents vsmc
proliferation and migration and endothelial dysfunction, shown by improved
vasoreactivity and NO production, rendering the vessel less susceptible to
inflammatory infiltration and free radical formation.
Progression through the cell cycle is controlled by the assembly and
disassembly of the different cyclin-cyclin dependent kinase complexes. These
complexes phosphorylate retinoblastoma protein leading to the release of the
sequestered transcription factors, E2F and EIf 1. The cyclin dependent
kinase inhibitors (CKIs) modulate the enzymatic activity of cyclin/CDK
complexes necessary for G.sub.1 progression. In vivo, Ad-p21 infection of
porcine iliofemoral and rat carotid arteries following balloon injury
reduces BrdU incorporation by 35% and I/M area ratio by 37%. Likewise, Gax
homeobox gene overexpression, as an upstream regulator of p21, in the rat
carotid artery injury model inhibited neointimal formation and luminal
narrowing by 59 and 56 percent, respectively. Adenovirus-mediated
overexpression of p27 in balloon-injured rat and porcine arteries
significantly attenuated intimal lesion formation.
The effects of many cyclin-CDK and CKI interactions are mediated through
their effect on the phosphorylation status and therefore activity of
retinoblastoma gene product (Rb). Rb inhibits cell cycle progression from
G.sub.1 into S phase by sequestering and inactivating a set of cellular
transcription factors. Localized infection of porcine endothelial cells and
vsmc with Ad-.DELTA.Rb, an unphosphorylatable, constitutively active Rb,
results in a significant reduction in cell proliferation and
[.sup.3Hlthymidine incorporation, yet the cells remain viable. In the rat
carotid artery injury as well as in the pig balloon injury model, .DELTA.Rb
expression results in a 42-47% decrease in the neointima/media area ratio
relative to control arteries.
Alternatively, inhibition of the cell cycle in human vein grafts with ex
vivo treatment of E2F decoy oligodeoxynucleotide reduces not only graft
susceptibility to atherosclerosis, and enhances medial hypertrophy, which
renders the graft more resistant to increased hemodynamic stress and
improves vein graft patency.
Metalloproteinases degrade the extracellular matrix, promote growth factor
release and cell activation and are therefor essential for cell migration.
Overexpression of tissue inhibitor of metalloproteinases (TIMP) was shown to
inhibit invasive and metastatic behavior of tumor cells. The effects of TIMP
protein expression has been evaluated in an organ culture model of
neointimal formation, which lends itself for the study of smc migration
rather than proliferation. Overexpression of TIMPi and 2 reduced neointima
formation and neointimal cell numbers by 54-79% and 71% respectively, but
did not alter smc proliferation and viability. These data confirm the
importance of metalloproteinases and smc migration to the development of
neointimal hyperplasia and suggest that a combined anti-proliferative and
anti-migratory gene therapy approach may optimize lesion reduction.
Other methods aim to reconstitute endothelial derived inhibitory signals,
which prevent leukocyte adhesion and platelet aggregation, relax local
muscle tone and inhibit vsmc proliferation by gene transfer of iNOS or eNOS.
The NOS pathway has been shown to play a significant role in a number of
cardiovascular disorders including atherosclerosis, systemic and pulmonary
hypertension, ischemia-reperfusion, hypercholesterolemia, and vasospasm. L-arginine
feeding, iNOS and eNOS gene transfer and various NO donors have shown to
successfully reduce lesion formation in hypercholesterolemic rabbits and
neointimal hyperplasia following arterial balloon injury model in pigs and
Thus, studies in various animal models demonstrate that genetic approaches
are feasible and effective in limiting cell proliferation, migration and
extracellular matrix deposition. The nucleic acids and proteins or
polypeptides of the present invention may be particularly useful in methods
of treating cardiovascular disease. For example, a nucleic acid encoding a
transdominant hKIS or mutated CKI may be introduced locally into an injured
artery to prevent restenosis. In a preferred embodiment, a vector comprising
both a trandominant hKIS and a mutated p27 is used to treat restenosis. Of
course, both genes may be introduced together but in separate vectors.
In other embodiments, gene therapy using a nucleic acid of the present
invention may be combined with other gene and non-gene therapies to treat a
cardiovascular disease. Potential molecular targets for cardiovascular
disease are shown in Table 5 (see Original Patent).
Pharmaceutically Acceptable Carriers
Aqueous compositions of the present invention comprise an effective amount
of a compound dissolved or dispersed in a pharmaceutically acceptable
carrier or aqueous medium. Aqueous compositions of gene therapy vectors are
also contemplated. The phrases "pharmaceutically or pharmacologically
acceptable" refer to molecular entities and compositions that do not produce
an adverse, allergic or other untoward reaction when administered to an
animal, or a human, as appropriate.
As used herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal agents
isotonic and absorption delaying agents and the like. The use of such media
and agents for pharmaceutical active substances is well known in the art.
Except insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is contemplated.
Supplementary active ingredients can also be incorporated into the
For human administration, preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of Biologics
The biological material should be extensively dialyzed to remove undesired
small molecular weight molecules and/or lyophilized for more ready
formulation into a desired vehicle, where appropriate. The active compounds
will then generally be formulated for parenteral administration, e.g.,
formulated for injection via the intravenous, intramuscular, subcutaneous,
intralesional, or even intraperitoneal routes.
The pharmaceutical forms suitable for local administration of a composition
of the present invention include sterile aqueous solutions or dispersions;
formulations including sesame oil, peanut oil or aqueous propylene glycol;
and sterile powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy syringeability exists. It must be stable under
the conditions of manufacture and storage and must be preserved against the
contaminating action of microorganisms, such as bacteria and fungi.
Solutions of the active compounds as free base or pharmacologically
acceptable salts can be prepared in water suitably mixed with a surfactant,
such as hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
A composition of the present invention can be formulated into a composition
in a neutral or salt form. Pharmaceutically acceptable salts, include the
acid addition salts (formed with the free amino groups of the protein) and
which are formed with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic, tartaric,
mandelic, and the like. Salts formed with the free carboxyl groups can also
be derived from inorganic bases such as, for example, sodium, potassium,
ammonium, calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the like.
The carrier can also be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, an
liquid polyethylene glycol, and the like), suitable mixtures thereof, and
vegetable oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol, sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or sodium
chloride. Prolonged absorption of the injectable compositions can be brought
about by the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active
compounds in the required amount in the appropriate solvent with various of
the other ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the
various sterilized active ingredients into a sterile vehicle which contains
the basic dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation are
vacuum-drying and freeze-drying techniques which yield a powder of the
active ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
In terms of using peptide therapeutics as active ingredients, the technology
of U.S. Pat. Nos. 4,601,903; 4,559,231; 4,559,230; 4,596,792; and 4,578,770,
each incorporated herein by reference, may be used.
The preparation of more, or highly, concentrated solutions for direct
administration is also contemplated, where the use of DMSO as solvent is
envisioned to result in extremely rapid penetration, delivering high
concentrations of the active agents to a small tumor area.
The composition of the present invention may be included in kits. Kits may
comprise compositions comprising various components made using the present
invention such as for example, cells, expression vectors, virus stocks,
proteins, antibodies, catheters, coated stents, and drugs. These components
may be in a form appropriate for the intended application. Generally, this
will entail preparing compositions that are essentially free of pyrogens, as
well as other impurities that could be harmful to humans or animals. Such
kits may include a nucleic acid encoding a CKI serine/threonine mutant
and/or a trandominant hKIS mutant.
Claim 1 of 17 Claims
1. A method of inhibiting proliferation
of a mammalian cell at a site comprising contacting the mammalian cell
with a nucleic acid encoding a mutant p27 protein having a mutation at a
serine/threonine phosphorylation site at amino acid number ten that
prevents or reduces phosphorylation by a serine/threonine kinase, wherein
the nucleic acid is delivered locally to the site by a medical device.
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