Note: Descriptions are shown in the official language in which they were submitted.
CA 02341705 2001-02-26
WO 00/12525 PCT/US99120394
SEQUENCES CHARACTERISTIC OF
HYPOXIA-REGULATED GENE TRANSCRIPTION
BACKGROUND OF THE INVENTION
TECHNICAL FIELD
The present invention relates to the
identification of genes that are differentially expressed
in hypoxia and use of the genes and gene products for
diagnosis and therapeutic intervention. The invention
further relates to identification of polynucleotide
sequences that are differentially expressed in hypoxia and
the use of the sequences for diagnosis and probes.
BACKGROUND ART
The level of tissue oxygenation plays an
important role in normal development as well as in
pathologic processes such as ischemia. Tissue oxygenation
plays a significant regulatory/inducer role in both
apoptosis and in angiogenesis (Bouck et al, 1996; Bunn et
al, 1996; Dor et al, 1997; Carmeliet et al, 1998).
Apoptosis (see Duke et al, 1996 for review) and growth
arrest occur when cell growth and viability are reduced
due to oxygen deprivation (hypoxia). Angiogenesis (i.e.
blood vessel growth, vascularization) is stimulated when
hypo-oxygenated cells secrete factors which stimulate
proliferation and migration of endothelial cells in an
attempt to restore oxygen homeostasis (for review see
Hanahan et al, 1996).
Hypoxia plays a critical role in the selection
of mutations that contribute to more severe tumorogenic
phenotypes (Graeber et al., 1996). Identifying activated
or inactivated genes and gene products in hypoxia and
ischemia is needed.
- 1 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
Ischemic disease pathologies involve a decrease
in the blood supply to a bodily organ, tissue or body part
generally caused by constriction or obstruction of the
blood vessels, as for example retinopathy, myocardial
infarction and stroke. Therefore, apoptosis and/or
angiogenesis as induced by the ischemic condition are also
involved in these disease states. Neoangiogenesis is seen
in some forms of retinopathy and in tumor growth. These
processes are complex cascades of events controlled by
many different genes reacting to the various stresses such
as hypoxia.
The ability to monitor hypoxia-triggered
activation of genes can provide a tool to identify not
immediately evident ischemia in a patient. Identification
of hypoxia-regulated genes permits the utilization of gene
therapy or direct use of gene products, or alternatively
inactivation of target genes for therapeutic intervention
in treating the diseases and pathologies associated with
hypoxia, ischemia and tumor growth.
SUI~ARY OF THE INVENTION
The present invention provides purified,
isolated and cloned polynucleotides (nucleic acid
sequences) associated with hypoxia-regulated activity and
having sequences designated as any one of SEQ ID NOS. 1-9,
or having complementary or allelic variation sequences
thereto.
The present invention provides a method of
regulating angiogenesis or apoptosis in a patient in need
of such treatment by administering to such patient a
therapeutically effective amount of an antagonist of at
least one protein as encoded by the nucleic acid sequences
as set forth in any of SEQ ID NOS. 1-9.
- 2 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
Also provided is the diagnostic tool for
identifying genes modulated by hypoxic conditions having a
detector for detecting the presence of a polynucleotide
having a nucleic acid sequence according to any of SEQ. ID
NOS. 1-9. A pharmaceutical composition for modulating
hypoxia and ischemia having an effective amount of a
polynucleotide having the nucleic acid sequence according
to SEQ. ID NOS. 1-9 and a pharmaceutically acceptable
carrier is also provided.
Also provided is a method of regulating hypoxia-
associated pathologies by administering an effective
amount of at least one antisense oligonucleotide against
one of the nucleic acid sequences (SEQ. ID NOS. 1-9) or
their proteins. There is provided a method of regulating
hypoxia associated pathology by administering an effective
amount of a protein encoded by the polynucleotides (SEQ.
ID NOS. 1-9) as active ingredients in the pharmaceutically
acceptable carrier.
Further, there are provided hypoxia response
regulating genes.
BRIEF DESCRIPTION OF THE FIGURES
Other advantages of the present invention will
be readily appreciated as the same becomes better
understood by reference to the following detailed
description when considered in connection with the
accompanying drawings wherein:
Figure 1 shows the nucleic acid sequence of the 92
gene (Seq. I.D. No. 1);
Figure 2 shows the nucleic acid sequence of the 95
gene (Seq. I.D. No. 2);
- 3 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
Figure 3 shows the nucleic acid sequence of the 98
gene (Seq. I.D. No. 3);
Figure 4 shows the nucleic acid sequence of the 60F6
gene (Seq. I.D. No. 4);
Figures 5 A-C show the nucleic acid sequence of the
648 gene (Seq. I.D. No. 5);
Figure 6 shows the nucleic acid sequence of the 24D4
gene (Seq. I.D. No. 6);
Figures 7 A and B show the nucleic acid sequence of
the 77H4 gene (Seq. I.D. No. 7);
Figure 8 shows the nucleic acid sequence of the 1462
gene (Seq. I.D. No. 8); and
Figure 9 shows the nucleic acid sequence of the 29F3
gene (Seq. I.D. No. 9).
DESCRIPTION OF THE INVENTION
The present invention identifies polynucleotides
(nucleic acid sequences) with sequences as set forth
herein in SEQ. ID Nos. 1-9, that can be utilized
diagnostically in hypoxia and ischemia and that can be
used as targets for therapeutic intervention, or can be
used to identify genes that are regulated and respond to
hypoxic conditions, SEQ ID Nos. 1-4 and 6-8 have not
previously been identified. SEQ ID No. 5 was found to
match sequences in data banks but has not been reported to
be associated with hypoxia regulation.
- 4 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
The present invention further provides candidate
genes and gene products that can be utilized
therapeutically and diagnostically in hypoxia and ischemia
and that can regulate apoptosis or angiogenesis.
By regulate or modulate or control, it is meant
that the process is either induced or inhibited to the
degree necessary to effect a change in the process and the
associated disease state in the patient. Whether induction
or inhibition is being contemplated will be apparent from
the process and disease being treated and will be known to
those skilled in the medical arts.
The present invention identifies genes for gene
therapy, diagnostics and therapeutics that have direct
causal relationships between a disease and its related
pathologies and up- or down-regulator (responder) genes.
That is, the present invention is initiated by a
physiological relationship between cause and effect.
The present invention also provides a method of
regulating angiogenesis or apoptosis in a patient in need
of such treatment by administering to such patient a
therapeutically effective amount of an antagonist of at
least one protein as encoded by the nucleic acid sequences
as set forth in any of SEQ ID NOS. 1-9.
As shown below, some sequences are partial gene
sequences which are markers/probes for genes that are
upregulated under hypoxic conditions. These partial
sequences can be designated "Expressed Sequence Tags"
(ESTs) and are markers for the genes actually expressed in
vivo and are ascertained as described herein in the
Examples or as is known in the art.
Generally, ESTs comprise DNA sequences
corresponding to a portion of nuclear encoded mRNA. The
EST has a length that allows for PCR (polymerase chain
reaction), for use as a hybridization probe and is a
- 5 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
unique designation for the gene with which it hybridizes
(generally under conditions sufficiently stringent to
require at least 95% base pairing). For a detailed
description and review of ESTs and their functional
utility see, WO 93/00353 PCT Application which is
incorporated herein in its entirety by reference, as well
as the references by Zweiger et al, 1997; Okubo et al,
1997 and Braren et al, 1997.
The WO 93/00353 PCT application further
describes how the EST sequences can be used to identify
the transcribed genes.
The present invention also provides a method of
diagnosing the presence of ischemia or other hypoxia-
associated pathologies in a patient including the steps of
analyzing a tissue sample from the patient for the
presence of at least one expressed gene (up-regulated)
identified by the sequences of the present invention
utilized as probes. Methods of identification of
hybridization can include immunohistochemical staining of
the tissue samples. Further, for identification of the
gene, in situ hybridization, Southern blotting, single
strand conformational polymorphism, restriction
endonuclease fingerprinting (REF), PCR amplification and
DNA-chip analysis using the nucleic acid sequences of the
present invention as probes/primers can be used.
Further, according to the present invention,
purified, isolated and cloned hypoxia-responding genes
identified by the probes/sequences hybridizing under
stringent conditions with 95% homology set forth herein or
a complementary or allelic variation sequence and human
homologies as needed thereto are disclosed. The present
invention further provides proteins as encoded by the
identified genes. The present invention further provides
antibodies directed against these proteins. The present
- 6 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
invention further provides transgenic animals and cell
lines carrying at least one expressible gene identified by
the present invention. The present invention further
provides knock-out eucaryotic organisms in which at least
one nucleic acid sequences as .identified by the probes of
the present invention.
The present invention provides a method of
regulating angiogenesis, apoptosis or other hypoxia-
associated pathologies in a patient in need of such
treatment by administering to a patient a therapeutically
effective amount of an antagonist of at least one protein
as encoded by the nucleic acid sequences or sequences
identified herein or by the probes of the present
invention, or alternatively by a non-protein product of
the gene's activity, or inactivation of a gene by chemical
compound. Alternatively, the present invention provides a
method of regulating angiogenesis, apoptosis or other
hypoxia-associated pathologies in a patient in need of
such treatment by administering to a patient a
therapeutically effective amount of at least one antisense
oligonucleotide against the nucleic acid sequences or
dominant negative peptide directed against the sequences
or their proteins.
The present invention further provides a method
of regulating angiogenesis or apoptosis in a patient in
need of such treatment by administering to a patient a
therapeutically effective amount of a protein encoded by
the identified genes as active ingredients in a
pharmaceutically acceptable carrier.
The present invention provides a method of
providing an apoptosis-regulating gene, an angiogenesis-
regulating gene or hypoxia-response regulating gene
identified by the probes of the present invention, by
administering directly to a patient in need of such
therapy an expressible vector comprising expression
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
control sequences operably linked to one of the identified
genes and its human homolog if appropriate.
The proteins of the present invention can be
produced recombinantly (see generally Marshak et al, 1996
"Strategies for Protein Purification and Characterization.
A laboratory course manual." CSHL Press) and analogues
can be due to post-translational processing. The term
Analogue as used herein is defined as a nucleic acid
sequence or protein which has some differences in their
amino acid/nucleotide sequences as compared to the native
sequence of SEQ. ID NOS. 1-9. Ordinarily, the analogue
will be generally at least 70% homologous over any portion
that is functionally relevant. In more preferred
embodiments the homology will be at least 80% and can
approach 95% homology to the protein/nucleotide sequence.
The amino acid or nucleotide sequence of an
analogue can differ from that of the primary sequence when
at least one residue is deleted, inserted or substituted,
but the protein or nucleic acid molecule remains
functional. Differences in glycosylation can provide
protein analogues.
Functionally relevant refers to the biological
property of the molecule and in this context means an in
vivo effector or antigenic fmnetinn nr ~..+-~...;~.. ~L_~ _ _
directly or indirectly performed by a naturally occurring
protein or nucleic acid molecule. Effector functions
include but are not limited to include receptor binding,
any enzymatic activity or enzyme modulatory activity, any
carrier binding activity, any hormonal activity, any
activity in promoting or inhibiting adhesion of cells to
extracellular matrix or cell surface molecules, or any
structural role as well as having the nucleic acid
sequence encode functional protein and be expressible. The
antigenic functions essentially mean the possession of an
_ g _
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
epitope or antigenic site that is capable of cross-
reacting with antibodies raised against a naturally
occurring protein. Biologically active analogues share an
effector function of the native which can, but need not,
in addition possess an antigenic function.
The antibodies can be either monoclonal,
polyclonal or recombinant and be used in immunoassays.
Conveniently, the antibodies can be prepared against the
immunogen or portion thereof for example a synthetic
peptide based on the sequence, or prepared recombinantly
by cloning techniques or the natural gene product and/or
portions thereof can be isolated and used as the
immunogen. Immunogens can be used to produce antibodies
by standard antibody production technology well known to
those skilled in the art as described generally in Harlow
and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY, 1988 and
Borrebaeck, Antibody Engineering - A Practical Guide, W.H.
Freeman and Co., 1992. Antibody fragments can also be
prepared from the antibodies and include Fab, F(ab')2, and
Fv by methods known to those skilled in the art.
For producing polyclonal antibodies a host, such
as a rabbit or goat, is immunized with the immunogen or
immunogen fragment, generally with an adjuvant and, if
necessary, coupled to a carrier; antibodies to the
immunogen are collected from the sera. Further, the
polyclonal antibody can be absorbed such that it is
monospecific. That is, the sera can be absorbed against
related immunogens so that no cross-reactive antibodies
remain in the sera rendering it monospecific.
For producing monoclonal antibodies the
technique involves hyperimmunization of an appropriate
donor with the immunogen, generally a mouse, and isolation
of splenic antibody producing cells. These cells are
_ g _
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
fused to a cell having immortality, such as a myloma cell,
to provide a fused cell hybrid which has immortality and
secretes the required antibody. The cells are then
cultured, in bulk, and the monoclonal antibodies harvested
from the culture media fox use.
For producing recombinant antibody (see
generally Huston et al, 1991; Johnson and Bird, 1991;
Mernaugh and Mernaugh, 1995), messenger RNAs from antibody
producing B-lymphocytes of animals, or hybridoma are
reverse-transcribed to obtain complimentary DNAs (cDNAs).
Antibody cDNA, which can be full or partial length, is
amplified and cloned into a phage or a plasmid. The cDNA
can be a partial length of heavy and light chain cDNA,
separated or connected by a linker. The antibody, or
antibody fragment, is expressed using a suitable
expression system to obtain recombinant antibody. Antibody
cDNA can also be obtained by screening pertinent
expression libraries.
The antibody can be bound to a solid support
substrate or conjugated with a detectable moiety or be
both bound and conjugated, as is well known in the art.
(For a general discussion of conjugation of fluorescent or
enzymatic moieties see Johnstone & Thorpe, Immunochemistry
in Practice, Blackwell Scientific Publications, Oxford,
1982.) The binding of antibodies to a solid support
substrate is also well known in the art. (See for a
general discussion Harlow & Lane Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Publications, New
York, 1988 and Borrebaeck, Antibody Engineering - A
Practical Guide, W.H. Freeman and Co., 1992). The
detectable moieties contemplated with the present
invention can include, but are not limited to,
fluorescent, metallic, enzymatic and radioactive markers
such as biotin, gold, ferritin, alkaline phosphatase,
- 10 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
galactosidase, peroxidase, urease, fluoresce in, rh.~dam~_ne,
tritium, '°C and iodination.
These transgenics and knock-cauts of the pre::ent
invention are constructed using standard methods knowr_ in
the art and as set forth in United St~~tES Patents
5,487,992, 5,464,764, 5,387,742, 5,360,735, 5,347,075,
5,298,422, 5,288,846, 5,221,778, 5,7.75,885, 5,175,384,
5,175,383, 4,736,866 as well as Burke and Olson (1991),
Capecchi (1989), Davies et al. (1992), Dickinson et. al.
(1993), Duff and Lincoln (1995), Huxley et al. (1991),
Jakobovits et al. (1993), Lamb et al. (19~~3), Pearson and
Choi (1993) , Rothstein (1991) , SchE:dl et a7.. (1993) ,
Strauss et al. (1993). Further, patent appJ_icatior~s WO
94/23049, WO 93/14200, WO 94!06908, WO 94/?8123 also
provide information.
More specifically, any tecnniquss known in the
art can be used to introduce the rrar_sgene expressibly
into animals to produce the parental lines of animals.
Such techniques include, but are not limited to,
pronuclear microinjection (U. S. patent 4,8%3,191);
retrovirus mediated gene transfer into germ lines (Van der
Putten et al., 1985); gene targeting in embryonic stem
cells (Thompson et al., 1989; Mansaur, 1990 and U.S.
patent 5,614,396); electroporation of embryos (Lo, 1983);
and sperm-mediated gene transfer (Lavi.trano et al . , 1989) .
For a review of such techniques see Gordon (1989).
Further, one parent strain instead of carrying a
direct human transgene can have the homologous endogenous
gene modified by gene targeting such that it approximates
the transgene. That is, the endogenous gene has been
"humanized" and/or mutated (Reaume et al, 1996). It
should be noted that if the animal and human sequence are
essentially homologous a "humanized" gene is not required.
The transgenic parent can also carry an over expressed
sequence, either the non-mutant or a mutant sequence and
- 11 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
humanized or not as required. The term transgene is
therefore used to refer to all these possibilities.
Additionally, cells can be isolated from the
offspring which carry a transgene from each transgenic
parent and that are used to establish primary cell
cultures or cell lines as is known in the art.
Where appropriate, a parent strain will be
homozygous for the transgene. Additionally, where
appropriate, the endogenous non--transgene in the genome
that is homologous to the transgene will be non-
expressive. By non-expressive is meant that the
endogenous gene will not be expressed and that this non-
expression is heritable in the offspring. For example,
the endogenous homologous gene could be "knocked-out" by
methods known in the art. Alternatively, the parental
strain that receives one of the transgenes could carry a
mutation at the endogenous homologous gene rendering it
non-expressed.
The antagonist/regulating agent/active
ingredient is dosed and delivered in a pharmaceutically
acceptable carrier as described herein below. The term
antagonist or antagonizing is used in its broadest sense.
Antagonism can include any mechanism or treatment which
results in inhibition, inactivation, blocking or reduction
in gene activity or gene product. It should be noted that
the inhibition of a gene or gene product can provide for
an increase in a corresponding function that the gene or
gene product was regulating. The antagonizing step can
include blocking cellular receptors for the gene products
and can include antisense treatment as discussed herein
below. For example, a patient can be in need of inducing
apoptosis in tumorogenic cells or angiogenesis in trauma
situations where for example a limb must be reattached or
in a transplant where revascularization is needed.
Many reviews have covered the main aspects of
- 12 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
antisense (AS) technology and its enormous therapeutic
potential (Wright and Anazodo, 1995). There are reviews
on the chemical (Crooke, 1995; Uhlmann et al, 1990),
cellular (Wagner, 1994) and therapeutic (Hanania, et al,
1995; Scanlon, et a1, 1995; Gewirtz, 1993) aspects of this
rapidly developing technology. Within a relatively short
time, ample information has accumulated about the in vitro
use of AS nucleotide sequences in cultured primary cells
and cell lines as well as for in vivo administration of
such nucleotide sequences for suppressing specific
processes and changing body functions in a transient
manner. Further, enough experience is now available in
vitro and in vivo in animal models and human clinical
trials to predict human efficacy.
Antisense intervention in the expression of
specific genes can be achieved by the use of synthetic AS
oligonucleotide sequences (for recent reports see
Lefebvre-d'Hellencourt et a1, 1995; Agrawal, 1996; Lev-
Lehman et a1, 1997). AS oligonucleotide sequences can be
short sequences of DNA, typically 15-30 mer but can be as
small as 7 mer (Wagner et al, 1996), designed to
complement a target mRNA of interest and form an RNA: AS
duplex. This duplex formation can prevent processing,
splicing, transport or translation of the relevant mRNA.
Moreover, certain AS nucleotide sequences can elicit
cellular RNase H activity when hybridized with their
target mRNA, resulting in mRNA degradation (Calabretta et
al, 1996). In that case, RNase H will cleave the RNA
component of the duplex and can potentially release the AS
to further hybridize with additional molecules of the
target RNA. An additional mode of action results from the
interaction of AS with genomic DNA to form a triple helix
which can be transcriptionally inactive.
- 13 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
The sequence target segment for the antisense
oligonucleotide is selected such that the sequence
exhibits suitable energy related characteristics important
for oligonucleotide duplex formation with their
complementary templates, and shows a low potential for
self-dimerization or self-complementation [Anazodo et al.,
1996]. For example, the computer program OLIGO (Primer
Analysis Software, Version 3.4), can be used to determine
antisense sequence melting temperature, free energy
properties, and to estimate potential self-dimer formation
and self-complimentary properties. The program allows the
determination of a qualitative estimation of these two
parameters (potential self-dimer formation and self-
complimentary) and provides an indication of "no
potential" or "some potential" or "essentially complete
potential". Using this program target segments are
generally selected that have estimates of no potential in
these parameters. However, segments can be used that have
"some potential" in one of the categories. A balance of
the parameters is used in the selection as is known in the
art. Further, the oligonucleotides are also selected as
needed so that analogue substitution do not substantially
affect function.
Phosphorothioate antisense oligonucleotides do
not normally show significant toxicity at concentrations
that are effective and exhibit sufficient pharmacodynamic
half-lives in animals (Agarwal et al., 1996) and are
nuclease resistant. Antisense induced loss-of-function
phenotypes related with cellular development were shown
for the glial fibrillary acidic protein (GFAP), for the
establishment of tectal plate formation in chick (Galileo
et al., 1991) and for the N-myc protein, responsible for
the maintenance of cellular heterogeneity in
neuroectodermal cultures (epithelial vs. neuroblastic
- 14 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
cells, which differ in their colony forming abilities,
tumorogenicity and adherence) {Rosalen et al., 1990;
Whitesell et a1, 1991). Antisense oligonucleotide
inhibition of basic fibroblast growth factor (bFgF),
having mitogenic and angiogenic properties, suppressed 80%
of growth in glioma cells (Morrison, 1991) in a saturable
and specific manner. Being hydrophobic, antisense
oligonucleotides interact well with phospholipid membranes
(Akhter et al., 1991). Following their interaction with
the cellular plasma membrane, they are actively (or
passively) transported into living cells (Loke et al.,
1989), in a saturable mechanism predicted to involve
specific receptors (Yakubov et al., 1989).
Instead of an antisense sequence as discussed
herein above, ribozymes can be utilized. This is
particularly necessary in cases where antisense therapy is
limited by stoichiometric considerations (Sarver et al.,
1990, Gene Regulation and Aids, pp. 305-325). Ribozymes
can then be used that will target the same sequence.
Ribozymes are RNA molecules that possess RNA catalytic
ability (see Cech for review) that cleave a specific site
in a target RNA. The number of RNA molecules that are
cleaved by a ribozyme is greater than the number predicted
by stochiochemistry. (Hampel and Tritz, 1989; Uhlenbeck,
1987) .
Ribozymes catalyze the phosphodiester bond
cleavage of RNA. Several ribozyme structural families
have been identified including Group I introns, RNase P,
the hepatitis delta virus ribozyme, hammerhead ribozymes
and the hairpin ribozyme originally derived from the
negative strand of the tobacco ringspot virus satellite
RNA (sTRSV) (Sullivan, 1994; U.S. Patent No. 5,225,347,
columns 4-5). The latter two families are derived from
viroids and virusoids, in which the ribozyme is believed
- 15 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
to separate monomers from oligomers created during rolling
circle replication (Symons, 1989 and 1992). Hammerhead
and hairpin ribozyme motifs are most commonly adapted for
trans-cleavage of mRNAs for gene therapy (Sullivan, 1994).
The ribozyme type utilized in the present invention is
selected as is known in the art. Hairpin ribozymes are
now in clinical trial and are the preferred type. In
general the ribozyme is from 30-100 nucleotides in length.
Modifications or analogues of nucleotides can be
introduced to improve the therapeutic properties of the
nucleotides. Improved properties include increased
nuclease resistance and/or increased ability to permeate
cell membranes.
Nuclease resistance, where needed, is provided
by any method known in the art that does not interfere
with biological activity of the antisense oligodeoxy
nucleotides, cDNA and/or ribozymes as needed for the
method of use and delivery (Iyer et al., 1990; Eckstein,
1985; Spitzer and Eckstein, 1988; Woolf et al., 1990; Shaw
et al., 1991). Modifications that can be made to
oligonucleotides in order to enhance nuclease resistance
include modifying the phosphorous or oxygen heteroatom in
the phosphate backbone. These include preparing methyl
phosphonates, phosphorothioates, phosphorodithioates and
morpholino oligomers. In one embodiment it is provided by
having phosphorothioate bonds linking between the four to
six 3'-terminus nucleotide bases. Alternatively,
phosphorothioate bonds link all the nucleotide bases.
Other modifications known in the art can be used where the
biological activity is retained, but the stability to
nucleases is substantially increased.
The present invention also includes all
analogues of, or modifications to, an oligonucleotide~of
the invention that does not substantially affect the
function of the oligonucleotide. The nucleotides can be
- 16 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
selected from naturally occurring or synthetic modified
bases. Naturally occurring bases include adenine,
guanine, cytosine, thymine and uracil. Modified bases of
the oligonucleotides include xanthine, hypoxanthine, 2-
aminoadenine, 6-methyl, 2-propyl and other alkyl adenines,
5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza
thymine, pseudo uracil, 4-thiuracil, 8-halo adenine, 8-
aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-
hydroxyl adenine and other 8-substituted adenines, 8-halo
guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl
guanines, 8-hydroxyl guanine and other substituted
guanines, other aza and deaza adenines, other aza and
deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro
cytosine.
In addition, analogues of nucleotides can be
prepared wherein the structure of the nucleotide is
fundamentally altered and that are better suited as
therapeutic or experimental reagents. An example of a
nucleotide analogue is a peptide nucleic acid (PNA)
wherein the deoxyribose (or ribose) phosphate backbone in
DNA (or RNAO is replaced with a polyamide backbone which
is similar to that found in peptides. PNA analogues have
been shown to be resistant to degradation by enzymes and
to have extended lives in vivo and in vitro. Further,
PNAs have been shown to bind stronger to a complementary
DNA sequence than a DNA molecule. This observation is
attributed to the lack of charge repulsion between the PNA
strand and the DNA strand. Other modifications that can be
made to oligonucleotides include polymer backbones, cyclic
backbones, or acyclic backbones.
The active ingredients of the pharmaceutical
composition can include oligonucleotides that are nuclease
resistant needed for the practice of the invention or a
fragment thereof shown to have the same effect targeted
- 17 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
against the appropriate sequences) and/or ribozymes.
Combinations of active ingredients as disclosed in the
present invention can be used including combinations of
antisense sequences.
The antisense oligonucleotides (and/or
ribozymes) and cDNA of the present invention can be
synthesized by any method known in the art for ribonucleic
or deoxyribonucleic nucleotides. For example, an Applied
Biosystems 380B DNA synthesizer can be used. When
fragments are used, two or more such sequences can be
synthesized and linked together for use in the present
invention.
The nucleotide sequences of the present
invention can be delivered either directly or with viral
or non-viral vectors. When delivered directly the
sequences are generally rendered nuclease resistant.
Alternatively, the sequences can be incorporated into
expression cassettes or constructs such that the sequence
is expressed in the cell as discussed herein below.
Generally, the construct contains the proper regulatory
sequence or promoter to allow the sequence to be expressed
in the targeted cell.
Negative dominant peptide refers to a partial
cDNA sequence that encodes for a part of a protein, i.e. a
peptide (see Herskowitz, 1987). This peptide can have a
different function from the protein it was derived from.
It can interact with the full protein and inhibit its
activity or it can interact with other proteins and
inhibit their activity in response to the full protein.
Negative dominant means that the peptide is able to
overcome the natural proteins and fully inhibit their
activity to give the cell a different characteristics like
resistance or sensitization to killing. For therapeutic
intervention either the peptide itself is delivered as the
active ingredient of a pharmaceutical composition or the
- 18 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
cDNA can be delivered to the cell utilizing the same
methods as for antisense delivery.
By gene therapy as used herein refers to the
transfer of genetic material (e. g. DNA or RNA) of interest
into a host to treat or prevent a genetic or acquired
disease or condition phenotype. The genetic material of
interest encodes a product (e. g. a protein, polypeptide,
peptide, functional RNA, antisense) whose production in
vivo is desired. For example, the genetic material of
interest can encode a hormone, receptor, enzyme,
polypeptide or peptide of therapeutic value.
Alternatively, the genetic material of interest encodes a
suicide gene. For a review see, in general, the text
"Gene Therapy" (Advances in Pharmacology 40, Academic
Press, 1997).
Two basic approaches to gene therapy have
evolved: (1) ex vivo and (2) in vivo gene therapy. In ex
vivo gene therapy cells are removed from a patient, and
while being cultured are treated in vitro. Generally, a
functional replacement gene is introduced into the cell
via an appropriate gene delivery vehicle/method
(transfection, transduction, homologous recombination,
etc.) and an expression system as needed and then the
modified cells are expanded in culture and returned to the
host/patient. These genetically reimplanted cells have
been shown to express the transfected genetic material in
si tu.
In in vivo gene therapy, target cells are not
removed from the subject rather the genetic material to be
transferred is introduced into the cells of the recipient
organism in situ, that is within the recipient. In an
alternative embodiment, if the host gene is defective, the
gene is repaired in situ [Culver, 1998]. These
- 19 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
genetically altered cells have been shown to express the
transfected genetic material in situ.
The gene expression vehicle is capable of
delivery/transfer of heterologous nucleic acid into a host
cell. The expression vehicle can include elements to
control targeting, expression and transcription of the
nucleic acid in a cell selective manner as is known in the
art . It should be noted that of ten the 5 ' UTR and/or 3 '
UTR of the gene can be replaced by the 5' UTR and/or 3'
UTR of the expression vehicle. Therefore as used herein
the expression vehicle can, as needed, not include the S'
UTR and/or 3' UTR of the actual gene to be transferred and
only include the specific amino acid coding region.
The expression vehicle can include a promoter
for controlling transcription of the heterologous material
and can be either a constitutive or inducible promoter to
allow selective transcription. Enhancers that can be
required to obtain necessary transcription levels can
optionally be included. Enhancers are generally any non
translated DNA sequence which works contiguously with the
coding sequence (in cis) to change the basal transcription
level dictated by the promoter. The expression vehicle
can also include a selection gene as described herein
below.
Vectors can be introduced into cells or tissues
by any one of a variety of known methods within the art.
Such methods can be found generally described in Sambrook
et al., Molecular Cloning: A Laboratory Manual, Cold
Springs Harbor Laboratory, New York (1989, 1992), in
Ausubel et al., Current Protocols in Molecular Biology,
John Wiley and Sons, Baltimore, Maryland (1989), Chang et
al., Somatic Gene Therapy, CRC Press, Ann Arbor, MI
(1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor,
MI (1995), Vectors: A Survey of Molecular Cloning Vectors
- 20 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
and Their Uses, Butterworths, Boston MA (1988) and Gilboa
et al (1986) and include, for example, stable or transient
transfection, lipofection, electroporation and infection
with recombinant viral vectors. In addition, see United
States patent 4,866,042 for vectors involving the central
nervous system and also United States patents 5,464,764
and 5,487,992 for positive-negative selection methods.
Introduction of nucleic acids by infection
offers several advantages over the other listed methods.
Higher efficiency can be obtained due to their infectious
nature. Moreover, viruses are very specialized and
typically infect and propagate in specific cell types.
Thus, their natural specificity can be used to target the
vectors to specific cell types in vivo or within a tissue
or mixed culture of cells. Viral vectors can also be
modified with specific receptors or ligands to alter
target specificity through receptor mediated events.
A specific example of DNA viral vector for
introducing and expressing recombinant sequences is the
adenovirus derived vector Adenop53TK. This vector
expresses a herpes virus thymidine kinase (TK) gene for
either positive or negative selection and an expression
cassette for desired recombinant sequences. This vector
can be used to infect cells that have an adenovirus
receptor which includes most cancers of epithelial origin
as well as others. This vector as well as others that
exhibit similar desired functions can be used to treat a
mixed population of cells and can include, for example, an
in vitro or ex vivo culture of cells, a tissue or a human
subject.
Additional features can be added to the vector
to ensure its safety and/or enhance its therapeutic
efficacy. Such features include, for example, markers that
can be used to negatively select against cells infected
- 21 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
with the recombinant virus. An example of such a negative
selection marker is the TK gene described above that
confers sensitivity to the antibiotic gancyclovir.
Negative selection is therefore a means by which infection
can be controlled because it provides inducible suicide
through the addition of antibiotic. Such protection
ensures that if, for example, mutations arise that produce
altered forms of the viral vector or recombinant sequence,
cellular transformation will not occur.
Features that limit expression to particular
cell types can also be included. Such features include,
for example, promoter and regulatory elements that are
specific for the desired cell type.
In addition, recombinant viral vectors are
useful for in vivo expression of a desired nucleic acid
because they offer advantages such as lateral infection
and targeting specificity. Lateral infection is inherent
in the life cycle of, for example, retrovirus and is the
process by which a single infected cell produces many
progeny virions that bud off and infect neighboring cells.
The result is that a large area becomes rapidly infected,
most of which was not initially infected by the original
viral particles. This is in contrast to vertical-type of
infection in which the infectious agent spreads only
through daughter progeny. Viral vectors can also be
produced that are unable to spread laterally. This
characteristic can be useful if the desired purpose is to
introduce a specified gene into only a localized number of
targeted cells.
As described above, viruses are very specialized
infectious agents that have evolved, in many cases, to
elude host defense mechanisms. Typically, viruses infect
and propagate in specific cell types. The targeting
specificity of viral vectors utilizes its natural
- 22 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
specificity to specifically target predetermined cell
types and thereby introduce a recombinant gene into the
infected cell. The vector to be used in the methods of
the invention will depend on desired cell type to be
targeted and will be known to those skilled in the art.
For example, if breast cancer is to be treated then a
vector specific for such epithelial cells would be used.
Likewise, if diseases or pathological conditions of the
hematopoietic system are to be treated, then a viral
vector that is specific fox blood cells and their
precursors, preferably for the specific type of
hematopoietic cell, would be used.
Retroviral vectors can be constructed to
function either as infectious particles or to undergo only
a single initial round of infection. In the former case,
the genome of the virus is modified so that it maintains
all the necessary genes, regulatory sequences and
packaging signals to synthesize new viral proteins and
RNA. Once these molecules are synthesized, the host cell
packages the RNA into new viral particles which are
capable of undergoing further rounds of infection. The
vector's genome is also engineered to encode and express
the desired recombinant gene. In the case of non-
infectious viral vectors, the vector genome is usually
mutated to destroy the viral packaging signal that is
required to encapsulate the RNA into viral particles.
Without such a signal, any particles that are formed will
not contain a genome and therefore cannot proceed through
subsequent rounds of infection. The specific type of
vector will depend upon the intended application. The
actual vectors are also known and readily available within
the art or can be constructed by one skilled in the art
using well-known methodology.
The recombinant vector can be administered in
several ways. If viral vectors are used, for example, the
- 23 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
procedure can take advantage of their target specificity
and consequently, do not have to be administered locally
at the diseased site. However, local administration can
provide a quicker and more effective treatment,
administration can also be performed by, for example,
intravenous or subcutaneous injection into the subject.
Injection of the viral vectors into a spinal fluid can
also be used as a mode of administration, especially in
the case of neuro-degenerative diseases. Following
injection, the viral vectors will circulate until they
recognize host cells with the appropriate target
specificity for infection.
DNA can also be administered using a gene gun.
(Ziao & Brancksman, Nuceic Acids, Res. 24, 2630-2622
(1996)). The DNA is precipitated onto the surface of
microscopic metal beads. The microprojectiles are
accelerated with a shock wave or expanding helium gas, and
penetrate tissues to a depth of several cell layers. For
example, the AcalT"" Gene Delivery Device manufactured by
Aegacetus, Inc., Middleton, WI, is suitable.
Alternatively, nucleic DNA can pass through skin into the
bloodstream simply by spotting the DNA onto skin with
chemical or mechanical irritation (see WO 95/05853).
An alternate mode of administration can be by
direct inoculation locally at the site of the disease or
pathological condition or by inoculation into the vascular
system supplying the site with nutrients or into the
spinal fluid. Local administration is advantageous
because there is no dilution effect and, therefore, a
smaller dose is required to achieve expression in a
majority of the targeted cells. Additionally, local
inoculation can alleviate the targeting requirement
required with other forms of administration since a vector
can be used that infects all cells in the inoculated area.
- 24 -
CA 02341705 2001-02-26
WO 00/12525 PCTNS99/20394
If expression is desired in only a specific subset of
cells within the inoculated area, then promoter and
regulatory elements that are specific for the desired
subset can be used to accomplish this goal. Such non-
targeting vectors can be, for example, viral vectors,
viral genome, plasmids, phagernids and the like.
Transfection vehicles such as liposomes can also be used
to introduce the non-viral vectors described above into
recipient cells within the inoculated area. Such
transfection vehicles are known by one skilled within the
art.
The pharmaceutical compositions containing the
active ingredients of the present invention as described
herein above are administered and dosed in accordance with
good medical practice, taking into account the clinical
condition of the individual patient, the site and method
of administration, scheduling of administration, patient
age, sex, body weight and other factors known to medical
practitioners. The pharmaceutically "effective amount"
for purposes herein is thus determined by such
considerations as are known in the medical arts. The
amount must be effective to achieve improvement including
but not limited to improved survival rate or more rapid
recovery, or improvement or elimination of symptoms and
other indicators as are selected as appropriate measures
by those skilled in the medical arts. The pharmaceutical
compositions can be combinations of the active ingredients
but will include at least one active ingredient.
In the method of the present invention, the
pharmaceutical compositions of the present invention can
be administered in various ways taking into account the
nature of compounds in the pharmaceutical compositions. It
should be noted that they can be administered as the
compound or as pharmaceutically acceptable salt and can be
administered alone or as an active ingredient in
- 25 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99120394
combination with pharmaceutically acceptable carriers,
diluents, adjuvants and vehicles. The compounds can be
administered orally, subcutaneously or parenterally
including intravenous, intra-arterial, intramuscular,
intra-peritoneally, and intra-nasal administration as well
as intra-thecal and infusion techniques. Implants of the
compounds are also useful. The patient being treated is a
warm-blooded animal and, in particular, mammals including
man. The pharmaceutically acceptable carriers, diluents,
adjuvants and vehicles as well as implant carriers
generally refer to inert, non-toxic solid or liquid
fillers, diluents or encapsulating material not reacting
with the active ingredients of the invention.
It is noted that humans are treated generally
longer than the mice or other experimental animals
exemplified herein which treatment has a length
proportional to the length of the disease process and drug
effectiveness. The doses can be single doses or multiple
doses over a period of several days, but single doses are
preferred.
The doses can be single doses or multiple doses
over a period of several days. The treatment generally
has a length proportional to the length of the disease
process and drug effectiveness and the patient species
being treated.
When administering the compound of the present
invention parenterally, it will generally be formulated in
a unit dosage injectable form (solution, suspension,
emulsion). The pharmaceutical formulations suitable for
injection include sterile aqueous solutions or dispersions
and sterile powders for reconstitution into sterile
injectable solutions or dispersions. The carrier can be a
solvent or dispersing medium containing, for example,
water, ethanol, polyol (for example, glycerol, propylene
glycol, liquid polyethylene glycol, and the like),
- 26 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
suitable mixtures thereof, and vegetable oils.
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. Non-aqueous
vehicles such a cottonseed oil, sesame oil, olive oil,
soybean oil, corn oil, sunflower oil, or peanut oil and
esters, such as isopropyl myristate, can also be used as
solvent systems for compound compositions. Additionally,
various additives which enhance the stability, sterility,
and isotonicity of the compositions, including
antimicrobial preservatives, antioxidants, chelating
agents, and buffers, can be added. Prevention of the
action of microorganisms can be ensured by various
antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, sorbic acid, and the
like. In many cases, it will be desirable to include
isotonic agents, for example, sugars, sodium chloride, and
the like. Prolonged absorption of the injectable
pharmaceutical form can be brought about by the use of
agents delaying absorption, for example, aluminum
monostearate and gelatin. According to the present
invention, however, any vehicle, diluent, or additive used
would have to be compatible with the compounds.
Sterile injectable solutions can be prepared by
incorporating the compounds utilized in practicing the
present invention in the required amount of the
appropriate solvent with various of the other ingredients,
as desired.
A pharmacological formulation of the present
invention can be administered to the patient in an
injectable formulation containing any compatible carrier,
such as various vehicle, adjuvants, additives, and
diluents; or the compounds utilized in the present
invention can be administered parenterally to the patient
- 27 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
in the form of slow-release subcutaneous implants or
targeted delivery systems such as monoclonal antibodies,
vectored delivery, iontophoretic, polymer matrices,
liposomes, and microspheres. Examples of delivery systems
useful in the present invention include: 5,225,182;
5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603;
4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196.
Many other such implants, delivery systems, and modules
are well-known to those skilled in the art.
A pharmacological formulation of the compound
utilized in the present invention can be administered
orally to the patient. Conventional methods such as
administering the compounds in tablets, suspensions,
solutions, emulsions, capsules, powders, syrups and the
like are usable. Known techniques which deliver it orally
or intravenously and retain the biological activity are
preferred.
In one embodiment, the compound of the present
invention can be administered initially by intravenous
injection to bring blood levels to a suitable level. The
patient's levels are then maintained by an oral dosage
form, although other forms of administration, dependent
upon the patient's condition and as indicated above, can
be used. The quantity to be administered will vary for
the patient being treated and will vary from about 100
ng/kg of body weight to 100 mg/kg of body weight per day
and preferably will be from 10 ~g/kg to 10 mg/kg per day.
The present invention also provides a method of
diagnosing the presence of ischemia in a patient including
the steps of analyzing a bodily fluid or tissue sample
from the patient for the presence or gene product of at
least one expressed gene (up-regulated) or their proteins
and where ischemia is determined if the up-regulated gene
or gene product is ascertained as described herein in the
- 28 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
Example. The bodily fluids can include tears, serum,
urine, sweat or other bodily fluid where secreted proteins
from the tissue that is undergoing an ischemic event can
be localized. Additional methods for identification of the
gene or gene product are immunoassays, such as and ELISA
or radioimmunoassays (RIA), can be used as are known to
those in the art particularly to identify gene products in
the samples. Immunohistochemical staining of tissue
samples is also utilized for identification. Available
immunoassays are extensively described in the patent and
scientific literature. See, for example, United States
patents 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,8.67,517; 3,879,262; 3,901,654; 3,935,074;
3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;
5,011,771 and 5,281,521. Further fox identification of
the gene, in situ hybridization, Southern blotting, single
strand conformational polymorphism, restriction
endonuclease fingerprinting (REF), PCR amplification and
DNA-chip analysis using nucleic acid sequence of the
present invention as primers can be used.
The above discussion provides a factual basis
for the use of the sequences of the present invention to
identify hypoxia-regulated genes and provide diagnostic
probes to identify ischemia. The methods used with and
the utility of the present invention can be shown by the
non-limiting examples herein.
GENERAL METHODS
Most of the techniques used in molecular biology
are widely practiced in the art, and most practitioners
are familiar with the standard resource materials which
describe specific conditions and procedures. However, for
convenience, the following paragraphs can serve as a
- 29 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
guideline.
General methods in molecular biology: Standard
molecular biology techniques known in the art and not
specifically described were generally followed as in
Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Springs Harbor Laboratory, New York (1989), and in
Ausubel et al., Current Protocols in Molecular Biology,
John Wiley and Sons, Baltimore, Maryland (1989)
particularly for the Northern Analysis and In Situ
analysis and in Perbal, A Practical Guide to Molecular
Cloning, John Wiley & Sons, New York (1988), and in Watson
et al., Recombinant DNA, Scientific American Books, New
York. Polymerase chain reaction (PCR) was carried out
generally as in PCR Protocols: A Guide To Methods And
Applications, Academic Press, San Diego, CA (1990).
Reactions and manipulations involving other
nucleic acid techniques, unless stated otherwise, were
performed as generally described in Sambrook et al., 1989,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, and methodology as set forth in United
States patents 4,666,828; 4,683,202; 4,801,531; 5,192,659
and 5,272,057 and incorporated herein by reference.
Additionally, in situ (In cell) PCR in
combination with flow cytometry can be used for detection
of cells containing specific DNA and mRNA sequences
(Testoni et al, 1996, Blood 87:3822).
General methods in immunology: Standard methods
in immunology known in the art and not specifically
described are generally followed as in Stites et al.(eds),
Basic and Clinical Immunology (8th Edition), Appleton &
Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds),
Selected Methods in Cellular Immunology, W.H. Freeman and
Co., New York (1980). Available immunoassays are
- 30 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
extensively described in the patent and scientific
literature. See, for example, United States patents
3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;
3,867,517; 3,879.,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5, 281, 521 as well as Sambrook et al, Molecular Cloning: A
Laboratory Manual, Cold Springs Harbor, New York, 1989.
GENERAL METHODS OF THE INVENTION
The general methods of the invention are
generally as described in US patent application serial
number USSN 09/309,862 of same applicant which is by
reference incorporated herein in its entirety.
In brief, USSN 09/309,862 provides methods for
identifying genes regulated at the RNA level by cue-
induced gene expression. It relates to the rapid isolation
of differentially expressed or developmentally regulated
gene sequences through analysis of mRNAs obtained from
specific cellular compartments and comparing the changes
in the relative abundance of the mRNA in these
compartments as a result of applying a cue to the tested
biological samples. The cellular compartments include
polysomal and nonpolysomal fractions, nuclear fractions,
cytoplasmic fractions and splicesomal fractions. The
method includes the steps of exposing cells or tissue to a
cue or stimulus such as mechanical, chemical, toxic,
pharmaceutical or other stress, hormones, physiological
disorders or disease; fractionating the cells into
compartments such as polysomes, nuclei, cytoplasm and
splicesomes; extracting the mRNA from these fractions, and
subjecting the mRNA to differential analysis using
accepted methodologies, such as gene expression array
- 31 -
CA 02341705 2001-02-26
WO 00/12525 PCTNS99/20394
( GEM ) .
The method is designed for identifying and
cloning genes which are either up- or down-regulated
responsive to a specific pathology, stress, physiological
condition, and so on, and in general, to any factor that
can influence cells or organisms to alter their gene
expression.
Further in USSN 09/309,862, an example is
provided which shows the use of RNA isolation from nuclei
for isolating genes whose steady state levels show only
minor changes, but which show high differential expression
when detected by nuclear RNA probe. Most such genes are
regulated at the transcriptional level.
The specific mRNA of the invention is total
cellular mRNA, and regulation is specifically on the
transcriptional level.
DNA Microarrays
Preparation of custom hypoxia-specific microarrays
The cell system consisted of the C6 or A172
glioma cell lines. The cells were exposed to hypoxia for
4 or 16 hours and compared to cells grown under normal
conditions.
Subtracted libraries were made from the
following sample:
1. 16 hours hypoxia vs. normal (genes induced by
hypoxia after 16 hours).
2. Normal vs. 16 hours hypoxia (genes reduced by
hypoxia) .
3. 4 hours hypoxia vs. normal (genes induced by
hypoxia after four hours).
Three enriched libraries from the three groups
above were made by the SSH method using the "PCR select
- 32 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
cDNA subtraction kit" from Clontech. From library 1,
1, 000 colonies were grown and the plasmids prepared in 96
well format. From libraries 2 and 3, 500 colonies were
processed from each. Thus, a total of 2,000 individual
plasmids were prepared and used for the fabrication of a
Gene Expression Microarray (GEM). For this, the inserts
of each plasmid were amplified by PCR and robotically
fabricated on the glass.
Preparation of probes for microarrav hybridization
Isolated messenger RNA is labeled with
fluorescent dNTP's using a reverse transcription reaction
to generate a labeled cDNA probe. mRNA is extracted from
either C6 or A172 cells cultured in normoxia conditions
and labeled with Cy3-dCTP (Amersham) and mRNA extracted
from C6 or A172 cells cultured under hypoxia conditions is
labeled with Cy5-dCTP (Amersham). The two labeled cDNA
probes are then mixed and hybridized onto microarrays
(Schena et al, 1996). Following hybridization the
rnicroarray is scanned using a laser scanner and the amount
of fluorescence of each of the fluorescence dyes is
measured for each cDNA clone on the micro-array giving an
indication of the level of mRNA in each of the original
mRNA populations being tested. Comparison of the
fluorescence on each cDNA clone on the micro-array between
the two different fluorescent dyes is a measure for the
differential expression of the indicated genes between the
two experimental conditions.
The following probes were made from C6 and A172
for screening the GEM:
1. Normoxia (Cy3 labeled ) + 16 hours hypoxia (Cy5
labeled).
- 33 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
2. Normoxia (Cy3 labeled) + 4 hours hypoxia (Cy5
labeled) .
The following cDNA sequences were found to be
induced under hypoxia conditions. The sequences are
divided into three categories: 1. new genes; 2. known
genes not known before this publication to be hypoxia-
inducible; and 3. known genes known to be induced under
hypoxia conditions.
In Situ Analysis:
In situ analysis is performed for the candidate
genes identified by the differential response to exposure
to hypoxia conditions as described above. The expression
is studied in normal tissues and in pathological models as
described herein.
Utilizing microarray hybridization the sequences
set forth herein were identified and cloned as being
differentially expressed under hypoxia conditions (see
also Hraren et al, 1997).
In parallel experiments Northern Analysis
results and results obtained by the gene expression
microarray analysis where found to coincide and either can
be used to determine hypoxia-regulated response. As well
in other experiments, the results from in situ analysis
showed a high degree of correlation with the Northern
Analysis and microarray analysis.
The sequences are listed that were found, the
sequences are identified by clone number. In some cases
either end of the clone has been sequenced for use or the
entire clone sequence and protein sequence are provided.
Unigeml (Syntheni) was utilized for screening of
human glioma cell line A172 to identify genes whose
expression is modified by hypoxia.
- 34 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
A Retinopathv Model:
Three major biological processes occur in
nervous tissues under hypoxic conditions:
1. apoptotic death of hypoxia-damaged cells;
2. ancriogenesis induced by factors secreted by hypoxia-
suffering cells (a feedback control of oxygen
concentration in tissue); and
3. secretion of neurotrophic and neuroprotective
factors.
Therefore, it was assumed that among novel genes
transcriptionally regulated by hypoxia in C6 and A172
glioma cells, there are those with pro- and antiapoptotic
function as well as secreted neurotrophic, neuroprotective
and angiogenic factors. It is worth noting, that
regulation of apoptosis and angiogenesis is closely linked
to cancerogenesis.
As initial step of biological characterization,
candidate genes were tested for their ability to
induce/protect cells from apoptosis, for neurotrophic
activity and for angiogenic/antiangiogenic activity.
The sequences of the invention, the methods used
therewith and the utility of the present invention can be
shown by the following non-limiting examples:
EXAMPLE l: 92 (SE ID. No ~ 1L
Northern Blot Analysis:
Gene 92 is found upregulated after 16 hours of
hypoxia. On Northern blots, it appears as a single 5 Kb
transcript.
- 35 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
Cloning:
Several partial human cDNA clones corresponding
to gene 92 were isolated from human A172 cDNA library. The
length of available contig is 2212 by and it contains an
ORF potentially coding for a 437 amino acids (265-1576 bp)
protein. The putative initiating ATG codon is preceded by
in frame stop codon.
Bioinformatic Analysis:
Similarity search with 92 cDNA sequence against
the public databases have shown 60 % similarity to unknown
Drosophila DNA sequence (AC004283) and mainly encompasses
the 3' UTR and a part of the coding sequence. The search
against the protein public databases gave partial
similarity to hypothetical C. elegans protein (1703624)
(77% similarity and 46% Identity).
The 92 cDNA sequence contains a region of 55
nucleotides (336-390 bp) that is constituted of CGG
repeats. On the level of amino acids it appears as a
GGD/SFGG repeated unit (aa 24-44). Two of the isolated
cDNA clones contain a 30 nucleotides in frame deletion
within this region, indicating that the amount of repeats
can be variable. 44 of these nucleotides form a strong
stem and loop secondary structure. When 92 cDNA was in
vitro translated, the obtained protein had much smaller
size than expected (30 kD instead of 45 kD). This, means
that the stem and loop structure formed right downstream
to the putative initiation codon prevents the proper
progression of ribosome and the initiation actually starts
from the next in frame ATG located at position 820 - 822.
EXAMPLE 2: 95 (SEO ID No 2)
- 36 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
Northern Blot Analysis:
Expression of this gene is upregulated by
hypoxia. In normoxic conditions it appears as a single
moderately expressed 3,9 Kb transcript. After four hours
of hypoxia, an additional transcript of 4.3 Kb becomes
evident. After 16 hours of hypoxia, an increase in the
amount of the major 3.9 Kb transcript takes place and
there appears the second additional transcript 4.5 Kb in
size.
Clonincx
A complete human 95 cDNA clone, corresponding to
the major 3.9 Kb mRNA was isolated. The contig is 3,535 by
long and it potentially codes for a 480 as (ORF: nt 323-
1763) protein. All the attempts to clone the gene 95
splice variants had thus far failed, although numerous
different approaches were employed.
Bioinformatic And Literature Analysis:
Search against public databases revealed that
gene 95 is similar to but not identical with a recently
cloned human P53-responsive gene PA26 that codes for a
nuclear protein (Oncogene, 18, 127 - 137, 1999). Like gene
95, the PA26 one is expressed in three splice variants.
The major transcript is 2.6 Kb in size (T2) and is
ubiquitously expressed, the second transcript is ~4 Kb
(T1) and displays a certain tissue specificity of
expression pattern. The third transcript is approximately
2.3 Kb (T3) and is very minor. Expression of 2.6 and 2.3
Kb transcripts is p53-regulated and is increased in
response to irradiation, W, doxorubicin etc. The 4 Kb
transcript is not induced by p53. The PA26-specific
transcripts share a set of C-terminal common exons, while
the first exon is individual for each splice form. Gene 95
- 37 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
encoded putative protein shares the maximal amino acid
homology with the common region of PA26-specific proteins.
In its N-terminus, the putative protein 95 mostly
resembles the T2 (major p53-inducible variant) variant of
PA26.
PA26, maps to chromosome 6q21. Biologically it
shares properties common to the GADD family of Growth
Arrest and DNA Damage-inducible stress-response genes.
Domain Analysis:
A short sequence with features of second
paroxysmal targeting signal was found in protein 95
between amino acids 353-361. A putative coiled coil
region is found between amino acid positions 253-283.
Further Analysis Of Gene ~5 Ext~ression Pattern:
By analogy with gene PA26, it was assumed that
alternative splicing in gene 95 also occurs in the first
exons. To identify the mRNA species where the cDNA clone
was derived from, two 95-specific probes were synthesized:
(1) corresponding to the region homologous to the first
exon of PA26 transcript T2 (Probe 1), and (2)
corresponding to the region homologous to a common set of
PA26 exons (but encompassing the sequences that are mostly
diverged between the two genes) (Probe 2). On Northern
blots, both probes recognized the same major 3.9 Kb 95-
specific transcript that is up-regulated by hypoxia. No
additional transcripts were identified by Probe 2. The 95-
specific probe that was used for the initial set of
hybridizations did not recognize any additional
transcripts when hybridized to this newly prepared
Northern blot. Therefore, the occurrence of 95-specific
additional mRNA species seem to vary among different mRNA
preparations.
- 38 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
The sizes of PA26 and gene 95 encoded
transcripts are different. However, in order to exclude
the possibility that the presence of alternative 95 RNA
species stem from cross-hybridization, a PA26 specific
cDNA probe was synthesized originating from the common
exons region. On the Northern blot used for the previous
experiment, the PA26 hybridized to two typical mRNA
species of 2.6 and 4 Kb. Their levels, unlike the level of
95 mRNA, were not affected by hypoxia.
There is contradictory data in the literature
about the nature of p53 protein present in A172 cells:
according to some sources, it is wild type, according to
others, mutated. The experiments performed thus far,
conclude that hypoxia-induced expression of gene 95 is
p53-independent.
The mammalian 95 expression vector was prepared
and effects of this gene overexpression in mammalian cells
were studied.
Summary:
95 is a novel hypoxia-induced human gene coding
for a protein similar to p53-induced GADD protein PA26.
However, the latter is inert to hypoxia stimulation.
Therefore, there appears to exist two closely related
proteins encoded by different genes that are upregulated
by different genotoxic stresses. Protein 95 contributes
to hypoxia-induced growth regulation in a way similar to
PA26, but in a p53 independent manner. It is known that
hypoxia-induced growth arrest that can take place in the
core regions of p53-negative tumors is one of the
obstacles for success of full chemotherapy. Therefore,
inactivation of gene 95 can be a potent adjuvant for
chemotherapeutic treatment of cancers.
- 39 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
EXAMPLE 3: 98 (SEQ ID No 3)
Northern Blot Analysis:
Expression of gene 98 is strongly up-regulated
by hypoxia already after four hours of exposure. On
Northern blots, it appears as a single mRNA species of
4.4. Kb.
Clonina:
A full-length 98 cDNA was cloned. It is 4138 by
long and contains an single ORF encompassing the
nucleotides 204 - 1445. The putative protein is 414 amino
acid a long.
Bioinformatic Analysis:
Search of the public databases revealed that 98
encoded protein is similar to two other human proteins:
(1) a putative protein encoded by anonymous human 24945
mRNA sequence (AF131826) and (2) VDUP1 (protein induced in
HL-60 cells by dihydroxy vitamin D3 treatment) (573591).
No significant structural features were found by existing
protein analysis tools within the 98 putative protein.
It was previously demonstrated that treatment
with vitamin D3 can induce apoptosis in C6 rat glioma
cells (J. Neurosci Res,46, 540 - 550). Therefore, the
relationship between the vitamin D-induced cell killing
and 98 gene expression and function in glioma cells was
studied.
The mammalian 98 expression vector was then and
its effects studied.
EXAMPLE 4: 60F6 (SE ID No 4)
- 40 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
Northern Blot Analysis:
Expression of this gene is moderately up-
regulated after 16 hours of hypoxia. On Northern blot, it
appears as a single 3.0 Kb species.
Clonincr
A complete 60F6 human cDNA clone was isolated
from A172 cDNA library. The contig is 2675 by long and
contains a single ORF (bp 134 - 866) able to code for a
putative protein of 244 amino acids.
Bioinformatic Analysis:
A similarity search against the public databases
revealed that the N-terminal half of 60F6 sequence exactly
corresponds to a human cDNA coding for RhoE/RhoB small
GTP-binding protein (P52199, HSRH08GRN). The identity of
gene 60F6 was not determined before, because the small
sequenced fragment that was initially possessed,
originated from the Rho8 long 3' UTR. All the sequence
information available in public databases did not include
the long 3' UTR of Rho8. Structurally, Rho8 belongs to a
family of Ras-related GTPases that regulate the actin
cytoskeleton. However, this protein is unique in that it
is constitutively active: GTPase deficient and in vivo
farnesylated (Mol Cell Biol. 1996 Jun; 16(6): 2689-99).
Therefore, it is intriguing to find that this
constitutively active G-protein is regulated on the level
of transcription. Hypoxia regulation of Rho8 was not
previously described.
EXAMPLE 5: 648 (lysvl hvdroxylase 2) (SE ID No ~ 5)
Northern analysis
Probe 648 has detected a single 3.8 Kb
- 41 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
transcript on Northern blots. Expression was induced in C6
glioma cells already after 9 hours of hypoxia,
Cloning
After extension of initial cDNA probe by RACE it
became evident that identified rat sequence is able to
code for protein that represents a rat homologue of human
lysyl hydroxylase 2 (PLOD2). The full- length open
reading frames was cloned for both human and rat lysyl
hydroxylase 2 homologues (by PCR, using primers built on
the basis of known sequence, for human variant, and
degenerative primers, for rat variant). The encoded
proteins have well defined signal peptides.
Bioinformatics data
The cloned rat 648 cDNA contains an ORF coding
for a putative protein that is 88~ identical to the
published human PLOD2 sequences. The least conserved
sequences are within the signal peptide, however its
functional features are completely preserved. The cloned
human cDNA is almost identical to published human PLOD2
sequence. The word "almost" in the previous sentence stems
from the fact that both in human and in rat cDNA species
cloned in QBI a stretch of amino acids between positions
501- 521 of published sequence PLOD2 sequence was absent.
Therefore, QBI's PLOD2 variants are differentially
spliced. Both rat and human homologues were amplified
from RNA extracted from glioma cell lines cultured in
hypoxic conditions.
Literature review
Lysyl hydroxylases are the enzymes that catalyze
the formation of hydroxylysine in collagens and other
proteins with collagen-like amino-acid sequences, by the
hydroxylation of lysine residue in X-K-G sequences. The
- 42 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
hydroxilysine residues have two important functions: (1)
serve as sites of attachment of carbohydrate units, and
(2) they are essential for the stability of the
intermolecular collagen crosslinks. Congenital deficiency
of lysyl hydroxylase in humans leads to increased
solubility of collagens and, consequently, to numerous
defects in organization of connective tissue in various
organs. There are three known isoforms of lysyl
hydroxilase, encoded by different genes. In humans, PLOD2
was found to be highly expressed in pancreas, skeletal
muscle, heart and placenta (by Northern blot). Nothing is
known either about the regulation of PLOD2 expression by
hypoxia or about its involvement in angiogenesis and
tumorigenesis. Induction of PLOD2 by hypoxia can probably
account for hypoxia-induced tissue fibrosis. Indeed,
specific lysyl hydroxylase inhibitor, minoxidil, was able
to suppress both cellular collagen production and
fibroblasts proliferation (J. Biol. Chem., 262, 11973 -8,
1987; Graefes Arch. Clin. Exp. Ophthalmol. 233, 347 -55,
1995). There were suggestions in literature to use
modified lysyl hydroxylase inhibitor for treatment of
vitreoretinopathy (Invest. Ophthalmol. Vis .Sci. 34, 567-
75, 1993).
Analysis of alternatively s lived versions of gene 64_8
In order to establish whether the observed
alternative splicing of PLOD2 is regulated by hypoxia, a
set of PCR primers were synthesized that flank the
alternatively spliced region. The expected sizes of RT-PCR
products are: 216 bp, for published sequence and 156 bp,
for the present sequence. Semi-quantitative RT-PCR was
performed on RNA template extracted from human glioma A172
cells culture in either normoxia or in hypoxia for 4 and
16 hours. The obtained results clearly demonstrate that
both PLOD2 forms are hypoxia regulated, but the form of
- 43 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
the invention appears only in hypoxic conditions.
Testing potential pro- and antia o totic activity in
transient transfection assays
pcDNA3-648 was transiently co-transfected
together with pcDNA3-GFP in Hela and 293 cells. 24 and 48
hours later the cells were fixed and stained with DAPI. No
apoptotic effect was observed in the transfected cells. In
order to evaluate the anti-apoptotic properties of the 648
protein, a co-transfection assay was conducted using the
pcDNA3-GFP and the FAS plasmids. No anti-apoptotic effect
was observed.
Obtaining stable cell clones overexpressing 648 cDNA
C6 were stably transfected with 5 ug of the
pcDNA3-648 plasmid. Following 6418 selection the level of
expression was measured using Northern blot analysis in
comparison to its level in C6 cells after 16 hours under
hypoxic conditions. Out of 18 independent clones from the
pcDNA3-648 transfection, no one was positive.
In situ hybridization analysis
Retinopathy model
Probe 648 demonstrates clear hybridization signal
throughout the inner nuclear layer of "hypoxic" pup's
retina while "normoxic" retina is negative for the
expression. No hybridization signal was detected in adult
retina.
In mouse embryo sections hybridization signal was
detected in some apoptotic cells in the roof of the fourth
brain ventricle and in developing retina ganglia, where
expressing cells had no apoptotic features.
Multi-tissue block hybridization shows expression
of 648 gene (rat PLOD2) in visceral smooth muscles in
oviduct, uterus, stomach and intestine. Vascular smooth
- 44 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
muscles do not display hybridization signal.
Most prominent cell type hybridizing to 648
probe in ovary are granulosa cells of larger secondary
follicles. No hybridization signal is detected in
granulosa cells of primary and small secondary follicles.
Significantly, hybridization signal is weakened in
postovulatory follicles and completely disappears in
corpora lutea. This shows that expression in granulosa
cells is established at later stages of follicular
maturation and it is abruptly down-regulated upon
ovulation and the onset of conversion into lutein cells.
On the other hand, follicular involution is not
accompanied by the changes in 648 expression since strong
hybridization signal is preserved in granulosa cells of
atretic follicles.
Weak hybridization signal can be seen in some
stromal cells surrounding large secondary follicles and
corpora lutea as well as in cells of theca internal of
secondary follicles. Prominent signal is found in
"interstitial glands". This shows distinct regulation of
648 expression in theca cells undergoing "luteinization"
in different locations: it is down regulated in corpora
lutea but preserved or even upregulated in interstitial
glands.
As to the germinal cells, an oocyte that
expresses 648 was found only in one primary follicle while
many other primary and secondary follicles had no
hybridization signal. This shows a transient expression of
648 in oocytes at some stage of their development.
Discrepancy in the hybridization patterns of
human (published) and rat PLOD2 (648) genes is explained by
different sensitivities of different detection methods
(Northern blot vs. in situ hybridization). The rat probe
used in the present invention does not span an
alternatively spliced region.
- 45 -
CA 02341705 2001-02-26
WO 00/12525 PCf/US99/20394
EXAMPLE 6: 24D4 (SE . ID. No : 6)
Northern blot analysis
Expression of gene 29D4 is down-regulated after
16 hours of hypoxia. On Northern blots, it appears as a
single 1.5 Kb mRNA species.
Cloning
A partial 24D4 human cDNA clone was isolated
from A172 cDNA library. The available sequence is 1486 by
long and contains an N-terminal truncated ORF (bp 1-397).
Bioinformatic analysis
The sequence has no analogs in public databases.
The available protein sequence contains three consequent
Zn-finger motifs, all of C2H2 type (aa 52-72, 80-100 and
108-128). Zinc finger domains of this type are usually
found in nucleic acid-binding proteins.
EXAMPLE 7: 77H4 ~,SEQ ID No. s 7~,
Northern blot analysis
Expression of gene 77H4 is up-regulated after 16
hours of hypoxia. On Northern blots, it appears as a
single mRNA species 0.6 -0.7 Kb in size.
Cloning
Several EST cDNA clones from public databases,
corresponding to clone 77H4, were sequenced. All clones
possess a poly A tail and a polyadenylation signal at
their 3' end.
- 46 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
Bioinformatic analysis
EST clone 18E contains 580 bp, that include an
ORF for a 120 as protein (bp 92 - 452 ) . The EST clone 3D
contains 486 bp. Comparison of nucleotide sequences of
clone 18E and 3D reveal that the latter has a out-of-frame
deletion of 71 nucleotides between by 279-339 of clone 18E
(its putative coding region)(Fig.68). This raised the
question whether 77H4 cDNA is at all coding. The sequence
was analyzed by Genscan program, that predicts the
potential coding sequences on the basis of codon usage.
The only potentially coding segment was found in clone 18E
between by 93 and 235. An exhaustive search was performed
of public databases for all 77H4-related sequences.
Several independent contigs were identified in TIGR THC
database. All of them are not completely identical to one
another and contain nucleotide deletions of various
length. This shows a certain variability in 77H4
nucleotide sequence.
Recently, a novel steroid receptor
transcriptional coactivator, SRA, was found to be present
as an RNA molecule in the transcription activating complex
SRC-1 (Cell, 97, 17 - 27, 1999) . Although no similarity
was found between clone 77H4 and SRA RNA on the sequence
level, several characteristic features seem to be shared
by both sequences:
~1 both mRNAs, 77H4 and SRA, are approximately of the same
size - 0.7 Kb;
~2 sequencing multiple cDNA clones corresponding to either
mRNA revealed extensive variability in certain regions;
~3 hybridization signals of both mRNA, therefore, appear as
fuzzy bands on Northern blots;
~4 both mRNA do not exhibit characteristics of protein.
Therefore, the 77H4 cDNA clone has similar to
- 47 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
SRA function and can serve a coactivator in some
transcriptional complexes.
EXAMPLE 8: 1462 (SE ID No.: 8)
Northern blot analysis
Expression of gene 1462 is regulated within 16
hours of hypoxia. On Northern blots, it appears as a
single mRNA species.
Cloning
A partial 1462 human cDNA clone was isolated.
The available sequence was then characterized and cloned
as shown in Figure 8.
EXAMPLE 9: 29F3 (SE ID No.~ 9)
Northern blot analysis
Expression of gene 29F3 is regulated within 16
hours of hypoxia. On Northern blots, it appears as a
single mRNA species.
Cloning
A partial 29F3 human cDNA clone was isolated.
The available sequence was then characterized and cloned
as shown in Figure 9.
Throughout this application, various
publications, including United States patents, are
referenced by author and year and patents by number. Full
citations for the publications are listed below. The
disclosures of these publications and patents in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of
the art to which this invention pertains.
- 48 -
CA 02341705 2001-02-26
WO 00/12525 PCTNS99/20394
The invention has been described in an
illustrative manner, and it is to be understood that the
terminology which has been used is intended to be in the
nature of words of description rather than of limitation.
Obviously, many modifications and variations of
the present invention are possible in light of the above
teachings. It is, therefore, to be understood that within
the scope of the described invention, the invention can be
practiced otherwise than as specifically described.
- 49 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
REFERENCES
Alon, et al. (1995). Vascular endothelial growth factor
acts as a survival factor for newly formed retinal vessels
and has implications for retinopathy of prematurity. Nat
Med. 1(10):1024-1028.
Benjamin, et al. (1997). Conditional switching of vascular
endothelial growth factor (VEGF) expression in tumors:
induction of endothelial cell shedding and regression of
hemangioblastoma-like vessels by VEGF withdrawal. Proc
Natl Acad Sci USA. 94(16):8761-8766.
Bouck, et al. (1996). How tumors become angiogenic. Adv.
Cancer Res. 69:135-174.
Braren et al. (1997) . Use of the EST database resource to
identify and clone novel mono(ADP-ribosyl)transferase gene
family members. Adv. Exp. Med. Bio. 419:163-168.
Bunn, et al. (1996). Oxygen sensing and molecular
adaptation in hypoxia. Physiol Rev. 76:839-885.
Carmeliet, et al. (1998). Role of HIF-lalpha in hypoxia-
mediated apoptosis, cell proliferation and tumor
angiogenesis. Nature. 394(6692):485-490.
Dor, et al. (1997). Ischemia-driven angiogenesis. Trends
Cardiovasc. Med. 7:289-294.
Duke, et al. (1996). Cell Suicide in Health and Disease.
Scientific American. 80-87.
Fyodorov, et al. (1998). Pet-l, a novel ETS domain factor
that can activate neuronal nAchR gene transcription. J
Neurobiol. 34(2):151-163.
Gallagher et al., (1997). Identification of p53 Genetic
Suppresser Elements Which Confer Resistance to Cisplatin.
Oncogene 14:185-193.
Hanahan, et al. (1996). Patterns and Emerging Mechanisms of
Angiogenic Switch During Tumorigenesis. Cell. 86:353-364.
Obeso, et al. (1990). A Hemangioendothelioma-Derived Cell.
Line: Its Use as a Model for the Study of Endothelial Cell
Biology. Laboratory Investigation. 83:259-264.
- 50 -
CA 02341705 2001-02-26
WO 00/12525 PCTlUS99/20394
Okubo et al. (1997). Complementary DNA sequence (EST)
collections and the expression information of the human
genome. FEBS Lett 403:225-229.
Schena et al., (1996) Parallel Human Genome Analysis:
Microarray-based Expression Monitoring of 1000 genes. PNAS
(USA) 93 (20) :10614-10619.
Spence, et al. (1998). Glucose metabolism in human
malignant gliomas measured quantitatively with PET, 1-[C-
ll]glucose and FDG: analysis of the FDG lumped constant. J
Nucl Med. 39(3):440-448.
Watanabe, et al. (1996). Tumor cell autocrine motility
factor is the neuroleukin/phosphohexose isomerase
polypeptide. Cancer Res. 56(13):2960-2963.
Zweiger et al. (1997) From Expressed Sequence Tags to
"epigenomics": An Understanding of Disease Processes. Curr
Opin Biotechnol 8(6):684-687.
Burke and Olson, "Preparation of Clone Libraries in Yeast
Artificial-Chromosome Vectors" in Methods in Enzymoloctv,
Vol. 194, "Guide to Yeast Genetics and Molecular Biology",
eds. C. Guthrie and G. Fink, Academic Press, Inc., Chap.
17, pp. 251-270 (1991).
Capecchi, "Altering the genome by homologous recombination"
Science 244:1288-1292 (1989).
Davies et al., "Targeted alterations in yeast artificial
chromosomes for inter-species gene transfer", Nucleic Acids
Research, Vol. 20, No. 11, pp. 2693-2698 (1992).
Dickinson et al., "High frequency gene targeting using
insertional vectors", Human Molecular Genetics, Vol. 2, No.
8, pp. 1299-1302 (1993).
Duff and Lincoln, "Insertion of a pathogenic mutation into
a yeast artificial chromosome containing the human APP gene
and expression in ES cells", Research Advances in
Alzheimer's Disease and Related Disorders, 1995.
Huxley et al., "The human HPRT gene on a yeast artificial
chromosome is functional when transferred to mouse cells by
cell fusion", Genomics, 9:742-750 (1991).
- 51 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
Jakobovits et al., "Germ-line transmission and expression
of a human-derived yeast artificial chromosome", Nature,
Vol. 362, pp. 255-261 (1993).
Lamb et al., "Introduction and expression of the 400
kilobase precursor amyloid protein gene in transgenic
mice", Nature Genetics, Vol. 5, pp. 22-29 (1993).
Pearson and Choi, Expression of the human ~-amyloid
precursor protein gene from a heast artificial chromosome
in transgenic mice. Proc. Natl. Scad. Sci. USA, 1993.
90:10578-82.
Rothstein, "Targeting, disruption, replacement, and allele
rescue: integrative DNA transformation in yeast" in Methods
in Enzymolocrv, Vol. 194, "Guide to Yeast Genetics and
Molecular Biology", eds. C. Guthrie and G. Fink, Academic
Press, Inc., Chap. 19, pp. 281-301 (1991).
Schedl et al., "A yeast artificial chromosome covering the
tyrosinase gene confers copy number-dependent expression in
transgenic mice", Nature, Vol. 362, pp. 258-261 (1993).
Strauss et al., "Germ line transmission of a yeast
artificial chromosome spanning the murine al (I) collagen
locus", Science, Vol. 259, pp. 1904-1907 (1993).
Gilboa, E, Eglitis, MA, Kantoff, PW, Anderson, WF: Transfer
and expression of cloned genes using retroviral vectors.
BioTechniques 4(6):504-512, 1986.
Cregg JM, Vedvick TS, Raschke WC: Recent Advances in the
Expression of Foreign Genes in Pichia pastoris,
Bio/Technology 11:905-910, 1993
Culver, 1998. Site-Directed recombination for repair of
mutations in the human ADA gene. (Abstract) Antisense DNA &
RNA based therapeutics, February, 1998, Coronado, CA.
Huston et al, 1991 "Protein engineering of single-chain Fv
analogs and fusion proteins" in Methods in Enzymology (JJ
Langone, ed.; Academic Press, New York, NY) 203:46-88.
Johnson and Bird, 1991 "Construction of single-chain Fvb
derivatives of monoclonal antibodies and their production
in Escherichia coli in Methods in Enzymology (JJ Langone,
ed.; Academic Press, New York, NY) 203:88-99.
- 52 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
Mernaugh and Mernaugh, 1995 ~~An overview of phage-displayed
recombinant antibodies' in Molecular Methods In Plant
Pathology (RP Singh and US Singh, eds.; CRC Press Inc.,
Boca Raton, FL) pp. 359-365.
Agrawal, 1996. Antisense oligonucleotides: towards
clinical trials, TIBTECH, 14:376.
Akhter et al, 1991. Interactions of antisense DNA
oligonucleotide analogs with phospholipid membranes
(liposomes). Nuc. Res. 19:5551-5559.
Blaesse, 1997. Gene Therapy for Cancer. Scientific
American 276(6):111-115.
Calabretta, et al, 1996. Antisense strategies in the
treatment of leukemias. Semin. Oncol. 23:78.
Crooke, 1995. Progress in antisense therapeutics, Hematol.
Pathol. 2:59.
Felgner, 1997. Nonviral Strategeies for Gene Therapy.
Scientific American. June, 1997, pgs 102-106.
Gewirtz, 1993. Oligodeoxynucleotide-based therapeutics for
human leukemias, Stem Cells Dayt. 11:96.
Hanania, et al 1995. Recent advances in the application of
gene therapy to human disease. Am. J. Med. 99:537.
Lefebvre-d'Hellencourt et al, 1995. Immunomodulation by
cytokine antisense oligonucleotides. Eur. Cytokine Netw.
6:7.
Lev-Lehman et al., 1997. Antisense Oligomers in vitro and
in vivo. In Antisense Therapeutics, A. Cohen and S.
Smicek, eds (Plenum Press, New York)
Loke et al, 1989. Characterization of oligonucleotide
transport into living cells. PNAS USA 86:3474.
Morrison, 1991. Suppression of basic fibroblast growth
factor expression by antisense oligonucleotides inhibits
the growth of transformed human astrocytes. J. Biol. Chem.
266:728.
Rosolen et al., 1990. Cancer Res. 50:6316.
- 53 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
Uhlmann and Peyman, 1990. Antisense Oligonucleotides: A
New Therapeutic Principle. Chem Rev 90(4):543-584.
Wagner et al., 1996. Potent and selective inhibition of
gene expression by an antisense heptanucleotide. Nature
Biotechnology 14:840-844.
Wagner, 1994. Gene inhibition using antisense
oligodeoxynucleotides. Nature 372:333.
Whitesell et al., 1991. Episome-generated N-myc antisense
RNA restricts the differentiation potential of primitive
neuroectodermal cell lines. Mol. Cell. Biol. 11:1360.'
Yakubov et al, 1989. PNAS USA 86:6454.
Wright & Anazodo, 1995. Antisense Molecules and Their
Potential For The Treatment Of Cancer and AIDS. Cancer J.
8:185-189.
Scanlon et al., 1995. Oligonucleotides-mediated modulation
of mammalian gene expression. FASEB J. 9:1288.
Galileo et al., 1991. J. Cell. Biol., 112:1285.
Gewirtz, 1993. Oligodeoxynucleotide-based therapeutics for
human leukemias, Stem Cells Dayt. 11:96.
Eckstein 1985. Nucleoside Phosphorothioates. Ann. Rev.
Biochem. 54:367-402.
Iyer et al. 1990. J. Org. Chem. 55:4693-4699.
Radhakrishnan et al., 1990. The automated synthesis of
sulfur-containing oligodeoxyribonucleotides using 3H-1,2-
Benzodithiol-3-One 1,1 Dioxide as a sulfur-transfer
reagent. J. Org. Chem. 55:4693-4699.
Shaw et al., 1991. Modified deoxyoligonucleotides stable to
exonuclease degradation in serum. Nucleic Acids Res.
19:747-750.
Spitzer and Eckstein 1988. Inhibition of deoxynucleases by
phosphorothioate groups in oligodeoxyribonucleotides.
Nucleic Acids Res. 18:11691-11704.
- 54 -
CA 02341705 2001-02-26
WO 00/12525 PCT/US99/20394
Woolf et al., 1990. The stability, toxicity and
effectiveness of unmodified and phosphorothioate antisense
oligodeoxynucleotides in Xenopus oocytes and embryos.
Nucleic Acids Res. 18:1763-1769.
Cell, 97, 17 - 27, 1999
- 55 -
