U.S. patent application number 10/044070 was filed with the patent office on 2002-11-07 for prevention of cell migration initiation with cmv us28 receptor antagonists.
Invention is credited to Nelson, Jay, Ruchti, Fronziska, Smith, Patricia, Soderberg-Naucler, Cecilia, Streblow, Daniel.
Application Number | 20020164579 10/044070 |
Document ID | / |
Family ID | 22270487 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020164579 |
Kind Code |
A1 |
Nelson, Jay ; et
al. |
November 7, 2002 |
Prevention of cell migration initiation with CMV US28 receptor
antagonists
Abstract
There is disclosed an assay system for determining therapeutic
activity for treating restenosis, atherosclerosis, chronic
rejection syndrome and graft versus host disease (GVHD) by
measuring inhibition of cell migration activity in smooth muscle
cells expressing a US28 receptor from the CMV genome. Specifically,
there is disclosed a method for measuring inhibition of cell
migration in isolated cells transfected with US28 or infected with
CMV and stimulated with a ligand. There is further disclosed a
method for treating atherosclerosis, restenosis, chronic rejection
syndrome and graft versus host disease (GVHD), comprising
administering an effective amount of an agent that is a US28
receptor antagonist, wherein a US28 receptor antagonist comprises
an inhibitor compound that prevents transduction of US28 receptor
signal stimulated by a US28 receptor ligand, wherein a US28
receptor ligand is selected from the group consisting of RANTES,
MIP-1.alpha. and MCP. The invention further provides a method for
treating restenosis, atherosclerosis, chronic rejection syndrome
and GVHD by administering KHSV encoded vMIP-2, fractalkine or
herbimycin.
Inventors: |
Nelson, Jay; (Tualitin,
OR) ; Streblow, Daniel; (Tigard, OR) ;
Soderberg-Naucler, Cecilia; (Bromma, SE) ; Smith,
Patricia; (Portland, OR) ; Ruchti, Fronziska;
(Portland, OR) |
Correspondence
Address: |
DAVIS WRIGHT TREMAINE, LLP
2600 CENTURY SQUARE
1501 FOURTH AVENUE
SEATTLE
WA
98101-1688
US
|
Family ID: |
22270487 |
Appl. No.: |
10/044070 |
Filed: |
January 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10044070 |
Jan 11, 2002 |
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09387044 |
Aug 31, 1999 |
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6420121 |
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60098689 |
Aug 31, 1998 |
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Current U.S.
Class: |
435/4 ;
435/40.5 |
Current CPC
Class: |
G01N 33/5061 20130101;
A61K 31/00 20130101; A61K 31/395 20130101; G01N 33/566 20130101;
G01N 33/5008 20130101; G01N 33/5029 20130101 |
Class at
Publication: |
435/4 ;
435/40.5 |
International
Class: |
G01N 033/48; C12Q
001/00 |
Claims
We claim:
1. An assay for determining therapeutic activity of US28 receptor
antagonists, comprising: (a) obtaining and isolating smooth muscle
cells into a first chamber of a migration device, wherein the first
migration chamber comprises growth media chambers and is defined by
a first side of a membrane and chamber walls, and wherein the
migration device comprises a second chamber defined by the second
side of the membrane and having an enclosed space; (b) infecting
the smooth muscle cells with human cytomegalovirus (HCVM)
containing a gene encoding the US28 receptor; (c) adding a
candidate therapeutic agent to the first chamber; and (d)
determining the amount of cellular migration into the second
chamber, whereby inhibition of cellular migration of infected
smooth muscle cells indicates therapeutic activity.
2. The assay of claim 1 wherein the smooth muscle cells are
isolated from pulmonary arteries.
3. The assay of claim 1 wherein the membrane has a pore size of
from about 2 to about 10 microns.
4. The assay of claim 3 wherein the membrane pore size is about 3
microns.
5. The assay of claim 1 wherein the amount of cellular migration is
determined by an assay for counting the number of smooth muscle
cells in the second chamber wherein the assay for counting the
number of smooth muscle cells is selected from the group consisting
of microscopic cell counting per unit area, radiolabeling the
smooth muscle cells and counting radioactivity in the second
chamber, attaching a fluorescent probe to the smooth muscle cells
and measuring fluorescence within the second chamber, and
combinations thereof.
6. A method for treating atherosclerosis, restenosis, chronic
rejection syndrome and graft versus host disease (GVHD), comprising
administering an effective amount of an agent that is a US28
receptor antagonist, wherein a US28 receptor antagonist comprises
an inhibitor compound that prevents transduction of US28 receptor
signal stimulated by a US28 receptor ligand, wherein a US28
receptor ligand is selected from the group consisting of RANTES,
MIP-1.alpha. and MCP.
7. The method of claim 6 wherein the US28 receptor antagonist is
selected from the group consisting of an antibody that binds to an
extracellular portion of the US28 receptor, an antisense
oligonucleotide having a nucleic acid sequence antisense to the
US28 cDNA and inhibiting translation of US28 expression in infected
smooth muscle cells, and US28 receptor antagonist is selected from
the group consisting of an antibody that binds to an extracellular
portion of the US28 receptor, and a US28 binding antagonist,
wherein the US28 binding antagonist is selected from the group
consisting of KHSV encoded vMIP-2, fractalkine, and herbimycin.
8. The method of claim 6 wherein the monoclonal antibody is
chimeric or humanized by means for humanizing non-human
antibodies.
9. The method of claim 6 wherein the US28 antisense sequences are
selected from the group consisting of SEQ ID NOS. 2-28.
10. A method for enhancing cellular migration, comprising infecting
a cell with a viral nucleic acid containing a gene encoding CVM
US28 receptor or tansfecting a cell with a vector comprising the
cDNA sequence for US28 operably linked to a viral promoter
sequence, and stimulating the transfected or infected cell with a
US28 receptor ligand, selected from the group consisting of RANTES,
MIP-1.alpha. and MCP1.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention provides an assay system for
determining therapeutic activity for treating restenosis,
atherosclerosis, chronic rejection syndrome and graft versus host
disease (GVHD) by measuring inhibition of cell migration activity
in smooth muscle cells expressing a US28 receptor from the CMV
genome. Specifically, the present invention provides a method for
measuring inhibition of cell migration in isolated cells
transfected with US28 or infected with CMV and stimulated with a
ligand. The invention further provides a method for treating
restenosis, atherosclerosis, chronic rejection syndrome and GVHD by
administering KHSV encoded vMIP-2, fractalkine or herbimycin.
BACKGROUND OF THE INVENTION
[0002] Atherosclerosis, Restenosis, Chronic Rejection Syndrome and
GVHD
[0003] Atherosclerosis is a major cause of morbidity in the
industrialized world. Atherosclerotic lesions usually become
apparent in adult patients as a result of complete occlusion of a
strategic blood vessel and the resulting complication. However,
such lesions begin much earlier in the life of the patient. It was
later noticed that there was a statistical association with viral
infection, particularly CMV.
[0004] It has been postulated that CMV and possibly herpes virus
are involved in the inducement of atherosclerotic lesions. Several
investigators have demonstrated the presence of CMV nucleic acids
and/or antigens in the human arterial wall using DNA hybridization
techniques (Melnick et al., Lancet 11:644-647, 1983),
immunohistochemistry (Petrie et al., J. Infect. Dis. 155:158-159,
1987), dot blot and in situ hybridization techniques (Hendrix et
al., Am. J Path. 134:1151-1157, 1989), and by polymerase chain
reaction (PCR) techniques using probes derived from immediate early
and late genomic regions (Hendrix et al., Am. J Path. 136:23-28,
1990). Thus, there has been finding of viral antigens and nucleic
acid sequences in arterial smooth muscle cells that suggest that
CMV infection of the arterial wall may be a common occurrence in
patients with atherosclerosis.
[0005] Soon after renal transplantation became an accepted
treatment, an association was noted between CMV infection,
glomerulopathy, and rejection of the transplanted kidney. Thus, CMV
was investigated to determine if it played a role in graft
atherosclerosis that frequently occurs after heart transplantation
(Grattan et al., J. Am. Med. Assn. 261:3561-3566, 1989). The
findings show that heart transplant patients who are
immunosuppressed and become infected with CMV are particularly
prone to develop atherosclerosis in the transplanted organ. It is
postulated that the artery wall may be the site of CMV latency
because CMV DNA but not infectious virus was found in the artery
wall.
[0006] Role of Chemokines
[0007] Chemokines are chemoattratants for neutrophils, monocytes,
lymphocytes and bone marrow progenitors, as well as other cell
types. The family of chemokines comprises four subfamilies, defined
by the distribution of cysteine residues in the N terminus of these
factors, the CXC, CC, C, and CX3C subfamilies. The chemokines are
related by primary structure, particularly by conservation of a
four-cysteine motif. C-C chemokines include such members as human
monocyte chemotactic protein 1 (MCP-1), RANTES, and the macrophage
inflammatory proteins 1.alpha. and 1.beta. (MIP-1.alpha. and
MIP-1.beta.). These ligands exhibit chemoattractant potential for
monocytes but not neutrophils. CMV infection can also modify the
level of chemokines. The level of RANTES (a chemokine) produced by
cells recovered by bronchoalveolar lavage from lung transplant
patients with CMV pneumonitis shows that cells from infected
patients secreted greater amounts of RANTES than did cells
recovered from either patients undergoing acute rejection or from
control subjects (Monti et al., Transplantation 61:1757-1762,
1996). AIDS patients with CMV encephlitis have higher
concentrations of MCP-1 (a chemokine) but not other chemokines in
their spinal fluid than do HIV seropositive persons who are
asymptomatic or AIDS patients with a number of other opportunistic
infections of the central nervous system (Bernasconi et al., J.
Infec. Dis. 174:1098-1101, 1996). When fibroblasts were infected
with CMV, RANTES mRNA and protein expression are induced early, but
extracellular RANTES accumulation, but not transcription is
down-regulated late during CMV infection (Michelson et al., J
Virol. 71:6495-6500, 1997). Therefore, CMV infection has the
capacity to both induce cell migration and enhance chemokine
production early during the infection process.
[0008] Chemokine receptors tend to be multiple membrane-spanning
proteins, generally 7 or 8 membrane-spanning proteins and tend to
transduce signal through G-coupled protein signal transduction.
Human C-C chemokines tend to bind to the US28 receptor of CMV
(Neote et al., Cell 72:415-425, 1993). There is also a sequence
homology between the C-CKR-1 receptor (normal human gene) and the
CMV US28 sequence in the open reading frame region. (Neote et al.,
1993). Thus, Neote et al. speculated that "the protein encoded by
the US28 open reading frame of Towne strain CMV can bind C-C
chemokines but not the C-X-C chemokine IL-8. However, none of the
earlier chemokine receptor papers, including Neote et al., has made
the connection between US28 and it s role in mediating smooth
muscle cell proliferation.
SUMMARY OF THE INVENTION
[0009] The present invention provides an assay for determining
therapeutic activity of US28 receptor antagonists, comprising (a)
obtaining and isolating smooth muscle cells into a first chamber of
a migration device, wherein the first migration chamber comprises
growth media chambers and is defined by a first side of a membrane
and chamber walls, and wherein the migration device comprises a
second chamber defined by the second side of the membrane and
having an enclosed space; (b) infecting the smooth muscle cells
with human cytomegalovirus (HCVM) containing a gene encoding the
US28 receptor; (c) adding a candidate therapeutic agent to the
first chamber; and (d) determining the amount of cellular migration
into the second chamber, whereby inhibition of cellular migration
of infected smooth muscle cells indicates therapeutic activity.
Preferably, the smooth muscle cells are isolated from pulmonary
arteries. Preferably, the membrane has a pore size of from about 2
to about 10 microns. Most preferably, the membrane pore size is
about 3 microns. Preferably the amount of cellular migration is
determined by an assay for counting the number of smooth muscle
cells in the second chamber wherein the assay for counting the
number of smooth muscle cells is selected from the group consisting
of microscopic cell counting per unit area, radiolabeling the
smooth muscle cells and counting radioactivity in the second
chamber, attaching a fluorescent probe to the smooth muscle cells
and measuring fluorescence within the second chamber, and
combinations thereof.
[0010] The present invention further provides a method for treating
atherosclerosis, restenosis, chronic rejection syndrome and graft
versus host disease (GVHD), comprising administering an effective
amount of an agent that is a US28 receptor antagonist, wherein a
US28 receptor antagonist comprises an inhibitor compound that
prevents transduction of US28 receptor signal stimulated by a US28
receptor ligand, wherein a US28 receptor ligand is selected from
the group consisting of RANTES, MIP-1.alpha. and MCP. Preferably,
the US28 receptor antagonist is selected from the group consisting
of an antibody that binds to an extracellular portion of the US28
receptor, and an antisense oligonucleotide having a nucleic acid
sequence antisense to the US28 cDNA and inhibiting translation of
US28 expression in infected smooth muscle cells, or a US28 binding
antagonist, wherein the US28 binding antagonist is selected from
the group consisting of KHSV encoded vMIP-2, fractalkine, and
herbimycin. Preferably, the monoclonal antibody is chimeric or
humanized by means for humanizing non-human antibodies. Preferably,
the US28 antisense sequences are selected from the group consisting
of SEQ ID NOS. 2-28.
[0011] The present invention further provides a method for
enhancing cellular migration, comprising infecting a cell with a
viral nucleic acid containing a gene encoding CVM US28 receptor or
tansfecting a cell with a vector comprising the cDNA sequence for
US28 operably linked to a viral promoter sequence, and stimulating
the transfected or infected cell with a US28 receptor ligand,
selected from the group consisting of RANTES, MIP-1.alpha. and
MCP1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows the ability of HCMV-GFP (human cytomegalovirus
GFP) to infect pulmonary smooth muscle cells (SMC) in vitro. SMC
were infected with HCMV-GFP (MOI 10) for 2 days and then examined
for the presence of GFOHCMV immediate early expression.
Colocalization of GFP and IE (intermediate-early) were observed
only in HCMV-GFP infected cells.
[0013] FIG. 2 shows that HCMV infected SMCs were examined by
electron microscopy for the presence of virus. The photos show
numerous virus capsids were found in the nucleus and mature virions
were observed on the plasma membrane of HCMV infected cells.
[0014] FIG. 3 shows a one-step growth curve showing HCMV
replication in SMCs. SMC were infected with HCMV Towne strain at
MOI1. These data show HCMV growth and release in SMC exhibited
normal kinetics.
[0015] FIG. 4 shows that HCMV infection induced actin
reorganization in SMC as an indication of migration activity. The
cells were treated with either PDGF (100 ng/ml) of infected with
HCMV Towne strain at MOI1. Actin distribution was visualized at 5
days post-treatment by fluorescence using TRITC conjugated
phalloidin.
[0016] FIG. 5 shows an SMC migration assay scheme. SMCs are
cultured in the upper chamber and infected with HCMV (preferably at
or near MOI1). Only cells that are infected will migrate through a
filter (preferably 3 micron) to the lower chamber. The cells in the
lower chamber are counted by microscopy or labeled with a
radioactive or fluorescent label.
[0017] FIG. 6 shows the presence of HCMV in migrating SMCs. SMC
were infected with HCMV-GFP. The migrating cells were analyzed for
the presence of GFP and HCMV glycoprotein gB by immunofluorescence
using anti-gB antibodies. All of the migrating SMCs exhibited GFP
and gB expression.
[0018] FIG. 7 shows that HCMV induced migration of SMCs. Cellular
mobility assays were used to determine the specificity of HCMV
induced SMC migration. SMCs that were treated with PDGF
(Platelet-derived growth factor, an inducer of cellular migration)
did not cause cell migration in SMCs to nearly the extent as HCMV
infection. Moreover, HCMV neutralizing antibodies reduced cellular
migration to mock levels.
[0019] FIG. 8 shows that HCMV-induced migration was specific for
SMCs. Both SMCs and human foreskin fibroblasts (HFF) were infected
and analyzed in migration assays. The data provided in FIG. 8 show
that HCMV induced cellular migration occurred only in SMCs but not
in similarly-infected HFFs.
[0020] FIG. 9 shows that protein synthesis was required for
HCMV-induced SMC migration. SMC mobility was blocked by
cyclohexamide (a general protein synthesis inhibitor). Moreover,
foscarnet (an inhibitor of HCMV late gene production) did not
inhibit SMC migration.
[0021] FIG. 10 shows that HCMV genome encodes four putative
chemokine receptors, including US27, US28, UL33 and UL78.
[0022] FIG. 11 shows that HCMV infection of HFFs induced RANTES
(chemokine) expression. HFF cell culture supernatants were
collected every 8 hours from infected HFFs and RANTES
concentrations in the supernatants were determined by an ELISA
assay.
[0023] FIG. 12 shows that the addition of RANTES at the
concentrations shown to HCMV-GFP-infected SMCs increased SMC
migration in a dose-dependent manner.
[0024] FIG. 13 shows that that a HCMV having the US28 receptor gene
deleted affected cell motility.
[0025] FIG. 14 shows the construction scheme for human CMV GFP
recombinants.
[0026] FIG. 15 shows the results of inhibition of a PTK pathway
effect of US28 SMC migration with several PTK inhibitors including
herimycin A, pertussis toxin and genistein at the concentrations
indicated. Pertussis toxin had no effect.
DETAILED DESCRIPTION OF THE INVENTION
[0027] US28 Receptor
[0028] The sequence characterization of the US28 receptor is
provided in Neote et al. (Cell 72:415-425, 1993) and also the cDNA
sequence is SEQ ID No. 1. The present invention is based upon the
discovery that the CMV effect in causing smooth muscle
proliferation and an initiating event in the diseases
atherosclerosis, restenosis, chronic organ rejection and GVHD, is
mediated primarily through signal transduction in infected smooth
muscle cells through the US28 receptor. Based upon this discovery,
described herein, the claimed invention is provided that provides
US28 receptor antagonist molecules that have therapeutic effect.
Moreover, the present invention provides an assay procedure to
screen of other US28 antagonist molecules that, based upon the
findings reported here, are effective for treating atherosclerosis,
restenosis, chronic organ rejection and GVHD.
[0029] Role of US28 in Smooth Muscle Cell Migration
[0030] The present invention is based upon the discovery of the
role of US28 in mediating the properties of CMV to stimulate smooth
muscle cells that can ultimately lead to atherosclerosis,
restenosis, chronic rejection syndrome or GVHD in susceptible
patients. It is clear to a skill practitioner that a patient must
first have been a transplant recipient before he or she is at risk
for either GVHD or chronic rejection syndrome. Moreover, restenosis
first requires an angioplasty-type procedure or other chemical or
surgical intervention in clearing occluded or partially occluded
arteries before the patient is at risk for restenosis.
[0031] The data provided in the FIGS. (1-13) show that CMV virus
infects smooth muscle cells in vitro and that intermediate early
expression of viral protein can be seen (FIGS. 1-3). The affect of
CMV infection in smooth muscle cells is shown affect actin
reorganization as a market for migration activity (FIG. 4).
Moreover, the infected cells were able to migrate in a migration
chamber, such as the one shown in FIG. 6. The scheme shown in FIG.
5 provides that only infected cells have the capability to migrate
through a filter in a migration chamber. Thus, smooth muscle cells
infected with CMV showed the ability to migrate, even to a much
greater extent than non-infected smooth muscle cells treated with
the migration enhancing growth factor, PDGF (FIG. 7).
[0032] The next set of experiments were designed to determine which
protein or proteins, encoded by the VMC genome, was responsible for
conferring the migration activity on infected smooth muscle cells.
It was first found that protein synthesis was required to confer
the migration activity on infected smooth muscle cells (FIG. 9).
Moreover, the suspect protein or proteins encoded by the CMV genome
were not late gene production genes as evidenced by the fact that
foscarnet (an inhibitor of HCMV late gene production) did not
inhibit migration of infected smooth muscle cells (FIG. 9). This
left four putative chemokine receptors that are encoded by the CMV
genome, US27, US28, UL33 and UL78 (FIG. 10). In knock-out
experiments, wherein each of the four foregoing chemokine receptor
genes were knocked out, it was only a US28 knock out that was able
to inhibit smooth muscle cell migration activity when smooth muscle
cells were infected with the US28 knock out variety of CMV (FIG.
13). However, the ability of CMV infection to increase cell
migration of smooth muscle cells may not be only as US28 affect and
there are also ligand activity that needs to activate the US28
receptor. Moreover, CMV infection seems to also have an autocrine
function in enhancing certain C-C chemokine production, such as
RANTES (FIGS. 11-12). Accordingly, the foregoing data provides the
basis for the present invention.
[0033] Screening Assay
[0034] The present invention provides an assay for determining
therapeutic activity of US28 receptor antagonists, comprising (a)
obtaining and isolating smooth muscle cells into a first chamber of
a migration device, wherein the first migration chamber comprises
growth media chambers and is defined by a first side of a membrane
and chamber walls, and wherein the migration device comprises a
second chamber defined by the second side of the membrane and
having an enclosed space; (b) infecting the smooth muscle cells
with human cytomegalovirus (HCVM) containing a gene encoding the
US28 receptor; (c) adding a candidate therapeutic agent to the
first chamber; and (d) determining the amount of cellular migration
into the second chamber, whereby inhibition of cellular migration
of infected smooth muscle cells indicates therapeutic activity.
Preferably, the smooth muscle cells are isolated from pulmonary
arteries. Preferably, the membrane has a pore size of from about 2
to about 10 microns. Most preferably, the membrane pore size is
about 3 microns. Preferably the amount of cellular migration is
determined by an assay for counting the number of smooth muscle
cells in the second chamber wherein the assay for counting the
number of smooth muscle cells is selected from the group consisting
of microscopic cell counting per unit area, radiolabeling the
smooth muscle cells and counting radioactivity in the second
chamber, attaching a fluorescent probe to the smooth muscle cells
and measuring fluorescence within the second chamber, and
combinations thereof. Preferably, the infected smooth muscle cells
are further stimulated with ligand to enhance migration activity,
wherein the ligand is a C-C chemokine. Preferably, the C-C ligand
is selected from the group consisting of RANTES, MCP-1,
MIP-1.alpha., MIP-1.beta., and combinations thereof.
[0035] KHSV-encoded vMIP-2
[0036] KSHV-(Kaposi's sarcoma-associated herpes virus) encoded vMip
alpha and beta has been described as having angiogenic and HIV
inhibitory functions (Boshoff et al., Science 278:290-294, 1997).
It has also been described as a broad-spectrum chemokine antagonist
(Kledal et al., Science 277:1656-1659, 1997). The present invention
adds to the therapeutic uses for KHSV-encoded MIP for treating
atherosclerosis, restenosis, chronic rejection syndrome and
GVHD.
[0037] Fractalkine
[0038] Results from several studies showed that the polypeptide
fractalkine is a ligand for US28 receptor and functions as a US28
antagonist through competitive binding. Fractalkine has been
described in Kledal et al. FEBS Lett. 441:209-214, 1998. The
present invention adds to the therapeutic uses for fractalkine for
treating atherosclerosis, restenosis, chronic rejection syndrome
and GVHD.
[0039] Herbimycin
[0040] Herbimycin A is a PTK (protein tyrosine kinase) pathway
inhibitor. It is available commercially (Sigma). In FIG. 15, the
effect of herbimycin A on US28 SMC (smooth muscle cell) migration
showed herbimycin A was effective in inhibiting US28 transfected
smooth muscle cell migration. Thus, it appears that US28 SMC
migration is mediated through a PTK pathway. The present invention
adds to the therapeutic uses for herbimycin for treating
atherosclerosis, restenosis, chronic rejection syndrome and
GVHD.
[0041] Antisense
[0042] US28 is made off of two different transcripts, one only
contains the US28 ORF and the other contains US27/28 ORF's. Both
use the same poly-A signal. Antisense oligo sequences as US28
antagonists for both US27 and US28 are as follows:
[0043] US27-5'-1---ATT TGT AGA GGT GGT CAT [SEQ ID NO. 9]
[0044] US27-5'-2---GCT CAC CTG CGT TAA GGT [SEQ ID NO. 10]
[0045] US27-5'-3---GTG CTG TTT AAG GTG TGG [SEQ ID NO. 11]
[0046] US27-5'-4---AGT GTA CTC GAA CAA CTG [SEQ ID NO. 12]
[0047] US27-5'-5---CAA CCA TAC CCC GTT GGC [SEQ ID NO. 13]
[0048] US27-3'-1---TTC ACG CAG CAA CAG GCG [SEQ ID NO. 14]
[0049] US27-3'-2---CCT GGT AAG GTA TAT CCT [SEQ ID NO. 15]
[0050] US27-3'-3---GTA GCT CAA TAT CAA TGT [SEQ ID NO. 16]
[0051] US27-3'-4---GCC CTT CTT TGT ATG TCC [SEQ ID NO. 17]
[0052] US27-3'-5---ATG GGT ACG TTT GGT GTG [SEQ ID NO. 18]
[0053] US28-5'-1---CGT CGT CGT CGG TGT CAT [SEQ ID NO. 19]
[0054] US28-5'-2---CGT CGT GAG TTC CGC GGT [SEQ ID NO. 20]
[0055] US28-5'-3---CAG GGA GTC GCT TCA TCG [SEQ ID NO. 21]
[0056] US28-5'-4---TGA TTA AGC ACG TCG GTG [SEQ ID NO. 22]
[0057] US28-5'-5---GAA GAG AAA GAC AAC GCC [SEQ ID NO. 23]
[0058] US28-3'-1---GCT GTG GTA CCA GGA TAC [SEQ ID NO. 24]
[0059] US28-3'-2---CTC CGA CGC GAA AAG CTC [SEQ ID NO. 25]
[0060] US28-3'-3---GTC TCT CTT CGG CTC GGC [SEQ ID NO. 26]
[0061] US28-3'-4---CGG ACA GCG TGT CGG AAG [SEQ ID NO. 27]
[0062] US28-3'-5---GAG ACG CGA CAC GCC TCG [SEQ ID NO. 28]
[0063] Additional antisense sequences are provided as SEQ ID NOS
2-8.
[0064] Pharmaceutical Formulation
[0065] The inventive method in the form of a pharmaceutical
composition comprising a US28 antagonist can be administered to a
patient either by itself (complex or combination) or in
pharmaceutical compositions where it is mixed with suitable
carriers and excipients. A US28 antagonist can be administered
parenterally, such as by intravenous injection or infusion,
intraperitoneal injection, subcutaneous injection, or intramuscular
injection. A US28 antagonist can be administered orally or rectally
through appropriate formulation with carriers and excipients to
form tablets, pills, capsules, liquids, gels, syrups, slurries,
suspensions and the like. A US28 antagonist can be administered
topically, such as by skin patch, to achieve consistent systemic
levels of active agent. A US28 antagonist is formulated into
topical creams, skin or mucosal patch, liquids or gels suitable to
topical application to skin or mucosal membrane surfaces. A US28
antagonist can be administered by inhaler to the respiratory tract
for local or systemic treatment of HIV infection.
[0066] The dosage of the US28 antagonist suitable for use with the
present invention can be determined by those skilled in the art
from this disclosure. The US28 antagonist will contain an effective
dosage (depending upon the route of administration and
pharmacokinetics of the active agent) of the US28 antagonist and
suitable pharmaceutical carriers and excipients, which are suitable
for the particular route of administration of the formulation
(i.e., oral, parenteral, topical or by inhalation). The active US28
antagonist is mixed into the pharmaceutical formulation by means of
mixing, dissolving, granulating, dragee-making, emulsifying,
encapsulating, entrapping or lyophilizing processes. The
pharmaceutical formulations for parenteral administration include
aqueous solutions of the active US28 antagonist in water-soluble
form. Additionally, suspensions of the active US28 antagonist may
be prepared as oily injection suspensions. Suitable lipophilic
solvents or vehicles include fatty oils such as sesame oil, or
synthetic fatty acid ester, such as ethyl oleate or triglycerides,
or liposomes. Aqueous injection suspensions may contain substances,
which increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol, or dextran. The suspension may
optionally contain stabilizers or agents to increase the solubility
of the complex or combination to allow for more concentrated
solutions.
[0067] Pharmaceutical formulations for oral administration can be
obtained by combining the active compound with soild excipients,
such as sugars (e.g. lactose, sucrose, mannitol of sorbitol),
cellulose preparations (e.g., starch, methyl cellulose,
hydroxypropylmethyl cellulose, and sodium carboxymethyl cellulose),
gelaten, gums, or polyvinylpyrrolidone. In addition, a
disintegrating agent may be added, and a stabilizer may be added.
Sequence CWU 1
1
28 1 1087 DNA Human cytomegalovirus 1 aaacgtcatc tcgccgacgt
ggtgaaccgc tcatatagac caaaccggac gctgcctcag 60 tctctcggtg
cgtggaccag acggcgtcca tgcaccgagg gcagaactgg tgctatcatg 120
acaccgacga cgacgaccgc ggaactcacg acggagtttg actacgatga agacgcgact
180 ccttgtgttt tcaccgacgt gcttaatcag tcaaagccag ttacgttgtt
tctgtacggc 240 gttgtctttc tcttcggttc catcggcaac ttcttggtga
tcttcaccat cacctggcga 300 cgtcggattc aatgctccgg cgatgtttac
tttatcaacc tcgcggccgc cgatttgctt 360 ttcgtttgta cactacctct
gtggatgcaa tacctcctag atcacaactc cctagccagc 420 gtgccgtgta
cgttactcac tgcctgtttc tacgtggcta tgtttgccag tttgtgtttt 480
atcacggaga ttgcactcga tcgctactac gctattgttt acatgagata tcggcctgta
540 aaacaggcct gccttttcag tattttttgg tggatctttg ccgtgatcat
cgccattcca 600 cactttatgg tggtgaccaa aaaagacaat caatgtatga
ccgactacga ctacttagag 660 gtcagttacc cgatcatcct caacgtagaa
ctcatgcttg gtgctttcgt gatcccgctc 720 agtgttatca gctactgcta
ctaccgcatt tccagaatcg ttgcggtgtc tcagtcgcgc 780 cacaaaggtc
gcattgtacg ggtacttata gcggtcgtgc ttgtctttat catcttttgg 840
ctgccgtacc acctaacgct gtttgtggac acgttaaaac tcctcaaatg gatctccagc
900 agctgcgagt tcgaaagatc gctcaaacgt gcgctcatct tgaccgagtc
gctcgccttt 960 tgtcactgtt gtctcaatcc gctgctgtac gtcttcgtgg
gcaccaagtt tcggcaagaa 1020 ctacactgtc tgctggccga gtttcgccag
cgactctttt cccgcgatgt atcctggtac 1080 cacagca 1087 2 20 DNA
Artificial Sequence US28 receptor antisense receptor/specific
antisense molecule 2 ctggctttga ctgattaagc 20 3 20 DNA Artificial
Sequence US28 receptor antisense receptor/specific antisense
molecule 3 catgatagca ccagttctgc 20 4 20 DNA Artificial Sequence
US28 receptor antisense receptor/specific antisense molecule 4
ccggagcatt gaatccgacg 20 5 20 DNA Artificial Sequence US28 receptor
antisense receptor/specific antisense molecule 5 gctggctagg
gagttgtgat 20 6 20 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 6 ctggctttga ctgattaagc 20 7
20 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 7 aaacaatagc gtagtagcga 20 8
20 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 8 ttggtcacca ccataaactg 20 9
18 DNA Artificial Sequence US27 receptor antisense
receptor/specific antisense molecule 9 atttgtagag gtggtcat 18 10 18
DNA Artificial Sequence US27 receptor antisense receptor/specific
antisense molecule 10 gctcacctgc gttaaggt 18 11 18 DNA Artificial
Sequence US27 receptor antisense receptor/specific antisense
molecule 11 gtgctgttta aggtgtgg 18 12 18 DNA Artificial Sequence
US27 receptor antisense receptor/specific antisense molecule 12
agtgtactcg aacaactg 18 13 18 DNA Artificial Sequence US27 receptor
antisense receptor/specific antisense molecule 13 caaccatacc
ccgttggc 18 14 18 DNA Artificial Sequence US27 receptor antisense
receptor/specific antisense molecule 14 ttcacgcagc aacaggcg 18 15
18 DNA Artificial Sequence US27 receptor antisense
receptor/specific antisense molecule 15 cctggtaagg tatatcct 18 16
18 DNA Artificial Sequence US27 receptor antisense
receptor/specific antisense molecule 16 gtagctcaat atcaatgt 18 17
18 DNA Artificial Sequence US27 receptor antisense
receptor/specific antisense molecule 17 gcccttcttt gtatgtcc 18 18
18 DNA Artificial Sequence US27 receptor antisense
receptor/specific antisense molecule 18 atgggtacgt ttggtgtg 18 19
18 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 19 cgtcgtcgtc ggtgtcat 18 20
18 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 20 cgtcgtgagt tccgcggt 18 21
21 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 21 caaggagtcg cgtcttcatc g 21
22 18 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 22 tgattaagca cgtcggtg 18 23
18 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 23 gaagagaaag acaacgcc 18 24
18 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 24 gctgtggtac caggatac 18 25
18 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 25 ctccgacgcg aaaagctc 18 26
18 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 26 gtctctcttc ggctcggc 18 27
18 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 27 cggacagcgt gtcggaag 18 28
18 DNA Artificial Sequence US28 receptor antisense
receptor/specific antisense molecule 28 gagacgcgac acgcctcg 18
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