Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR INHIBITING
HUMAN IMMUNODEFICIENCY VIRUS INFECTION
BY DOWN-REGULATING HUMAN CELLULAR GENES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part application of PCT International
Application No. PCT/US98/11452, filed June 2, 1998 and U.S. patent application
No.
09/087,609 filed May 29, 1998, both of which are continuations-in-part of U.S.
patent
application No. 08/867,314, filed June 2, 1997. Each of the patent
applications referred
to in this section is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
The present invention relates to the identification of certain human genes as
cellular targets for the design of therapeutic agents for suppressing human
immunodeficiency virus (HIV) infection. These genes encode products which are
necessary for HIV infection, because HIV infection is inhibited when
expression of these
genes is down-regulated. Therefore, compounds that inhibit expression of these
genes
or function of the encoded gene products can be used as therapeutic agents for
the
treatment and/or prevention of HIV infection. In addition, the invention
relates to
2 0 methods for identifying additional cellular genes as therapeutic targets
for suppressing
HIV infection, and methods of using such cellular genes and their encoded
products in
screening assays for selecting protective compounds that inhibit HIV
infection.
BACKGROUND OF THE INVENTION
2 5 The primary cause of acquired immunodeficiency syndrome (AIDS) has been
shown to be HIV (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et
al., 1984,
Science 224:500-503). HIV causes immunodeficiency in an individual by
infecting
important cell types of the immune system, which results in their depletion.
This, in
turn, leads to opportunistic infections, neoplastic growth and death.
3 0 HIV is a member of the lentivirus family of retroviruses (Teich et
al.,1984, RNA
TUMOR VIRUSES, Weiss et al., eds., CSH-Press, pp. 949-956). Retroviruses are
small
enveloped viruses that contain a diploid, single-stranded RNA genome, and
replicate via
a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-
dependent DNA polymerase (Varmus,1988, Science 240:1427-1439). There are at
least
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two distinct subtypes ofHIV: HIV-1 (Bane-Sinoussi et al., ibid.; Gallo et al.,
ibid.) and
HIV-2 (Clavel et a1.,1986, Science 233:343-346; Guyader et al., 1987, Nature
326:662-
669). Genetic heterogeneity exists within each of these HIV subtypes.
CD4+ T cells are the major targets of HIV infection because the CD4 cell
surface
protein acts as a cellular receptor for HIV attachment (Dalgleish et al.,
1984, Nature
312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986,
Cell
47:333-348). Viral entry into cells is dependent upon viral protein gp 120
binding to the
cellular CD4 receptor molecule (McDougal et al., 1986, Science 231:382-385;
Maddon
et al., 1986, Cell 47:333-348).
HIV infection is pandemic and HIV-associated diseases have become a world-
wide health problem. Despite considerable efforts in the design of anti-HIV
modalities,
there is, thus far, no successful prophylactic or therapeutic regimen against
AIDS.
However, several stages of the HIV life cycle have been considered as
potential targets
for therapeuticintervention(Mitsuyaetal.,1991,FASEBJ.5:2369-2381). For
example,
virally-encoded reverse transcriptase has been a major focus of drug
development. A
number of reverse-transcriptase-targeted drugs, including 2', 3'-
dideoxynucleotide
analogs such as AZT, ddI, ddC, and ddT have been shown to be active against
HIV
(Mitsuya et al., 1990, Science 249:1533-1544). While beneficial, these
nucleotide
analogs are not curative, probably due to the rapid appearance of drug
resistant HIV
2 0 mutants (Larder et al., 1989, Science 243:1731-1734). In addition, these
drugs often
exhibit toxic side effects, such as bone marrow suppression, vomiting, and
liver
abnormalities.
Another stage of the HIV life cycle that has been targeted is viral entry into
cells,
the earliest stage of HIV infection. This approach has primarily utilized
recombinant
2 5 soluble CD4 protein to inhibit infection of CD4+ T cells by some HIV-1
strains (Smith
et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however,
are
relatively less sensitive to inhibition by recombinant CD4 (Daar et al. ,1990,
Proc. Natl.
Acad. Sci. USA 87:6574-6579). To date, clinical trials of recombinant, soluble
CD4
have produced inconclusive results (Schooley et al., 1990, Ann. Int. Med.
112:247-253;
30 Kahn et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan et al., 1989, Proc.
Vth Int.
Conf. on AIDS, p. 564, MCP 137).
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Additionally, the later stages of HIV replication (which involve crucial virus-
specific processing of certain viral proteins and enzymes) have been targeted
for anti-
HIV drug development. Late-stage processing is dependent on the activity of a
virally-
encoded protease, and drugs including saquinavir, ritonavir, and indinavir
have been
developed to inhibit this protease (Pettit et al., 1993, Persp. Drug Discov.
Design 1:69-
83). With this class of drugs, the emergence of drug resistant HIV mutants is
also a
problem; resistance to one inhibitor often confers cross-resistance to other
protease
inhibitors (Condra et al., 1995, Nature 374:569-571). Also, these drugs often
exhibit
toxic side-effects such as nausea, altered taste, circumoral parethesias, fat
deposits,
diarrhea and nephrolithiasis.
Antiviral therapy of HIV using different combinations, of nucleoside analogs
and
protease inhibitors have recently been shown to be more effective than the use
of a
single drug alone (Torres et al., 1997, Infec. Med. 14:142-160). However,
despite the
ability to achieve significant decreases in viral burden, there is no evidence
to date that
combinations of available drugs will afford a curative treatment for AIDS.
Other potential approaches for developing treatment for AIDS include the
delivery of exogenous genes into infected cells. One such gene therapy
approach
involves the use of genetically-engineered viral vectors to introduce toxic
gene products
to kill HIV-infected cells. Another form of gene therapy is designed to
protect virally-
2 0 infected cells from cytolysis by specifically disrupting viral
replication. Stable
expression of RNA-based (decoys, antisense and ribozymes) or protein-based
(transdominant mutants) HIV-1 antiviral agents can inhibit certain stages of
the viral life
cycle. A number of anti-HIV suppressors have been reported, such as decoy RNA
of
TAR or RRE (Sullenger et al., 1990, Cell 63:601-608; Sullenger et al., 1991,
J. Virol.
65:6811-6816; Lisziewicz et al., 1993, New Biol. 3:82-89; Lee et al., 1994, J.
Virol.
68:8254-8264), ribozymes (Sarver et al., 1990, Science 247:1222-1225;
Wecrasinghe
et al., 1991, J. Virol. 65:5531-5534; Dropulic et al., 1992, J. Virol. 66:1432-
1441;
Ojwang et a1.,1992, Proc. Natl. Acad. Sci. USA 89:10802-10806; Yu et a1.,1993,
Proc.
Natl. Acad. Sci. USA 90:6340-6344; Yu et a1.,1995, Proc. Natl. Acad. Sci. USA
92:699-
3 0 703; Yamada et a1.,1994, Gene Therapy 1:38-45), antisense RNA
complementaryto the
mRNA of viral gag, tat, rev or env genes (Sezakiel et al., 1991, J. Virol.
65:468-472;
Chatterjee et al., 1992, Science 258:1485-1488; Rhodes et al., 1990, J. gen.
Virol.
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71:1965. Rhodes et a1.,1991, AIDS 5:145-151; Sezakiel et al., 1992, J. Virol.
66:5576-
5581; Joshi et al., 1991, J. Virol. 65:5524-5530) and transdominant mutants
including
Rev (Bevec et al., 1992, Proc. Natl. Acad. Sci. USA 89:9870-9874), Tat
(Pearson et al.,
1990, Proc. Natl. Acad. Sci. USA 87:5079-5083; Modesti et a1.,1991, New Biol.
3:759-
768), Gag (Trono et al., 1989, Cell 59:113-120), Env (Bushschacher et al.,
1995, J.
Virol. 69:1344-1348) and protease (Junker et al., 1996, J. Virol. 70:7765-
7772).
Antisense polynucleotides have been designed to complex with and sequester the
HIV-1 transcripts (Holmes et al., WO 93/11230; Lipps et al., WO 94/10302;
Kretschmer
et al., EP 594,881; and Chatterjee et al., 1992, Science 258:1485).
Furthermore, an
enzymatically active RNA, termed ribozyme, has been used to cleave viral
transcripts.
The use of a ribozyme to generate resistance to HIV-1 in a hematopoietic cell
line has
been reported (Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA 89:10802-06;
Yamada
et al., 1994, Gene Therapy 1:38-45; Ho et al., WO 94/26877; and Cech and
Sullenger,
WO 95/13379). In preclinical studies, RevMlO, a transdominant Rev protein, has
been
transfected ex vivo into CD4+ cells of HIV-infected individuals and shown to
confer
survival advantage over cells transfected with vector only (Woffendin et
a1.,1996, Proc.
Natl. Acad. Sci. USA 93:2889-2894).
Despite enormous efforts in the art, reliable, curative anti-HIV therapeutic
agents
and regimens have not been developed.
2 0 In nature, evolution of an intracellular pathogen such as HIV requires the
development of interactions of its genes and gene products with multiple
cellular
components. For instance, the interactions of a virus with a host cell
involves binding
of the virus to a specific cellular receptor(s), translocation through the
cellular
membrane, uncoating, replication of the viral genome, transcription of the
viral genes,
2 5 etc. Each of these events occurs in a cell and involves interactions with
a cellular
component. Thus, the life cycle of a virus can be completed only if the cell
is
"permissive" for viral infection. Availability of amino acids and nucleotides
for
replication of the viral genome and protein synthesis, energy status of the
cell, the
presence of cellular transcription factors and enzymes all contribute to the
propagation
3 0 of the virus in the cell. Consequently, the cellular components, in part,
determine host
cell susceptibility to infection, and can be used as potential targets for the
development
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of new therapeutic interventions. In the case of HIV, one cellular component
which has
been used towards this end is the cell surface molecule for HIV attachment,
CD4.
Recently, it was reported that HIV entry into a susceptible cell requires the
expression of a second type of receptor, the chemokine receptors (CCR2, CCR3,
CCRS
or CXCR4), in addition to CD4 (Moore, 1997, Science 276:51-52). A chemokine
receptor normally binds RANTES, MIP-1 cc and MIP-1 ~3 as its natural ligand.
In the case
of HIV infection, it has been proposed that CD4 first binds to the HIV gp 120
protein on
the cell surface followed by binding of this complex to a chemokine receptor,
resulting
in viral entry into the cells (Cohen, 1997, Science 275:1261). Therefore,
chemokine
receptors can present an additional cellular target for the design of HIV
therapeutic
agents. Inhibitors of HIV/chemokine receptor interactions are being tested as
anti-HIV
agents. However, there remains a need for the discovery of additional cellular
targets
for the design of anti-HIV therapeutics, particularly intracellular targets
for disrupting
viral replication after viral entry into a cell.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods for inhibiting HIV
infection by down-regulating expression of certain human cellular genes and/or
inhibiting the activity of products encoded by such genes. In particular, it
relates to a
2 0 number of human cell-derived nucleic acid molecules which inhibit HIV
infection in
susceptible cells. The isolated nucleic acid molecules correspond to portions
of cellular
genes or complements thereof, and are referred to herein as genetic suppressor
elements
(GSEs). The cellular genes encode intracellular products necessary for
productive HIV
infection. Additionally, small molecule inhibitors of the same cellular genes
and their
2 5 encoded products are also within the scope of the present invention. The
invention also
relates to methods for identifying additional cellular genes as therapeutic
targets for
suppressing HIV infection, and methods for using such cellular genes and their
encoded
products for selecting additional inhibitors of HIV.
The invention is based, in part, on the Applicants' discovery that nucleic
acid
3 0 molecules isolated from human cells can prevent both the activation of
latent HIV-1 in
a CD4+ cell line and productive HIV infection in such cells, and that such
nucleic acid
molecules correspond to fragments of certain human cellular genes. In that
regard, any
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cellular or viral marker associated with HIV infection can be used to select
for such
nucleic acid molecules. An example of such a marker is CD4, which is
conveniently
monitored by using a specific antibody.
Based on substantial sequence identity (90%-100%), a number of the isolated
GSEs correspond to portions of human cellular genes which encode different
subunits
of a mitochondrial enzyme complex, NADH dehydrogenase. In addition, inhibitors
of
this enzyme also inhibit HIV infection in susceptible host cells, including
freshly
isolated human CD4+ T cells. Furthermore, additional GSEs have been selected
which
have substantial sequence identity (90% - 100%) with the following human
cellular
genes: 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase/ cytosolic
thyroid
hormone binding protein, calnexin, ADP-ribosylation factor 3, eukaryotic
initiation
factor 3, protein tyrosine phosphatase, herpesvirus-associated ubiquitin-
specific protease,
eukaryotic initiation factor 4B, CD44, phosphatidyl-inositol 3 kinase,
elongation factor
1 alpha, bone morphogenic protein-1, double-strand break DNA repair gene
protein, rat
guanine nucleotide releasing protein, anti-proliferative factor (BTG-1),
lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase,
eukaryotic
release factor 1, GTP binding protein, importin beta subunit, cell adhesion
molecule Ll,
U-snRNP associated cyclophilin, recepin, Arg/Abl interacting protein
(ArgBP2A),
keratin-related protein, p18 protein, p40 protein, glucosidase II, alpha
enolase,
2 0 macrophage inflammatory protein 1 alpha, tumor protein translationally-
controlled 1
(TCTP1), BBC1, Nef interacting protein, Na+-D-glucose cotransport regulator
gene
protein, hsp90 chaperone protein, FK506-binding protein A1, Rox, beta signal
sequence
receptor, tumorous imaginal disc protein and cell surface heparin binding
protein.
Among the GSEs selected to inhibit HIV infection, several function in the
sense
2 5 orientation, while others function in the antisense orientation. Not
intending to be bound
by any particular theory, the GSEs of the invention are believed to down-
regulate a
cellular gene by different mechanisms. The GSEs are expressed in a host cell
by
encoding RNA molecules that do or do not encode protein products. GSEs in the
sense
orientation can exert their effects as transdominant mutants or RNA decoys.
3 0 Transdominant mutants are expressed proteins or peptides that
competitively inhibit the
normal function of a wild-type protein in a dominant fashion. RNA decoys are
protein
binding sites that titrate out these proteins. GSEs in the antisense
orientation can exert
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their effects as antisense RNA; i. e. nucleic acid molecules complementary to
the mRNA
of the target gene. These nucleic acid molecules bind to mRNA and block the
translation of the mRNA. Some antisense nucleic acid molecules can act
directly at the
DNA level to inhibit transcription. The down-regulation of a cellular gene by
a GSE,
in turn, removes a cellular component necessary for, for example HIV
replication,
resulting in an inhibition of HIV infection.
A wide range of uses are encompassed by the invention including, but not
limited
to, HIV treatment and prevention by transfernng protective GSE compounds as
inhibitory compositions into HIV-susceptible cell types. For example, GSEs can
be
transferred into T cells, particularly CD4+ T cells which are the major cell
population
targeted by HIV. Alternatively, GSEs can be transferred into hematopoietic
stem cells
in vitro followed by their engraftment in an autologous or histocompatible or
even
histoincompatible recipient. In another embodiment, any cells susceptible to
HIV
infection can be directly transduced or transfected with GSEs in vivo. In yet
another
embodiment, inhibitors of NADH dehydrogenase, 2-oxoglutarate dehydrogenase, M2-
type pyruvate kinase/ cytosolic thyroid hormone binding protein, calnexin, ADP-
ribosylation factor 3, eukaryotic initiation factor 3, protein tyrosine
phosphatase,
herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation
factor 4B, CD44,
phosphatidyl-inositol 3 kinase, elongation factor 1 alpha, bone morphogenic
protein-1,
2 0 double-strand break DNA repair gene protein, rat guanine nucleotide
releasing protein,
anti-proliferative factor (BTG-1), lymphocyte-specific protein 1, protein
phosphatase
2A, squalene synthetase, eukaryotic release factor l, GTP binding protein,
importinbeta
subunit, cell adhesion molecule L1, U-snRNP associated cyclophilin, recepin,
Arg/Abl
interacting protein (ArgBP2A), keratin related protein, p 18 protein, p40
protein,
2 5 glucosidase II, alpha enolase, macrophage inflammatory protein 1 alpha,
tumor protein
translationally-controlled 1 (TCTPl), BBC1, Nef interacting protein, Na+-D-
glucose
cotransport regulator gene protein, hsp90 chaperone protein, FK506-binding
protein A1,
Rox, beta signal sequence receptor, tumorous imaginal disc protein and cell
surface
heparin binding protein can be used as inhibitory compositions in vivo to
suppress or
3 0 prevent HIV infection.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the percentage of CD4+ OM10.1 cells that diminish after
TNF-eG induction; TNF-induced cells, -~-; uninduced cells,-1-.
Figure 2 illustrates the percentage of intracellular p24+ CEM-ss cells
containing
the CF-315 sequence (SEQ ID NO:1 ) after infection with HIV-1 S~ at a TCIDSO
of 1000.
CEM-ss cells (106) containing the C-315 construct or control vector DNA
(denoted as
LNGFRM) were harvested on the indicated days post infection, stained with FITC-
conjugated anti-p24 monoclonal antibody and analyzed by flow cytometry. Mock
infected cells, -0-; LNGFRM vector-infected cells, -0-; and C-315 infected
cells, -0-.
Figure 3 illustrates the percentage of intracellular p24+ CEM-ss cells
containing
various GSEs: CF-004 (SEQ ID N0:7), CF-025 (SEQ ID NO:S), CF-113 (SEQ ID
N0:8) and CF-204 (SEQ ID N0:9) after infection with HIV-ls~ at a TCIDso of
1000.
Controls include mock-infected, vector (LNGFRM)-infected and HIV-infected CEM-
ss
cells.
Figure 4 illustrates the percentage of intracellular p24+ CEM-ss cells
containing
CF-001 (SEQ ID NO:10) after infection with HIV-ls~ at a TCIDSO of 1000.
Controls
include mock-infected and vector (LNGFRM)-infected cells.
Figure 5 illustrates the percentage of CD4+ OM10.1 cells after treatment with
amytal following TNF-cx induction. TNF induction, -~-; no TNF induction, -~-.
2 0 Figure 6 illustrates the percentage of CD4+ OM10.1 cells after treatment
with
mofarotene following TNF-a induction. TNF induction, -~-; no TNF induction, -1-
.
Figure 7 illustrates the percentage of intracellular p24+ CEM-ss cells
containing
various GSEs: CF-527 (SEQ ID N0:41), CF-529 (SEQ ID N0:45) and CF-531 (SEQ
ID N0:47) after infection with HIV-ls~ at a TCIDSO of 1000. Controls include
mock-
2 5 infected, vector (LNGFRM)-infected and CEM-ss cells transfected with RevM
10.
Figure 8 illustrates the percentage of intracellular p24+ CEM-ss cells
containing
the GSE CF-579 (SEQ ID N0:61) after infection with HIV-ls~ at a TCIDSO of
1000.
Controls include mock-infected, vector (LNGFRM)-infected and CEM-ss cells
transfected with RevMlO.
3 0 Figure 9 illustrates the percentage of intracellular p24+ CEM-ss cells
containing
various GSEs: CF-619 (SEQ ID N0:53), CF-620 (SEQ ID NO:55) and CF-624 (SEQ
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ID N0:57) after infection with HIV-ls~ at a TCIDSO of 1000. Controls include
mock-
infected, vector (LNGFRM)-infected and CEM-ss cells transfected with RevMlO.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention includes novel methods to identify genetic suppressor
elements capable of inhibiting HIV infection, genetic suppressor elements
identified by
such methods, nucleic acid molecules representing host cellular genes involved
in HIV
infection and inhibitory compositions that inhibit HIV infection by down-
regulating the
expression of such cellular genes or inhibit the activity of the products of
such cellular
genes. As used herein, the term "HIV infection" refers to the ability of HIV
to enter a
host cell and/or replicate in the host cell.
One embodiment of the present invention is a method for isolating genetic
suppressor elements, referred to herein as GSEs, comprising the steps of: 1)
randomly
fragmenting cell-derived cDNA into fragments; 2) inserting the fragments into
expression vectors to form a random fragment expression (RFE) library; 3)
transfernng
the expression library into a population of cells containing an inducible
latent HIV-1
provirus or susceptible to HIV infection; 4) selecting a subpopulation of
cells which
contain a subset of the expression library enriched for GSEs by monitoring the
expression of a cellular or viral marker associated with HIV infection; and 5)
recovering
2 0 the GSEs from the selected cell population. In preferred embodiments, the
cell-derived
cDNA is randomly-fragmented into 100-700 base pair (bp) fragments. The method
further includes repetition of the aforementioned steps so that manyrounds of
successive
selection can be performed. The method can further comprise the step of
selecting GSEs
by determining the continued expression of a cellular marker such as CD4 or
the
decreased expression of a viral marker such as p24 or gp120 using, for
example, an
antibody.
The invention is discussed in more detail in the subsections below, solely for
purposes of description and not by way of limitation. For clarity of
discussion, the
specific procedures and methods described herein are exemplified using OM10.1
cells,
3 0 CEM-ss cells, tumor necrosis factor-alpha (TNF-oc), an anti-CD4 antibody,
and an anti-
p24 antibody, but they are merely illustrative for the practice of the
invention.
Analogous procedures and techniques are equally applicable to isolating GSEs
from
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other cellular DNA, utilizing any cell line and any marker associated with HIV
infection
that can be easily assayed.
A cell-derived RFE library can be constructed from nucleic acid molecules of
any mammalian cells preferably from cDNA of HIV-susceptible cells. In that
regard,
Example 1 demonstrates that GSEs can be selected from HL-60 cells that are
naturally
susceptible to HIV infection and from HeLa cells which are not naturally
susceptible to
HIV infection due to the lack of CD4 expression. However, it has been shown
that
expression of CD4 on the surface of HeLa cells by means of a retroviral vector
renders
the cells susceptible to HIV infection. Therefore, cell types not normally
susceptible to
HIV infection can still be useful as a source of genetic material for the
construction of
RFE libraries. It is also preferred that a normalized cDNA library is prepared
(Gudkov
and Roninson, 1996, Methods in Molecular Biology 69:229-231 ). DNA is first
treated
with enzymes to produce randomly cleaved fragments. This can be conveniently
performed by DNase I cleavage in the presence of Mn~ (Roninson et al., U.S.
Patent
No. 5,217,889, column 5, lines 5-20). Thereafter, the randomly-cleaved DNA is
size
fractionated by gel electrophoresis. Fragments of between 100 and 700 by are
the
preferred lengths for constructing RFE libraries. Single strand breaks of the
size-
selected fragments are repaired by methods well known in the art.
The fragments are ligated with 5' and 3' adaptors, which are selected to have
non-
2 0 cohesive restriction sites so that each fragment can be inserted into an
expression vector
in an oriented fashion. Further, the 5' adaptor contains a start (ATG) codon
to allow the
translation of the fragments which contain an open reading frame in the
correct phase.
The fragments are then inserted into appropriate expression vectors. Any
expression
vector that results in efficient expression of the fragments in host cells can
be used. In
2 5 a preferred embodiment viral-based vectors such as the retroviral vectors
LNCX (Miller
and Rosman,1989, BioTechniques 7:980) and LNGFRM are exemplified.
Alternatively,
adenovirus, adeno-associated virus and herpes virus vectors can also be used
for this
purpose.
When viral-based vectors are used, the ligated vectors are first transfected
into
3 0 a packaging cell line to produce viral particles. For retroviral vectors,
any amphotropic
packaging line such as PA317 (Miller and Buttimore, 1986, Mol. Cell. Biol.
6:2895-
2902; ATCC CRL #9078) can be used to efficiently produce virus. In a preferred
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embodiment of the invention, the viral vector also contains a selectable gene,
such as the
neo' gene or a truncated nerve growth factor receptor (NGFR) gene, which
allows
isolation of the cells that contain the vector.
The number of independent clones present in each RFE expression library can
vary. In a preferred embodiment, libraries of cell-derived cDNA of about 106
to 108
independent clones can be used.
In a specific embodiment illustrated by way of example in Example l, OM10.1
cells are used to select for GSEs, and are maintained in conventional tissue
culture as
described in Butera, U.S. Patent No. 5,256,534. The purpose of using OM10.1
cells for
the selection of GSEs is that they contain a latent HIV-1 provirus which is
inducible by
TNF-a. Other cell lines can be similarly engineered with an inducible HN
provirus.
Examples of cell lines that are infected with latent HIV include, but are not
limited to,
Ul, U33, 8E5, ACH-2, LL58, THP/HIV and UHC4 (Bednarik and Folks, 1992, AIDS
6:3-16). A variety of agents have been shown to be capable of inducing latent
HIV-
infected cells, and these include TNF-oc, TNF-~3, interleukins-l, -2, -3, -4
and -6,
granulocyte-macrophage colony stimulating factors, macrophage-colony
stimulating
factors, interferon-'y, transforming growth factor-(3, PMA, retinoic acid and
vitamin D3
(Poli and Fauci, 1992, AIDSRes. Human Retroviruses 9:191-197). Alternatively,
GSEs
can be selected on the basis of their ability to directly protect HIV-
susceptible cells from
2 0 HIV infection using methods described herein.
The cell-derived RFE library can be introduced into latently HIV-infected
cells
or HIV-susceptible cells by any technique well known in the art that is
appropriate to the
vector system employed. In one embodiment of the invention, the viral vector
also
contains a selectable marker in addition to a random fragment of cellular DNA.
A
2 5 suitable marker is the neo' gene, which permits selection of cells
containing RFE library
members using the drug G-418. In a preferred embodiment, the viral vector
contains a
truncated low affinity nerve growth factor receptor (NGFR) which permits
selection of
the cells using an anti-NGFR monoclonal antibody. In alternative embodiments,
the
multiplicity of infection of the virions of the library is adjusted so that
pre-selection for
3 0 cells that are transduced by the vector is not needed.
In the case of OM10.1 cells, the transduced cell population is treated with 10
U/mL TNF-cG for a period of 24-72 hours and preferably about 24 hours
according to the
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method of Butera. The activation of the latent HIV-1 provirus in OM10.1 can be
detected by the suppression of the cell surface CD4. (It is believed that
viral protein
gp 120 binds to CD4 in the cytoplasm, which prevents subsequent expression of
CD4 on
the cell surface.) Clones that are resistant to HIV replication continue to
express cell
surface CD4. Such clones can be selected, for example, by cell sorting using
any
antibody staining technique for CD4 and a fluorescence activated cell sorter
(FACS).
The fraction of CD4+ cells that have been transduced with the RFE library can
be compared with cells transduced with an expression library consisting of the
vector
only. An increased relative difference between the cell-derived RFE library
and the
control library can be found with each additional round of TNF-oc induction.
Thus, in
the preferred embodiment of the invention there are at least two cycles of
induction,
selection and recloning before the GSEs are recovered from the cells for
further
characterization.
After selection, specific nucleic acid molecules corresponding to the GSEs can
be recovered from cells that continue to express CD4 following induction of
the latent
HIV provirus by TNF-cx. The specific GSEs are recovered from genomic DNA
isolated
from CD4+ cells sorted by FACS after TNF-cc induction. The GSEs in this
population
are preferably recovered by PCR amplification using primers designed from the
sequences of the vector.
2 0 The recovered GSEs can be introduced into an expression vector as
discussed in
the Examples section herein. The resultant GSEs expression library is known as
a
secondary library. The secondary library can utilize the same or a different
vector from
that used for the construction of the primary library. The secondary library
can be
transduced into another cell population and the resultant population selected,
recloned
2 5 and processed as described herein.
Additionally, each individually recovered GSE can be inserted into elanirrg
vectors for determining its specific nucleotide sequence and its orientation.
The
sequence of the GSE is then compared with sequences of known genes to
determine the
portion of the cellular gene with which it corresponds. Alternatively, the PCR
products
3 0 themselves can be directly sequenced to determine their nucleotide
sequences.
Concurrently, the isolated GSEs can be analyzed to determine their minimal
core
sequences. A "core sequence" is a common sequence found by comparison of GSEs
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with overlapping sequences. The GSEs are further tested for their ability to
protect
previously uninfected cells from HIV infection.
Another embodiment of the present invention includes a method for determining
the core sequence of a GSE. This can be done by comparing overlapping
sequences of
independently derived GSEs. Alternatively, GSEs can be altered by additions,
substitutions or deletions and assayed for retention of HIV-suppressive
function.
Alterations in the GSEs sequences can be generated using a variety of chemical
and
enzymatic methods which are well known to those skilled in the art. For
example,
oligonucleotide-directed mutagenesis can be employed to alter the GSE sequence
in a
defined way and/or to introduce restriction sites in specific regions within
the sequence.
Additionally, deletion mutants can be generated using DNA nucleases such as
Bal 31 or
Exo III and S 1 nuclease. Progressively larger deletions in the GSE sequences
can be
generated by incubating the DNA with nucleases for increased periods of time
(see
Ausubel, et al., ibid., for a review of mutagenesis techniques).
The altered sequences can be evaluated for their ability to suppress
expression
of HIV proteins such as p24 in appropriate host cells. It is within the scope
of the
present invention that any altered or shortened GSE nucleic acid molecules
that retain
their ability to suppress HIV infection can be incorporated into recombinant
expression
vectors for further use.
2 0 In order to confirm that the selected GSEs can protect uninfected cells
from HIV
infection, the GSEs can be transferred into latently HIV infected or into HIV-
susceptible
host cells followed by HIV infection. In this connection, GSEs also can be
directly
selected from a RFE library for their ability to prevent productive infection
by HIV, as
shown in a specific embodiment exemplified in the Examples section herein.
Protection
2 5 experiments can be performed in any cell type that takes up the potential
GSEs and
which is otherwise susceptible to HIV infection. In a preferred embodiment by
way of
example, the CEM-ss cell line is used (Foley et al. 1965, Cancer 18:522-529).
The use
of CEM-ss cells as targets for quantitative infectivity of HIV-1 has been
described by
Nara & Fischinger (1988, Nature 322:469-470). Other cell lines that are
susceptible to
3 0 HIV infection include, but are not limited to, HUT-78, H9, Jurkat E6-1,
A3.01, U-937,
AA-2, HeLa CD4+ and C8166. In addition, freshly isolated peripheral blood
leukocytes
can be used.
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The test of the potential GSEs can be performed using the same expression
vector system as that employed in the RFE library transduction of cells during
initial
selection steps. In other embodiments, the vector system can be modified to
achieve
higher levels of expression, e.g., the linkers can be employed to introduce a
leader
sequence that increases the translational efficiency of the message. One such
sequence
is disclosed by Kozak, 1994, Biochemie 76:815-821.
Another way of testing the effectiveness of a potential GSE against HN
infection
is to determine how rapidly HN-1 variants develop that can negate the effects
of that
element. Such a test includes infection of a culture of susceptible cells such
as CEM-ss
cells at a low multiplicity of infection and repeatedly assaying the culture
to determine
whether and how quickly HN-1 infection becomes widespread. The range of useful
multiplicities of infection is between about 100 to 1000 tissue culture
infectious units
(TCIDSO) per 106 CEM-ss cells. The TC~SO is determined by an endpoint method
and
is important for determining the input multiplicity of infection (moi).
A parameter that correlates with the development in the test culture of HN-1
strains that are resistant to the effects of the potential GSEs is the
fraction of cells that
are infected in the culture. This fraction can be determined by
immunofluorescent
staining with an antibody specific for the HN-1 p24 antigen of fixed
permeabilized
cells. Commercially available reagents are suitable for performing such tests
(Lee et al.,
2 0 1994, J. Virol. 68:8254-8264).
One embodiment of the present invention is an isolated nucleic acid molecule
comprising a human cellular gene, or at least a portion thereof, that is
necessary for HN
infection. These isolated nucleic acid molecules are referred to herein as
"cell-derived
nucleic acid molecules." It is to be noted that the term "a" or "an" entity
refers to one
2 5 or more of that entity; for example, a protein refers to one or more
proteins or at least
one protein. As such, the terms "a" (or "an"), "one or more" and "at least
one" can be
used interchangeably herein. It is also to be noted that the terms
"comprising",
"including", and "having" can be used interchangeably. Furthermore, a compound
"selected from the group consisting of" refers to one or more of the compounds
in the
30 list that follows, including mixtures (i.e., combinations) of two or more
of the
compounds. In accordance with the present invention, an isolated nucleic acid
molecule
is a nucleic acid molecule that has been removed from its natural milieu (i.
e., that has
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been subj ect to human manipulation). As such, "isolated" does not reflect the
extent to
which the nucleic acid molecule has been purified. An isolated nucleic acid
molecule
can include DNA, RNA, or derivatives or hybrids of either DNA or RNA. An
isolated
nucleic acid molecule of the present invention can be obtained from its
natural source
either as an entire (i. e., complete) gene or a portion thereof corresponding
to at least a
portion of the gene that encodes a product necessary for productive HIV
infection or
necessary to inhibit HIV infection. As used herein, the phrase "at least a
portion of an
entity refers to an amount of the entity that is at least sufficient to have
the functional
aspects of that entity. For example, at least a portion of a nucleic acid
sequence, as used
herein, is an amount of a nucleic acid sequence necessary for HIV infection.
An isolated
nucleic acid molecule of the present invention can also be produced using
recombinant
DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning)
or
chemical synthesis. Isolated nucleic acid molecules of the present invention
include
natural nucleic acid molecules and homologs thereof, including, but not
limited to,
natural allelic variants and modified nucleic acid molecules in which
nucleotides have
been inserted, deleted, substituted, and/or inverted in such a manner that
such
modifications do not substantially interfere with the nucleic acid molecule's
ability to
promote HIV infection or inhibit HIV infection.
A nucleic acid sequence complement of any nucleic acid sequence of the present
2 0 invention refers to the nucleic acid sequence of the nucleic acid strand
that is
complementary to (i. e., can form a complete double helix with) the strand for
which the
sequence is cited. It is to be noted that a double-stranded nucleic acid
molecule of the
present invention for which a nucleic acid sequence has been determined for
one strand
that represented by a SEQ ID NO also comprises a complementary strand having a
2 5 sequence that is a complement of that SEQ m NO. As such, nucleic acid
molecules of
the present invention, which can be either double-stranded or single-stranded,
include
those nucleic acid molecules that form stable hybrids under stringent
hybridization
conditions with either a given SEQ ID NO denoted herein and/or with the
complement
of that SEQ ID NO, which may or may not be denoted herein. Methods to deduce a
3 0 complementary sequence are known to those skilled in the art.
One embodiment of a cell-derived nucleic acid molecule is a GSE nucleic acid
molecule that is capable of inhibiting HIV infection in a susceptible cell. A
preferred
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GSE nucleic acid molecule of the present invention comprises a nucleic acid
sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3,
SEQ
ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ
ID NO:1 S, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID
N0:20, SEQ ID N0:25, SEQ ID N0:27, SEQ ID N0:29, SEQ ID N0:31, SEQ ID
N0:33, SEQ ID N0:35, SEQ ID N0:37, SEQ ID N0:39, SEQ ID N0:41, SEQ ID
N0:43, SEQ ID N0:45, SEQ ID N0:47, SEQ ID N0:49, SEQ ID NO:51, SEQ ID
N0:53, SEQ ID NO:55, SEQ ID N0:57, SEQ ID N0:59, SEQ ID N0:61, SEQ ID
l0 N0:63, SEQ ID N0:65, SEQ 117 N0:67, SEQ ID N0:69; SEQ ID N0:71; SEQ ID
N0:73; SEQ ID N0:75; SEQ ID N0:77, SEQ ID N0:79, SEQ ID N0:81, SEQ ID
N0:83, SEQ ID N0:85; SEQ ID N0:87; SEQ 117 N0:89; SEQ ID N0:91; SEQ ID
N0:93 and/or SEQ ID N0:95, as well as complements of any of these sequences,
homologs thereof, or nucleotide sequences capable of hybridizing to these
sequences or
their complements under highly or moderately stringent hybridization
conditions. Also
included are GSEs with conservative nucleotide substitutions which produce the
same
protein products. Highly stringent hybridization conditions can be defined as
hybridization to filter-bound DNA in 0.5 M NaHP04, 7% sodium dodecyl sulfate
(SDS),
1mM EDTA at 65°C, followed by washing in 0.1 x SSC/0.1% SDS at
68°C (Ausubel
2 0 F.M. et al., eds, 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Vol. I,
Green
Publishing Associates, Inc., and John Wiley & Sons, Inc., New York at p.
2.10.3).
Moderately stringent conditions can be defined as hybridizations earned out as
described
above, followed by washing in 0.2 x SSC/0.1% SDS at 42°C (Ausubel et
al., 1989,
CURRENT PROTOCOLS FOR MOLECULAR BIOLOGY).
2 5 Another embodiment of a cell-derived nucleic acid molecule of the present
invention comprises a cellular gene that encodes an intracellular product
necessary for
productive HIV infection, referred to herein as a "target gene." Preferably, a
target gene
of the present invention comprises a nucleic acid molecule that corresponds to
a GSE
having a nucleic acid sequence selected from the group consisting of SEQ ID
NO:l,
3 0 SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID
N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12,
SEQ ID N0:13, SEQ ID N0:14, SEQ ID NO:15, SEQ ID N0:16, SEQ ID N0:17, SEQ
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m N0:18, SEQ m N0:19, SEQ m N0:20, SEQ m N0:25, SEQ m N0:27, SEQ m
N0:29, SEQ >D N0:31, SEQ ~ N0:33, SEQ >D N0:35, SEQ m N0:37, SEQ m
N0:39, SEQ m N0:41, SEQ m N0:43, SEQ ~ N0:45, SEQ m N0:47, SEQ m
N0:49, SEQ m N0:51, SEQ m N0:53, SEQ m N0:55, SEQ m N0:57, SEQ m
N0:59, SEQ m N0:61, SEQ m N0:63, SEQ m N0:65, SEQ m N0:67, SEQ m
N0:69; SEQ m N0:71; SEQ m N0:73; SEQ >D N0:75; SEQ m N0:77, SEQ m
N0:79, SEQ m N0:81, SEQ a7 N0:83, SEQ m N0:85; SEQ m N0:87; SEQ >D
N0:89; SEQ m N0:91; SEQ m N0:93 and/or SEQ D7 N0:95, and complements
thereof, and any other GSE sequence disclosed herein and their complements.
Particularly preferred GSE nucleic acid molecules of the present invention
include plasmids CF-315, CF-319, CF-101, CF-117, CF-025, CF-128, CF-004, CF-
113, CF-204, CF-001, CF-273, CF-311, CF-313, CF-210, CF-266, CF-302, CF-317,
CF-286, CF-061, CF-280, CF-537, CF-320, CF-321, CF-322, CF-332, CF-335, CF-
42, CF-50, CF-527, CF-528, CF-529, CF-531, CF-545, CF-547, CF-619, CF-620,
CF-624, CF-630, CF-579, CF-676, CF-675, CF-653, CF-674, CF-675, CF-673, CF-
693, CF-287, CF-658, CF-672, CF-679, CF-681, CF-622, CF-683, CF-684, CF-685
and CF-686, as defined and identified herein.
As used herein, the term "corresponds to" refers a nucleic acid sequence that
is
at least about 75%, more preferably about 80%, more preferably about 85%, more
2 0 preferably about 90%, more preferably about 95% and more preferably about
100%
identical to nucleic acid sequence SEQ >D NO:1, SEQ m N0:2, SEQ >D N0:3, SEQ m
N0:4, SEQ >D N0:5, SEQ m N0:6, SEQ >D N0:7, SEQ m N0:8, SEQ ~ N0:9, SEQ
>D NO:10, SEQ m NO:1 l, SEQ m N0:12, SEQ )17 N0:13, SEQ a7 N0:14, SEQ m
N0:15, SEQ m N0:16, SEQ >D N0:17, SEQ >D N0:18, SEQ m N0:19, SEQ )D
N0:20, SEQ ~ N0:25, SEQ m N0:27, SEQ m N0:29, SEQ m N0:31, SEQ m
N0:33, SEQ m N0:35, SEQ >D N0:37, SEQ m N0:39, SEQ m N0:41, SEQ >D
N0:43, SEQ m N0:45, SEQ m N0:47, SEQ >D N0:49, SEQ m N0:51, SEQ m
N0:53, SEQ m N0:55, SEQ m N0:57, SEQ >D N0:59, SEQ a7 N0:61, SEQ >D
N0:63, SEQ m N0:65, SEQ m N0:67, SEQ >D N0:69; SEQ >D N0:71; SEQ >D
3 0 N0:73; SEQ m N0:75; SEQ m N0:77, SEQ m N0:79, SEQ >D N0:81, SEQ m
N0:83, SEQ m N0:85; SEQ m N0:87; SEQ >D N0:89; SEQ >D N0:91; SEQ m
N0:93 or SEQ m N0:95 and complements thereof. The lack of complete identity
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between the GSEs and the target gene sequences can result from genetic
polymorphism
between different individuals or mutations introduced during cloning or PCR
amplification.
A preferred embodiment of the present invention includes a target gene that
encodes at least a portion a protein selected from the group consisting of
NADH
dehydrogenase, 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase/
cytosolic
thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3,
eukaryotic
initiation factor 3, protein tyrosine phosphatase, herpesvirus-associated
ubiquitin-
specific protease, eukaryotic initiation factor 4B, CD44, phosphatidyl-
inositol 3 kinase,
elongation factor 1 alpha, bone morphogenic protein-1, double-strand break DNA
repair
gene protein, rat guanine nucleotide releasing protein, anti-proliferative
factor (BTG-1 ),
lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase,
eukaryotic
release factor 1, GTP binding protein, importin beta subunit, cell adhesion
molecule L1,
U-snRNP associated cyclophilin, recepin, Arg/Abl interacting protein
(ArgBP2A),
keratin related protein, p18 protein, p40 protein, glucosidase II, alpha
enolase,
macrophage inflammatory protein 1 alpha, tumor protein translationally-
controlled 1
(TCTP1), BBC1, Nef interacting protein, Na+-D-glucose cotransport regulator
gene
protein, hsp90 chaperone protein, FK506-binding protein A1, Rox, beta signal
sequence
receptor, tumorous imaginal disc protein or cell surface heparin binding
protein, in
2 0 which the portion of the nucleic acid molecule encodes an intracellular
product that is
necessary for HIV infection.
The subsections below describe such cellular genes that have been identified
herein as important targets for the development of HIV therapeutics.
In aerobic organisms, adenosine triphosphate (ATP) provides the major source
2 5 of energy. For the generation of ATP, energy rich molecules such as NADH
and FADHz
are first formed in glycolysis, fatty acid oxidation and the citric acid
cycle. When these
molecules donate their electrons to molecular oxygen, free energy is released
to generate
ATP.
Oxidative phosphorylation is the process by which ATP is formed as electrons
3 0 are transferred from NADH or FADHZ to OZ by a series of electron carriers
(Stryer,
1988, Biochemistry, Freeman). This process occurs in the mitochondria of
eukaryotic
cells. More specifically, the enzymes that catalyze the electron transport
chain reside in
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the inner membrane of mitochondria, and they are encoded by both nuclear and
mitochondria) DNA. These enzymes exist as large protein complexes, and the
first
complex of the chain is known as NADH dehydrogenase or NADH-Q reductase. It
has
a molecular weight of 850,000 daltons and consists of over 40 polypeptide
subunits,
seven of which are encoded by the mitochondria) genome. (Anderson et al.,
1981,
Nature 290:457; Chomyn et al., 1985, Nature 314:592; Chomyn et al., 1986,
Science
234:619). The nucleotide sequences of the cDNAs of nuclear genes for the
subunits
have been described (Walker et al., 1992, J. Mol. Biol. 226:1051; Fearnley et
al., 1989,
EMBO J. 8:665; Pilkington et al., 1989, Biochem. 28:3257). NADH dehydrogenase
catalyzes the transfer of electrons from NADH to an electron carrier termed
ubiquinone.
The 2-oxoglutarate dehydrogenase complex catalyzes oxidative decarboxylation
of 2-oxoglutarate to succinyl-CoA and CO2, and is the rate-limiting enzyme
which
controls the flux of substrates through the Krebs cycle (Delvin, T.M., ed,
1992,
Textbook of Biochemistry, Wiley-Liss, Inc.). This enzyme complex is located in
the
inner membrane/matrix compartment of the mitochondria. The complex consists of
multiple copies of 2-oxoglutarate dehydrogenase (lipoamide) (OGDH or ELO; 2-
oxoglutarate: lipoamide 2-oxidoreductase (decarboxylating and acceptor
succinylating),
EC 1.2.4.2), dihydrolipoamide succinyltransferase (designated E20; EC
2.3.1.61) and
dihydrolipoamide dehydrogenase (E3; EC 1.8.1.4). The coding sequence of 2-
2 0 oxoglutarate dehydrogenase has been described (GenBank Accession Nos. D
10523 and
D90499; Koike et al., 1992, Proc. Nat). Acad. Sci. USA 89:1963-1967; Koike,
1995,Gene 159:261-266).
Pyruvate kinase/thyroid hormone binding protein p58 (TBP) is a monomer of
pyruvate kinase (ATP pyruvate OZ-phosphotransferase, EC 2.7.1.40) subtype M2.
Its
2 5 conversion to the tetrameric pyruvate kinase is regulated by fructose 1,6,-
bisphosphate
(Fru-1,6-PZ). At low glucose concentrations mammalian cells contain low Fru-
1,6-PZ
andpyruvate kinase is inactive. At high glucose concentration (regularmedium
contains
5-10 mM glucose), high levels of Fru-1,6-PZ are found in proliferating and
tumor cells,
which require high pyruvate kinase activity for growth. It has been
demonstrated that
3 0 low Fru-1,6-Pz favors formation of p58 and high concentrations convert it
to the
tetrameric enzyme. An increase in glucose concentration could lead to
multimerization
of p5 8 which in turn activates pyruvate kinase and glycolysis. At the same
time thyroid
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hormone is released from the complex with TBP and might bind to nuclear and
mitochondrial receptors and activate oxidative phosphorylation. The coding
sequence
of pyruvate kinase/thyroid hormone binding protein has been described (GenBank
Accession No. M26252; Kato et al., 1989, Proc. Natl. Acad. Sci. USA 86:7861-
7865).
Calnexin is a type I membrane protein which functions as a molecular chaperon
for secretory glycoproteins in the endoplasmic reticulum (ER) with ATP and
Ca~'~" as two
cofactors involved in the substrate binding (0u et al., 1995, J. Biol. Chem.
270:18051 ).
It has been demonstrated that folding of gp120 is mediated by calnexin during
the
translocation of the newly synthesized gp120 into ER (Li et al., 1996, Proc.
Natl. Acad.
Sci. USA 93:9606). The coding sequence of calnexin has been described (GenBank
Accession No. L10284; David et al., 1993, J. Biol. Chem. 268:9585-9592).
ADP-ribosylation factors (ARFs) are guanine nucleotide binding proteins of
about 20kDa molecular weight that stimulate ADP-ribosyltransferase activity of
cholera
toxin in vitro (Tsai et a1.,1991, J. Biol. Chem. 266:23053-23059). Five
different ARFs
from human cDNA have been cloned. ARF3 is represented by two mRNAs of 3.7 and
1.2 kb that are generated through the use of alternative polyadenylation
signals (Tsai et
al., 1991, supra).
Ubiquitin-specific protease (USP) plays an important role in several cellular
processes, including the regulation of gene expression, control of the cell
cycle, DNA
2 0 repair and differentiation (Hochtrasser, 1995, Curr. Opin. Cell. Biol.
7:215-223;
Wilkinson,1995, Ann. Rev. Nutr. 15:161-189). One of the best characterized
ubiquitin-
dependent pathways involved in the control of gene expression is activation of
NF-KB.
Cleavage of p105, a precursor of the p50 subunit of NF-KB requires ubiquitin
conjugation (Palombella et al., 1994, Cell 78:773-785) and secondly the
destruction of
2 5 IKB (a process which allows NF-xB to migrate to the nucleus in an active
form) requires
ubiquitination of the inhibitor in a phosphorylation-dependent manner (Scherer
et al.,
1995, Proc. Natl. Acad. Sci. USA 92:11259-11263).
USP is characterized by the presence of two conserved active site domains and
has been shown to cleave ubiquitin from model substrates (Everett et al.,
1997, EMBO
3 0 J. 16: 556-577). There are two classes of USP. The first includes proteins
involved in
the generation of free ubiquitin from precursor fusion proteins or from
peptide-linked
polyubiquitin after proteolysis of the substrate by the proteosome
(Hochstrasser, 1995
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ibid.). The second comprises an increasing number of de-ubiquitinating
proteins which
can recognize and stabilize specific substrates byremoving ubiquitin adducts.
Examples
of this class include the Drosophila fat facets protein, whose de-
ubiquitination is
required for proper eye development (Huang et al., 1995, Science 270:1828-
1831).
Another example is the DUB-1 gene which is an immediate early gene that
regulates cell
growth (Zhu et al., 1996, Proc. Natl. Acad. Sci. USA 93:3275-3279).
The coding sequence for herpesvirus-associated ubiquitin-specific protease has
been described (GenBank accession No. 272499; Everett et al., 1997, EMBO J.
16:566-
577).
CD44 is a broadly distributed surface receptor glycoprotein implicated in
multiple physiologic cellular functions, such as extracellular matrix-cell
adhesion,
lymphocyte homing, lympho-hematopoiesis, T cell activation, and tumor
metastasis
(Shimuzu et al., 1989, J. Immunol. 143:2457-2463; Huet et al., 1989, J.
Immunol.
143:798-801 ). Some of these functions depend on the ability of CD44 to
recognize the
extracellular matrix component hyaluronic acid. CD44 is expressed as several
different
isoforms, varying between 85 to 200 kDa, depending on differential usage of 10
exons
encoding aportion ofthe extracellular domain and cell type specific
glycosylation. Each
isoform can display some degree of functional uniqueness (Stamenkovich et al.,
1989,
Cell 56:1057-1062). The most widely expressed molecule is the 85-90 kDa
2 0 glycoprotein, commonly referred to as CD44H, which has been demonstrated
to be the
major receptor for hyaluronic acid (Bartolazzi et a1.,1996, J. Cell Biol.
132:1199-1208).
CD44H represents the principle isoform found on hematopoietic cells. It has
been
shown that CD44, along with other HIV receptors like CD4, can play a role in
viral
tropism and affects infectivity of the virus. HIV causes CD44 downmodulation
in
2 5 monocytes, but on a post-translational level (Guo and Hildreth, 1993, J.
Immunol.
151:2225-2236).
The coding sequence for human CD44 has been described (GenBank accession
No. X551 S0; Stamenkovich et al., 1991, EMBO J. 10:243-248).
Phosphorylation of serine, threonine and tyrosine residues is one of the
3 0 significant regulatory mechanisms in gene expression and post-
translational
modifications. Tyrosine phosphorylation is important in the control of normal
cellular
processes such as cell proliferation and differentiation, as well as
pathological events
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such as malignant transformation (Cantley et al., 1991, Cell 64:281-301). The
overall
levels of tyrosine phosphorylation are modulated by the complementary and
antagonistic
actions ofprotein tyrosine kinases and protein tyrosine phosphatases. In the
steady state,
less that 1 % of the total cellular phosphorylation is due to tyrosine
phosphorylation.
However, the phosphotyrosine content can increase dramatically upon cellular
transformation (Walton and Dixon, 1993, Ann. Rev. Biochem. 62:101-120). There
are
about 40 known phosphatases (Zolnierowiez and Hemmings, 1994, Trends Cell
Biol.
4:61-64).
Several viruses are known to contain protein tyrosine kinases and
phosphatases.
A putative protein tyrosine phosphatase is found in the HIV-1 5'LTR (Nandi and
Banerjee,1995, Med. Hypothesis 45:476-480). Phosphorylation plays an important
role
in tat-mediated transactivation (Ensoli et al., 1990, Nature 345:84-86), nef
protein
function (Balliet et al., 1994, Virology 200:623-631; Venkatesh et al., 1990,
Virology
176:39-47), and viral matrix assembly (Camaur et al., 1997, Virology 71:6834-
6841).
The coding sequence of protein tyrosine phosphatases from Brain Derived
Phosphatase (BDPI) mRNA and CL 100 mRNA have been described (GenBank
accession No. X79568; Kim et al., 1996, Oncogene 13:2275-2279 and GenBank
accession No. X68277; Keyse and Emslie, 1992, Nature 359:644-647).
Phosphatidylinositol 3-kinases (PI3K) have been characterized as enzymes
2 0 involved in receptor signal transduction. Multiple forms with different
substrate
specificities exist. They have been associated with a diverse range of cell
surface
receptors including those for growth factors, thrombin, chemotactic peptides
and
cytokines. It has been proposed that PI3Ks act as second messengers, possibly
via the
activation of certain protein kinase C isotypes (Liscovitch and Cantley, 1994,
Cell
2 5 77:329-334; Toker et a1.,1994, J. Biol. Chem. 269:323598-32367). PI3K can
also play
a role in the regulation of protein trafficking from the Golgi to the lysosome
(Volinia et
al., 1995, EMBO J. 14:3339-3348).
HIV-1 nef expression severely impairs PI3K association with a receptor
suggesting that Nef selectively affects the PI3K signaling pathway resulting
in adverse
3 0 effects on host cell function (Graziani et al., 1996, J. Biol. Chem.
271:6590-6593). Nef
expression is also accompanied by a decrease in basal intracellular PIK,
suggesting a
role for PI3K in HIV replication (Garcia, 1997, C.R. Acad. Sci. III320:505-
508). PI3K
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can be activated by gp 160 and also has been implicated in Tat-mediated
apoptosis
(Mazerolles et al., 1997, Eur. J. Immunol. 27:2457-2465; Borgatti et al.,
1997, Eur. J.
Immunol. 27:2805-2811). Known inhibitors of PI3K include wortmannin and
theophylline.
The coding sequence of phosphatidylinositol 3-kinase has been described
(GenBank accession No. 246973; Volinia et al., 1995, EMBO J. 14:3339-3348).
Elongation of the polypeptide chain occurs following initiation of
translation.
Elongation factors utilized the energy released by GTP hydrolysis to ensure
selection of
the proper aminoacyl-tRNA and to move the message and associated tRNAs through
the
decoding region of the ribosome (Devlin, ed, 1992, Textbook of Biochemistry,
Wiley-
Liss, Inc.). EF-1-cx is an evolutionarily conserved universal cofactor
ofprotein synthesis
in all living cells. It carries aminoacyl-tRNAs to the A-site of the ribosome
in GTP-
dependent manner.
The expression levels of EF-1 cx are regulated in various stages of cell life
such
as growth arrest, transformation and aging. Levels of EF-lcx can be a key
regulator in
modulating the rate of apoptosis. Reduction of EF-1-cx expression decelerates
apoptosis
while overexpression accelerates the process (Duttaroy et al., 1998, Exp. Cell
Res.
238:168-176).
The coding sequence for human EF-1 cc has been described (GenBank accession
2 0 No. J04617; Uetsuki et al., 1989, J. Biol. Chem. 264:5791-5798).
Initiation of translation occurs by the binding of the 40S ribosomal subunit
at or
near the cap structure of mRNA followed by ribosome scanning of the 5'
untranslated
region until an initiator AUG is encountered. This process is promoted by a
complex
group of proteins known as initiation factors. These factors participate only
in initiation
2 5 of translation (Devlin, ed, 1992, Textbook of Biochemistry, Wiley-Liss,
Inc.).
Eukaryotic initiation factor 3 (eIF3) is the largest multisubunit complex
involved
in initiation of protein synthesis. It has a mass of 600 kDa and 10 subunits.
The factor
prevents association of ribosomal subunits, stabilizes methionyl-tRNA binding
to the
405 subunits and promotes mRNA binding (Mernck and Hershey, l 996, in
Translational
3 0 Control, Hershey, Mathews and Sonenberg eds, pp31-69, Cold Spring Harbor,
Cold
Spring Harbor, NY). The sequence for human eukaryotic initiation factor 3 has
been
described (GenBank accession No. U78525).
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Eukaryotic initiation factor 4B (eIF4B) is an 80 kDa polypeptide that is
essential
for the binding of mRNA to ribosomes. eIF4B has RNA binding activity,
stimulates
ATPase and RNA helicase (Mehot et al., 1996, RNA 2:38-50; Abramson et al.,
1987,
J. Biol. Chem. 262:3826-3832; Ray et al., 1985, J. Biol. Chem. 260:7651-7658;
Rozen
et al., 1990, Mol. Cell. Biol. 10:1134-1144). Additionally, eIF4B has been
reported to
have RNA annealing activity, promoting base pairing between complementary
sequences
in RNA strands (Altmann et al., 1995, EMBO J. 14:3820-3827). It also interacts
with
eIF4A and eIF3 (Methot et al., 1996, Mol. Cell. Biol. 16:5328-5334).
Overproduction
of eIF4B results in a general inhibition of translation (Milburn et al., 1990,
EMBO J.
9:2783-2790).
The sequence for human eIF4B has been described (GenBank accession No.
X55733; Milburn et al., 1990, EMBO J. 9:2783-2790).
Protein extracts derived from bone can initiate the process that begins with
cartilage formation and ends in de novo bone formation. The protein extract is
referred
to as bone morphogenic protein (BMP). It is not known what are the critical
components of BMP that direct cartilage and bone formation, and constitutive
elements
supplied by the animal during cartilage and bone formation (Wozney et al.,
1988,
Science 242:1528-1534). Amino acid sequence has been derived from a highly
purified
preparation of BMP from bovine bone. Human complementary DNA clones
2 0 corresponding to three polypeptides present in a BMP preparation have been
isolated,
and expression of the recombinant human proteins have been obtained. Each of
the
three expressed human proteins, BMP-1, BMP-2A, and BMP-3, appears to be
independently capable of inducing the formation of cartilage in vivo. BMP-2A
and
BMP-3 are new members of the TGF-beta supergene family, while the third, BMP-
1, is
2 5 a regulatory molecule.
Human and mouse homologs of the rad21 gene of SchizosacchaYOmyces pombe
(which is involved in the repair of ionizing radiation-induced DNA double-
strand
breaks) havebeen described (McKayet a1.,1996, Genomics 36:305-315). The
predicted
amino acid sequences of mHR21(sp) (mouse homolog of Rad2l, S. pombe) and
3 0 hHR21 (sp) (human homolog of Rad2l, S. pombe) were 96% identical, whereas
the
human and S. pombe proteins were 25% identical and 47% similar. RNA blot
analysis
showed that mHR21 sp mRNA was abundant in all adult mouse tissues examined,
with
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the highest expression in testis and thymus. In addition to a 3.1 kb
constitutive mRNA
transcript, a 2.2 kb transcript was present at a high level in postmeiotic
spermatids, while
expression of the 3.1 kb mRNA in testis was confined to the meiotic
compartment.
hHR21 sp mRNA was cell cycle regulated in human cells, increasing in late S
phase and
peaking in G2 phase. In situ hybridization showed that mHR21 sp resided on
chromosome 1 SD3, whereas hHR21 sp localized to the syntenic 8q24 region.
Elevated
expression of mHR21 sp in testis and thymus indicates a role for the rad21
mammalian
homologs in V(D)J immunoglobulin gene and meiotic recombination, respectively.
Cell
cycle regulation of rad2l, conserved in S. pombe and humans, is consistent
with a
conservation of function between S. pombe and human rad21 genes.
Small GTP-binding proteins of the ras superfamily are important for exocytosis
from eukaryotic cells. These GTP-binding proteins can exist in two different
conformations, depending on whether they are bound to GDP or GTP, and function
as
molecular switches that regulate a variety of cellular processes. The GTP-GDP
cycle is
controlled by accessory proteins that promote the exchange of bound GDP or the
hydrolysis of GTP. cDNA encoding a mammalian GDP releasing protein, mss4, has
been cloned (Burton et al., 1993, Nature 361:464-467). Mss4 is a guanine
nucleotide
exchange factor that specifically binds to and promotes GDP-GTP exchange on a
subset
of the Rab GTPases (Burton et a1.,1994, EMBO J. 13:5547-5558). The Mss4
protein
2 0 also stimulates GDP release from Yptl and from the mammalian protein
Rab3a, but not
from Ras2. Mss4 shows sequence similarity to Dss4, a yeast protein with
similar
biochemical properties.
The product of the B-cell translocation gene 1 (BTG1), a member of an
antiproliferative protein family including Tis-21/PC3 and Tob, regulates cell
cycle
2 5 progression (Rodier et a1.,1999, Exp Cell Res. 249:337-348). The BTG1 gene
locus has
been shown to be involved in a t(8;12)(q24;q22) chromosomal translocation in a
certain
B-cell chronic lymphocytic leukemia (Rouault et al., 1992, EMBO J. 11:1663-
1670).
The cDNA for BTGlwas isolated (Rouault et al., ibid.) and contains an open
reading
frame of 171 amino acids. BTG1 expression is maximal in the GO/G1 phases of
the cell
3 0 cycle and is down-regulated when cells progress throughout G1.
Furthermore,
transfection experiments of NIH3T3 cells indicate that BTG1 negatively
regulates cell
proliferation. The BTG1 open reading frame is 60% homologous to PC3, an
immediate
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early gene induced by nerve growth factor in rat PC 12 cells. Sequence and
Northern blot
analyzes indicate that BTG1 and PC3 are not cognate genes. Triiodothyronine
(T3) or
8-Br-cAMP increases BTGI nuclear accumulation in confluent myoblast cultures
(Rodier et al., ibid.). It has been demonstrated that AP-1 activity, a crucial
target
involved in the triiodothyronine myogenic influence, repressed BTG1
expression, thus
explaining the low BTG1 expression level in proliferating myoblasts. An AP-1-
like
sequence located in the BTG1 promoter was shown to be involved in the negative
regulation of BTG-1 expression.
The mouse and human lymphocyte-specific proteinl (LSP 1 ) genes are expressed
in normal B cells and T cells, including Thyl+ thymocytes and in normal
macrophages
and neutrophils (Pulford et al., 1999, Immunology 96:262-271). LSP1 is found
in all
hematopoietic cells, and its function is unclear. In intact cells, mitogen-
activated protein
kinase-activated protein (MAPKAP) kinase 2 is rapidly activated by various
cytokines,
stresses, and chemotactic factors. Recently, it was shown that LSP1 is a
substrate for
MAPKAP kinase 2 in human neutrophils (Zu et a1.,1996, Blood 87:5287-5296).
LSP1
was also identified as one of the major substrates of protein kinase C in B
cells (Carballo
et al., 1996, J. Immunol. 156:1709-1713).
Protein phosphatases can control the activity of various protein kinases.
Protein
phosphatase 2A (PP2A) regulates cell growth and division. Investigators have
suggested
2 0 that HIV infection activates protein phosphatase 2A (Han et al., 1992, J.
Virol. 66:4065-
4072) . Protein from other viruses are known to interact with PP2A . SV40
small tumor
antigen (small-t) was used as a model to identify structural elements involved
in the
interaction between regulatory proteins and PP2A (Mateer et al., 1998, J.
Biol. Chem.
273: 35339-35346). NCp7 and Vpr form a tight complex which becomes a more
potent
2 5 activator of PP2A than NCp7 alone. The ability of NCp7 to activate protein
PP2A is
regulated by Vpr. The C-terminal portion of Vpr prevents NCp7 from activating
protein
PP2A while the N-terminal portion of Vpr potentiates the effect of NCp7 on the
activity
of PP2A. These findings indicate that Vpr acts as a targeting subunit which
directs
NCp7 to activate protein PP2A (Tung et al., 1997, FEBS Lett. 401:197-201 ).
3 0 The reaction catalysed by squalene synthetase (SQS) shows many
similarities to
that performed by another polyisoprene synthase, phytoene synthetase (PhS). By
identifying sequences conserved between yeast SQS (ySQS) and PhS, a 2 kb cDNA
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(hSQS) encoding human SQS, a protein of 417 amino acids with a predicted M(r)
of
48,041 was cloned (Summers et al., 1993, Gene 136:185-192). Two hSQS mRNA
species of 2.0 and 1.55 kb have been identified which differ in their 3'
untranslated
sequences. The two mRNAs are present in roughly equal amounts in heart,
placenta,
lung, liver, kidney and pancreas, but the 2 kb mRNA predominates in brain and
skeletal
muscle. In HepG2 cells, both mRNAs are induced 2-fold to 4-fold by the 3-
hydroxy-3-
methylglutaryl-coenzyme A reductase inhibitor, lovastatin. In contrast,
Northern blot
analysis of rat tissues reveals only a 2.0 kb mRNA, which is considerably up-
regulated
in vivo by lovastatin.
The termination of protein synthesis in ribosomes is governed by termination
codons in messenger RNAs and by polypeptide chain release factors (RFs). Amino
acid
sequences of members of the eRF 1 family are highly conserved. These RF
proteins are
directly implicated in the termination of translation in eukaryotes ( Frolova
et al., 1994,
Nature 372: 701-703).
Prokaryotic and eukaryotic cells incorporate the amino acid selenocysteine at
a
UGA codon, which conventionally serves as a termination signal. Translation of
eukaryotic selenoprotein mRNA requires a nucleotide selenocysteine insertion
sequence
in the 3'-untranslated region. Erb-1 can recognize a selenocysteine insertion
sequence
element.
2 0 It has also been demonstrated that eRFl associates with the catalytic
subunit of
protein phosphatase 2A (Andjelkovic et al., 1996, EMBO J. 15:7156-67). It was
postulated that eRFI also functions to recruit PP2A into polysomes, thus
bringing the
phosphatase into contact with putative targets among the components of the
translational
apparatus. It was also demonstrated that retinoic acid induction of
granulocyte
2 5 differentiation of HL60 cells results a transient and reversible
interconversion of
phosphatase 2A holoenzyme and that the C-terminus of PP2A catalytic subunit is
transiently methylated in S phase of HL-60 cells (Zhu, 1997, Arch Biochem
Biophys.
339:210-217).
G-proteins are a family of heterotrimeric guanine nucleotide-binding proteins
3 0 that play important roles in signal transduction and whose expression is
regulated in a
tissue-specific manner. Golf) alpha is a G-protein originally believed to
mediate signal
transduction exclusively within the olfactory neuroepithelium and subsequently
found
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to be a major stimulatory G-protein in the basal ganglia and several other
tissues
(Zigman et al., 1993, Endocrinology 133:2508-2514 ).
Nucleocytoplasmic transport takes place through nuclear pores. Peripheral pore
structures interact with transport receptors and their cargo when these
receptor
complexes first encounter the pore. Protein nuclear import is mediated by
basic nuclear
localization signals (NLSs) that bind to the importin alpha (Imp alpha) NLS
receptor.
Imp beta is also necessary for nuclear import.
The human immunodeficiency virus type 1 (HIV-1) Rev protein binds to
unspliced HIV-1 pre-mRNA and exports it from the nucleus. Rev itself can
"shuttle"
l0 between the nucleus and cytoplasm. This bi-directional transport is
mediated by two
specific Rev sequences: a NLS, which overlaps the RNA-binding domain, and a
distinct
nuclear export signal (NES). Imp beta supports Tat or Rev nuclear import
(Truant et al.,
1999, Mol Cell Biol, 19:1210-7) through the classical NLS pathway as
demonstrated by
inhibition of Imp beta interaction with Tat and Rev by RanGTP but not RanGDP.
Importin beta also interacts with the HIV-1 protein Vpr (Jenkins et al., 1998,
J
Cell Biol,143:875-885). Vpr contains two discrete nuclear targeting signals
that use two
different import pathways, both of which are distinct from the classical NLS
pathway.
Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists
under
conditions of low energy. Vpr bypasses many of the soluble receptors involved
in
2 0 import of cellular proteins. Vpr directly accesses the NPC, a property
that can help to
ensure the capacity of HIV to replicate in nondividing cellular hosts (Jenkins
et al.,
ibid.). Overexpression of either Vpr or importin beta in yeast blocks nuclear
transport
of mRNAs. A mutant form of Vpr, Vpr F34I, that does not localize at the
nuclear
envelope, or bind to importin alpha and nucleoporins, renders HIV-1 incapable
of
2 5 infecting macrophages efficiently. Vpr F34I, however, still causes G2
arrest,
demonstrating that the dual functions of Vpr are genetically separable
(Vodicka et al.,
1998, Genes Dev, 12:175-185).
The rodent, avian, and insect L1-like cell adhesion molecules are members of
the
immunoglobulin superfamily that have been implicated in axon growth. The
entire
3 0 coding region of human L1 CAM has been cloned and found to have a high
degree of
homology to mouse L1CAM, with 92% identity at the amino acid level (Hlavin et
al.,
1991, Genomics 11:416-423). This similarity suggests that L1CAM is an
important
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molecule in normal human nervous system development and nerve regeneration.
This
molecule has never been associated with HIV-1 life-cycle.
Cyclophilins have been proposed to act as chaperones in a variety of cellular
processes. U4/LJ6 snRNP-associated cyclophilin has been cloned and sequenced
(Horowitz et al., 1997, RNA 3:1374-1387).
The nucleotide sequence for the recepin gene, a novel human liver cDNA
encoding a serpin-like molecule has been directly submitted to GenBank.
Arg and c-Abl represent the mammalian members of the Abelson family of
protein-tyrosine kinases. The Arg/Abl-binding protein, ArgBP2, was isolated
using a
l0 segment of the Arg COOH-terminal domain as bait in the yeast two-hybrid
system
(Wang et. al., 1997, J. Biol Chem. 272:17542-17550). ArgBP2 contains three
COOH-
terminal Src homology 3 domains, a serine/threonine-rich domain, and several
potential
Abl phosphorylation sites. ArgBP2 associates with and is a substrate of Arg
and v-Abl,
and is phosphorylated on tyrosine in v-Abl-transformed cells. ArgBP2 is widely
expressed in human tissues and extremely abundant in heart. In epithelial
cells, ArgBP2
is located in stress fibers and the nucleus, similar to the reported
localization of c-Abl.
In cardiac muscle cells, ArgBP2 is located in the Z-disks of sarcomeres. These
observations indicate that ArgBP2 functions as an adapter protein to assemble
signaling
complexes in stress fibers, and that ArgBP2 is a link between Abl family
kinases and the
2 0 actin cytoskeleton.
Interferon (IFN)-gamma has been implicated in the pathogenesis of several
autoimmune disorders and inflammatory skin diseases. A cDNA detecting a 1.6 kb
mRNA that accumulated in response to IFN-gamma but not in response to IFN-
alpha or
IFN-beta has been cloned and sequenced (Flohr et al., 1992, Eur Jlmmunol.
22:975-
2 5 979). The gene is regulated by IFN-gamma in human cell lines of epithelial
origin. The
mRNA encodes a predicted protein of 432 amino acids and the primary structure
of the
protein demonstrates that it is a member of developmentally regulated keratin
class I
genes.
The oncoprotein 18 (0p18) gene encodes a proliferation-related cytosolic
3 0 phosphoprotein which is induced in normal lymphocytes following mitogenic
stimulation. The cDNA for this gene has been cloned (Zhu et al., 1989, JBiol
Chem.
264:14556-14560). It is encoded by two different-sized full-length cDNAs. The
two
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cDNAs differ in their 3'-noncoding regions as a result of alternative
polyadenylation.
The Opl8 gene, which is 6.3 kilobases in length, is comprised of five exons
and four
introns and exhibits features that are common to other genes involved in
cellular growth
and proliferation. This gene is highly conserved in several animal species and
low
stringency hybridization studies suggest that the p 18 gene can be a member of
a family
of partially homologous genes in the human genome. The increase in Op 18
polypeptide
in leukemia is associated with increased RNA transcription without gene
amplification
or rearrangement (Melhem et al., 1991, J. Biol Chem. 266:17747-17753).
Treatment of
K562 leukemia cell line with hemin that induces terminal differentiation
resulted in
decreased expression of Opl8.
Putative G-protein coupled receptor has been cloned recently (Mayer et al.,
1998, Biochim. Biophys. Acta 1395:301-308). The cDNA sequence encodes a
protein
of 399 amino acids. Northern and RNA dot blot analyzes demonstrated that the
major
4.8 kb transcript is predominantly expressed in brain. In situ hybridization
studies of
tissue sections revealed high expression in neurons of the brain and spinal
cord,
thymocytes, megakaryocytes, and macrophages.
Glucosidase alpha II is a glycoprotein involved in the processing of N-linked
glycans. It resides in the endoplasmic reticulum (ER) and controls the
formation of
glycoproteins in the ER. The glucosidase alpha II gene was cloned and the
encoded
2 0 protein has been shown not to contain known ER retention signals or
hydrophobic
regions that could represent a transmembrane domain. Glucosidase alpha II,
however,
has been shown to contain a single N-glycosylation site close to the amino
terminus.
HIV-1 contains two heavily glycosylated envelope proteins, gp120 and gp4l,
which
mediate attachment of virions to the glycosylated cell surface receptor
molecule, CD4.
2 5 It has also been shown that gp 120 and gp41 can be involved in syncytium
formation and
associated cytopathic effects of HIV. The alpha-glucosidase inhibitor N-
butyldeoxynojirimycin (NB-DNJ) is an inhibitor of HIV replication and HIV-
induced
syncytium formation in vitro. The NB-DNJ-mediated retention of glycosylated -
glycans
inhibits HIV entry by a combined effect of a reduction in virion gp120 content
and a
3 0 qualitative defect within the remaining gp 120, preventing it from
undergoing
conformational changes after CD4 binding (Fischer et a1.,1996, J. Virol.
70:7153-7160).
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In mammals there are at least three isoforms of the glycolytic enzyme enolase
encoded by three similar genes: alpha, beta and gamma. Structure of the human
gene
for alpha-enolase locus has been described (Giallongo et al., 1990, Eur J
Biochem,
190:567-573). The gene is composed of 12 exons distributed over more than
18,000
bases. The structure of this gene has a high degree of similarity to that of
the human and
rat gamma-enolase genes, with identical positions for all the intron regions.
The putative
promoter region, like that of other house-keeping genes, lacks canonical TATA
and
CART boxes, is extremely G + C-rich and contains several potential SP 1
binding sites.
It has been demonstrated that a 48 kDa protein (p48), that specifically reacts
with an
antiserum directed against the 12 carboxyl-terminal amino acids of the c-myc
gene
product, is alpha enolase (Giallongo et al., 1986, Proc Natl Acad Sci USA
83:6741-
6745).
The murine gene, macrophage inflammatory protein 1 alpha (MIP1 alpha) is a
cytokine that inhibits proliferation ofbone marrow stem cells (Russell et
a1.,1993, DNA
Cell Biol. 12:157-175). MIP1 alpha has been shown to suppress HIV-1
replication in
human peripheral blood mononuclear cells. MIP 1 alpha can also suppress
transcription
from the HIV-1 LTR in transient transfection assays in cells of the Jurkat
acute T
leukemia cell line (Harden et al., 1997, FEBS Lett. 410:301-302). MIP1 alpha
is a
ligand of CCRS and can prevent M-tropic HIV infection in vitro (Cochi, et al.,
1995,
2 0 Science, 270:1811-1815; Gong et al., 1998, J. Biol. Chem, 271:2599-2603).
Certain
individuals with elevated levels of MIP 1 alpha expression appear to be
resistant to HIV
infection (Cho et al., 1997, Biomed. Pharmacother. 51:221-229). It is not
known,
however, how downmodulation of intracellular expression of MIP 1 alpha can
affect HIV
replication in T cells.
2 5 The translationally-controlled tumor protein (TCTP) is a growth-related
protein
which is regulated at the translational level. It is present in mammals,
higher plants and
Saccharomyces cerevisiae (Sanchez et al., 1997, Electrophoresis 18:150-155).
It has
been shown that macrophage activation by PHA results in up-regulation of TCTP
(Walsh et al., 1995, J. Leukoc Biol. 57:507-512). TCTP is also differentially
expressed
3 0 in C6.9 glioma cells during vitamin D-induced cell death (Baudet et al., l
998, Cell Death
Differ, 5:116-125). TCTP is localized on chromosome 13q12-ql4 (MacDonald et
al.,
1999, Cytogenet Cell Genet, 84:128-129). Both vitamin D and PHA can induce
latent
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HIV-1. This indicates that the TCTP1 pathway overlaps with signal transduction
pathways involving critical for the HIV-1 life-cycle.
The nef gene of human and simian immunodeficiency virus is a key factor in
acquired immunodeficiency syndrome pathogenesis and virus replication. Several
Nef
induced phenomena, including the down-regulation of CD4 in T cells, have been
previously reported. Nafl (Nef associated factor 1 ) has been cloned (Fukushi,
1999,
FEBS Lett. 442: 83). The Nafl gene generates two isoforms, Naflalpha and beta,
containing four coiled-coil structures. Nafl mRNA is ubiquitously expressed in
human
tissues with strong expression in peripheral blood lymphocytes and spleen.
Nafl
overexpression in T cells increases surface CD4 expression. Expression of Nef
suppressed this Nafl-induced augmentation of CD4 expression, providing a novel
mode
of Nef action in CD4 down-regulation.
The human gene of a protein that modifies Na+-D-glucose co-transport, Na+-D-
Glucose cotransport regulator gene (hRSI), has been cloned and sequenced
(Lambotte
et al., 1996, DNA Cell Biol, 15:769-777). It is an intronless gene designated
hRSl
(6,743 bp), which encodes a 617 amino acid protein with 74% identity to pRS 1.
By
fluorescence in situ hybridization, hRSl was localized to chromosome 1p36.1.
It is
homologous to the nucleic acid sequence of pRSl, which was cloned from pig
kidney
cortex and encodes a membrane-associated protein involved in Na+-coupled sugar
2 0 transport. pRS 1 alters sugar transport by SGLT 1 from rabbit intestine or
by SMIT from
dog kidney which is homologous to SGLT1. In contrast, pRSl does not influence
transporters from other genetic families. The function of hRS 1 has been
demonstrated
by co-expression experiments of hRSI and SGLTl, from human intestine, in
oocytes
fromXenopus laevis. It was demonstrated that hRS 1-protein inhibits Na+-D-
glucose co-
t 5 transport expressed by human SGLT1 by decreasing both the Vm~ and the
apparent K",
value of the transporter. The analysis of the 5'-noncoding sequence of hRS 1
revealed
different enhancer consensus sequences that are absent in the SGLT1 gene,
e.g., several
consensus sequences for steroid-binding proteins.
Hsp90 is an abundant molecular chaperone that is involved in the folding of a
3 0 defined set of signaling molecules including steroid-hormone receptors and
kinases. In
vitro experiments suggest that Hsp90 contains two different binding sites for
non-native
proteins, which allow it to combine the properties of a promiscuous chaperone
with
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those of a dedicated folding-helper protein. The heat shock protein Hsp90 is
known as
an essential component of several signal transduction pathways and has now
been
identified as an essential host factor for hepatitis B virus replication.
Hsp90 interacts
with the viral reverse transcriptase to facilitate the formation of a
ribonucleoprotein
(RNP) complex between the polymerase and an RNA ligand (Hu et al., 1996, Proc
Natl
Acad Sci USA 93:1060-1064). Hsp90 has not been associated with the HIV life-
cycle.
Specific inhibitors of hsp90, e.g., anti-fungal macrolydes, geldanamycin and
radocicol
can be used against HIV-1 infection.
FK506-binding protein A1 is a potent immunosuppressive agent which is 100-
fold more active than cyclosporin A, a cyclic decapeptide that is used to
prevent
rejection after transplantation of bone marrow and organs, such as kidney,
heart, and
liver. It was shown that FK506 binds to a cellular protein distinct from
cyclophilin
which is known to bind cyclosporin A. A cDNA has been isolated from human
peripheral blood T-cells that encodes FK506-binding protein (FKBP) (Maki et
a1.,1990,
Proc Natl Acad Sci USA 87: 5440-5443). The isolated cDNA contained an open
reading
frame encoding 108 amino acid residues. The first 40 residues of the deduced
amino
acid sequence were identical to those of the reported amino-terminal sequence
ofbovine
FKBP, indicating that the DNA sequence isolated represents the gene coding for
FKBP.
It is expressed in brain, lung, liver, placental cells and leukocytes.
Induction of Jurkat
2 0 leukemic T cells with phorbol 12-myristate 13-acetate and ionomycin did
not affect the
level of FKBP mRNA.
Proteins of the Myc and Mad family are involved in transcriptional regulation
and mediate cell differentiation and proliferation. These molecules share a
basic-helix-
loop-helix leucine zipper domain (bHLHZip) and bind DNA at the E box (CANNTG)
2 5 consensus by forming heterodimers with Max (Meroni et al., 1997, EMBO J.
15-
16:2892-906). Rox transcriptional repressor heterodimerizes with Max and
weakly
homodimerizes. Interestingly, bandshift assays demonstrate that the Rox-Max
heterodimer shows a novel DNA binding specificity, having a higher affinity
for the
CACGCG site compared with the canonical E box CACGTG site. Transcriptional
3 0 studies indicate that Rox represses transcription in both human HEK293
cells and yeast.
Moreover, ROX expression appears to be induced in U937 myeloid leukemia cells
stimulated to differentiate with 12-O-tetradecanoylphorbol-13-acetate. These
data
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indicate that HIV-1 may use Rox to interfere with cellular transcription,
since an anti-
sense element derived from this gene is able to interfere with HIV-1
replication.
Human signal sequence receptor gene, S SR2, encodes an endoplasmic reticulum
(ER) membrane protein associated with protein translocation across the ER
membrane.
This gene has been cloned (Chinen et al., 1995, Cytogenet Cell Genet. 70:215-
217).
Northern blot analysis revealed its ubiquitous expression in all organs
examined (Chinen
et al., ibid.). The gene is located on chromosome bands 1q21 through q23.
Various
GSEs described herein affect expression of the ER proteins calnexin,
glucosidase alpha
and glucosyltransferase, and such expression has an inhibitory effect on HIV
replication.
The SSR2 gene inhibits HIV-1 replication in a similar manner, affecting
translocation
of gp160 into the ER.
A human tumorous imaginal disc protein, hTid-1, which is a homolog of the
Drosophila tumor suppressor protein Tid56, has been cloned (Schilling, 1998,
Virology
247:74-85). The hTid-1 protein is able to form complexes with the human
papillomavirus E7 oncoprotein. The carboxyl terminal cysteine-rich metal
binding
domain of E7 is the major determinant for interaction with hTid-1. The hTid-1
protein
is a member of the DnaJ-family of chaperones. The mRNA of hTid-1 is widely
expressed in human tissues, including the HPV-18-positive cervical carcinoma
cell line,
HeLa, and human genital keratinocytes, the normal host cells of the HPVs. The
hTid-1
2 0 gene has been mapped to the short arm of chromosome 16. The large tumor
antigens of
polyomaviruses encode functional J-domains that are important for viral
replication and
cellular transformation. The ability of HPV E7 to interact with a cellular
DnaJ protein
suggests that these two viral oncoproteins may target common regulatory
pathways
through J-domains (Schilling, ibid.).
2 5 Heparin sulfate proteoglycans are expressed on the cell surface and their
corresponding binding sites have been suggested to play an important role
during the
initial attachment of murine blastocysts to uterine epithelium, and human
trophoblastic
cell lines to uterine epithelial cell lines. Three major peptide fragments
from heparin-
binding protein have been isolated and partial amino-terminal amino acid
sequence for
3 0 each peptide fragment was obtained (Raboudi, et al., 1992, J. Biol. Chem.
267, 11930-
11939). A full-length cDNA for a heparin-binding protein, named HP/HS
interacting
protein (HIP), was cloned from RL95 cells (Liu et al., 1996, JBiol Chem.
271:11817-
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11823). The HIP cDNA encodes aprotein of 159 amino acids. Transfection ofHIP
full-
length cDNA into NIH-3T3 cells resulted in the cell surface expression of HIP
protein
having a size similar to HIP expressed by human cells. Predicted amino acid
sequence
indicates that HIP lacks a membrane spanning region and has no consensus sites
for
glycosylation. Northern blot analysis detected a single transcript of 1.3
kilobases in both
total RNA and poly(A+) RNA. Examination of human cell lines and normal tissues
using
both Northern blot and Western blot analyzes revealed that HIP is expressed at
different
levels in a variety of human cell lines and normal tissues.
The minimal size of a nucleic acid molecule of the present invention is the
size
required for the use of the nucleic acid molecule to inhibit HIV replication.
One of skill
in the art will recognize that the length can differ if the nucleic acid
molecule is in the
form of RNA or DNA. A nucleic acid molecule of the present invention can be
used in
a variety of applications including, but not limited to, as probes to identify
additional
nucleic acid molecules, as primers to amplify or extend nucleic acid molecules
or in
therapeutic applications to inhibit, for example, expression of a target gene
ofthe present
invention. Such therapeutic applications include the use of such nucleic acid
molecules
in, for example, anti-sense-RNA and -DNA molecules, triplex formation-,
ribozyme-
and/or RNA drug-based technologies, that function to inhibit HIV infection,
and are
referred to herein as "therapeutic nucleic acid molecules." For example, anti-
sense RNA
2 0 and DNA molecules can be used to directly block the translation of mRNA
encoded by
these cellular genes by binding to targeted mRNA and preventing protein
translation.
Polydeoxyribonucleotides can form sequence-specific triple helices by hydrogen
bonding to specific complementary sequences in duplexed DNA. Formation of
specific
triple helices can selectively inhibit the replication and/or expression of
targeted genes
2 5 by prohibiting the specific binding of functional trans-acting factors.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific
cleavage of RNA. The mechanism of ribozyme action involves sequence-specific
hybridization of the ribozyme molecule to complementary target RNA, followed
by
endonucleolytic cleavage. Within the scope of the invention are engineered
3 0 hammerhead motif ribozyme molecules that specifically and efficiently
catalyze
endonucleolytic cleavage of cellular RNA sequences. Antisense RNA showing high
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affinity binding to target sequences can also be used as ribozymes by addition
of
enzymatically active sequences known to those skilled in the art.
Nucleic acid molecules to be used in triplex helix formation should be single
stranded and composed of deoxynucleotides. The base composition of these
nucleic acid
molecules must be designed to promote triple helix formation via Hoogsteen
base
pairing rules, which generally require sizeable stretches of either purines or
pyrimidines
to be present on one strand of a duplex. Nucleic acid molecule sequences can
be
pyrimidine-based, which will result in TAT and CGC triplets across the three
associated
strands of the resulting triple helix. The pyrimidine-rich polynucleotides
provide base
complementarity to a purine-rich region of a single strand of the duplex in a
parallel
orientation to that strand. In addition, nucleic acid molecules can be chosen
that are
purine-rich, for example, containing a stretch of G residues. These nucleic
acid
molecules will form a triple helix with a DNA duplex that is rich in GC pairs,
in which
the maj ority of the purine residues are located on a single strand of the
targeted duplex,
resulting in GGC triplets across the three strands in the triplex.
Alternatively, the potential sequences that can be targeted for triple helix
formation can be increased by creating a so called "switchback"
polynucleotide.
Switchback polynucleotides are synthesized in an alternating 5'-3', 3'-5'
manner, such
that they base pair with first one strand of a duplex and then the other,
eliminating the
2 0 necessity for a sizeable stretch of either purines or pyrimidines to be
present on one
strand of a duplex.
Both anti-sense RNA and DNA molecules, and ribozymes of the invention can
be prepared by any method known in the art. These include techniques for
chemically
synthesizing polydeoxyribonucleotides well known in the art such as solid
phase
2 5 phosphoramidite chemical synthesis. Alternatively, RNA molecules can be
generated
by in vitro and in vivo transcription of DNA sequences encoding the antisense
RNA
molecule. Such DNA sequences can be incorporated into a wide variety of
vectors
which incorporate suitable RNA polymerise promoters such as the T7 or SP6
polymerise promoters. Alternatively, antisense cDNA constructs that synthesize
3 0 antisense RNA constitutively or inducibly, depending on the promoter used,
can be
introduced stably into host cells.
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The present invention, therefore, includes such therapeutic nucleic acid
molecules and methods to interfere with the production of proteins encoded by
a target
gene necessary for HIV infection of the present invention by use of one or
more of such
technologies.
A preferred therapeutic nucleic acid molecule comprises at least a portion of
a
target gene encoding a protein selected from the group consisting of NADH
dehydrogenase, 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase/
cytosolic
thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3,
eukaryotic
initiation factor 3, protein tyrosine phosphatase, herpesvirus-associated
ubiquitin-
specific protease, eukaryotic initiation factor 4B, CD44, phosphatidyl-
inositol 3 kinase,
elongation factor 1 alpha, bone morphogenic protein-1, double-strand break DNA
repair
gene protein, rat guanine nucleotide releasing protein, anti-proliferative
factor (BTG-1 ),
lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase,
eukaryotic
release factor 1, GTP binding protein, importin beta subunit, cell adhesion
molecule L1,
U-snRNP associated cyclophilin, recepin, Arg/Abl interacting protein
(ArgBP2A),
keratin related protein, p18 protein, p40 protein, glucosidase II, alpha
enolase,
macrophage inflammatory protein 1 alpha, tumor protein translationally-
controlled 1
(TCTP1), BBC1, Nef interacting protein, Na+-D-glucose cotransport regulator
gene
protein, hsp90 chaperone protein, FK506-binding protein A1, Rox, beta signal
sequence
2 0 receptor, tumorous imaginal disc protein or cell surface heparin binding
protein, or
homologs thereof, in which the portion is capable of down-regulating the
expression of
the associated target gene. A more preferred therapeutic nucleic acid molecule
is a GSE
nucleic acid molecule of the present invention, particularly comprising SEQ ID
NO: l,
SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID
2 5 N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:1 l, SEQ ID N0:12,
SEQ ID N0:13, SEQ ID N0:14, SEQ ID NO:15, SEQ ID N0:16, SEQ ID N0:17, SEQ
ID N0:18, SEQ II7 N0:19, SEQ ID N0:20, SEQ ID N0:25, SEQ ID N0:27, SEQ ID
N0:29, SEQ ID N0:31, SEQ )D N0:33, SEQ ID N0:35, SEQ ID N0:37, SEQ ID
N0:39, SEQ ID N0:41, SEQ ID N0:43, SEQ ID N0:45, SEQ )D N0:47, SEQ ID
3 0 N0:49, SEQ ID NO:51, SEQ ID N0:53, SEQ ID NO:55, SEQ 117 N0:57, SEQ ID
N0:59, SEQ >D N0:61, SEQ ID N0:63, SEQ ID N0:65, SEQ ID N0:67, SEQ ID
N0:69; SEQ ID N0:71; SEQ ID N0:73; SEQ ID N0:75; SEQ ID N0:77, SEQ ID
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N0:79, SEQ >D N0:81, SEQ m N0:83, SEQ JD N0:85; SEQ m N0:87; SEQ >D
N0:89; SEQ >D N0:91; SEQ m N0:93 and/or SEQ >D N0:95, or other GSE sequences
disclosed herein, as well as complements of any of these sequences or homologs
thereof.
The present invention also includes a recombinant vector, which includes a
nucleic acid molecule of the present invention inserted into any vector
capable of
delivering the nucleic acid molecule into a host cell. Such a vector contains
heterologous nucleic acid sequences, that is nucleic acid sequences that are
not naturally
found adjacent to a cell-derived nucleic acid molecule of the present
invention. The
vector can be either RNA or DNA, either prokaryotic or eukaryotic, and
typically is a
virus or a plasmid. Recombinant vectors can be used in the cloning,
sequencing, and/or
otherwise manipulating of nucleic acid molecules of the present invention. One
type of
recombinant vector, herein referred to as a recombinant expression molecule
and
described in more detail below, can be used in the expression of nucleic acid
molecules
of the present invention. Preferred recombinant vectors are capable of
replicating in the
transformed cell.
A preferred nucleic acid molecule to include in a recombinant vector of the
present invention is a cell-derived nucleic acid molecule of the present
invention. A
particularly preferred nucleic acid molecule to include in a recombinant
vector is at least
2 0 a portion of a target gene that encodes a protein selected from the group
consisting of
NADH dehydrogenase, 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase/
cytosolic thyroid hormone binding protein, calnexin, ADP-ribosylation factor
3,
eukaryotic initiation factor 3, protein tyrosine phosphatase, herpesvina-
associated
ubiquitin-specific protease, eukaryotic initiation factor 4B, CD44,
phosphatidyl-inositol
2 5 3 kinase, elongation factor 1 alpha, bone morphogenic protein-1, double-
strand break
DNA repair gene protein, rat guanine nucleotide releasing protein, anti-
proliferative
factor (BTG-1), lymphocyte-specific protein 1, protein phosphatase 2A,
squalene
synthetase, eukaryotic release factor 1, GTP binding protein, importin beta
subunit, cell
adhesion molecule L1, U-snRNP associated cyclophilin, recepin, Arg/Abl
interacting
30 protein (ArgBP2A), keratin related protein, p18 protein, p40 protein,
glucosidase II,
alpha enolase, macrophage inflammatory protein 1 alpha, tumor protein
translationally-controlled 1 (TCTPl), BBC1, Nef interacting protein, Na+-D-
glucose
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cotransport regulator gene protein, hsp90 chaperone protein, FK506-binding
protein A1,
Rox, beta signal sequence receptor, tumorous imaginal disc protein or cell
surface
heparin binding protein, or complements or homologs thereof. Other preferred
nucleic
acid molecules to include in a recombinant vector is a GSE nucleic acid
molecule having
a nucleic acid sequence selected from the group consisting of SEQ ~ NO:1, SEQ
m
N0:2, SEQ ID N0:3, SEQ >D N0:4, SEQ >D NO:S, SEQ m N0:6, SEQ m N0:7, SEQ
>D N0:8, SEQ m N0:9, SEQ m NO:10, SEQ >D NO:11, SEQ >D N0:12, SEQ m
N0:13, SEQ m N0:14, SEQ 1D NO:15, SEQ ~ N0:16, SEQ m N0:17, SEQ m
N0:18, SEQ >D N0:19, SEQ m N0:20, SEQ m N0:25, SEQ 1D N0:27, SEQ )D
N0:29, SEQ m N0:31, SEQ 117 N0:33, SEQ m N0:35, SEQ 1D N0:37, SEQ m
N0:39, SEQ B7 N0:41, SEQ >D N0:43, SEQ ~ N0:45, SEQ ~ N0:47, SEQ m
N0:49, SEQ ~ NO:S 1, SEQ >D N0:53, SEQ ~ NO:SS, SEQ m N0:57, SEQ 1D
N0:59, SEQ m N0:61, SEQ ~ N0:63, SEQ ~ N0:65, SEQ ID N0:67, SEQ B7
N0:69; SEQ m N0:71; SEQ m N0:73; SEQ m N0:75; SEQ m N0:77, SEQ m
N0:79, SEQ >D N0:81, SEQ )D N0:83, SEQ m N0:85; SEQ m N0:87; SEQ ID
N0:89; SEQ B7 N0:91; SEQ m N0:93 or SEQ m N0:95, or other GSE sequences
disclosed herein, as well as complements of any of these sequences or homologs
thereof.
A recombinant expression molecule of the present invention comprises one or
more nucleic acid molecules of the present invention operably linked to an
expression
2 0 vector containing one or more regulatory sequences. The phrase "operably
linked" refers
to insertion of a nucleic acid molecule into an expression vector in a manner
such that
the molecule is able to be expressed when transformed into a host cell. As
used herein,
an expression vector is a DNA or RNA vector that is capable of transforming a
host cell
and of effecting expression of a specified nucleic acid molecule. Preferably,
the
2 5 expression vector is also capable of replicating within the host cell.
Expression vectors
can be either prokaryotic or eukaryotic, and are typically viruses or
plasmids.
Expression vectors of the present invention include any vectors that function
(i. e., direct
gene expression) in recombinant cells of the present invention, including in
bacterial,
fungal, insect, animal, and/or plant cells. As such, nucleic acid molecules of
the present
3 0 invention can be operably linked to expression vectors containing
regulatory sequences
such as promoters, operators, repressors, enhancers, termination sequences,
origins of
replication, and other regulatory sequences that are compatible with the
recombinant cell
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and that control the expression of nucleic acid molecules of the present
invention. As
used herein, a regulatory sequence includes a sequence which is capable of
controlling
the initiation, elongation, and termination of transcription. Particularly
important
regulatory sequences are those which control transcription initiation, such as
promoter,
enhancer, operator and repressor sequences. Suitable regulatory sequences
include any
transcription control sequence that can function in a cell susceptible to
infection by HIV.
A variety of such transcription control sequences are known to those skilled
in the art.
Additional suitable regulatory sequences include tissue-specific promoters and
enhancers
as well as lymphokine-inducible promoters (e.g., promoters inducible by
interferons or
interleukins). Regulatory sequences of the present invention can also include
naturally-
occurnng transcription control sequences naturally associated with a DNA
sequence
encoding a nucleic acid molecule of the present invention. In cases where a
recombinant
molecule of the present invention is expressed as a protein, specific
initiation signals can
be required for efficient translation of inserted nucleic acid molecules.
Exogenous
translational control signals, including the ATG initiation codon, need to be
provided.
Furthermore, the initiation codon must be in phase with the reading frame of
the inserted
nucleic acid molecule to ensure translation of the entire insert. These
exogenous
translational control signals and initiation codons can be of a variety of
origins, both
natural and synthetic.
2 0 Preferred expression vectors of the present invention are derived from
viruses
such as retroviruses, adenovirus, adeno-associated virus, herpes viruses, or
papilloma
viruses. Methods which are well known to those skilled in the art can be used
to
construct a recombinant molecule of the present invention (Sambrook et al.,
1989,
MOLECULAR CLONING: ALABORATORYMANUAL, Cold Spring Harbor Laboratory, N.Y.
2 5 and Ausubel et al., 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene
Publishing Associates and Wiley Interscience, N.Y.). In cases where an
adenovirus is
used as an expression vector, a nucleic acid molecule of the present invention
can be
ligated to an adenovirus transcription-translation control complex, e.g., the
late promoter
and tripartite leader sequence. This chimeric gene can then be inserted in the
adenovirus
3 0 genome by in vitro or in vivo recombination. Insertion in a non-essential
region of the
viral genome, e.g., region El or E3, will result in a recombinant virus that
is viable and
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capable of expressing GSEs in infected hosts (Logan & Shenk, 1984, Proc. Natl.
Acad.
Sci. USA 81:3655-3659).
It will be appreciated by one skilled in the art that use of recombinant DNA
technologies can improve expression of transformed nucleic acid molecules by
manipulating, for example, the number of copies of the nucleic acid molecules
within
a host cell, the efficiency with which those nucleic acid molecules are
transcribed, the
efficiency with which the resultant transcripts are translated, and the
efficiency of
post-translational modifications. Recombinant techniques useful for increasing
the
expression of nucleic acid molecules of the present invention include, but are
not limited
to, operably linking nucleic acid molecules to high-copy number plasmids,
integration
of the nucleic acid molecules into one or more host cell chromosomes, addition
of vector
stability sequences to plasmids, substitutions or modifications of
transcription control
signals (e.g., promoters, operators, enhancers), substitutions or
modifications of
translational control signals (e.g., ribosome binding sites, Shine-Dalgarno
sequences),
modification of nucleic acid molecules of the present invention to correspond
to the
codon usage of the host cell, deletion of sequences that destabilize
transcripts, and use
of control signals that temporally separate recombinant cell growth from
recombinant
protein production during fermentation. The activity of an expressed
recombinant
protein of the present invention can be improved by fragmenting, modifying, or
2 0 derivatizing the resultant protein.
Various modifications to the nucleic acid molecules can be introduced as a
means of increasing intracellular stability and half life. Possible
modifications include,
but are not limited to, the addition of flanking sequences of ribonucleotides
or
deoxyribonucleotides to the 5' and/or 3' ends of the molecule or the use of
2 5 phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages
within the
polydeoxyribonucleotide backbone.
One embodiment of the present invention is an isolated protein that is
necessary
for HIV infection. Such a protein is referred to herein as a "cell-derived
protein." As
used herein, the term "protein" is intended to include both polypeptides and
peptides.
3 0 Preferably, the invention provides peptides or less than full-length
fragments of such
cell-derived proteins of the invention, wherein expression of said proteins in
an HIV-
susceptible cell inhibits HIV infection, replication or production of HIV
progeny virus.
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Also encompassed by this definition are full-length proteins having at least
one change
in a constituent amino acid residue whereby the protein no longer supports HIV
infection
in said cell, and most preferably wherein the altered protein inhibits HIV
infection,
replication or production of HIV progeny virus. According to the present
invention, an
isolated, or biologically pure, protein is a protein that has been removed
from its natural
milieu. As such, "isolated" and "biologically pure" do not necessarily reflect
the extent
to which the protein has been purified. An isolated cell-derived protein of
the present
invention can be obtained from its natural source, can be produced using
recombinant
DNA technology or can be produced by chemical synthesis. Preferably, an
isolated cell-
derived protein of the present invention comprises at least a portion of a
protein selected
from the group consisting ofNADH dehydrogenase, 2-oxoglutarate dehydrogenase,
M2-
type pyruvate kinase/ cytosolic thyroid hormone binding protein, calnexin, ADP-
ribosylation factor 3, eukaryotic initiation factor 3, protein tyrosine
phosphatase,
herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation
factor 4B, CD44,
phosphatidyl-inositol 3 kinase, elongation factor 1 alpha, bone morphogenic
protein-1,
double-strand break DNA repair gene protein, rat guanine nucleotide releasing
protein,
anti-proliferative factor (BTG-1), lymphocyte-specific protein l, protein
phosphatase
2A, squalene synthetase, eukaryotic release factor l, GTP binding protein,
importin beta
subunit, cell adhesion molecule Ll, U-snRNP associated cyclophilin, recepin,
Arg/Abl
2 0 interacting protein (ArgBP2A), keratin related protein, p 18 protein, p40
protein,
glucosidase II, alpha enolase, macrophage inflammatory protein 1 alpha, tumor
protein
translationally-controlled 1 (TCTP1), BBCI, Nef interacting protein, Na+-D-
glucose
cotransport regulator gene protein, hsp90 chaperone protein, FK506-binding
protein A1,
Rox, beta signal sequence receptor, tumorous imaginal disc protein or cell
surface
2 5 heparin binding protein or any homolog of such a protein. An isolated
protein of the
present invention, including a homolog, can be identified in a straight-
forward manner
by the protein's ability to inhibit HIV infection. Examples of homologs
include proteins
in which amino acids have been deleted (e.g., a truncated version of the
protein, such as
a peptide), inserted, inverted, substituted and/or derivatized (e.g., by
glycosylation,
3 0 phosphorylation, acetylation, myristoylation, prenylation, palmitoylation,
amidation
and/or addition of glycerophosphatidyl inositol) such that the homolog has at
least some
ability to inhibit HIV infection, wherein expression of the wildtype protein
is necessary
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for HIV infection. Homologs also include at least one epitope capable of
eliciting an
immune response against a cell-derived protein. Techniques to measure HIV
infection
or the inhibition thereof, are disclosed herein.
Cell-derived protein homologs of the present invention can be the result of
natural allelic variation or natural mutation. Cell-derived protein homologs
of the
present invention can also be produced using techniques known in the art
including, but
not limited to, direct modifications to the protein or modifications to the
gene encoding
the protein using, for example, classic or recombinant nucleic acid techniques
to effect
random or targeted mutagenesis.
The minimal size of a cell-derived protein homolog of the present invention is
a size sufficient to function as an inhibitor of HIV infection. The minimal
size of such
homolog is typically at least about 6 to about 10 residues in length. There is
no limit,
other than a practical limit, on the maximal size of such a homolog in that
the protein
homolog can include a peptide, a less than full length fragment of a full-
length protein,
a full-length protein, multiple proteins, or portions thereof, wherein said
full-length
proteins most preferably comprise an altered amino acid sequence whereby the
altered
protein inhibits HN infection, replication or production of infectious virus.
The present invention also includes mimetopes of cell-derived proteins of the
present invention. As used herein, a mimetope of a cell-derived protein of the
present
2 0 invention refers to any compound that is able to mimic the activity of
such a cell-derived
protein (e.g., abilityto inhibit HIV infection), often because the mimetope
has a structure
that mimics the cell-derived protein. It is to be noted, however, that the
mimetope need
not have a chemical structure similar to a cell-derived protein as long as the
mimetope
functionally mimics the protein. Mimetopes can be, but are not limited to:
peptides that
2 5 have been modified to decrease their susceptibility to degradation; anti-
idiotypic and/or
catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic
portions of
an isolated protein (e.g., carbohydrate structures); synthetic or natural
organic or
inorganic molecules, including nucleic acids; and/or any other peptidomimetic
compounds. Mimetopes of the present invention can be designed using computer-
3 0 generated structures of cell-derived proteins of the present invention.
Mimetopes can
also be obtained by generating random samples of molecules, such as
polynucleotides,
peptides or other organic molecules, and screening such samples by affinity
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chromatography techniques using the corresponding binding partner. A preferred
mimetope is a peptidomimetic compound that is structurally and/or functionally
similar
to a cell-derived protein of the present invention.
One embodiment of a cell-derived protein of the present invention is a fusion
protein that includes a cell-derived protein-containing domain attached to one
or more
fusion segments. Suitable fusion segments are known to those of skill in the
art
depending upon the use of the segment, such as for protein stability or
protein delivery
into a cell. Fusion proteins are preferably produced by culturing a
recombinant cell
transformed with a fusion nucleic acid molecule that encodes a cell-derived
protein
including the fusion segment.
Isolated cell-derived proteins of the present invention have the further
characteristic of being encoded by a cell-derived nucleic acid molecule of the
present
invention. A preferred cell-derived protein of the present invention is
encoded by a
nucleic acid molecule having a nucleic acid sequence selected from the group
consisting
of SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID
N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ
ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID N0:20, SEQ ID N0:25, SEQ ID
N0:27, SEQ ID N0:29, SEQ ID N0:31, SEQ ID N0:33, SEQ ID N0:35, SEQ ID
2 o N0:37, SEQ ID N0:39, SEQ ID N0:41, SEQ ID N0:43, SEQ ID N0:45, SEQ ID
N0:47, SEQ ID N0:49, SEQ ID N0:51, SEQ ID N0:53, SEQ ID N0:55, SEQ ID
N0:57, SEQ ID N0:59, SEQ ID N0:61, SEQ ID N0:63, SEQ ID N0:65, SEQ ID
N0:67, SEQ ID N0:69; SEQ ID N0:71; SEQ ID N0:73; SEQ ID N0:75; SEQ ID
N0:77, SEQ ID N0:79, SEQ ID N0:81, SEQ ID N0:83, SEQ ID N0:85; SEQ ID
2 5 N0:87; SEQ ID N0:89; SEQ ID N0:91; SEQ ID N0:93 and/or SEQ ID N0:95, or
other
sequences disclosed herein, as well as complements of any of these sequences
or
homologs thereof.
An isolated GSE nucleic acid molecule of the present invention can suppress
HIV activity by either encoding a protein or RNA product. The present
invention
3 0 encompasses any such protein product, including a fusion protein, a leader
peptide and
a localization signal.
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One embodiment of the present invention is an inhibitory composition that,
when administered to an animal in an effective manner, is capable of
protecting that
animal from HIV infection, prophylactically or therapeutically. An inhibitory
composition of the present invention can include a protective compound that
down-
s regulates the expression of a gene that encodes NADH dehydrogenase, 2-
oxoglutarate
dehydrogenase, M2-type pyruvate kinase/ cytosolic thyroid hormone binding
protein,
calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, protein
tyrosine
phosphatase, herpesvirus-associated ubiquitin-specific protease, eukaryotic
initiation
factor 4B, CD44, phosphatidyl-inositol 3 kinase, elongation factor 1 alpha,
bone
morphogenic protein-1, double-strand break DNA repair gene protein, rat
guanine
nucleotide releasing protein, anti-proliferative factor (BTG-1), lymphocyte-
specific
protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic release
factor l, GTP
binding protein, importin beta subunit, cell adhesion molecule Ll, U-snRNP
associated
cyclophilin, recepin, Arg/Abl interacting protein (ArgBP2A), keratin related
protein, p 18
protein, p40 protein, glucosidase II, alpha enolase, macrophage inflammatory
protein 1
alpha, tumor protein translationally-controlled 1 (TCTP1), BBC1, Nef
interacting
protein, Na+-D-glucose cotransport regulator gene protein, hsp90 chaperone
protein,
FK506-binding protein A1, Rox, beta signal sequence receptor, tumorous
imaginal disc
protein or cell surface heparin binding protein. Such a protective compound
comprises
2 0 an isolated cell-derived nucleic acid molecule of the present invention
operably linked
to a regulatory sequence that controls its expression. The protective compound
can be
an RNA- or DNA-based molecule as described herein. Particularly preferred are
GSE
nucleic acid molecules of the present invention. A functionally-active
fragment of a
GSE, and a GSE containing conservative nucleotide substitutions as functional
2 5 equivalents of a GSE, are within the scope of the present invention.
Preferably, a GSE
nucleic acid molecule, or a functional equivalent thereof, is operably linked
to a
regulatory sequence that controls its expression.
An inhibitory composition of the present invention can include a protective
compound that inhibits the activity of a product of a gene that encodes NADH
3 0 dehydrogenase, 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase/
cytosolic
thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3,
eukaryotic
initiation factor 3, protein tyrosine phosphatase, herpesvirus-associated
ubiquitin-
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specific protease, eukaryotic initiation factor 4B, CD44, phosphatidyl-
inositol 3 kinase,
elongation factor 1 alpha, bone morphogenic protein-1, double-strand break DNA
repair
gene protein, rat guanine nucleotide releasing protein, anti-proliferative
factor (BTG-1 ),
lymphocyte-specific protein l, protein phosphatase 2A, squalene synthetase,
eukaryotic
release factor 1, GTP binding protein, importin beta subunit, cell adhesion
molecule L1,
U-snRNP associated cyclophilin, recepin, Arg/Abl interacting protein
(ArgBP2A),
keratin related protein, p 18 protein, p40 protein, glucosidase II, alpha
enolase,
macrophage inflammatory protein 1 alpha, tumor protein translationally-
controlled 1
(TCTP1), BBC1, Nef interacting protein, Na+-D-glucose cotransport regulator
gene
protein, hsp90 chaperone protein, FK506-binding protein A1, Rox, beta signal
sequence
receptor, tumorous imaginal disc protein or cell surface heparin binding
protein. Such
a protective compound can include an isolated cell-derived protein of the
present
invention, in particular a peptide of a cell-derived protein, a mimetope of a
cell-derived
protein, and small molecule inhibitors of the activity of target gene
products. Examples
of protective compounds, e.g., proteins, mimetopes, nucleic acid molecules,
and
inhibitors, are disclosed herein. It is within the scope of the present
invention that an
inhibitory composition can contain one or more protective compounds.
Suitable inhibitors of the activity of target gene products include compounds
that interact directly with the active sites of such products, usually by
binding to or
2 0 otherwise interacting with or otherwise modifying the products's active
site. Product
inhibitors can also interact with other regions of the product to inhibit its
activity, for
example, by allosteric interaction. The inhibitor of a product is identified
by its ability
to bind to, or otherwise interact with the product, thereby inhibiting the
activity of the
product and/or HIV infection.
2 5 In the case of NADH dehydrogenase, a large number of small molecule
inhibitors are available in the art. Such inhibitors can be used in the
methods of the
present invention. An in vitro assay can be established to screen for
additional NADH
dehydrogenase complex I inhibitors by measuring membrane potential of live
cells with
DiC6, a dye which accumulates in the mitochondria) and cytoplasmic membrane
3 0 depending on mitochondria) functional activities and ATP concentrations
(Methods in
Enz~molo~y, 1995, 260:448). Compounds are selected for their ability to
decrease
membrane potential by methods well known in the art. Examples of NADH
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dehydrogenase inhibitors include, but are not limited to, Amytal, Annonin VI,
Aurachin
A, Aurachin B, Aureothin, Benzimidazole, Bullactin, Capsaicin, Ethoxyformic
anhydride, Ethoxyquin, Fenpyroximate, Mofarotene (Ro 40-8757; arotinoids),
Molvizarin, Myxalamide PI, Otivarin (annonaceous acetogenins), Pethidine,
Phenalamid A2, Phenoxan, Piericidin A, p-chloromercuribenzoate, Ranolazine (RS-
43285), Rolliniasatin-1, Rolliniasatin-2, Rotenone, Squamocin, and Thiangazole
(Singer
et al., 1992, Mol. Mechan. in Bioenergetics, Chap. 6, p.153; Degli et al.,
1994, Biochem.
J. 301:161; Friedrich et al., 1994, Eur. J. Biochem. 219:691; Uchida et al.,
1994, Int. J.
Cancer 58:891; Wyatt et al., 1995, Biochem. Pharmacol. 50:1599; Shimomura et
al.,
1989, Arch. Biochem Biophy. 270:573).
Examples of inhibitors of 2-oxoglutarate dehydrogenase have been described
by Majamaa et al., 1985, Biochem. J. 229:127-133.
Examples of inhibitors of protein tyrosine phosphatase include, but are not
limited to,
ortho-vanadate, pervanadate, sodium vanadate, and phenylarsine oxide.
Inhibitors of
protein tyrosine phosphatase have been described by Yousefi et al., 1994,
Proc. Natl.
Acad. Sci. USA 91:10868-10872 and Lund-Johansen et a1.,1996, Cytometry 25:182-
190.
Examples of inhibitors of EF-1 cc include, but are not limited to, include
quercetin
(3,3',4'5,7-pentahydrozyflavone) and didemnin B.
Once a cellular gene has been identified as a potentially important target for
2 0 supporting the HIV life cycle, assay systems can be established using such
gene for
screening and selection of additional compounds as anti-HIV therapeutics based
on their
ability to down-regulate the expression of the gene or inhibit the activities
of its gene
product. For example, a cell line which naturally expresses the gene of
interest or has
been transfected with it can be incubated with various compounds. A reduction
of the
2 5 expression of the gene of interest or an inhibition of the activities of
its encoded product
can be used as an indication that the compound is effective in inhibiting
expression
and/or the function of said gene. The compounds are retested in other assays
such as in
OM10.1 cells or in productive HIV infection to confirm their activities
against HN
infection. These compounds can be screened from known organic compounds,
products
3 0 of peptide libraries and products of chemical combinatorial libraries.
One embodiment of the present invention is a method to identify a compound
capable of inhibiting HIV infection. Such a method includes the steps of (a)
contacting,
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e.g., combining or mixing, an isolated cell-derived protein with a putative
inhibitory
compound under conditions in which, in the absence of the compound, the
protein has
activity, and (b) determining if the putative inhibitory compound inhibits the
activity of
the cell-derived protein. Putative inhibitory compounds to screen include
small organic
molecules, antibodies (including mimetopes thereof) and substrate analogs.
Methods
to determine the activity if a cell-derived protein are known to those skilled
in the art.
According to the present invention, cell-derived nucleic acid molecules, in
particular GSE nucleic acid molecules, can be used to design polypeptides or
peptides
capable of inhibiting HIV infection. A method to test inhibitorypolypeptides
or peptides
for use as a therapeutic compound can include the steps of (a) delivering a
putative
inhibitory peptide to a cell susceptible to HIV infection; and (b) determining
the ability
of such peptide to inhibit HIV infection. Methods to deliver and determine HIV
infection are disclosed herein.
One preferred embodiment of the present invention is the use of protective
compounds of the present invention to protect an animal from HIV infection.
Preferred
protective compounds of the present invention have been disclosed herein.
Additional
protection can be obtained by administering additional protective compounds,
including
other reagents known to inhibit HIV infection.
A protective compound comprising a cell-derived nucleic acid molecule can be
2 0 transferred into any HIV-susceptible host cells such as CD4+ T cells or
hematopoietic
progenitor cells such as CD34+ cells obtained from bone marrow or mobilized
peripheral
blood, by any DNA transfer techniques well known in the art such as
electroporation,
transfection or transduction, followed by transplantation of the cells into a
recipient.
When the cell-derived nucleic acid molecule-containing progenitor cells
differentiate in
2 5 vivo, the progeny cells express the nucleic acid molecule product and
become resistant
to HIV. Preferably, such cell-derived nucleic acid molecule is a GSE nucleic
acid
molecule of the present invention.
Alternatively, a cell-derived nucleic acid molecule can be directly
administered
in vivo using a gene therapy expression vector. In particular, the molecules
can be
3 0 delivered or transferred into CD4+ T cells in both HIV-infected or
uninfected individuals
to protect against development of HIV infection. The recombinant molecules can
also
be transferred into stromal cells, including macrophages.
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Alternatively, a cell-derived nucleic acid molecule can be delivered into a
target
cell by a non-viral delivery system. For example, a GSE nucleic acid molecule
can be
reconstituted into liposomes for delivery to susceptible cells. Liposomes are
spherical
lipid bilayers with aqueous interiors. All molecules that are present in an
aqueous
solution at the time of liposome formation (in this case, oligonucleotides)
are
incorporated into this aqueous interior. The liposomal contents are both
protected from
the external microenvironment and, because liposomes fuse with cell membranes,
are
efficiently delivered into the cell cytoplasm, obviating the need to
neutralize the
oligonucleotides' negative charge.
Methods for introducing cell-derived nucleic acid molecules into cells or
tissues
include the insertion of naked nucleic acid molecule, i.e. by injection into
tissue, the
introduction of a GSE in a cell ex vivo, i.e., for use in autologous cell
therapy, the use
of a vector such as a virus, retrovirus, phage or plasmid, etc. or techniques
such as
electroporation which can be used in vivo or ex vivo.
Protective compounds of the present invention can be formulated and
administered through a variety of means, including systemic, localized, or
topical
administration. Techniques for formulation and administration can be found in
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA. The
mode
of administration can be selected to maximize delivery to a desired target
site in the
2 o body.
For systemic administration, route of injection include, but are not limited
to,
intramuscular, intravenous, intraperitoneal, and subcutaneous. The cell-
derived nucleic
acid molecules of the invention are formulated in aqueous solutions,
preferably in
physiologically compatible buffers such as Hanks's solution, Ringer's
solution, or
2 5 physiological saline buffer. In addition, the cell-derived nucleic acid
molecules can be
formulated in solid or lyophilized form, then redissolved or suspended
immediatelyprior
to use.
Protective compounds that inhibit the expression of genes encoding NADH
dehydrogenase, 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase/
cytosolic
3 0 thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3,
eukaryotic
initiation factor 3, protein tyrosine phosphatase, herpesvirus-associated
ubiquitin-
specific protease, eukaryotic initiation factor 4B, CD44, phosphatidyl-
inositol 3 kinase,
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elongation factor 1 alpha, bone morphogenic protein-1, double-strand break DNA
repair
gene protein, rat guanine nucleotide releasing protein, anti-proliferative
factor (BTG-1 ),
lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase,
eukaryotic
release factor 1, GTP binding protein, importin beta subunit, cell adhesion
molecule L1,
U-snRNP associated cyclophilin, recepin, Arg/Abl interacting protein
(ArgBP2A),
keratin related protein, p18 protein, p40 protein, glucosidase II, alpha
enolase,
macrophage inflammatory protein 1 alpha, tumor protein translationally-
controlled 1
(TCTP1), BBCl, Nef interacting protein, Na+-D-glucose cotransport regulator
gene
protein, hsp90 chaperone protein, FK506-binding protein A1, Rox, beta signal
sequence
receptor, tumorous imaginal disc protein and cell surface heparin binding
protein, as
well as inhibitors of their encoded products, can be administered to a human
patient in
need of such treatment, by themselves, or in pharmaceutical compositions where
an
inhibitor is mixed with suitable carriers or excipient(s). A therapeutically
effective dose
refers to that amount of the compound sufficient to result in an inhibition of
HIV
infection as compared to the pre-treatment condition. Techniques for
formulation and
administration of the compounds of the instant application can be found in
"Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, PA.
Suitable routes of administration can, for example, include oral, rectal,
transmucosal, transcutaneous, or intestinal administration; parenteral
delivery, including
2 o intramuscular, subcutaneous, intramedullary injections, as well as
intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or intraocular
injections.
Alternatively, one can administer the compound in a local rather than systemic
manner,
for example, via injection of the compound directly into a specific tissue,
often in a
depot or sustained release formulation.
2 5 Furthermore, one can administer the compound in a targeted drug delivery
system, for example, in a liposome and/or conjugated with a cell-specific
antibody. The
liposomes and cell-specific antibody will be targeted to and taken up
selectivelyby HIV-
infected cells.
The pharmaceutical compositions of the present invention can be manufactured
3 0 in a manner that is itself known, e.g., by means of a conventional mixing,
dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping
or
lyophilizing processes.
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Pharmaceutical compositions for use in accordance with the present invention
thus can be formulated in conventional manner using one or more
physiologically
acceptable Garners comprising excipients and auxiliaries which facilitate
processing of
the active compounds into preparations which can be used pharmaceutically.
Proper
formulation is dependent upon the route of administration chosen.
For inj ection, the compounds of the invention can be formulated in
appropriate
aqueous solutions, such as physiologically compatible buffers such as Hanks's
solution,
Ringer's solution, or physiological saline buffer. For transmucosal and
transcutaneous
administration, penetrants appropriate to the barrier to be permeated are used
in the
formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readilyby combining
the active compounds with pharmaceutically acceptable Garners well known in
the art.
Such Garners enable the compounds of the invention to be formulated as
tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like,
for oral
ingestion by a patient to be treated. Pharmaceutical preparations for oral use
can be
obtained with solid excipient, optionally grinding a resulting mixture, and
processing the
mixture of granules, after adding suitable auxiliaries, if desired, to obtain
tablets or
dragee cores. Suitable excipients are, in particular, fillers such as sugars,
including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize
2 0 starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth,
methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added,
such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof
such as sodium
alginate.
2 5 Dragee cores are provided with suitable coatings. For this purpose,
concentrated
sugar solutions can be used, which can optionally contain gum arabic, talc,
polyvinyl
pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide,
lacquer
solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments can
be added to the tablets or dragee coatings for identification or to
characterize different
3 0 combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of gelatin, as well as soft, sealed capsules made of gelatin and a
plasticizer, such
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as glycerol or sorbitol. The push-fit capsules can contain the active
ingredients in
admixture with filler such as lactose, binders such as starches, and/or
lubricants such as
talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the
active
compounds can be dissolved or suspended in suitable liquids, such as fatty
oils, liquid
paraffin, or liquid polyethylene glycols. In addition, stabilizers can be
added. All
formulations for oral administration should be in dosages suitable for such
administration. For buccal administration, the compositions can take the form
of tablets
or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the
present
invention are conveniently delivered in the form of an aerosol spray
presentation from
pressurized packs or a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon
dioxide or other suitable gas. In the case of a pressurized aerosol the dosage
unit can be
determined by providing a valve to deliver a metered amount. Capsules and
cartridges
of e.g., gelatin for use in an inhaler or insufflator can be formulated
containing a powder
mix of the compound and a suitable powder base such as lactose or starch.
The compounds can be formulated for parenteral administration by injection,
e.g., by bolus injection or continuous infusion. Formulations for injection
can be
presented in unit dosage form, e.g., in ampoules or in mufti-dose containers,
with an
2 0 added preservative. The compositions can take such forms as suspensions,
solutions or
emulsions in oily or aqueous vehicles, and can contain formulatory agents such
as
suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous
solutions of the active compounds in water-soluble form. Additionally,
suspensions of
the active compounds can be prepared as appropriate oily injection
suspensions.
Suitable lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic
fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
Aqueous injection
suspensions can contain substances which increase the viscosity of the
suspension, such
as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the
suspension can
3 0 also contain suitable stabilizers or agents which increase the solubility
of the compounds
to allow for the preparation of highly concentrated solutions. Alternatively,
the active
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ingredient can be in powder form for constitution with a suitable vehicle,
e.g., sterile
pyrogen-free water, before use.
The compounds can also be formulated in rectal compositions such as
suppositories or retention enemas, e.g., containing conventional suppository
bases such
as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds can also
be formulated as a depot preparation. Such long acting formulations can be
administered by implantation (for example subcutaneously or intramuscularly)
or by
intramuscular injection. Thus, for example, the compounds can be formulated
with
suitable polymeric or hydrophobic materials (for example as an emulsion in an
acceptable oil) or ion exchange resins, or as sparingly soluble derivatives,
for example,
as a sparingly soluble salt.
A pharmaceutical carrier for the hydrophobic compounds of the invention is a
cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-
miscible
organic polymer, and an aqueous phase. The cosolvent system can be the VPD co-
solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the
nonpolar
surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to
volume in
absolute ethanol. The VPD co-solvent system (VPD:SW) consists of VPD diluted
1:1
with a 5% dextrose in water solution. This co-solvent system dissolves
hydrophobic
2 0 compounds well, and itself produces low toxicity upon systemic
administration.
Naturally, the proportions of a co-solvent system can be varied considerably
without
destroying its solubility and toxicity characteristics. Furthermore, the
identity of the co-
solvent components can be varied: for example, other low-toxicity nonpolar
surfactants
can be used instead of polysorbate 80; the fraction size of polyethylene
glycol can be
2 5 varied; other biocompatible polymers can replace polyethylene glycol, e.g.
polyvinyl
pyrrolidone; and other sugars or polysaccharides can substitute for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical
compounds can be employed. Liposomes and emulsions are well known examples of
delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents
such as
3 0 dimethylsulfoxide also can be employed, although usually at the cost of
greater toxicity.
Additionally, the compounds can be delivered using a sustained-release system,
such as
semipermeable matrices of solid hydrophobic polymers containing the
therapeutic agent.
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Various sustained-release materials have been established and are well known
by those
skilled in the art. Sustained-release capsules can, depending on their
chemical nature,
release the compounds for a few weeks up to over 100 days. Depending on the
chemical
nature and the biological stability of the therapeutic reagent, additional
strategies for
protein and nucleic acid stabilization can be employed.
The pharmaceutical compositions also can comprise suitable solid or gel phase
carriers or excipients. Examples of such carriers or excipients include but
are not
limited to calcium carbonate, calcium phosphate, various sugars, starches,
cellulose
derivatives, gelatin, and polymers such as polyethylene glycols.
The compounds of the invention can be provided as salts with pharmaceutically
compatible counterions. Pharmaceutically compatible salts can be formed with
many
acids, including but not limited to hydrochloric, sulfuric, acetic, lactic,
tartaric, malic,
succinic, etc. Salts tend to be more soluble in aqueous or other protonic
solvents that
are the corresponding free base forms.
Pharmaceutical compositions suitable for use in the present invention include
compositions wherein the active ingredients are contained in an effective
amount to
achieve its intended purpose. More specifically, a therapeutically effective
amount
means an amount effective to prevent development of or to alleviate the
existing
symptoms of the subject being treated. Determination of the effective amounts
is well
2 0 within the capability of those skilled in the art, especially in light of
the detailed
disclosure provided herein.
For any compound used in the method of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays. For
example, a dose
can be formulated in animal models to achieve a circulating concentration
range that
includes the EC50 (effective dose for 50% increase) as determined in cell
culture, i.e.,
the concentration of the test compound which achieves a half maximal
inhibition of HIV
replication as assayed by the infected cells to retain CD4 expression, to
reduce viral p24
or gp 120, and to prevent syncytia formation. Such information can be used to
more
accurately determine useful doses in humans.
3 0 Toxicity and therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental animals,
e.g., for
determining the LD50 (the dose lethal to 50% of the population) and the ED50
(the dose
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therapeutically effective in SO% of the population). The dose ratio between
toxic and
therapeutic effects is the therapeutic index and it can be expressed as the
ratio between
LD50 and EDSO. Compounds which exhibit high therapeutic indices are preferred.
The
data obtained from these cell culture assays and animal studies can be used in
formulating a range of dosage for use in humans. The dosage of such compounds
lies
preferably within a range of circulating concentrations that include the ED50
with little
or no toxicity. The dosage can vary within this range depending upon the
dosage form
employed and the route of administration utilized. The exact formulation,
route of
administration and dosage can be chosen by the individual physician in view of
the
patient's condition. (See, e.g. Fingl et al., 1975, in "The Pharmacological
Basis of
Therapeutics", Ch.l, p.1).
Dosage amount and interval can be adjusted individually to provide plasma
levels of the active moiety which are sufficient to maintain the inhibitory
effects. Usual
patient dosages for systemic administration range from 100 - 2000 mg/day.
Stated in
terms of patient body surface areas, usual dosages range from 50 - 910
mg/mz/day.
Usual average plasma levels should be maintained within 0.1-1000 ~M.
In cases of local administration or selective uptake, the effective local
concentration of
the compound can not be related to plasma concentration.
The amount of composition administered will, of course, be dependent on the
2 0 subj ect being treated, on the subj ect's body surface area, the severity
of the affliction, the
manner of administration and the judgment of the prescribing physician.
The following examples are provided for the purposes of illustration and are
not
intended to limit the scope of the present invention. The present invention is
not to be
limited in scope by the exemplified embodiments, which are intended as
illustrations of
2 5 individual aspects of the invention. Indeed, various modifications of the
invention in
addition to those shown and described herein will become apparent to those
skilled in
the art from the foregoing description and accompanying drawings. Such
modifications
are intended to fall within the scope of the appended claims.
All publications cited herein are incorporated by reference in their entirety.
EXAMPLES
Example 1
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This example describes isolation and identification of human cell-derived GSEs
exhibiting HIV-suppressive activities.
A. Construction of RFE libraries
Two RFE libraries were constructed from cDNAs of two human cell lines
according to the method described by Gudkov et al. (1994, Proc. Natl. Acad.
Sci. USA
91:3744). The cDNA prepared from HL-60 cells and HeLa cells was partially
digested
with DNase I in the presence of Mn~'~" (Sambrook et al., 1989, MOLECULAR
CLONING:
A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y.). Under these
conditions, DNase I is known to produce mostly double-stranded breaks.
The resulting fragments were repaired with the Klenow fragment of DNA
polymerise I and T4 polymerise and ligated to synthetic double-stranded
adaptors. The
5' adaptors (SEQ ID NOS: 21 and 22) were:
5'-CTCGGAATTCAAGCTTATGGATGGATGG-3'
3'-CCTTAAGTTCGAATACCTACCTAC-S'
The 3' adaptors (SEQ ID NOS: 23 and 24) were:
S'TGAGTGAGTGAATCGATGGATCCGTCT-3'
3'-ACTCACTCACTTAGCTACCTAGGCAGATCCT-5'
In the case of the library made from HL-60 cells, mRNA from uninduced cells
was first subtracted from mRNA from cells induced with TNF-cx. The subtracted
HL-60
2 0 library represents a modification of the procedure described in Coche et
al., 1994,
Nucleic Acids Res. 22:1322-1323. The tracer mRNA was purified from HL-60 cells
containing the LNCX plasmid at different time points after induction with TNF-
a. The
LNCX gene was used as an internal standard to monitor the enrichment of the
sequences
present in the tracer after subtraction. The mRNAs isolated from induced and
uninduced
2 5 cells were annealed separately to oligo dT magnetic beads (available from
Dyna) and the
first cDNA strand was synthesized using reverse transcriptase and oligo dT as
the
primer. The RNA strand was hydrolyzed and the second strand was synthesized on
the
induced population using a primer containing three ATG codons and 10 random
nucleotides on the 3' end. Single stranded cDNA fragments were annealed to an
excess
3 0 of driver cDNA attached to the magnetic beads. This procedure was repeated
several
times until substantial enrichment in the spiked LNCX sequence was seen. The
final
population of single-stranded DNA (ssDNA) molecules was amplified using
primers
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with three TGA codons in all three reading frames with 10 random nucleotides
on the
3' end. The resulting population of cDNA fragments were cloned into LNCX. This
step
was taken to enrich for mRNA encoded by cellular genes that might be important
in
supporting certain stages of the HIV life cycle in order to compensate for the
low
efficiency of retroviral transfer into OM10.1 cells. This library was
represented by 106
recombinant clones.
The random fragments of cDNA from HeLa cells were subjected to a
normalization procedure to provide for uniform abundance of different DNA
sequences
(Gudkov and Roninson,1997, Methods in Molecular Biology 69:221, Humana Press,
Inc., Totowa, N.J.). This procedure was used to increase the probability of
isolating
GSEs from rare cDNAs, since total polyA+ RNA was a mixture of unequally-
represented
sequences. In brief, the method first denatured 20 ~,g of cDNA by boiling for
5 min. in
25 ~L of TE buffer, followed by immediate cooling on ice. Then, 25 ~.L of 2X
hybridization solution was added, and the mixture was divided into four
aliquots in
Eppendorf tubes, 12.5 ~L each. One to two drops of mineral oil were added to
each
sample to avoid evaporation, and the tubes were placed into a 68°C
water bath for
annealing. One tube was frozen every 12 hours. After the last time-point, each
of the
annealing mixtures was diluted with water to a final volume of 500 ~L and
subjected
to hydroxylapatite (HAP) chromatography. HAP suspension equilibrated with 0.01
M
2 0 phosphate-buffered saline (PBS) was placed into Eppendorf tubes so that
the volume of
HAP pellet was approximately 100 ~L. The tubes with HAP and all the solutions
used
below were preheated and kept at 65°C. The excess of PBS was removed,
and diluted
annealing solution was added. After mixing by shaking in a 65°C water
bath, the tubes
were left in the water bath until HAP pellet was formed (a 15-s centrifugation
was used
2 5 to collect the pellet without exceeding 1000g in the microcentrifuge to
avoid damage of
HAP crystals). The supernatant was carefully replaced with 1 mL of preheated
0.01 M
PBS, and the process was repeated. To elute the ssDNA, the HAP pellet was
suspended
in 500 ~L of PBS at the single-strand elution concentration determined, e.g.,
0.16M, the
supernatant was collected, and the process was repeated. The supernatants were
3 0 combined and traces of HAP were removed by centrifugation. The ssDNA was
concentrated by centrifugation, and washed three times using 1 mL of water on
Centricon-100. The isolated ssDNA sequences were amplified by PCR with the
sense
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primer from the adapter, using a minimal number of cycles to obtain 10 ~,g of
the
product. The size of the PCR product that remained within the desired range
(200-500
bp) was ascertained. The normalization quality was tested by Southern or slot-
blot
hybridization with 32P-labeled probes for high, moderate- and low-expressing
genes
using 0.3-1.0 ~,g of normalized cDNA/lane. ~3-actin and (3-tubulin cDNAs were
used
as probes for high-expressing genes, c-myc and topo II cDNAs for moderately-
expressing genes, and c fos cDNA for low-expressing genes. The cDNAs isolated
after
different annealing times were compared with the original unnormalized cDNA.
The
probes were ensured to have a similar size and specific activity. The best-
normalized
ssDNA fraction was used, which produced the most uniform signal intensity with
different probes, for large-scale PCR amplification to synthesize at least 20
~,g of the
product for cloning. More ssDNA template was used to obtain the desired amount
by
scaling up the number of PCR or the reaction volume.
After normalization, the mixture of fragments ligated to adaptors was digested
with BamHI and EcoRI, column purified and ligated to the retroviral vector
pLNCX
(Miller and Rosman,1989, BioTechniques 7:980-990) orpLNGFRM cut withEcoRI and
BamHI. The pLNGFRM vector is the same as the pLNCX vector except that the neo'
gene has been replaced with a truncated low-affinity NGFR gene. The cells
transduced
with pLNGFRM express the truncated receptor on their surface which can be
easily
2 0 selected by an anti-NGFR antibody and FACS. The ligation mixture was
transformed
into E. coli. The total plasmid was purified from ~ 100,000 recombinant
clones. The
size distribution of the cloned fragments was tested by PCR amplification
using primers
derived from the vector sequences adjacent to the adapters.
2 5 B. Cell Lines and Reagents
The OM10.1 cells are available from the American Type Culture Collection,
Manassas, VA as CRL 10850 (Butera, U.S. Patent No. 5,256,534). The CEM-ss
cells
are available from the NIH AIDS Research and Reference Reagent Program as Cat.
No.
776. HIV-ls~ is available from NIH AIDS Research and Reference Reagent Program
3 0 as Cat. No. 275. HL-60 cells are available from American Tissue Culture
Collection as
CCL 240. HIV-l~ is available from AIDS Program as Cat. No. 398.
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The anti-CD4 (Q4120PE) and anti-p24 (KC-57 FITC) antibodies were purchased
from Sigma and Coulter, respectively. TNF-cc was obtained from Boehringer
Mannheim. 6418 was purchased from Gibco/BRL as Geneticin. The anti-NGFR
antibody was obtained from the ATCC (HB-8737) as hybridoma 20.4 (U.S. Patent
Nos.
4,786,593 and 4,855,241). Two anti-CD4 antibodies (L77 and L120) were obtained
from Becton Dickenson.
C. Transduction and Selection of GSEs
The libraries prepared from HL-60 cells according to the method of Section A
l0 of this Example, were transfected into the packaging cell line, PA317 (ATCC
CRL
#9078), and converted into retrovirus for infection of OM10.1 cells. Libraries
in
pLNCX vector were co-cultured and selected with 6418. Libraries in pLNGFRM
vector
were transduced by spinoculation (centrifugation of target cells at 1200 x g
for 90 min.
in the presence of filtered retroviral supernatant; Dunn et al., 1999, Gene
Therapy 6:
133-137) and selected by FACS sorting of the NGFR+ population. After
selection, the
OM10.1 cells harboring the entire RFE library were induced with 10 U/mL of TNF-
cx
at 37°C, and 24 hours later, were stained with an antibody and sorted
for CD4
expression. Genomic DNA from the CD4+ cells was purified and used for PCR
amplification of inserts with the vector-derived primers. The amplified
mixture was
2 0 digested with EcoRI and BamHI and cloned back into the retroviral vector.
The
selection was repeated for additional rounds.
Additionally, a normalized RFE library made from HeLa cells was transferred
into CEM-ss cells and the neo resistant population was infected with TCmso of
3000/1 O6
cellsofHIV-1~. ThisRFElibrarywasrepresentedby50x106independentrecombinant
2 5 clones. Four and seven days after infection, a purified anti-CD4
monoclonal antibody,
L77 (available from Becton Dickinson), was added at 5 ~,g/mL to prevent
syncytia
formation. Syncytia formation is thought to be prevented by blocking the
interaction
between gp120 expressed on the surface of an infected cell and CD4 on the
surface of
an uninfected cell. Antibody L77 does not prevent HIV infection of a cell. At
10-12
3 0 days after infection, CD4+, p24- cell population representing uninfected
cells were
sorted. Genomic DNA from the CD4+, p24~ cells was purified and used for PCR
amplification of inserts with the vector-derived primers. The amplified
mixture was
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digested with EcoRI and BamHI and cloned back into the retroviral vector. The
selection was repeated for additional rounds.
D. Immunofluorescence and Flow Cytometry
For the immunofluorescent staining of CD4+ cells for selection, 10' cells were
washed twice with Assay Buffer (500 ml PBS, 1 mL of 0.5 mM of EDTA at pH 8,
0.5
mL of 10% sodium azide and 10 mL of fetal bovine serum), and resuspended in
500 ~,L
PBS to which 50 ~L of anti-CD4 antibody (Q4120 PE, available from Sigma, St.
Louis,
MO) was added. After incubation at 4°C for 30 min., 5 mL of Assay
Buffer was added
and the cells centrifuged at 1200 rpm for 4 min. The cells were washed twice
with
Assay Buffer before sorting by FACS. The aforementioned procedure was
performed
under sterile conditions.
In order to determine p24 expression in HIV-infected cells, the cells were
first
washed twice with Assay Buffer. About 106 cells were suspended in 100 ~,L
Assay
Buffer, mixed with 2 mL of Ortho PermeaFix Solution (available from Ortho
Diagnostics), and incubated for 40 min. at room temperature. After
centrifugation at
1200 rpm for 4 min. at 4°C, the cells were resuspended in 2 ml Wash
Buffer (500 mL
PBS, 25 mL fetal bovine serum, 1.5% bovine serum albumin and 0.0055% EDTA) for
10 min. at room temperature. After centrifugation, the cells were resuspended
in 50 ~L
2 0 Wash Buffer and mixed with 1:500 dilution of an IgGZa antibody for 20 min.
at 4°C,
followed by incubation with 5-10 ~.L of anti-p24 antibody (KC57-FITC,
available from
Coulter) for 30 min. at 4°C. The cells were then washed twice with Wash
Buffer and
analyzed by flow cytometry.
For the selection of NGFR+ cells, 10' cells were washed twice with Assay
buffer
2 5 and resuspended in 200 ~L Assay buffer plus 5% normal mouse serum, and 2
mL of
anti-NGFR-PE antibody was added. After incubation at 4°C for 30 min., 5
mL of Assay
buffer was added and the cells were centrifuged at 1200 rpm for 4 min. The
cells were
washed twice with Assay buffer before sorting by FACS.
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E. Recovery of GSEs and Seduence Analysis
Genomic DNA was isolated from the selected population of OM 10.1 or CEM-ss
cells harboring putative GSEs by resuspending the cell pellet in 0.1% Triton X-
100,
20~,g/mL proteinase K in lx PCR buffer, incubating at 55°C for 1 hour,
and boiling for
10 minutes. Genomic DNA was used for PCR amplification using vector-derived
primers, cloned into the retroviral vector, and transformed into E. coli using
techniques
well known in the art. Individual plasmids were purified from E. coli clones
using
QIAGEN plasmid kits. Inserts were sequenced by the dideoxy procedure
(available
from AutoRead Sequencing Kit, Pharmacia Biotech) and run on a Pharmacia LKB
A.L.F. DNA sequencer. Sequences were analyzed using the DNASTAR program.
F. Identification of GSEs
Two selection strategies were used to isolate human cell-derived GSEs with
HIV-suppressive activities from RFE libraries. One strategy selected for GSEs
which
suppressed productive infection of cells by HIV. The second strategy selected
for GSEs
which suppressed induction of the latent provirus in OM 10.1 cells. When OM
10.1 cells
were treated with TNF-cx and stained with an antibody specific for the cell
surface
molecule CD4, a rapid loss of CD4 expression was observed (Figure 1 ). In
contrast, the
vast majority of the uninduced OM10.1 cells retained CD4 expression. It is
believed
2 0 that activation of the latent virus in OM10.1 cells by TNF-oc leads to the
production of
viral protein gp 120, which binds to cytoplasmic CD4, thereby preventing its
translocation to the cell surface. A diminution of CD4+ OM10.1 cells also
correlates
with an increased production of viral protein p24 in the cells following TNF-
cx
induction.
2 5 GSEs derived from cDNAs representing expressed human cellular genes were
identified and isolated from a RFE library made from HL-60 cells using HIV
provirus
activation in OM 10.1 cells as a read-out. Following transfection of the
entire library into
a packaging cell line, retrovirus carrying the library was used to infect
OM10.1 cells by
co-cultivation or spinoculation, and NGFR selection was performed to ensure
the
3 0 retention of the viral vector. When the infected cells were treated with
TNF-cx, a small
number of residual CD4+ cells were detected by an anti-CD4 antibody and sorted
by
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FACS. The GSEs contained in these cells were recovered by PCR amplification
and
their nucleotide sequences determined.
A total of twenty GSEs were isolated by the two aforementioned selection
strategies using OM10.1 and CEM-ss cells. Six of these GSEs were shown to have
substantial sequence identity with cDNAs of genes encoding different subunits
of the
NADH dehydrogenase enzyme complex. For example, CF-315 (SEQ ID NO:l) is a
GSE which suppresses HIV replication as an antisense molecule, which in its
sense
orientation has sequence identity with a gene encoding a subunit, ND6, of a
mitochondrial enzyme, NADH dehydrogenase (Chomyn et a1.,1988, Science
234:614).
CF-315 was further shown to protect uninfected human T cells from aproductive
HN-1
infection (Figure 2). In this experiment, the retroviral vector, pLNGFRM,
containing
the CF-315 nucleic acid molecule was transferred into CEM-ss cells followed by
NGFR
selection. Vector containing plasmid DNA (denoted LNGFRM) was used as negative
control. The NGFR+ cells were 99% CD4+, and they were then infected with
TCIDSO of
1000 of HIV-ls~. The infected cells were removed at 1 l, 14, 18, 21, 25, 28,
32, 35 and
39 days after infection, and stained with a fluorescinated-anti-p24 antibody
as an
indicator of HIV infection. Figure 2 shows that CF-315 inhibited infection of
human T
cells by HIV-ls~, as compared with negative control of vector plasmid DNA.
CF-319 (SEQ ID N0:2) is a GSE which suppresses HIV replication in the sense
2 0 orientation and has substantial sequence identity with another portion of
the gene
encoding the ND6 subunit of NADH dehydrogenase. CF-101 (SEQ ID N0:3) also
exhibits its HIV-suppressive activities in the sense orientation and has
substantial
sequence identity with a gene encoding the ND2 subunit of NADH dehydrogenase
(Chomyn et al., 1985, Nature 314:592). CF-117 (SEQ ID N0:4) suppresses HIV
2 5 activities as an antisense molecule, and in its sense orientation, it has
substantial
sequence identity with a gene encoding the ND6 subunit of NADH dehydrogenase.
CF-
025 (SEQ ID NO:S) suppresses HIV infection as an antisense molecule, and in
its sense
orientation, it has substantial sequence identity with a gene encoding the ND2
subunit
of NADH dehydrogenase. CF-128 (SEQ ID N0:6) suppresses HIV infection in the
3 0 sense orientation and it has substantial sequence identity with a gene
encoding the ND4
subunit of NADH dehydrogenase.
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Since both selection strategies produced GSEs having substantial sequence
identity with different subunits of NADH dehydrogenase, this enzyme complex
plays
an important role during HIV infection, and thus, methods to down-regulate the
expression of this complex or any of its subunits can be used to inhibit HIV
replication
in infected cells.
The selection of GSEs from HeLa cell library using productive infection of
CEM-ss cells isolated twelve additional nucleic acid molecules which have
substantial
sequence identity with other cellular genes. CF-004 (SEQ ID N0:7) suppresses
HIV
infection as a sense molecule and has substantial sequence identity with a
gene encoding
human 2-oxoglutarate dehydrogenase (Koike, 1995, Gene 158:261-266; Koike et
al.,
1992, Proc. Natl. Acad. Sci. USA 89:1963-1967). CF-113 (SEQ ID N0:8)
suppresses
HIV infection as a sense molecule and has substantial sequence identity with a
gene
encoding human M2-type pyruvate kinase/ cytosolic thyroid hormone binding
protein
(Kato et al., 1989, Proc. Natl. Acad. Sci. USA 86:7861-7865). CF-204 (SEQ ID
N0:9)
suppresses HIV infection as an antisense molecule, and in its sense
orientation, it has
substantial sequence identity with a gene encoding human calnexin (David et
al., 1993,
J. Biol. Chem. 268:9585-9592). CF-001 (SEQ ID NO:10) suppresses HIV infection
as
an antisense molecule, and in its sense orientation, it has substantial
sequence identity
with a gene encoding human ADP-ribosylation factor 3 (Tsai et al., 1991, J.
Biol. Chem.
266:23053-23059). CF-273 (SEQ ID NO: 11) suppresses HIV infection as a sense
molecule and has substantial sequence identity with a gene encoding human
eukaryotic
translation initiation factor 3 (Merrick and Hershey, 1996, Translational
Control, Cold
Spring Harbor, NY, pp. 31-69). CF-311 (SEQ ID NO: 12) suppresses HIV infection
as
an antisense molecule, and in its sense orientation, it has substantial
sequence identity
2 5 with a gene encoding a human protein tyrosine phosphatase (Kim et
a1.,1996, Oncogene
13:2275-2279). CF-313 (SEQ ID NO: 13) suppresses HIV infection as a sense
molecule
and has substantial sequence identity with a gene encoding a human protein
tyrosine
phosphatase (Keyse and Emslie,1992, Nature 359:644-647). CF-210 (SEQ ID NO:
14)
suppresses HIV infection as a sense molecule and has substantial sequence
identity with
3 o a gene encoding herpesvirus-associated ubiquitin-specific protease
(Everett et a1.,1997,
EMBO J. 16:566-577). CF-266 (SEQ ID NO: 15) suppresses HIV infection as an
antisense molecule, and in its sense orientation, it has substantial sequence
identity with
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a gene encoding human eIF4B (Milburn et al., 1990, EMBO J., 9:2783-2790). CF-
302
(SEQ ID NO: 16) suppresses HIV infection as an antisense molecule, and in its
sense
orientation, it has substantial sequence identity with a gene encoding human
CD44
(Stamenkovic et a1.,1991, EMBOJ.10:243-248). CF-317 (SEQ ID NO: 17) suppresses
HIV infection as an antisense molecule, and in its sense orientation, it has
substantial
sequence identity with a gene encoding human phosphatidylinositol 3-kinase
(Volinia
et al., 1995, EMBO J. 14:3339-3348). CF-286 (SEQ ID NO: 18) suppresses HIV
infection as a sense molecule and has substantial sequence identity with a
gene encoding
human elongation factor-la (Uetsuki et al., 1989, J. Biol. Chem. 264:5791-
5798).
In addition, two isolated GSEs did not share sequence homologywith any known
genes by sequence comparison. CF-061 (SEQ ID NO: 19) was selected in OM 10.1
cells.
CF-280 (SEQ ID NO: 20) was selected in CEM-ss cells. These two nucleic acid
molecules represent portions of unknown human cellular genes which play a role
in
supporting HIV infection.
Figure 3 shows the ability of four exemplary GSEs mentioned above in
preventing productive infection of CEM-ss cells by HIV. While the GSEs were
isolated
as fragments of distinct cellular genes, they all inhibited HIV infection as
shown by a
reduction of p24 levels in the infected cells when compared with controls.
Additionally,
Figure 4 shows the HIV-suppressive activities of CF-001 which functions as an
2 0 antisense molecule.
The selection of GSEs from a HeLa cell library using productive infection of
CEM-ss cells isolated 21 additional nucleic acid molecules which have
substantial
sequence identity with other cellular genes. CF-537 (SEQ ID N0:25) is A GSE
that
interferes with the HIV life cycles in the sense orientation and has
substantial sequence
identity with a gene encoding human bone morphogenic protein-1 (BMP1-6; Wozney
et a1.,1988, Science 242:1528-1534). The complement of SEQ ID N0:25 is
represented
by SEQ ID N0:26. CF-320 (SEQ ID N0:27) is A GSE that interferes with HIV
infection in the sense orientation and has substantial sequence identity with
a gene
encoding double-strand break DNA repair gene protein (McKay et al., 1996,
Genomics
3 0 36:305-31 S). The complement of SEQ ID N0:27 is represented by SEQ ID
N0:28. CF-
321 (SEQ ID N0:29) is A GSE that interferes with HIV replication in the sense
orientation and has substantial sequence identity with a gene encoding rat
guanine
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nucleotide releasing protein (Burton et al., 1993, Nature 361: 464-467). The
complement of SEQ ID N0:29 is represented by SEQ TD N0:30. CF-322 (SEQ ID
N0:31) is A GSE that interferes with HIV replication in the sense orientation
and has
substantial sequence identity with a gene encoding anti-proliferative factor
protein
(BTG-1; Rouault et al., 1992, EMBO J, 11:1663-1670). The complement of SEQ ID
N0:31 is represented by SEQ ID N0:32. CF-332 (SEQ ID N0:33) is A GSE that
interferes with HIV replication as an antisense molecule and has substantial
sequence
identitywith a gene encoding lymphocyte-specific protein 1 (Jongstra-Bilen et
a1.,1990,
J. Immunol. 144: 1104-1110). The complement of SEQ ID N0:33 is represented by
SEQ ID N0:34. CF-335 (SEQ ID N0:35) is A GSE that interferes with HIV
replication
as an antisense molecule and has substantial sequence identity with a gene
encoding
protein phosphatase 2A protein (Mayer et al., 1991, Biochemistry 30:3589-
3597). The
complement of SEQ ID N0:35 is represented by SEQ ID N0:36. CF-42 (SEQ ID
N0:37) is A GSE that interferes with HIV replication in the sense orientation
and has
substantial sequence identity with a gene encoding squalene synthetase protein
(Summers et al., ibid.). The complement of SEQ ID N0:37 is represented by SEQ
ID
N0:38. CF-50 (SEQ ID N0:39) also exhibits its HIV-suppressive activities in
the sense
orientation and has substantial sequence identity with a gene encoding
squalene
synthetase protein (Summers et al., ibid.). The complement of SEQ ID N0:39 is
represented by SEQ ID N0:40. CF-527 (SEQ ID N0:41) is a GSE, a peptide from
which interferes with HIV replication, and has substantial sequence identity
with a gene
encoding Eukaryotic release factor 1 protein (ERF-1; Andjelkovic et al., 1996,
EMBO
J. 15:7156-7167). The complement of SEQ ID N0:41 is represented by SEQ ID
N0:42.
CF-528 (SEQ ID N0:43) is A GSE that interferes with HIV infection in the sense
2 5 orientation and has substantial sequence identity with a gene encoding GTP
binding
protein (Zigman et a1.,1993, Endocrinology 133:2508-2514). The complement of
SEQ
ID N0:43 is represented by SEQ ID N0:44. CF-529 (SEQ ID N0:45) is A GSE that
interferes with HIV replication as an antisense molecule and has substantial
sequence
identity with a gene encoding Importin beta subunit protein (Gorlich et al.,
1995, Curr
3 0 Biol. 5:383-392). The complement of SEQ ID N0:45 is represented by SEQ ID
N0:46.
CF-531 (SEQ ID N0:47) is A GSE that interferes with HIV replication as an
antisense
molecule and has substantial sequence identity with a gene encoding cell
adhesion
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molecule L1 protein (LCAM1; Hlavin et al., 1991, Genomics 11:416-23). The
complement of SEQ 117 N0:47 is represented by SEQ ID N0:48. CF-545 (SEQ ID
N0:49) is A GSE that interferes with HIV replication as an antisense molecule
and has
substantial sequence identity with a gene encoding U-snRNP associated
cyclophilin
protein (Horowitz et al., ibid.). The complement of SEQ ID N0:49 is
represented by
SEQ ID NO:50. CF-547 (SEQ ID NO:51 ) is A GSE that interferes with HIV
replication
as an antisense molecule and has substantial sequence identity with a gene
encoding
recepin endoprotease protein (GenBank Accession No. U03644). The complement of
SEQ ID NO:51 is represented by SEQ ID N0:52. CF-619 (SEQ ID N0:53) is A GSE
that interferes with HIV replication in the sense orientation and has
substantial sequence
identity with a gene encoding Arg/Abl interacting protein (ArgBP2A; GenBank
Accession No. AF049884). The complement of SEQ ID N0:53 is represented by SEQ
ID N0:54. CF-620 (SEQ ID NO:55) is A GSE that interferes with HIV replication
as
an antisense molecule and has substantial sequence identity with a gene
encoding keratin
related protein (IFN-'y regulated; Flohr et al., ibid. ). The complement of
SEQ 117 NO:55
is represented by SEQ ID N0:56. CF-624 (SEQ ID N0:57) is A GSE that interferes
with HIV replication in a sense orientation and has substantial sequence
identity with a
gene encoding p18 protein (Zhu et al., ibid.). The complement of SEQ ID N0:57
is
represented by SEQ ID N0:58. CF-630 (SEQ ID N0:59) is A GSE that interferes
with
2 0 HIV replication as an antisense molecule and has substantial sequence
identity with a
gene encoding p40 protein (Mayer et al., 1988, Biochim Biophys Acta 1395:301-
308).
The complement of SEQ ID N0:59 is represented by SEQ ID N0:60. CF-579 (SEQ ID
N0:61) is A GSE that interferes with HIV replication in a sense orientation
and has
substantial sequence identity with a gene encoding glucosidase alpha II
protein
2 5 (GenBank Accession No. AJ000332). The complement of SEQ ID N0:61 is
represented
by SEQ ID N0:62. CF-287 (SEQ ID N0:77) is A GSE that interferes with HIV
replication in a sense orientation and has substantial sequence identity with
a gene
encoding Na+-D-Glucose cotransport regulator protein (Lambotte et a1.,1996,
DNA Cell
Biol. 15:769-77). The complement of SEQ ID N0:77 is represented by SEQ ID
N0:78.
3 0 CF-622 (SEQ ID N0:87) is A GSE that interferes with HIV replication as an
antisense
molecule and has substantial sequence identity with a gene encoding Rox
protein
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(Meroni et al., 1997, EMBO J. 16:2892-2906). The complement of SEQ ID N0:87 is
represented by SEQ ID N0:88.
Figures 7, 8 and 9 show the ability of seven exemplary GSEs mentioned
immediately above in preventing productive infection of CEM-ss cells by HIV.
While
the GSEs were isolated as fragments of distinct cellular genes, they all
inhibited HIV
infection as shown by a reduction of p24 levels in the infected cells when
compared with
controls.
Example 2
This Example demonstrates the suppression of HN infection by NADH
dehydrogenase inhibitors
NADH dehydrogenase inhibitors, amytal (available from Sigma, St. Louis, MO)
and mofarotene (Uchida et al., 1994, Int. J. Cancer 58:891-897) were diluted
in sterile
culture medium and used according to the indicated concentrations .
OM10.1 cells were cultured in RPMI 1640 glucose-free media prior to and
during incubation with NADH dehydrogenase inhibitors and TNF-oc induction. The
inhibitors were added to the cells followed by TNF-cx induction 1-2 hours
later. The
expression of CD4 by the cells was assessed by anti-CD4 antibody staining and
flow
cytometry after 24 hour incubation at 37°C.
2 0 Human peripheral blood leukocytes (PBLs) were isolated using Ficoll-
Hypaque
density gradient separation. Cells were washed twice and resuspended in RPMI +
10%
FBS + 2% human AB serum + penicillin/streptomycin/glutamine + 100 Units/mL of
IL-
2 at a concentration of 0.5 x 106 cells/mL. PBLs were then activated with
phytohemagglutinin at 0.5 ~g/mL and placed in a humidified incubator at
37°C/5% COz.
2 5 After two days of activation, 106 cells were infected with HIV-lSF3s at
TCIDSO of 1000,
in the presence of various concentrations of mofarotene: 0 ~,M, 1 ~,M, 0.5
~,M, 0.1 ~M,
and 0.05 ~M. A separate set of uninfected samples under identical
concentrations of
mofarotene were also maintained as controls. The samples were analyzed by flow
cytometry at day 4 and day 6 post infection. The cells were gated for CD3
expression
3 0 (for T cells). Then the expression of CD4 and viral p24 and CD4 was
examined by
bivariate dot plot.
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Since several GSEs have been selected and shown to have substantial sequence
identity with cellular genes which encode different subunits of NADH
dehydrogenase,
two compounds with known NADH dehydrogenase-inhibitory activities were tested
for
their ability to suppress HIV infection. Figure 5 shows that amytal inhibited
the
induction of latent HIV provirus in OM10.1 cells in a dose-dependent manner,
as shown
by its ability to retain CD4 expression by TNF-a-induced OM10.1 cells. In the
same
assay, mofarotene, which down-regulates mitochondrial gene expression, also
inhibited
HIV-1 induction at even lower concentrations (Figure 6). At 100 nM of
mofarotene,
80% of the cells retained CD4 expression, and greater than 80% of the cells
remained
viable. In addition, when p24 expression was measured as an indication by HIV
infection, both amytal and mofarotene also suppressed intracellular p24 levels
in the
treated cells as compared with untreated controls.
In order to test the ability of a NADH dehydrogenase inhibitor to prevent
productive infection of T cells by HIV, human PBLs were isolated and infected
with
HTVSFS3 in the presence of various concentrations of mofarotene. The CD3+ T
cells were
analyzed with respect to their expression of CD4 and viral p24 and the results
are shown
in Table 1.
Table 1
HIV Mofarotene %CD4+ in % p24' in CD4' T
( M o ulation o ulation
2 0 - 1.0 36% 0%
- 0.5 34% 0%
- 0.1 34% 0%
- 0.05 33% 0%
- 0.0 32% 0%
+ 1.0 25% 10%
+ 0.5 24% 12.5%
+ 0.1 28% 11.4%
+ 0.05 18% 20.5%
3 0 + 0.0 15% 33%
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On day 4 post-infection, there was little detectable difference between PBLs
infected with HIV whether or not mofarotene was added. Mafarotene alone did
not
significantly alter the percentage of CD4+ T cells, nor did it alter the
expression level of
CD3 on the cell surface.
On day 6, a significant difference was detected with respect to the percentage
of
CD4+ T cells and percentage of p24+ CD4+ T cells. In the HIV-infected sample
without
mofarotene, 33% of CD4+ T cells were p24+ and only 1 S% of T cells were CD4+.
In the
HIV-uninfected control sample, ~ 32% of T cells were CD4+, suggesting a
dramatic
depletion of CD4+ T cells by HIV infection. In the HIV-infected sample with 1
~M of
mofarotene, only 10% of CD4+ T cells were p24+. In addition, ~25% of T cells
were
CD4+, indicating that mofarotene inhibited the depletion, of CD4+ T cells by
HIV
infection. The level of protection by mofarotene, as measured by the
percentage of p24+
CD4+ T cells and percentage of CD4+ cells in the CD3+ T cell population,
diminished
with decreased concentrations of mofarotene. While mofarotene alone increased
the
percentage of CD4+ T cells in uninfected samples, the effect was minimal (up
to 4%
higher). At day 6, mofarotene did not alter the expression level of CD3 on the
cell
surface. Hence, a NADH dehydrogenase inhibitor prevented productive infection
by
HIV of primary cultures of human T cells.
2 0 Example 3
This example describes the production of a PBMC library and isolation of GSEs
therefrom.
A. PBMC Library
A cell-derived random fragment library (RFL) was constructed using cDNA from
2 5 peripheral blood mononuclear cells (PBMC). Four buffy coats were obtained
from four
healthy blood donors and PBMCs were purified by Ficoll gradient centrifugation
followed by stimulation with PHA (l~.g/mL). Cells were removed at 5, 10, and
24
hours after the addition of PHA and total RNA was isolated by Trizol
extraction. All
time points from all donors were then pooled. Polyadenylated mRNA was purified
from
3 0 the total RNA by passages through an oligo-dT column. cDNA was synthesized
from
the polyadenylated RNA with random primers using the Gibco Superscript Choice
system for cDNA Synthesis (Gibco BRL, Rockville, MD). The cDNA was normalized
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using the PCR-Select cDNA Subtraction kit (Clontech, Palo Alto, CA) based on
the
suppression subtractive hybridization methods of Diatchenko et al. (1996,
Proc. Natl.
Acad. Sci. USA 93, 6025-6030, 1996). The primers used for the normalization
were:
5'-TAGGGCTCGAGCCGCCACCATG-3' (XhoS'S#2; SEQ ID N0:97)
5'-ATCCCTGCAGGTCACTCACTCA-3' (Sse3'AS#1; SEQ ID N0:98)
The normalized random fragments were digested with XhoI and Sse8387I, purified
on
quick spin columns (available from Qiagen, Valencia, CA) and ligated into the
SseIlXhoI
site of a bicistronic retroviral vector, EMCVNgfrMPBIN. This vector was based
on
l0 LXSNgfr as described in Dunn et al. (1999, Gene Therapy 6:130-137).
Modifications
included substitution of the Moloney murine leukemia virus LTR with the hybrid
LTR
described by Baum et al., 1995, (J. Virol. 69:7541-7547) and replacement of
the SV40
promoterwith encephalomyocarditis virus (EMCV) internal ribosomal entry site
isolated
from the plasmid, pCITE (available from Amersham, Arlington Heights, IL). The
ligation mix was then transformed into competent cells. The total plasmid was
purified
from approximately 50 million clones.
B. Isolation of GSEs
The PBMC library was screened using the methods described above in Section
2 0 1F. A total of 14 GSEs were isolated using CEM-ss cells. CF-674 (SEQ ID
N0:69) is
GSE that interferes with HIV replication in a sense orientation and has
substantial
sequence identity with a gene encoding human translationally controlled tumor
protein
1 (GenBank Accession No. HUMCH13C4A). The complement of SEQ ID N0:69 is
represented by SEQ ID N0:70. CF-675 (SEQ ID N0:71) is another GSE that has
2 5 substantial sequence identity with a gene encoding human translationally
controlled
tumor protein 1 (GenBank Accession No. HUMCH13C4A). The complement of SEQ
ID N0:71 is represented by SEQ ID N0:72. CF-679 (SEQ ID N0:83) is another GSE
that has substantial sequence identity with a gene encoding human
translationally
controlledtumorprotein 1 (GenBankAccessionNo.HLTMCH13C4A). The complement
3 0 of SEQ ID N0:83 is represented by SEQ ID N0:84. CF-693 (SEQ ID N0:75) is
GSE
that interferes with HIV replication in a sense orientation and has
substantial sequence
identity with a gene encoding NEF interacting protein (GenBank Accession No.
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HSU83843). The complement of SEQ ID N0:75 is represented by SEQ ID N0:76. CF-
660 (SEQ m N0:79) is a GSE that interferes with HIV replication as an
antisense
molecule and has substantial sequence identity with a gene encoding human
hsp90
chaperone protein (Rebbe et al., 1989, J Biol Chem. 264:15006-15011). The
complement of SEQ ID N0:79 is represented by SEQ ID NO:80. CF-653 (SEQ ID
N0:67) is a GSE that interferes with HIV replication as an antisense molecule
and has
substantial sequence identity with a gene encoding MIP-1 alpha protein (Obata
et al.,
1988, J Yirol. 62:4381-4386). The complement of SEQ ID N0:67 is represented by
SEQ ID N0:68. CF-676 (SEQ ID N0:63) is a GSE that interferes with HIV
replication
as an antisense molecule and has substantial sequence identity with a gene
encoding
alpha enolase protein (Yu et al., 1997, Genome Res. 7:353-358). The complement
of
SEQ ID N0:63 is represented by SEQ ID N0:64. CF-675 (SEQ ID N0:65) is another
GSE that has substantial sequence identity with a gene encoding alpha enolase
protein.
The complement of SEQ ID N0:65 is represented by SEQ ID N0:66. CF-673 (SEQ ID
N0:73) is a GSE that interferes with HIV replication as an antisense molecule
and has
substantial sequence identity with a gene encoding BBC-1 satellite DNA protein
(GenBank AccessionNo. AJ223209). The complement of SEQ ID N0:73 is represented
by SEQ ID N0:74. CF-672 (SEQ ID N0:81 ) is another GSE that interferes with
HIV
replication as an antisense molecule and has substantial sequence identity
with a gene
2 0 encoding BBC-1 satellite DNA protein. The complement of SEQ ID N0:81 is
represented by SEQ ID N0:82. CF-681 (SEQ ID N0:85) is a GSE that interferes
with
HIV replication as an antisense molecule and has substantial sequence identity
with a
gene encoding FK506-binding protein Al (Maki et a1.,1990, Proc. Natl. Acad.
Sci. USA
87:5440-5443). The complement of SEQ ID N0:85 is represented by SEQ ID N0:86.
2 5 CF-683 (SEQ ID N0:89) is a GSE that interferes with HIV replication as an
antisense
molecule and has substantial sequence identity with a gene encoding beta-
signal
sequence receptor protein (Chinen et al., ibid.). The complement of SEQ ID
N0:89 is
represented by SEQ ID N0:90. CF-684 (SEQ ID N0:91) is a GSE that interferes
with
HIV replication in a sense orientation and has substantial sequence identity
with a gene
30 encoding human tumorous imaginal disc protein (Schilling et al., ibid.).
The
complement of SEQ ID N0:91 is represented by SEQ ID N0:92. CF-685 (SEQ ID
N0:93) is representative of a cluster of GSEs that interfere with HIV
replication as an
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antisense molecule and has substantial sequence identity with a gene encoding
cell
surface heparin binding protein (Liu et al., ibid.). The complement of SEQ ID
N0:93
is represented by SEQ ID N0:94. Another member of the cluster is CF-686 (SEQ
ID
N0:95), also having substantial sequence identity with a gene encoding cell
surface
heparin binding protein (Liu et al., ibid.). The complement of SEQ ID N0:95 is
represented by SEQ ID N0:96.
Example 4
This example describes the identification of GSEs that inhibit translocation
of
the HIV protein Rev.
The methods generally described in Stauber et al. (1998, Virology 251:38-48),
are used to identify GSEs that inhibit the translocation of Rev HIV protein in
a cell.
Plasmids CF-24 and CF-367 (vector without insert), and CF-203, CF-261, CF-527,
CF-
529, CF-537, CF-545, CF-619, CF-653, CF-659, CF-660, CF-662 and CF-674 are
transfected into cells that express a Rev protein fused to a green fluorescent
protein
(Rev-GFP). Confocal microscopy is used to determine whether transport of Rev-
GFP
fusion protein between the nucleus and the cytoplasm of a cell transfected
with a
plasmid containing a GSE nucleic acid sequence is inhibited by expression of
the GSE.
2 0 The observations using cells transfected with GSE are compared with cells
transfected
with vector alone.
While various embodiments of the present invention have been described in
detail, it is apparent that modifications and adaptations of those embodiments
will occur
to those skilled in the art. It is to be expressly understood, however, that
such
2 5 modifications and adaptations are within the scope of the present
invention, as set forth
in the following claims.
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SEQUENCE LISTING
<110> Holzmayer, Tanya
Dunn, Steve
<120> Compositions and Methods for Inhibiting Human
Immunodeficiency Virus Infection by Down-Regulating
Human Cellular Genes
<130> PPD
<140> PCT
<141> 2000-09-O1
<160> 98
<170> PatentIn Ver. 2.1
<210> 1
<211> 135
<212> DNA
<213> Homo Sapiens
<400> 1
agatcctatt ggtgcgtggg ctttgtatga ttatgggcgt agattagtag tagttactgg 60
ttgaacattg tttgttggtg tatatattgt aattgggatt gctcggggga ataggttatg 120
tgattaggag taggg 135
<210> 2
<211> 100
<212> DNA
<213> Homo Sapiens
<400> 2
gatcctcccg aatcaaccct gacccctctc cttcataaat tattcagctt cctacactat 60
taaagtttac cacaaccacc acgccatcat actccttcac 100
<210> 3
<211> 143
<212> DNA
<213> Homo Sapiens
<400> 3
catcagccct tcttaacatc tacttctacc tacgcctaat ctactccacc tcaatcacac 60
tactccccat atctaacaac gtaaaaataa aatgacagtt tgagcataca aaacccaccc 120
cattcctccc cacactcatc gcc 143
<210> 4
<211> 100
<212> DNA
<213> Homo sapiens
<400> 4
gtgaaagagt atgatggggt ggtggttgtc gtaaacttta atagtgtagg aagctgaata 60
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atttataaag gagaggggac agggttgatt cgggaggatc 100
<210> 5
<211> 116
<212> DNA
<213> Homo Sapiens
<400> 5
gaagaagcag gccggatgtc agaggggtgc cttgggtaac ctctgggact cagaagtgaa 60
agggggctat tcctagtttt attgctatag ccattatgat tattaatgat gagtat 116
<210> 6
<211> 127
<212> DNA
<213> Homo sapiens
<400> 6
tcctagtcct cacaatcatg gcaagccagc gccacttatc cagtgaacca ctatcacgaa 60
aaaaactcta cctctctata ctaatctccc tacaaatctc attaattata atattcacaa 120
ccacaga 127
<210> 7
<211> 105
<212> DNA
<213> Homo Sapiens
<400> 7
gcgcaaagca cgcgacatgg tggggcaggt ggccatcaca aggattgagc agctgtcgcc 60
attccccttt gacctcctgc tgaaggaggt gcagaagtac cccaa 105
<210> 8
<211> 105
<212> DNA
<213> Homo Sapiens
<400> 8
gaaatcctgg aggccagtga tgggatcatg gtggctcgcg gtgatctagg cattgagatt 60
cctgcagaga aggtcttcct tgctcagaag atgataattg gacgg 105
<210> 9
<211> 107
<212> DNA
<213> Homo Sapiens
<400> 9
ggttgtcaat cacaggtcgc caggagacac cacatccagg agctgactca catctagggt 60
tggcaatctg aggagcctcc cattctccat ccatgtcttc atcccaa 107
<210> 10
<211> 121
<212> DNA
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3/18
<213> Homo Sapiens
<400> 10
ccacattgaa cccaatggta gggatggtgg tgacgatctc ccccagtttc agcttgtata 60
ggatggtggt ctttcctgcg gcatccaggc ccaccatcag gatgcgcatc tccttcttcc 120
C 121
<210> 11
<211> 114
<212> DNA
<213> Homo Sapiens
<400> 11
gctcgtcagt gtccacccct cctcgcaact ccaggcgctc ctttttctgc tccatataga 60
gctcctgggc cattttccgg tacttccgga aatcttccat catggtgcgc cttc 114
<210> 12
<211> 78
<212> DNA
<213> Homo sapie.ns
<400> 12
gggggctctg tttggtggtc tctctagctg cactggtcta tcaagctgtt ggctggtctc 60
tctctctggc tggggatc 78
<210> 13
<211> 95
<212> DNA
<213> Homo Sapiens
<400> 13
cattttgtta cataaggatg acttttttat acaatggaat aaattatggc atttctattg 60
aaatttcaac gcttttgttt ctttggcaac cacac 95
<210> 14
<211> 114
<212> DNA
<213> Homo Sapiens
<400> 14
gcattacaaa gagtgttcta tgaattacag catagtgata aacctgtagg aacaaaaaag 60
ttaacaaagt catttgggtg ggaaacttta gatagcttca tgcaacatga tgtt 114
<210> 15
<211> 97
<212> DNA
<213> Homo Sapiens
<400> 15
gccgttcttc tacttctctt tctcaagcag cagtgtcaac aggctttgcc cctccaaaga 60
tagaagcagc tcgagtggac tgggaggtac tagcaga 97
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<210> 16
<211> 73
<212> DNA
<213> Homo Sapiens
<400> 16
gatccctggg cgcggggcag gggccggcgg aggacgggac gaggatggcg gaccgaacct 60
ggcagaggct ggg 73
<210> 17
<211> 103
<212> DNA
<213> Homo Sapiens
<400> 17
ccaagcttcc tatcttaagc tgggcgttga tatccaggtc acaactatag atgtagtgaa 60
acttctctgc ttcccccatc gcaccgtctg caaaggtacc acc 103
<210> 18
<211> 145
<212> DNA
<213> Homo Sapiens
<400> 18
cagttggggt gggtgtcatc aaagcagtgg acaagaaggc tgctggagct ggcaaggtca 60
ccaagtctgc ccagaaagct cagaaggcta aatgaatatt atccctaata cctgccaccc 120
cactcttaat cagtggtgga agaac 145
<210> 19
<211> 173
<212> DNA
<213> Homo Sapiens
<400> 19
acacacagac acacagacac agagagacac acagacacac acacagagat acacagagac 60
acagacacag aaacactctg agagacacac acacagagac acacagacac acagacacag 120
agagacacac agacacacac acacagagat acacagagac acagacacag aaa 173
<210> 20
<211> 233
<212> DNA
<213> Homo Sapiens
<400> 20
gtgccgtatg aatatacaaa ataatggcat cagggatccc tgtgctcatt cacatagcta 60
gggacaacag gatttcatct ccaggaaact cagtagtata cttttgtgac ttctctcttt 120
aagcaccaaa gcatactttc agggaaaaac aaaaaagaga ttaaaaatgt aaagaattct 180
ttcatgctgc ttggagaggt gagggaaggt agcccactga aagtgacaga gaa 233
<210> 21
<211> 28
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<212> DNA
<213> Homo Sapiens
<400> 21
ctcggaattc aagcttatgg atggatgg 28
<210> 22
<211> 24
<212> DNA
<213> Homo sapiens
<400> 22
catccatcca taagcttgaa ttcc 24
<210> 23
<211> 27
<212> DNA
<213> Homo Sapiens
<400> 23
tgagtgagtg aatcgatgga tccgtct 27
<210> 24
<211> 31
<212> DNA
<213> Homo Sapiens
<400> 24
tcctagacgg atccatcgat tcactcactc a 31
<210> 25
<211> 57
<212> DNA
<213> Homo Sapiens
<400> 25
ggtccaccat ggccctgctg cactccggcc gcgtcctccc cgggatcgcc gccgcct 57
<210> 26
<211> 57
<212> DNA
<213> Homo Sapiens
<400> 26
aggcggcggc gatcccgggg aggacgcggc cggagtgcag cagggccatg gtggacc 57
<210> 27
<211> 78
<212> DNA
<213> Homo sapiens
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<400> 27
gcctgcacat gacgatatgg atgaggatga taatgtatca atgggtgggc ctgatagtcc 60
tgattcagtg gatcccgt 78
<210> 28
<211> 78
<212> DNA
<213> Homo Sapiens
<400> 28
acgggatcca ctgaatcagg actatcaggc ccacccattg atacattatc atcctcatcc 60
atatcgtcat gtgcaggc 78
<210> 29
<211> 54
<212> DNA
<213> Homo Sapiens
<400> 29
agttcctttt actttttaat ctttccttaa agcacgcctg tgttgggcta acga 54
<210> 30
<211> 54
<212> DNA
<213> Homo Sapiens
<400> 30
tcgttagccc aacacaggcg tgctttaagg aaagattaaa aagtaaaagg aact 54
<210> 31
<211> 77
<212> DNA
<213> Homo Sapiens
<400> 31
cgacagctgc agaccttcag ccagagcctg caggagctgc tggcagaaca ttataaacat 60
cactggttcc cagaaaa 77
<210> 32
<211> 77
<212> DNA
<213> Homo Sapiens
<400> 32
ttttctggga accagtgatg tttataatgt tctgccagca gctcctgcag gctctggctg 60
aaggtctgca gctgtcg 77
<210> 33
<211> 43
<212> DNA
<213> Homo Sapiens
CA 02382030 2002-02-14
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<400> 33
aacacccata gtaggcctaa aagcagccac caattaagaa agc 43
<210> 34
<211> 43
<212> DNA
<213> Homo Sapiens
<400> 34
gctttcttaa ttggtggctg cttttaggcc tacgatgggt gtt 43
<210> 35
<211> 89
<212> DNA
<213> Homo Sapiens
<400> 35
ggtaagggta gggcactttt aatttaaatg acttcttgca ccatcttgcc taatggacta 60
gattggactg tatcaacatt gatttactc 89
<210> 36
<211> 89
<212> DNA
<213> Homo Sapiens
<400> 36
gagtaaatca atgttgatac agtccaatct agtccattag gcaagatggt gcaagaagtc 60
atttaaatta aaagtgccct acccttacc 89
<210> 37
<211> 121
<212> DNA
<213> Homo Sapiens
<400> 37
tggctgggac ctttaggaaa gtgaaatgca ggtgagaaga acctaaacat gaaaggaaag 60
ggtgcctcat cccagcaacc tgtccttgtg ggtgatgatc actgtgctgc ttgtggctca 120
t 121
<210> 38
<211> 121
<212> DNA
<213> Homo sapiens
<400> 38
atgagccaca agcagcacag tgatcatcac ccacaaggac aggttgctgg gatcaggcac 60
cctttccttt catgtttagg ttcttctcac ctgcatttca ctttcctaaa ggtcccagcc 120
a 121
<210> 39
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<211> 123
<212> DNA
<213> Homo Sapiens
<400> 39
aagtgaaatg caggtgagaa gaacctaaac atgaaaggaa agggtgcctc atcccagcaa 60
cctgtccttg tgggtgatga tcactgtgct gcttgtggct catggcagag cattcagtgc 120
caa 123
<210> 40
<211> 123
<212> DNA
<213> Homo Sapiens
<400> 40
ttggcactga atgctctgcc atgagccaca agcagcacag tgatcatcac ccacaaggac 60
aggttgctgg gatgaggcac cctttccttt catgtttagg ttcttctcac ctgcatttca 120
ctt 123
<210> 41
<211> 118
<212> DNA
<213> Homo Sapiens
<400> 41
cgtttaagaa tggaaaagcg acataactat gttcggaaag tagcagagac tgctgtgcag 60
ctgtttattt ctggggacaa agtgaatgtg gctggtctag ttttagctgg atccgctc 118
<210> 42
<211> 118
<212> DNA
<213> Homo Sapiens
<400> 42
gagcggatcc agctaaaact agaccagcca cattcacttt gtccccagaa ataaacagct 60
gcacagcagt ctctgctact ttccgaacat agttatgtcg cttttccatt cttaaacg 118
<210> 43
<211> 59
<212> DNA
<213> Homo Sapiens
<400> 43
agtgctttaa cgatgtcaca gctatcattt acgtcgcagc ctgcagtagc tacaacatg 59
<210> 44
<211> 59
<212> DNA
<213> Homo sapiens
<400> 44
catgttgtag ctactgcagg ctgcgacgta aatgatagct gtgacatcgt taaagcact 59
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<210> 45
<211> 135
<212> DNA
<213> Homo sapiens
<400> 45
ggagtgtgca cggatgctga atgtttggga atgagaggat gagtgagtga ggcttgaaaa 60
cacaccacat tgaaaatcct gccacagcag cagccgcagc cgccaacagc agcgctgtta 120
gtgagctaag taagc 135
<210> 46
<211> 135
<212> DNA
<213> Homo Sapiens
<400> 46
gcttacttag ctcactaaca gcgctgctgt tggcggctgc ggctgctgct gtggcaggat 60
tttcaatgtg gtgtgttttc aagcctcact cactcatcct ctcattccca aacattcagc 120
atccgtgcac actcc 135
<210> 47
<211> 89
<212> DNA
<213> Homo sapiens
<400> 47
ctgggtcccc aaggagggcc agtgcaactt caggttccat atcttgttca aagccttggg 60
agaagagaag ggtggggctt ccctttcgc 89
<210> 48
<211> 89
<212> DNA
<213> Homo Sapiens
<400> 48
gcgaaaggga agccccaccc ttctcttctc ccaaggcttt gaacaagata tggaacctga 60
agttgcactg gccctccttg gggacccag 89
<210> 49
<211> 181
<212> DNA
<213> Homo Sapiens
<400> 49
cttcttggtg gtgttcttga gtaagataat ctggactggc ccccgtcttt gcttccctgc 60
ctgctgctgc cccatttgat caagagacca tggaagtgtc agagattcag aatccaagat 120
tgtctttaag ttttcaactg taaataaagt ttttttgtat gcgtaaaaaa agctcgtgcc 180
t 181
<210> 50
CA 02382030 2002-02-14
WO 01/16322 PCT/US00/24262
10/18
<211> 181
<212> DNA
<213> Homo sapiens
<400> 50
aggcacgagc tttttttacg catacaaaaa aactttattt acagttgaaa acttaaagac 60
aatcttggat tctgaatctc tgacacttcc atggtctctt gatcaaatgg ggcagcagca 120
ggcagggaag caaagacggg ggccagtcca gattatctta ctcaagaaca ccaccaagaa 180
g 181
<210> 51
<211> 97
<212> DNA
<213> Homo sapiens
<400> 51
acagaaggag agaccgaata caaatttgaa tggcagaaag gagccccacg agaaaaatat 60
gccaaagatg acatgaacat cagagatcag cccttta 97
<210> 52
<211> 97
<212> DNA
<213> Homo Sapiens
<400> 52
taaagggctg atctctgatg ttcatgtcat ctttggcata tttttctcgt ggggctcctt 60
tctgccattc aaatttgcat tcggtctctc cttctgt 97
<210> 53
<211> 74
<212> DNA
<213> Homo Sapiens
<400> 53
gttccacctc cagtcccgcc gcttcgacca agagatcggt cttcaacaga aaagcatgac 60
tgggatcctc caga 74
<210> 54
<211> 74
<212> DNA
<213> Homo Sapiens
<400> 54
caaggtggag gtcagggcgg cgaagctggt tctctagcca gaagttgtct tttcgtactg 60
accctcggag gtct 74
<210> 55
<211> 141
<212> DNA
<213> Homo sapiens
<400> 55
CA 02382030 2002-02-14
WO 01/16322 PCT/US00/24262
11/18
gcagcgtgga ggagcagctg gcccagcttc gctgcgagat ggagcagcag aaccaggaat 60
acaaaatcct gctggatgtg aagacgcggc tggagcagga gattgccacc taccgccgcc 120
tgctggaggg agaggatgcc c 141
<210> 56
<211> 141
<212> DNA
<213> Homo Sapiens
<400> 56
gggcatcctc tccctccagc aggcggcggt aggtggcaat ctcctgctcc agccgcgtct 60
tcacatccag caggattttg tattcctggt tctgctgctc catctcgcag cgaagctggg 120
ccagctgctc ctccacgctg c 141
<210> 57
<211> 91
<212> DNA
<213> Homo Sapiens
<400> 57
tgttctgaga actgactttc tccccatccc cttcctaaat atccaaagac tgtactggcc 60
agtgtcattt tattttttcc ctcctgacaa t 91
<210> 58
<211> 91
<212> DNA
<213> Homo Sapiens
<400> 58
attgtcagga gggaaaaaat aaaatgacac tggccagtac agtctttgga tatttaggaa 60
ggggatgggg agaaagtcag ttctcagaac a 91
<210> 59
<211> 120
<212> DNA
<213> Homo Sapiens
<400> 59
gcttagagta tggagaacat ggatgcagaa caccagacac ccctttctct ctctttgaag 60
gaatggctgg aacaatatat ttcctggctg acctgctagt ccccacaaaa gccaggttcg 120
<210> 60
<211> 120
<212> DNA
<213> Homo Sapiens
<400> 60
cgaacctggc ttttgtgggg actagcaggt cagccaggaa atatattgtt ccagccattc 60
cttcaaagag agagaaaggg gtgtctggtg ttctgcatcc atgttctcca tactctaagc 120
<210> 61
CA 02382030 2002-02-14
WO 01/16322 PCT/US00/24262
12/18
<211> 139
<212> DNA
<213> Homo Sapiens
<400> 61
agttggtatc aatggagtgg gagaggtgag gagatcacaa gaggaatttg gagcccaggg 60
tcagccctct catcctgccc aaatgttggc agaatctggg tcttagacta gcataagtga 120
agtctgggga gggccgaac 139
<210> 62
<211> 139
<212> DNA
<213> Homo sapiens
<400> 62
gttcggccct ccccacactt cacttatgct agtctaagac ccagattctg ccaacatttg 60
ggcaggatga gagggctgac cctgggctcc aaattcctct tgtgatctcc tcacctctcc 120
cactccattg ataccaact 139
<210> 63
<211> 125
<212> DNA
<213> Homo Sapiens
<400> 63
gtacaaccag ctcctcagaa ttgaagagga gctgggcagc aaggctaagt ttgccggcag 60
gaacttcaga aaccccttgg ccaagtaagc tgtgggcagg caagcccttc ggtcacctgt 120
tggct 125
<210> 64
<211> 125
<212> DNA
<213> Homo Sapiens
<400> 64
agccaacagg tgaccgaagg gcttgcctgc ccacagctta cttggccaag gggtttctga 60
agttcctgcc ggcaaactta gccttgctgc ccagctcctc ttcaattctg aggagctggt 120
tgtac 125
<210> 65
<211> 158
<212> DNA
<213> Homo Sapiens
<400> 65
tacaaccagc tcctcagaat tgaagaggag ctgggcagca aggctaagtt tgccggcagg 60
aacttcagaa accccttggc caagtaagct gtgggcaggc aagcccttcg gtcacctgtt 120
ggctacacag acccctcccc tcgtgtcagc tcaggcag 158
<210> 66
<211> 158
<212> DNA
CA 02382030 2002-02-14
WO 01/16322 PCT/US00/24262
13/18
<213> Homo sapiens
<400> 66
ctgcctgagc tgacacgagg ggaggggtct gtgtagccaa caggtgaccg aagggcttgc 60
ctgcccacag cttacttggc caaggggttt ctgaagttcc tgccggcaaa cttagccttg 120
ctgcccagct cctcttcaat tctgaggagc tggttgta 158
<210> 67
<211> 114
<212> DNA
<213> Homo sapiens
<400> 67
cccacattcc gtcacctgct cagaatcatg caggtctcca ctgctgccct tgctgtcctc 60
ctctgcacca tggctctctg caaccagttc tctgcatcac ttgctgctga cacg 114
<210> 68
<211> 114
<212> DNA
<213> Homo sapiens
<400> 68
cgtgtcagca gcaagtgatg cagagaactg gttgcagaga gccatggtgc agaggaggac 60
agcaagggca gcagtggaga cctgcatgat tcagagcagg tgacggaatg tggg 114
<210> 69
<211> 82
<212> DNA
<213> Homo Sapiens
<400> 69
ctctgttctt caagtttccc tttgattgat ttcatgtaat ctttgatgta cttcttgtag 60
gcttcttttg tgaaacttgt tt 82
<210> 70
<211> 82
<212> DNA
<213> Homo Sapiens
<400> 70
aaacaagttt cacaaaagaa gcctacaaga agtacatcaa agattacatc aaatcaatca 60
aagggaaact tgaagaacag ag 82
<210> 71
<211> 82
<212> DNA
<213> Homo Sapiens
<400> 71
ctctgttctt caagtttccc tttgattgat ttcatgtaat ctttgatgta cttcttgtag 60
gcttcttttg tgaaacttgt tt 82
CA 02382030 2002-02-14
WO 01/16322 PCT/US00/24262
14/18
<210> 72
<211> 82
<212> DNA
<213> Homo Sapiens
<400> 72
aaacaagttt cacaaaagaa gcctacaaga agtacatcaa agattacatc aaatcaatca 60
aagggaaact tgaagaacag ag 82
<210> 73
<211> 89
<212> DNA
<213> Homo Sapiens
<400> 73
tcatcactga ggaagagaag aatttcaaag ccttcgctag tctccgtatg gcccgtgcca 60
acgcccggct cttcggcaca cgggcaaaa 89
<210> 74
<211> 89
<212> DNA
<213> Homo Sapiens
<400> 74
ttttgcccgt gtgccgaaga gccgggcgtt ggcacgggcc atacggagac tagcgaaggc 60
tttgaaattc ttctcttcct cagtgatga 89
<210> 75
<211> 56
<212> DNA
<213> Homo Sapiens
<400> 75
gattctcagc tggtagctgg tgttgcattc aagaagactt tctcttacgc tgggtt 56
<210> 76
<211> 56
<212> DNA
<213> Homo Sapiens
<400> 76
aacccagcgt aagagaaagt cttcttgaat gcaacaccag ctaccagctg agaatc 56
<210> 77
<211> 114
<212> DNA
<213> Homo Sapiens
<400> 77
caggttgtct ttaagatgtt cttttagaca gctgcacatt gtagaccctt tcacctgccc 60
tacaccaaag atgtacgatg cactaggaaa ctgctcatag gatttctgtc agct 114
CA 02382030 2002-02-14
WO 01/16322 PCT/US00/24262
15/18
<210> 78
<211> 114
<212> DNA
<213> Homo Sapiens
<400> 78
agctgacaga aatcctatga gcagtttcct agtgcatcgt acatctttgg tgtagggcag 60
gtgaaagggt ctacaatgtg cagctgtcta aaagaacatc ttaaagacaa cctg 114
<210> 79
<211> 66
<212> DNA
<213> Homo sapiens
<400> 79
gagagggggg atctcatcag gaactgcagc attgggttcc tctgctgcca cttcatcttc 60
atcaat 66
<210> 80
<211> 66
<212> DNA
<213> Homo Sapiens
<400> 80
attgatgaag atgaagtggc agcagaggaa cccaatgctg cagttcctga tgagatcccc 60
cctctc 66
<210> 81
<211> 109
<212> DNA
<213> Homo sapiens
<400> 81
aagccctcgg cccccaagaa gggagacagt tctgctgaag aactgaaact ggccacccag 60
ctgaccggac cggtcatgcc cgtccggaac gtctataaga aggagaaag 109
<210> 82
<211> 109
<212> DNA
<213> Homo Sapiens
<400> 82
ctttctcctt cttatagacg ttccggacgg gcatgaccgg tccggtcagc tgggtggcca 60
gtttcagttc ttcagcagaa ctgtctccct tcttgggggc cgagggctt 109
<210> 83
<211> 84
<212> DNA
<213> Homo Sapiens
CA 02382030 2002-02-14
WO 01/16322 PCT/US00/24262
16/18
<400> 83
ggtctctgtt cttcaagttt ccctttgatt gatttcatgt atctttgatg tacttcttgt 60
aggcttcttt tgtgaaactt gttt 84
<210> 84
<211> 84
<212> DNA
<213> Homo Sapiens
<400> 84
aaacaagttt cacaaaagaa gcctacaaga agtacatcaa agatacatga aatcaatcaa 60
agggaaactt gaagaacaga gacc 84
<210> 85
<211> 320
<212> DNA
<213> Homo Sapiens
<400> 85
tgggggaagg gtgcagcaac gatttctcac caaatcacta cacaggacag caaaggggtg 60
agaaggggct gagggaggaa aagccaggaa actgagatca gcagagggag ccaagcatca 120
aaaaacagga gatgctgaag ctgcgatgac cagcatcatt ttcttaagag aacattcaag 180
gatttgtcat gatggctggg ctttcacttg gtgttaagtc tacaaacagc accttcaatt 240
ggaactgtca attaaagttc ttaagattta ggaagtggtg gagcttggaa agttatgaga 300
ttacaaaatt tctgaaagtc 320
<210> 86
<211> 320
<212> DNA
<213> Homo sapiens
<400> 86
gactttcaga aattttgtaa tctcataact ttccaagctc caccacttcc taaatcttaa 60
gaactttaat tgacagttcc aattgaaggt gctgtttgta gacttaacac caagtgaaag 120
cccagccatc atgacaaatc cttgaatgtt ctcttaagaa aatgatgctg gtcatcgcag 180
cttcagcatc tcctgttttt tgatgcttgg ctccctctgc tgatctcagt ttcctggctt 240
ttcctccctc agccccttct cacccctttg ctgtcctgtg tagtgatttg gtgagaaatc 300
gttgctgcac ccttccccca 320
<210> 87
<211> 123
<212> DNA
<213> Homo sapiens
<400> 87
ggaaaaaaaa aaaaactaca aaaaccctaa ttttgtacat actgtatttt tactattgaa 60
ctgtattcta gtggctgttc atgctccaag actttagtta ccgagacatg aatactatcc 120
atg 123
<210> 88
<211> 123
<212> DNA
CA 02382030 2002-02-14
WO 01/16322 PCT/US00/24262
17/18
<213> Homo Sapiens
<400> 88
catggatagt attcatgtct cggtaactaa agtcttggag catgaacagc cactagaata 60
cagttcaata gtaaaaatac agtatgtaca aaattagggt ttttgtagtt tttttttttt 120
tcc 123
<210> 89
<211> 110
<212> DNA
<213> Homo Sapiens
<400> 89
ctcattttct ggactgggca gcctttgggg tcatgaccct tccctccatc ggcatccccc 60
tgctattgtg gtactccagc aagaggaaat atgacactcc caaaacgaag 110
<210> 90
<211> 110
<212> DNA
<213> Homo Sapiens
<400> 90
cttcgttttg ggagtgtcat atttcctctt gctggagtac cacaatagca gggggatgcc 60
gatggaggga agggtcatga ccccaaaggc tgcccagtcc agaaaatgag 110
<210> 91
<211> 90
<212> DNA
<213> Homo Sapiens
<400> 91
aatcattgtt ttttcctttg taaatgttga ttcagaaaag gaaagcacag gctaagcagt 60
tgaaggttcc ccaccattca gtgagagcag 90
<210> 92
<211> 90
<212> DNA
<213> Homo Sapiens
<400> 92
ctgctctcac tgaatggtgg ggaaccttca actgcttagc ctgtgctttc cttttctgaa 60
tcaacattta caaaggaaaa aacaatgatt 90
<210> 93
<211> 103
<212> DNA
<213> Homo Sapiens
<400> 93
tcgggagccg cggcttatgg tgcagacatg gccaagtcca agaaccacac cacacacaac 60
cagtcccgaa aatggcacag aaatggtatc aagaaacccc gat 103
CA 02382030 2002-02-14
WO 01/16322 PCT/US00/24262
18/18
<210> 94
<211> 103
<212> DNA
<213> Homo Sapiens
<400> 94
atcggggttt cttgatacca tttctgtgcc attttcggga ctggttgtgt gtggtgtggt 60
tcttggactt ggccatgtct gcaccataag ccgcggctcc cga 103
<210> 95
<211> 82
<212> DNA
<213> Homo Sapiens
<400> 95
cgggagccgc ggcttatggt gcagacatgg ccaagtccaa gaaccacacc acacacaacc 60
agtcccgaaa atggcacaga as 82
<210> 96
<211> 82
<212> DNA
<213> Homo Sapiens
<400> 96
tttctgtgcc attttcggga ctggttgtgt gtggtgtggt tcttggactt ggccatgtct 60
gcaccataag ccgcggctcc cg 82
<210> 97
<211> 22
<212> DNA
<213> Homo sapiens
<400> 97
tagggctcga gccgccacca tg 22
<210> 98
<211> 22
<212> DNA
<213> Homo Sapiens
<400> 98
atccctgcag gtcactcact ca 22
