U.S. patent application number 13/650526 was filed with the patent office on 2013-05-09 for methods and compositions for the inhibition of hiv-1 replication.
This patent application is currently assigned to The United States of America, as represented by Secretary, Department of Health & Human Services. The applicant listed for this patent is The United States Of America, as Represented By The Secretary, Department Of Health & Human Services. Invention is credited to Teresa Greenwell-Wild, Nancy Vazquez-Maldonado, Sharon M. Wahl.
Application Number | 20130116305 13/650526 |
Document ID | / |
Family ID | 34590128 |
Filed Date | 2013-05-09 |
United States Patent
Application |
20130116305 |
Kind Code |
A1 |
Wahl; Sharon M. ; et
al. |
May 9, 2013 |
METHODS AND COMPOSITIONS FOR THE INHIBITION OF HIV-1
REPLICATION
Abstract
This invention relates to methods and compositions for the
attenuation of HIV-1 replication in human cells, and especially in
human macrophages. The invention particularly concerns the use of
inhibitors of P21 (CDKNIA) expression to attenuate such
replication. The invention particularly concerns the use of
antisense P21 oligonucleotides, siRNA and/or
2-cyano-3,12-dioxooleana-1,9-dien28-oic (CDDO) to attenuate such
replication.
Inventors: |
Wahl; Sharon M.; (N.
Potomac, MD) ; Vazquez-Maldonado; Nancy; (Chevy
Chase, MD) ; Greenwell-Wild; Teresa; (Gaithersburg,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Department Of Health & Human Services; The United States Of
America, as Represented By The Secretary, |
Rockville |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by Secretary, Department of Health & Human
Services
Rockville
MD
|
Family ID: |
34590128 |
Appl. No.: |
13/650526 |
Filed: |
October 12, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10578536 |
Mar 21, 2007 |
8288439 |
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PCT/US04/36492 |
Nov 3, 2004 |
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13650526 |
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60516734 |
Nov 4, 2003 |
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Current U.S.
Class: |
514/44A ;
435/375 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 15/1132 20130101; C12N 2310/14 20130101; C12N 2310/315
20130101; C12N 2310/11 20130101; A61P 31/18 20180101; C12N 15/113
20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/44.A ;
435/375 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0002] This invention was funded by the National Institutes of
Health, Department of Health and Human Services. The United States
Government has certain rights to this invention.
Claims
1. A method of attenuating the transmission or infection of an
immunodeficiency virus into a cell comprising providing to said
cell an inhibitor of p21, wherein said inhibitor is a
polynucleotide provided in an amount and duration sufficient to
cause an attenuation of at least about 50% in said transmission or
infection of said virus relative to an untreated cell.
2. The method of claim 1, wherein said immunodeficiency virus is
human immunodeficiency virus (HIV), and said cell is a human
cell.
3. (canceled)
4. The method of claim 2, wherein said polynucleotide is
complementary to a portion of a p21 gene or p21 cDNA molecule.
5. The method of claim 4, wherein said p21 gene or p21 cDNA is of a
human p21 gene or p21 cDNA molecule.
6. The method of claim 5, wherein said polynucleotide comprises at
least 10 contiguous nucleotides of SEQ ID NO.:4.
7. The method of claim 6, wherein said polynucleotide comprises at
least 10 contiguous nucleotides of SEQ ID NO.:8 or SEQ ID
NO.:10.
8. The method of claim 4, wherein said p21 gene or p21 cDNA is of a
non-human animal or is a variant of a non-human p21 gene or p21
cDNA molecule.
9. The method of claim 8, wherein said polynucleotide comprises at
least 10 contiguous nucleotides of SEQ ID NO.:6.
10. The method of claim 9, wherein said polynucleotide comprises at
least 10 contiguous nucleotides of SEQ ID NO.:7 or SEQ ID
NO.:9.
11.-12. (canceled)
13. A method of treating AIDS in an individual, comprising
providing to HIV-1 infected cells of said individual, or to HIV 1
susceptible cells of such individual, an amount of a p21 inhibitor
sufficient to attenuate the propagation of HIV, wherein said
inhibitor is a polynucleotide provided in an amount and duration
sufficient to cause an attenuation of at least 50% in said
propagation of HIV relative to untreated cells.
14. (canceled)
15. The method of claim 13, wherein said polynucleotide is
complementary to a portion of a p21 gene or p21 cDNA molecule.
16. The method of claim 15, wherein said p21 gene or p21 cDNA is of
a human p21 gene or p21 cDNA molecule.
17. The method of claim 16, wherein said polynucleotide comprises
at least 10 contiguous nucleotides of SEQ ID NO.:4.
18. The method of claim 17, wherein said polynucleotide comprises
at least 10 contiguous nucleotides of SEQ ID NO.:8 or SEQ ID
NO.:10.
19. The method of claim 15, wherein said p21 gene or p21 cDNA is of
a non-human animal or is a variant of a non-human p21 gene or p21
cDNA molecule.
20. The method of claim 19, wherein said polynucleotide comprises
at least 10 contiguous nucleotides of SEQ ID NO.:6.
21. The method of claim 20, wherein said polynucleotide comprises
at least 10 contiguous nucleotides of SEQ ID NO.:7 or SEQ ID
NO.:9.
22.-34. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Patent application
Ser. No. 60/516,734 (filed on Nov. 4, 2004), which application is
herein incorporated by reference.
FIELD OF THE INVENTION
[0003] This invention relates to methods and compositions for the
attenuation of HIV-1 replication in human cells, and especially in
human macrophages. The invention particularly concerns the use of
inhibitors of P21 (CDKN1A) expression to attenuate such
replication. The invention particularly concerns the use of
antisense P21 oligonucleotides and/or
2-cyano-3,12-dioxooleana-1,9-dien-28-oic (CDDO) to attenuate such
replication.
BACKGROUND OF THE INVENTION
[0004] Human immunodeficiency virus-1 (HIV-1) is the causative
agent of acquired immune deficiency syndrome (AIDS) and related
disorders (Gallo, R. C. et al. (1983) "Isolation of human T-cell
leukemia virus in acquired immune deficiency syndrome (AIDS),"
Science 220(4599):865-7; Barre-Sinoussi, F. et al. "ISOLATION OF A
T-LYMPHOTROPIC RETROVIRUS FROM A PATIENT AT RISK FOR ACQUIRED
IMMUNE DEFICIENCY SYNDROME (AIDS)," (1983) Science 220:868-870;
Gallo, R. et al. (1984) "FREQUENT DETECTION AND ISOLATION OF
CYTOPATHIC RETROVIRUSES (HTLV-III) FROM PATIENTS WITH AIDS AND AT
RISK FOR AIDS," Science 224:500-503; Teich, N. et. al. (1984) "RNA
TUMOR VIRUSES," Weiss, R. at al. (eds.) Cold Spring Harbor Press
(NY) pp. 949-956).
[0005] T lymphocytes and macrophages expressing CD4 and the seven
transmembrane chemokine co-receptors CXCR4 and CCR5 are susceptible
to HIV-1 infection (Berger, E. A. at al. (1999) "CHEMOKINE
RECEPTORS AS HIV-1 CORECEPTORS; ROLES IN VIRAL ENTRY, TROPISM, AND
DISEASE," Annu. Rev. Immunol. 17:657-700). In contrast to CD4.sup.+
lymphocytes, HIV-1 infected macrophages can resist cell death
despite viral infection. Viruses within and shed from infected
macrophages may serve as a reservoir for the infection of
additional cells (Wahl, S. M. et al. (1996) In: MACROPHAGE FUNCTION
IN HIV INFECTION, pages 303-336; Orenstein, J. W. (2001) "THE
MACROPHAGE IN HIV INFECTION," Immunobiology 204(5):598-602;
Balestra, E. at al. (2001) "MACROPHAGES: A CRUCIAL RESERVOIR FOR
HUMAN IMMUNODEFICIENCY VIRUS IN THE BODY," J. Biol. Regul. Homeost.
Agents 15:272-276; Igarashi, T. et al. (2001) "MACROPHAGE ARE THE
PRINCIPAL RESERVOIR AND SUSTAIN HIGH VIRUS LOADS IN RHESUS MACAQUES
AFTER THE DEPLETION OF CD 4+ T CELLS BY A HIGHLY PATHOGENIC SIMIAN
IMMUNODEFICIENCY VIRUS/HIV TYPE 1 CHIMERA (SHIV): IMPLICATIONS FOR
HIV-1 INFECTIONS OF HUMANS," Proc. Natl. Acad. Sci. U.S.A.
98:658-663; Garbuglia, A. R. et al., (2001) "DYNAMICS OF VIRAL LOAD
IN PLASMA AND HIV DNA IN LYMPHOCYTES DURING HIGHLY ACTIVE
ANTIRETROVIRAL THERAPY (HAART): HIGH VIRAL BURDEN IN MACROPHAGES
AFTER 1 YEAR OF TREATMENT," J Chemother 13:188-194).
[0006] The persistence of HIV during highly active antiviral
therapy, and poor susceptibility of macrophages to antiviral
therapy (Igarashi, T. at al. (2001) "MACROPHAGE ARE THE PRINCIPAL
RESERVOIR AND SUSTAIN HIGH VIRUS LOADS IN RHESUS MACAQUES AFTER THE
DEPLETION OF CD 4+ T CELLS BY A HIGHLY PATHOGENIC SIMIAN
IMMUNODEFICIENCY VIRUS/HIV TYPE 1 CHIMERA (SHIV): IMPLICATIONS FOR
HIV-1 INFECTIONS OF HUMANS," Proc Natl Acad Sci USA 98:658-63;
Garbuglia, A. R. at al. (2001) "DYNAMICS OF VIRAL LoAD IN PLASMA
AND HIV DNA IN LYMPHOCYTES DURING HIGHLY ACTIVE ANTIRETROVIRAL
THERAPY (HAART): HIGH VIRAL BURDEN IN MACROPHAGES AFTER 1 YEAR OF
TREATMENT," J Chemother 13, 188-94) has intensified the interest in
characterizing the mechanisms underlying infection and replication
in this cell population.
[0007] Attempts to treat HIV infection have focused on the
development of drugs that disrupt the viral infection and
replication cycle (see, Mitsuya, H. at al. (1991) "TARGETED THERAPY
OF HUMAN IMMUNODEFICIENCY VIRUS-RELATED DISEASE," FASEB J.
5:2369-2381). Such intervention could potentially inhibit the
binding of HIV to cell membranes, the reverse transcription of the
HIV RNA genome into DNA, the exit of the virus from the host cell
and infection of new cellular targets, or inhibition of viral
enzymes (see, U.S. Pat. No. 6,475,491). Thus, for example, soluble
CD4 has been developed in an effort to competitively block the
binding of HIV to lymphocytes (Smith, D. H. et al. (1987) "BLOCKING
OF HIV-1 INFECTIVITY BY A SOLUBLE, SECRETED FORM OF THE CD4
ANTIGEN," Science 238:1704-1707; Schooley, R. at al. (1990)
"RECOMBINANT SOLUBLE CD4 THERAPY IN PATIENTS WITH THE ACQUIRED
IMMUNODEFICIENCY SYNDROME (AIDS) AND AIDS-RELATED COMPLEX. A PHASE
I-II ESCALATING DOSAGE TRIAL," Ann. Int. Med. 112:247-253; Kahn, J.
O. at al. (1990) "THE SAFETY AND PHARMACOKINETICS OF RECOMBINANT
SOLUBLE CD 4 (RCD 4) IN SUBJECTS WITH THE ACQUIRED IMMUNODEFICIENCY
SYNDROME (AIDS) AND AIDS-RELATED COMPLEX. A PHASE 1 STUDY," Arm.
Int. Med. 112:254-261; Yarchoan, R. at al. (1989). Proc. V.sup.th
Int. Conf. on AIDS, p 564, MCP 137). Similarly, the ability of
antisense HIV-1 oligonucleotides to inhibit viral replication has
been investigated (Maeda N et al. (1998) "INHIBITION OF HUMAN
T-CELL LEUKEMIA VIRUS TYPE 1 REPLICATION BY ANTISENSE ENV
OLIGODEOXYNUCLEOTIDE," Biochem Biophys Res Common
243(1):109-112).
[0008] Unfortunately, although considerable effort has been
expended to design effective therapeutics, no curative
anti-retroviral drugs against AIDS currently exist. All available
therapies are marred by substantial adverse side effects, and by
the capacity of HIV to rapidly mutate into forms that are
refractive to treatment (Miller, V. et al. (2001) "MUTATIONAL
PATTERNS IN THE HIV GENOME AND CROSS-RESISTANCE FOLLOWING
NUCLEOSIDE AND NUCLEOTIDE ANALOGUE DRUG EXPOSURE," Antivir Ther. 6
Suppl 3:25-44; Lerma, J. G. et al. (2001)"RESISTANCE OF HUMAN
IMMUNODEFICIENCY VIRUS TYPE 1 TO REVERSE TRANSCRIPTASE AND PROTEASE
INHIBITORS: GENOTYPIC AND PHENOTYPIC TESTING," J Clin Virol.
21(3):197-212; O'Brien, W. A. (2000) "RESISTANCE AGAINST REVERSE
TRANSCRIPTASE INHIBITORS," Clin Infect Dis. 30 Suppl 2:S185-92;
Wain-Hobson, S. (1996) "RUNNING THE GAMUT OF RETROVIRAL VARIATION,"
Trends Microbiol. 4(4):135-41; Lange J. (1995) "COMBINATION
ANTIRETROVIRAL THERAPY. BACK TO THE FUTURE," Drugs. 49 Suppl
1:32-40). Thus, a continuing need exists for safe and effective
anti-HIV therapeutics. The present invention is directed to this
and other needs.
SUMMARY OF THE INVENTION
[0009] By monitoring virus production by multiple parameters
including RNA, p24 antigen expression and ultra-structural
detection of viral particles it has been possible to characterize
the temporal events associated with the initial virus-macrophage
encounter leading to massive viral replication. In parallel,
macrophage changes in gene expression subsequent to virus-receptor
interaction have been compared to uninfected cells by cDNA
expression array. Analysis of 1200 genes at multiple intervals from
initial HIV-1 binding through levels of massive replication (10-14
days) reveals a profile of gene modulation, which favored virus
life cycle, and could influence recruitment and infection of
additional HIV-1 host cells. One gene found to be consistently
expressed following virus binding and re-expressed at the peak of
HIV-1 replication is CDKN1A, also known as p21, Cip1 (Cdk
interacting protein), or Waf1 (wild type p53-activated fragment), a
protein associated with cell cycle regulation, anti-apoptotic
response and cell differentiation (Dotto, G. P. (2000)
"P21(WAF1/CIP1): MORE THAN A BREAK TO THE CELL CYCLE?" Biochim
Biophys Acta 1471: M43-56). Importantly, modulation of p21 in vitro
results in suppression of viral replication, and implicated this
cellular protein as an interventional target.
[0010] In contrast to CD4.sup.+ lymphocytes, HIV-1 infected
macrophages typically resist cell death, support viral replication,
and facilitate HIV-1 transmission. To elucidate how the virus
commandeers macrophage intracellular machinery for its benefit,
HIV-1 infected human monocyte-derived macrophages have been
analyzed for viral-induced gene transcription by cDNA expression
array. HIV-1 infection induces the transcriptional regulation of
genes associated with host defense, signal transduction, apoptosis
and cell cycle, including cyclin-dependent kinase inhibitor p21.
CDKN1A/p21 expression follows a bimodal pattern with maximum levels
occurring during HIV-1 replication. Treatment of macrophages with
p21 anti-sense oligonucleotides or siRNA directed against p21
inhibits HIV-1 replication. Furthermore, the synthetic triterpenoid
and peroxisome proliferator-activated receptor gamma (PPARg)
ligand, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), which
influences p21 expression, drives a dose dependent suppression of
viral replication. These data implicate p21 as a pivotal macrophage
facilitator of viral replication. Moreover, regulators of p21; such
as CDDO, provide an interventional approach to modulate HIV-1
replication.
[0011] This invention thus relates to methods and compositions for
the attenuation of immunodeficiency virus replication in cells, and
especially in macrophages. The invention particularly concerns the
use of inhibitors of P21 (CDKN1A) expression to attenuate such
replication. The invention particularly concerns the use of
antisense P21 oligonucleotides, siRNA and/or
2-cyano-3,12-dioxooleana-1,9-dien-28-oic (CDDO) to attenuate such
replication. The invention finds use in the treatment of AIDS, and
in the treatment of lymphoma, especially in HIV-infected
individuals.
[0012] In detail, the invention concerns a method of attenuating
the transmission or infection of an immunodeficiency virus into a
cell comprising providing to the cell an inhibitor of p21, wherein
the inhibitor is provided in an amount and duration sufficient to
cause an attenuation of at least 50% in the transmission or
infection of the virus relative to an untreated cell. The invention
particularly concerns the embodiments of such method wherein the
immunodeficiency virus is a human immunodeficiency virus (HIV), and
the cell is a human cell; wherein the immunodeficiency virus is a
feline immunodeficiency virus (FIV), and the cell is a feline cell;
or wherein the immunodeficiency virus is a simian immunodeficiency
virus (SIV), and the cell is a simian cell.
[0013] The invention further provides a method of treating AIDS in
an individual, comprising providing to HIV-1 infected cells of said
individual an amount of a p21 inhibitor sufficient to attenuate the
propagation of HIV, wherein said inhibitor is provided in an amount
and duration sufficient to cause an attenuation of at least 50% in
said propagation of HIV relative to untreated cells.
[0014] The invention particularly concerns the embodiments of such
methods wherein the inhibitor of p21 is a polynucleotide, and
especially wherein the polynucleotide is complementary to a portion
of a p21 gene or p21 cDNA molecule.
[0015] The invention particularly concerns the embodiments of such
methods wherein the p21 gene or p21 cDNA is of a human p21 gene or
p21 cDNA molecule, or of a non-human animal or is a variant of a
non-human p21 gene or p21 cDNA molecule.
[0016] The invention further concerns a method of treating AIDS in
an individual, comprising providing to HIV-1 infected cells of the
individual, or providing to HIV-1 susceptible cells of the
individual prior to the infection of such cells by HIV-1, an amount
of a p21 inhibitor sufficient to attenuate the propagation of HIV,
wherein the inhibitor is provided in an amount and duration
sufficient to cause an attenuation of at least 50% in the
propagation of HIV relative to untreated cells.
[0017] The invention particularly concerns the embodiments of such
methods wherein the polynucleotide comprises at least 10 contiguous
nucleotides of SEQ NO.:4 (and in particular wherein the
polynucleotide comprises at least 10 contiguous nucleotides of SEQ
ID NO.:8 or SEQ ID NO.:10) or at least 10 contiguous nucleotides of
SEQ ID NO.:6 (and in particular wherein the polynucleotide
comprises at least 10 contiguous nucleotides of SEQ ID NO.:7 or SEQ
NO.:9).
[0018] The invention further concerns the embodiments of the above
methods wherein the inhibitor of p21 is a protein or organic
molecule other than a polynucleotide, and in particular concerns
the embodiment of such method wherein the inhibitor is
2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), or a salt or
derivative thereof.
[0019] The invention further concerns a pharmaceutical composition
comprising an inhibitor of p21 and an excipient or carrier, wherein
the inhibitor is present in an amount sufficient to attenuate the
propagation of HIV, wherein the inhibitor is present in the
composition in an amount sufficient to cause an attenuation of at
least 50% in the propagation of HIV relative to untreated
cells.
[0020] The invention particularly concerns the embodiments of such
composition wherein the inhibitor of p21 is a polynucleotide, and
especially wherein the polynucleotide is complementary to a portion
of a p21 gene or p21 cDNA molecule. The invention particularly
concerns the embodiments of such compositions wherein the p21 gene
or p21 cDNA is of a human p21 gene or p21 cDNA molecule, or of a
non-human animal or is a variant of a non-human p21 gene or p21
cDNA molecule. The invention further particularly concerns an
inhibitor of an upstream or downstream modulator of p21 production
or action (i.e., a p21 inhibitor molecule.
[0021] The invention further particularly concerns the embodiments
of such compositions wherein the polynucleotide comprises at least
10 contiguous nucleotides of SEQ ID NO.:4 (and in particular
wherein the polynucleotide comprises at least 10 contiguous
nucleotides of SEQ ID NO.:8 or SEQ ID NO.:10) or at least 10
contiguous nucleotides of SEQ ID NO.:6 (and in particular wherein
the polynucleotide comprises at least 10 contiguous nucleotides of
SEQ NO.:7 or SEQ ID NO.:9).
[0022] The invention further concerns the embodiments of the above
compositions wherein the inhibitor of p21 is a protein or organic
molecule other than a polynucleotide, and in particular concerns
the embodiment of such method wherein the inhibitor is
2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), or a salt or
derivative thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIGS. 1A-1D illustrate the kinetics of infection in
monocyte-derived adherent macrophages. FIG. 1A: Macrophages are
exposed to an R5 strain of HIV-1 for 90 min, washed, and total RNA
extracted at the indicated time periods and examined by Northern
blot analysis with a .sup.32P-labeled cDNA probe for HIV-1 (Wahl,
S. M. et al. (1991) "MACROPHAGE-AND ASTROCYTE-DERIVED TRANSFORMING
GROWTH FACTOR BETA AS A MEDIATOR OF CENTRAL NERVOUS SYSTEM
DYSFUNCTION IN ACQUIRED IMMUNE DEFICIENCY SYNDROME," J Exp Med
173:981-991). Bands of 9.1 and 4.3 kb correspond to viral gag/pol
and env mRNA respectively. FIG. 1B: Supernatants are collected from
infected cultures (days 1-15) and examined by ELISA for p24. FIG.
1C: Macrophages are incubated for the indicated intervals (3-10
days) after infection, fixed in gluteraldehyde and process for
transmission electron microscopy (TEM). Original magnification
10,000.times.. Ultrastructural analysis of infected cells reveals
virions (FIG. 1C and FIG. 10) in macrophages by 5-7 days post
infection, with increasing viral numbers per cell (FIG. 1C) and
numbers of infected macrophages (FIG. 1D) most evident >day 10
as quantified by counting >200 cells/time point. Data correspond
to a representative experiment (n.gtoreq.4).
[0024] FIGS. 2A-2E illustrate HIV-induced alterations in macrophage
transcriptome. FIG. 2A: Distribution of transcription changes in
macrophages 3-6 hr after exposure to HIV. Numbers represent % of
total upregulated genes (134/1200) associated with the indicated
categories in .gtoreq.4 donors. FIG. 2B Transcription related genes
upregulated .gtoreq.2 fold after 3-6 hours in HIV-1 infected
macrophages. FIG. 2C: Signal transduction-related genes upregulated
.gtoreq.2 fold above parallel cultures in descending order. FIGS.
2D-2E depict the fold change in gene expression in HIV-1-treated
macrophages compared to gene expression levels from mock-infected
macrophages from the same donor at intervals from 0.25 to 14 days
(mean values, n=3; *p.ltoreq.0.05 RM-ANOVA). The legend for FIGS.
2D-2E is shown in FIG. 2E.
[0025] FIGS. 3A-3D demonstrate increased p21 (CDKN1A) gene
expression in HIV-1 infected macrophages. FIG. 3A: Kinetic profile
of p21 expression determined by cDNA expression array following HIV
infection from day 0.25-14 days (n=3). FIG. 3D: RPA analysis of
total mRNA from uninfected and HIV-1 infected macrophages at the
indicated time points confiiuis enhanced gene expression for p21,
with minimal effect on p53 (representative donor, n=2). FIG. 3C:
Graphic representation of densitometry analysis of RPA for p21 and
p53 genes (shown in FIG. 3D) normalized to GAPDH. FIG. 3C:
Macrophages are infected with HIV-1.sub.BAL, the laboratory viral
isolate ADA or primary clinical isolate 727, washed and total RNA
collected after 12 days and analyzed for p21 transcription by
PCR.
[0026] FIGS. 4A-4C illustrate that HIV-1-infected macrophages
express increased p21 protein. FIG. 4A: Overlay confocal images
from differential interference contrast (DIC) (1, 4) and
immunofluorescence (Texas Red) for p21 labeling in uninfected (1,
2, and 3) and virus infected cells (4, 5 & 6), (original image
400.times.). At higher magnification (original 1000.times., 3 &
6), infected-macrophages express increased nuclear and cytoplasmic
p21 protein when compared with control cells. FIG. 4B:
Densitometric fluorescence intensity (FI) analysis using confocal
microscopy and Metamorph (Universal Imaging) analysis confirm
enhanced nuclear and cytoplasmic p21 protein as represented by the
signal intensity across equal line segments sampling nuclear or
cytoplasmic regions (representative experiment, n=3). FIG. 4C:
Western blot analysis shows increased p21 protein expression in
HIV-1-infected macrophages. HIV-1 infected cells (12 days) show at
least a two fold increase in p21 protein expression as indicated by
immunoprecipitation as quantified by densitometry analysis relative
to uninfected cells (n=3).
[0027] FIGS. 5A-5F illustrate inhibition of HIV-1 replication in
macrophages by CDDO and di-CDDO, FIG. 5A: Macrophages were
pre-treated with CDDO or DMSO as a control for 45 min and then
infected with HIV-1.sub.BAL. CDDO-treated macrophages show reduced
viral replication as quantitated by p24 levels compared with DMSO
control and untreated cells (day 10) (n=3, P=0.01), Supernatants
were collected on day 12 for p24 Ag analysis by ELISA and cells
processed by TEM (FIG. 5B; FIG. 5C). Ultrastructural analysis
demonstrates dramatically reduced numbers of infected cells and in
the few remaining HIV positive cells, very few virions were
identified in cultures treated with CDDO. Analysis of >200
cells/treatment condition revealed the absence or near absence of
detectable virions. (FIG. 50) CDDO treated cells infected with
HIV-1 demonstrate reduced p21 transcription as determined by
ribonuclease protection assay (day 12 post-infection shown) (mean,
n=2). Inset: TdT-FITC and DAPI staining of cultures that were
infected with HIV-1 and were treated or not with CDDO (FIG. 5D).
FIG. 5E: Macrophages were infected with HIV-1.sub.BaL or ADA and
treated or not with CDDO (0.1 .mu.M) and analyzed by PCR for p21
and GAPDH. FIG. 5F: Supernatants (12 days) collected from
HIV-1.sub.BaL, ADA or 727 infected cells that were treated or not
with CDDO were analyzed for viral replication by p24 ELISA.
[0028] FIGS. 6A-6D show the results of siRNA for p2.1 and its
effect on HIV infection. FIG. 6A: p21 specific oligonucleotides (50
nM), but not control oligonucleotide inhibit HIV-1 growth in
replicate cultures as determined by p24 levels (day 12 shown) (% of
positive HIV control, no oligo treatment). FIG. 6B: Macrophages
were treated with p21 siRNA duplexes (5 .mu.M) five days prior to
HIV infection (% of positive HIV control, no siRNA treatment)
(representative experiment, n=3). Percent of HIV-1 infection was
determined comparing the p24 levels in untreated vs siRNA treated
macrophages. FIG. 6C: Cells treated with p21 and negative control
siRNA (5 days) were analyzed by flow cytometry for CD4 and CCR5
cell surface expression. FIG. 6D: Nested PCR to detect pro-viral
DNA on days 1 and 2 after HIV-1.sub.BaL infection in macrophages
treated with p21 or negative control si RNA. Control represents
uninfected cells.
[0029] FIGS. 7A-7D show the induction of p21 gene and protein
expression by Vpr. FIG. 7A: Cells treated with Vpr (6 .mu.g/ml) for
3 hr show increased gene transcription (FIG. 7A) and protein
expression for p21 (FIG. 7B). FIG. 7C: Macrophages were infected
with the wild type (wt) virus type clone pNLAD8, or pNLAD8 Vpr
minus (#1) or pNLAD8-delta R (#2) R5 macrophage tropic viruses and
12 day supernatants analyzed by p24 ELISA. FIG. 7D: Total RNA was
isolated from cells infected with the viruses as indicated in FIG.
7C and analyzed for p21 and GAPDH by PCR. Representative
experiment, n=2.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] The present invention relates to methods and compositions
for the attenuation of HIV-1 replication in human cells, and
especially in human macrophages. As used herein, such "attenuation"
is preferably of a magnitude sufficient to mediate a reduction of
at least 50%, more preferably 60%, most preferably 80%, or greater
in the replication, propagation or transmission of HIV.
[0031] The present invention derives in part from the recognition
that the replication of human cells has been found to be tightly
coordinated by the expression and interaction of an array of cell
cycle regulatory proteins. Any of the genes described herein that
are induced by HIV-1 are potentially able to serve as tagets for
achieving the inhibition of HIV-1. CDKN1A, also known as p21Cip1
(Cdk interacting protein), or Waf1 (wild type p53-activated
fragment), is one of these proteins (Dotto, G. P. (2000)
"p21(WAP1/CIP1): MORE THAN A BREAK TO THE CELL CYCLE?," Biochim.
Biophys. Acta 1471:M43-M56). The loss of CDKN1A expression has been
observed in many tumor cells, suggesting a role for the protein in
preventing malignant progression. Thus, in a preferred embodiment,
the present invention contemplates that agents that inhibit p21
expression or function serve to inhibit HIV-1. Such agents include
antibodies (including single chain immunoglobulins, chimeric
antibodies, etc.) that are immunologically reactive with p21 (i.e.;
able to bind to p21 epitopes), molecules that interfere with p21
function (e.g., CDDO and its analogues), polynucleotides capable of
inhibiting p21 transcription or translation (e.g., siRNA, antisense
molecules, etc.), and p21 inhibitor molecules. All such molecules
are inhibitors of p21, as that term is employed herein.
[0032] HIV-1 and p21 are discussed in U.S. Pat. Nos. 6,359,124;
5,965,722; 5,889,156; 6,548,657; 6,511,847; 6,489,163; 6,410,010;
6,379,965; 6,204,248; 6,133,444; 6,069,134; 5,866,698; 5,861,290;
5,834,440; 5,795,870; 5,766,882; 5,747,469; and 5,693,769.
[0033] Although macrophages express the requisite CD4 and chemokine
co-receptors making them susceptible targets, and R5 viral variants
are preferentially transmitted, it has remained a challenge to
identify HIV-1 positive macrophages early after viral exposure in
mucosal tissues (Schacker, T. et al. (2001) "PRODUCTIVE INFECTION
OFT CELLS IN LYMPHOID TISSUES DURING PRIMARY AND EARLY HUMAN
IMMUNODEFICIENCY VIRUS INFECTION," J Infect Dis 183:555-562) or in
the absence of co-pathogens (Orenstein, J. M. et al. (1997)
"MACROPHAGES AS A SOURCE OF HIV DURING OPPORTUNISTIC INFECTIONS,"
Science 276:1857-1861). When exposed to HIV-1, monocyte-derived
macrophages (MDM) bind and/or internalize the virus, but the
consequences of that interaction are ill defined. Whether the
macrophages are triggered by this encounter to modify their
phenotypic and functional repertoire or whether HIV-1 enters
stealthily, and transiently remains unrecognized by the immune
system, it is important to define the early stages when HIV-1 is
gaining a foothold on the immune system and identify key signals
which not only promote permissiveness for macrophage HIV-1
infection, but also promote replication.
[0034] One aspect of the present invention relates to the
recognition that HIV-1 infection stimulates the expression of p21
(CDKN1A) protein in human macrophages (Vazquez, N. et al. (October
2002) "HIV-1 Enhancement of CDKN1A (p21) in Human Macrophages Is
Associated with Viral Replication," 5.sup.th Intl. Workshop on HIV,
Cells of Macrophage/Dendritic Lineage and Other Reservoirs, Rome,
Italy).
[0035] The Vpr gene product of HIV-1 has been found to prevent cell
proliferation by activating p21 expression, suggesting that the
upregulation of p21 by HIV-1 Vpr may have important consequences in
HIV-1 pathogenesis (Chowdhury I. H. et al. (2003) "HIV-1 VPR
ACTIVATES CELL CYCLE INHIBITOR P21/WAF1/CIP1: A POTENTIAL MECHANISM
OF G2/M CELL CYCLE ARREST," Virol. 305:371-377). A similar
relationship has been disclosed between p21 and HIV-1 Nef (Fackler,
O. T. at al. (2000) "P21-ACTIVATED KINASE 1 PLAYS A CRITICAL ROLE
IN CELLULAR ACTIVATION BY NEF," Mol Cell Biol. 20:2619-2627;
Hiipakka M et al (2001) "INHIBITION OF CELLULAR FUNCTIONS OF HIV-1
NEF BY ARTIFICIAL SH3 DOMAINS," Virol. 286:152-159; Nunn M F at al
(1996) "HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 NEF ASSOCIATES WITH A
MEMBER OF THE P21-ACTIVATED KINASE FAMILY," 3 Virol. 70:6157-6161;
Renkema G H at al. (1999) "IDENTIFICATION OF THE NEF-ASSOCIATED
KINASE AS P21-ACTIVATED KINASE 2," Curr Biol. 9:1407-1410).
[0036] Additionally, Clark, E. et al. disclosed that the treatments
(such as gamma irradiation) that cause a loss of cell cycle control
at the G.sub.1/S checkpoint cause HIV-1 infected cells to lose p21
function, and undergo apoptosis (Clark E et al. (2000) "LOSS OF
G(1)/S CHECKPOINT IN HUMAN IMMUNODEFICIENCY VIRUS TYPE 1-INFECTED
CELLS IS ASSOCIATED WITH A LACK OF CYCLIN-DEPENDENT KINASE
INHIBITOR P21/WAF 1," J Virol. 74:5040-5052). Gomez, T. et al.
(http://www.retroconference.org/2002/Posters/13446.pdf;
"CYTOPLASMIC P21.sup.QAF1/CIP1 PROTECTS U937 PROMONOCYTIC CELLS
FROM HIV MEDIATED APOPTOSIS") disclose that the administration of
p21-antisense oligonucleotides to promonocytic cells suppressed p21
levels in the cells, and accelerated the death of the HIV-infected
cells. The results are stated to indicate that p21 confers
resistance to HIV-induced apoptosis in promonocytic cells, and to
suggest a possible mechanism for the persistence of its infection
in cells such as macrophages.
[0037] Antisense oligonucleotides of p21 have been used to affect
cell-cycle transit in astrocytoma cells. (Jinbo Liu, et al. (2000)
"ANTI-SENSE OLIGONUCLEOTIDE OF P21(WAF1/CIP1) PREVENTS INTERLEUKIN
4-MEDIATED ELEVATION OF P27(KIP1) IN LOW GRADE ASTROCYTOMA CELLS,"
Oncogene 19:661-669). The oligonucleotides had the sequences:
TABLE-US-00001 SEQ ID NO: 1: 5'-ucc gcg ccc agc ucc-3' and SEQ ID
NO: 2: 5'-ucc gcc cgc agc ucc-3'
[0038] The present invention thus particularly concerns the use of
one or more p21 inhibitors to prevent or attenuate the infection of
additional host cells, and as such to provide a therapy for AIDS
and its simian and feline counterparts.
[0039] As used herein, the term "p21 inhibitor" is intended to
denote any of a variety of molecules that function to suppress or
prevent p21 activity. Such inhibitors can be, for example,
transcriptional inhibitors, such as promoter blockers, RNAi
molecules, antisense polynucleotides of the p21 gene or cDNA, or
allelic or non-human species variants thereof. Alternatively, such
molecules can comprise translational inhibitors of p21, molecules
that inhibit or otherwise interfere with p21 function, etc.
[0040] As used herein, an allelic variant of a p21 polynucleotide
is a polynucleotide having the sequence of a naturally occurring
allele of the p21 gene or cDNA thereof, or of a non-naturally
occurring polynucleotide that is 80% homologous, and more
preferably 90% homologous, and most preferably comprises a sequence
that has at least 10, and more preferably, at least 20 contiguous
nucleotides that are identical in sequence to a portion of a
naturally occurring p21 gene or cDNA. Examples of such sequences
include NCBI accession Nos. BC000312 and BC013967. As used herein,
a non-human species variant of a p21 polynucleotide is a
polynucleotide having the sequence of the p21 gene or cDNA of a
non-human animal, for example a mouse or rat. Examples of such
sequences include NM 007669 and U24174.
[0041] Antisense molecules suitable for use in the present
invention can be identified as polynucleotide molecules having a
length of 10-250, or more preferably 10-150, and most preferably,
10-100 nucleotides that are complementary to a portion of SEQ ID
NO.:3 (the human p21 gene, see Xiong, Y. et al. (1993) "P21 IS A
UNIVERSAL INHIBITOR OF CYCLIN KINASES," Nature 366:701-704 (1993);
el-Deiry, W. S. et al. (1993) "WAF1, A POTENTIAL MEDIATOR OF P53
TUMOR SUPPRESSION," Cell 75:817-825 (1993); Harper, J. W. et al.
(1993) "THE P21 CDK-INTERACTING PROTEIN CIP1 IS A POTENT INHIBITOR
OF G1 CYCLIN-DEPENDENT KINASES," Cell 75:805-816) or allelic or
non-human species variants thereof, especially the murine p21 gene
(NM 007669).
TABLE-US-00002 SEQ ID NO.: 3: 1 gctgccgaag tcagttcctt gtggagccgg
agctgggcgc ggattcgccg 51 aggcaccgag gcactcagag gaggtgagag
agcggcggca gacaacaggg 101 gaccccgggc cggcggccca gagccgagcc
aagcgtgccc gcgtgtgtcc 151 ctgcgtgtcc gcgaggatgc gtgttcgcgg
gtgtgtgctg cgttcacagg 201 tgtttctgcg gcaggcgcca tgtcagaacc
ggctggggat gtccgtcaga 251 acccatgcgg cagcaaggcc tgccgccgcc
tcttcggccc agtggacagc 301 gagcagctga gccgcgactg tgatgcgcta
atggcgggct gcatccagga 351 ggcccgtgag cgatggaact tcgactttgt
caccgagaca ccactggagg 401 gtgacttcgc ctgggagcgt gtgcggggcc
ttggcctgcc caagctctac 451 cttcccacgg ggccccggcg aggccgggat
gagttgggag gaggcaggcg 501 gcctggcacc tcacctgctc tgctgcaggg
gacagcagag gaagaccatg 551 tggacctgtc actgtcttgt acccttgtgc
ctcgctcagg ggagcaggct 601 gaagggtccc caggtggacc tggagactct
cagggtcgaa aacggcggca 651 gaccagcatg acagatttct accactccaa
acgccggctg atcttctcca 701 agaggaagcc ctaatccgcc cacaggaagc
ctgcagtcct ggaagcgcga 751 gggcctcaaa ggcccgctct acatcttctg
ccttagtctc agtttgtgtg 801 tcttaattat tatttgtgtt ttaatttaaa
cacctcctca tgtacatacc 851 ctggccgccc cctgcccccc agcctctggc
attagaatta tttaaacaaa 901 aactaggcgg ttgaatgaga ggttcctaag
agtgctgggc atttttattt 951 tatgaaatac tatttaaagc ctcctcatcc
cgtgttctcc ttttcctctc 1001 tcccggaggt tgggtgggcc ggcttcatgc
cagctacttc ctcctcccca 1051 cttgtccgct gggtggtacc ctctggaggg
gtgtggctcc ttcccatcgc 1101 tgtcacaggc ggttatgaaa ttcaccccct
ttcctggaca ctcagacctg 1151 aattcttttt catttgagaa gtaaacagat
ggcactttga aggggcctca 1201 ccgagtgggg gcatcatcaa aaactttgga
gtcccctcac ctcctctaag 1251 gttgggcagg gtgaccctga agtgagcaca
gcctagggct gagctgggga 1301 cctggtaccc tcctggctct tgataccccc
ctctgtcttg tgaaggcagg 1351 gggaaggtgg ggtcctggag cagaccaccc
cgcctgccct catggcccct 1401 ctgacctgca ctggggagcc cgtctcagtg
ttgagccttt tccctctttg 1451 gctcccctgt accttttgag gagccccagc
tacccttctt ctccagctgg 1501 gctctgcaat tcccatctgc tgctgtccct
cccccttgtc ctttcccttc 1551 agtaccctct cagctccagg tggctctgag
gtgcctgtcc cacccccacc 1601 cccagctcaa tggactggaa ggggaaggga
cacacaagaa gaagggcacc 1651 ctagttctac ctcaggcagc tcaagcagcg
accgccccct cctctagctg 1701 tgggggtgag ggtcccatgt ggtggcacag
gcccccttga gtggggttat 1751 ctctgtgtta ggggtatatg atgggggagt
agatctttct aggagggaga 1801 cactggcccc tcaaatcgtc cagcgacctt
cctcatccac cccatccctc 1851 cccagttcat tgcactttga ttagcagcgg
aacaaggagt cagacatttt 1901 aagatggtgg cagtagaggc tatggacagg
gcatgccacg tgggctcata 1951 tggggctggg agtagttgtc tttcctggca
ctaacgttga gcccctggag 2001 gcactgaagt gcttagtgta cttggagtat
tggggtctga ccccaaacac 2051 cttccagctc ctgtaacata ctggcctgga
ctgttttctc tcggctcccc 2101 atgtgtcctg gttcccgttt ctccacctag
actgtaaacc tctcgagggc 2151 agggaccaca ccctgtactg ttctgtgtct
ttcacagctc ctcccacaat 2201 gctgaatata cagcaggtgc tcaataaatg
attcttagtg actttaaaaa 2251 aaaaaaaaaa aaaaa
[0042] Preferred p21 inhibitors of the present invention thus
include polynucleotide fragments of SEQ ID NO: 4 (the antisense
complement of SEQ ID NO.:3), or allelic or non-human species
variants thereof:
TABLE-US-00003 SEQ ID NO.: 4: 1 tttttttttt tttttttttt aaagtcacta
agaatcattt attgagcacc 51 tgctgtatat tcagcattgt gggaggagct
gtgaaagaca cagaacagta 101 cagggtgtgg tccctgccct cgagaggttt
acagtctagg tggagaaacg 151 ggaaccagga cacatgggga gccgagagaa
aacagtccag gccagtatgt 201 tacaggagct ggaaggtgtt tggggtcaga
ccccaatact ccaagtacac 251 taagcacttc agtgcctcca ggggctcaac
gttagtgcca ggaaagacaa 301 ctactcccag ccccatatga gcccacgtgg
catgccctgt ccatagcctc 351 tactgccacc atcttaaaat gtctgactcc
ttgttccgct gctaatcaaa 401 gtgcaatgaa ctggggaggg atggggtgga
tgaggaaggt cgctggacga 451 tttgaggggc cagtgtctcc ctcctagaaa
gatctactcc cccatcatat 501 acccctaaca cagagataac cccactcaag
ggggcctgtg ccaccacatg 551 ggaccctcac ccccacagct agaggagggg
gcggtcgctg cttgagctgc 601 ctgaggtaga actagggtgc ccttcttctt
gtgtgtccct tccccttcca 651 gtccattgag ctgggggtgg gggtgggaca
ggcacctcag agccacctgg 701 agctgagagg gtactgaagg gaaaggacaa
gggggaggga cagcagcaga 751 ggggaattgc agagcccagc tggagaagaa
gggtagctgg ggctcctcaa 801 aaggtacagg ggagccaaag agggaaaagg
ctcaacactg agacgggctc 851 cccagtgcag gtcagagggg ccatgagggc
aggcggggtg gtctgctcca 901 ggaccccacc ttccccctgc cttcacaaga
cagagggggg tatcaagagc 951 caggagggta ccaggtcccc agctcagccc
taggctgtgc tcacttcagg 1001 gtcaccctgc ccaaccttag aggaggtgag
gggactccaa agtttttgat 1051 gatgccccca ctcggtgagg ccccttcaaa
gtgccatctg tttacttctc 1101 aaatgaaaaa gaattcaggt ctgagtgtcc
aggaaagggg gtgaatttca 1151 taaccgcctg tgacagcgat gggaaggagc
cacacccctc cagagggtac 1201 cacccagcgg acaagtgggg aggaggaagt
agctggcatg aagccggccc 1251 acccaacctc cgggagagag gaaaaggaga
acacgggatg aggaggcttt 1301 aaatagtatt tcataaaata aaaatgccca
gcactcttag gaacctctca 1351 ttcaaccgcc tagtttttgt ttaaataatt
ctaatgccag aggctggggg 1401 gcagggggcg gccagggtat gtacatgagg
aggtgtttaa attaaaacac 1451 aaataataat taagacacac aaactgagac
taaggcagaa gatgtagagc 1501 gggcctttga ggccctcgcg cttccaggac
tgcaggcttc ctgtgggcgg 1551 attagggctt cctcttggag aagatcagcc
ggcgtttgga gtggtagaaa 1601 tctgtcatgc tggtctgccg ccgttttcga
ccctgagagt ctccaggtcc 1651 acctggggac ccttcagcct gctcccctga
gcgaggcaca agggtacaag 1701 acagtgacag gtccacatgg tcttcctctg
ctgtcccctg caggagagca 1751 ggtgaggtgc caggccgcct gcctcctccc
aactcatccc ggcctcgccg 1801 gggccccgtg ggaaggtaga gcttgggcag
gccaaggccc cgcacacgct 1851 cccaggcgaa gtcaccctcc agtggtgtct
cggtgacaaa gtcgaagttc 1901 catcgctcac gggcctcctg gatgcagccc
gccattagcg catcacagtc 1951 gcggctcagc tgctcgctgt ccactgggcc
gaagaggcgg cggcaggcct 2001 tgctgccgca tgggttctga cggacatccc
cagccggttc tgacatggcg 2051 cctgccgcag aaacacctgt gaacgcagca
cacacccgcg aacacgcatc 2101 ctcgcggaca cgcagggaca cacgcgggca
cgcttggctc ggctctgggc 2151 cgccggcccg gggtcccctg ttgtctgccg
ccgctctctc acctcctctg 2201 agtgcctcgg tgcctcggcg aatccgcgcc
cagctccggc tccacaagga 2251 actgacttcg gcagc
[0043] The murine p21 sequence is provided below:
TABLE-US-00004 SEQ ID NO. 5: 1 gagccgagag gtgtgagccg ccgcggtgtc
agagtctagg ggaattggag 51 tcaggcgcag atccacagcg atatccagac
attcagagcc acaggcacca 101 tgtccaatcc tggtgatgtc cgacctgttc
cgcacaggag caaagtgtgc 151 cgttgtctct tcggtcccgt ggacagtgag
cagttgcgcc gtgattgcga 201 tgcgatcatg gcgggctgtc tccaggaggc
ccgagaacgg tggaactttg 251 acttcgtcac ggagacgccg ctggagggca
acttcgtctg ggagcgcgtt 301 cggagcctag ggctgcccaa ggtctacctg
agccctgggt cccgcagccg 351 tgacgacctg ggaggggaca agaggcccag
tacttcctct gccctgctgc 401 aggggccagc tccggaggac cacgtggcct
tgtcgctgtc ttgcactctg 451 gtgtctgagc ggcctgaaga ttccccgggt
gggcccggaa catctcaggg 501 ccgaaaacgg aggcagacca gcctgacaga
tttctatcac tccaagcgca 551 gattggtctt ctgcaagaga aaaccctgaa
gtgcccacgg gagcccdgcc 601 ctcttctgdt gtgggtcagg aggcctcttc
cccatcttcg gccttagccc 651 tcactctgtg tgtcttaatt attatttgtg
ttttaattta aacgtctcct 701 gtatatacgc tgcctgccct ctcccagtct
ccaaacttaa agttatttaa 751 aaaaagaaca aaacaaaaca aaaaaaaacc
aaaacaaaac aaacctaaat 801 tagtaggacg gtagggccct tagtgtgggg
gatttctatt atgtagatta 851 ttattattta agcccctccc aacccaagct
ctgtgtttcc tataccggag 901 gaacagtcct actgatatca acccatctgc
atccgtttca cccaaccccc 951 ctccccccat tccctgcctg gttccttgcc
acttcttacc tgggggtgat 1001 cctcagacct gaatagcact ttggaaaaat
gagtaggact ttggggtctc 1051 cttgtcacct ctaaggccag ctaggatgac
agtgaagcag tcacagccta 1101 gaacagggat ggcagttagg actcaaccgt
aatatcccga ctcttgacat 1151 tgctcagacc tgtgaagaca ggaatggtcc
ccactctgga tcccctttgc 1201 cactcctggg gagcccacct ctcctgtggg
tctctgccag ctgcccctct 1251 attttggagg gttaatctgg tgatctgctg
ctcttttccc ccaccccata 1301 cttccccttc tgcaggtcgg caggaggcat
atctaggcac ttgccccaca 1351 gctcagtgga ctggaaggga atgtatatgc
agggtacact aagtgggatt 1401 ccctggtctt accttaggca gctccagtgg
caaccccctg cattgtgggt 1451 ctagggtggg tccttggtgg tgagacaggc
ctcccagagc attctatggt 1501 gtgtggtggt gggggtgggc ttatctggga
tggggacccc agttggggtt 1551 ctcagtgact tctcccattt cttagtagca
gttgtacaag gagccaggcc 1601 aagatggtgt cttgggggct aagggagctc
acaggacact gagcaatggc 1651 tgatcctttc tcagtgttga ataccgtggg
tgtcaaagca cttagtgggt 1701 ctgactccag ccccaaacat ccctgtttct
gtaacatcct ggtctggact 1751 gtctaccctt agcccgcacc ccaagaacat
gtattgtggc tccctccctg 1801 tctccactca gattgtaagc gtctcacgag
aagggacagc accctgcatt 1851 gtcccgagtc ctcacacccg accccaaagc
tggtgctcaa taaatacttc 1901 tcgatgatt
[0044] The antisense murine p21 sequence is provided below:
TABLE-US-00005 SEQ ID NO. 6: 1 aatcatcgag aagtatttat tgagcaccag
ctttggggtc gggtgtgagg 51 actcgggaca atgcagggtg ctgtcccttc
tcgtgagacg cttacaatct 101 gagtggagac agggagggag ccacaataca
tgttcttggg gtgcgggcta 151 agggtagaca gtccagacca ggatgttaca
gaaacaggga tgtttggggc 201 tggagtcaga cccactaagt gctttgacac
ccacggtatt caacactgag 251 aaaggatcag ccattgctca gtgtcctgtg
agctccctta gcccccaaga 301 caccatcttg gcctggctcc ttgtacaact
gctactaaga aatgggagaa 351 gtcactgaga accccaactg gggtccccat
cccagataag cccaccccca 401 ccaccacaca ccatagaatg ctctgggagg
cctgtctcac caccaaggac 451 ccaccctaga cccacaatgc agggggttgc
cactggagct gcctaaggta 501 agaccaggga atcccactta gtgtaccctg
catatacatt cccttccagt 551 ccactgagct gtggggcaag tgcctagata
tgcctcctgc cgacctgcag 601 aaggggaagt atggggtggg ggaaaagagc
agcagatcac cagattaacc 651 ctccaaaata gaggggcagc tggcagagac
ccacaggaga ggtgggctcc 701 ccaggagtgg caaaggggat ccagagtggg
gaccattcct gtcttcacag 751 gtctgagcaa tgtcaagagt cgggatatta
cggttgagtc ctaactgcca 801 tccctgttct aggctgtgac tgcttcactg
tcatcctagc tggccttaga 851 ggtgacaagg agaccccaaa gtcctactca
tttttccaaa gtgctattca 901 ggtctgagga tcacccccag gtaagaagtg
gcaaggaacc aggcagggaa 951 tggggggagg ggggttgggt gaaacggatg
cagatgggtt gatatcagta 1001 ggactgttcc tccggtatag gaaacacaga
gcttgggttg ggaggggctt 1051 aaataataat aatctacata atagaaatcc
cccacactaa gggccctacc 1101 gtcctactaa tttaggtttg ttttgttttg
gttttttttt gttttgtttt 1151 gttctttttt taaataactt taagtttgga
gactgggaga gggcaggcag 1201 cgtatataca ggagacgttt aaattaaaac
acaaataata attaagacac 1251 acagagtgag ggctaaggcc gaagatgggg
aagaggcctc ctgacccaca 1301 gcagaagagg gcggggctcc cgtgggcact
tcagggtttt ctcttgcaga 1351 agaccaatct gcgcttggag tgatagaaat
ctgtcaggct ggtctgcctc 1401 cgttttcggc cctgagatgt tccgggccca
cccggggaat cttcaggccg 1451 ctcagacacc agagtgcaag acagcgacaa
ggccacgtgg tcctccggag 1501 ctggcccctg cagcagggca gaggaagtac
tgggcctctt gtcccctccc 1551 aggtcgtcac ggctgcggga cccagggctc
aggtagacct tgggcagccc 1601 taggctccga acgcgctccc agacgaagtt
gccctccagc ggcgtctccg 1651 tgacgaagtc aaagttccac cgttctcggg
cctcctggag acagcccgcc 1701 atgagcgcat cgcaatcacg gcgcaactgc
tcactgtcca cgggaccgaa 1751 gagacaacgg cacactttgc tcctgtgcgg
aacaggtcgg acatcaccag 1801 gattggacat ggtgcctgtg gctctgaatg
tctggatatc gctgtggatc 1851 tgcgcctgac tccaattccc ctagactctg
acaccgcggc ggctcacacc 1901 tctcggctc
[0045] Of particular interest are antisense oligonucleotides that
have a nucleotide sequence of 10, and more preferably 20,
nucleotides within the sequence 1751-1850 or 1351-1450 of SEQ ID
NO.:6, or of variants or fragments thereof that possess the ability
to inhibit p21 expression and/or HIV replication or transmission.
Such variants include oligonucleotides that are complementary to a
corresponding region of the human p21 gene, or to non-human
homologs as well as oligonucleotides that are composed of at least
10 of the nucleotide residues of 1751-1850 or 1351-1450 of SEQ ID
NO.: 6. As an example, SEQ ID NO.:7: 5'-TGTCAGGCTGGTCTGCCTCC-3', is
an antisense oligonucleotide of SEQ ID NO.: 6 (shown underlined in
SEQ ID NO.: 6) that possesses the ability to inhibit p21 expression
and/or HIV replication or transmission. An example of a variant of
this sequence is the corresponding sequence of the human p21
antisense sequence: SEQ ID NO. 8: TGTCATGC TGGTCTGCCG CC, shown
underlined in SEQ ID NO.:4).
[0046] As a further example, the antisense oligonucleotide, SEQ ID
NO. 9: 5'-ACATCACCAGGATTGGACAT-3', is fragment of SEQ ID NO.: 6
(shown double underlined in SEQ ID NO.: 6) that possesses the
ability to inhibit human p21 expression and/or HIV replication or
transmission. An example of a variant of this sequence is the
corresponding sequence of the human p21 antisense sequence: SEQ ID
NO. 10: ACATCCCCAGCCGGTTCTGACAT, shown double underlined in SEQ ID
NO.:4).
[0047] Additional examples of preferred p21 inhibitors of the
present invention include polynucleotide fragments of SEQ ID NO.:
11 (the antisense complement of the promoter region of the p21 gene
(SEQ ID NO.:12)) or allelic or non-human species variants thereof,
as well as promoter blockers, and other transcriptional or
translational repressors of the p21 gene.
TABLE-US-00006 SEQ ID NO.: 11 1 accatcccct tcctcacctg aaaacaggca
gcccaaggac aaaatagcca 51 ccagcctctt ctatgccaga gctcaacatg
ttgggacatg ttcctgacgg 101 ccagaaagcc aatcagagcc acagcctgct
gcccaagcat gttcctggga 151 agcaggcagc atagggatgg agggaggctc
agcctggggg aacaagagtg 201 cc SEQ ID NO.: 12 1 ggcactcttg ttcccccagg
ctgagcctcc ctccatccct atgctgcctg 51 cttcccagga acatgcttgg
gcagcaggct gtggctctga ttggctttct 101 ggccgtcagg aacatgtccc
aacatgttga gctctggcat agaagaggct 151 ggtggctatt ttgtccttgg
gctgcctgtt ttcaggtgag gaaggggatg 201 gt
[0048] Additional examples of preferred p21 inhibitors of the
present invention include protein and other non-polynucleotide
inhibitors of transcription, translation of the p21 gene,
inhibitory p21 mimetics, or inhibitors of the transport or
processing of the expressed p21 gene product (SEQ ID NO.: 13).
TABLE-US-00007 SEQ ID NO.: 13: 1 MSEPAGDVRQ NPCGSKACRR LFGPVDSEQL
SRDCDALMAG CIQEARERWN 51 FDFVTETPLE GDFAWERVRG LGLPKLYLPT
GPRRGRDELG GGRRPGTSPA 101 LLQGTAEEDH VDLSLSCTLV PRSGEQAEGS
PGGPGDSQGR KRRQTSMTDF 151 YHSKRRLIFS
[0049] Preferred inhibitors thus include triterpenoids, especially
oleanane triterpenoids, and particularly the oleanane triterpenoid
2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), and analogs
thereof (see, for example, Stadheim, T. A. et al. (2002) "THE NOVEL
TRITERPENOID 2-CYANO-3,12-DIOXOOLEANA-1,9-DIEN-28-OIC ACID (CDDO)
POTENTLY ENHANCES APOPTOSIS INDUCED BY TUMOR NECROSIS FACTOR IN
HUMAN LEUKEMIA CELLS," J Biol. Chem. 277:16448-16455; Suh, N. et
al. (1999) "A NOVEL SYNTHETIC OLEANANE TRITERPENOID,
2-CYANO-3,12-DIOXOOLEAN-1,9-DIEN-28-OIC ACID, WITH POTENT
DIFFERENTIATING, ANTIPROLIFERATIVE, AND ANTI-INFLAMMATORY
ACTIVITY," Cancer Res. 59:336-341). Also suitable are derivatives
and salts of such compounds, for example, 1
[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im)
(Place, W. A. et al. (2003) "The Novel Synthetic Triterpenoid,
CDDO-Imidazolide,'Inhibits Inflammatory Response and Tumor Growth
in Vivo," Clinical Cancer Research 9:2798-2806).
[0050] Molecules that inhibit or otherwise interfere with p21
function include binding ligands of p21 protein, including
antibodies, or fragments thereof that bind to p21 protein. Suitable
antibodies may be derived from human antisera, from non-human
mammalian origin, or may be monoclonal, recombinant, single-chain,
or humanized. Antigen-binding fragments of such antibodies (e.g.,
Fab and F(ab).sub.2 fragments) may alternatively be employed. If
desired, such administration can be provided in concert with other
p21 inhibitors.
COMPOSITIONS OF THE PRESENT INVENTION
[0051] In one embodiment of the present invention,
non-polynucleotide p21 inhibitors may be employed. Alternatively,
or conjunctively, one or more of the above-described p21 inhibitor
molecules will comprise a polynucleotide or polynucleotide
construct that may be administered to a recipient prior to the
commencement of HIV infection, or subsequent to the onset of such
infection. In accordance with the methods of the present invention,
a single polynucleotide, polynucleotide construct, or composition
comprising a polynucleotide or polynucleotide construct containing
more than one polynucleotide sequence encoding one or more
molecules may be administered. Alternatively, more than one
polynucleotide, polynucleotide construct, or composition comprising
a polynucleotide or polynucleotide construct; each containing
polynucleotide sequences encoding one or more molecules may be
co-administered or sequentially administered.
[0052] The p21 inhibitor compound(s) of the present invention may
be provided to recipients using the principles of genetic therapy
or as pharmaceutical compositions.
[0053] In accordance with the principles of genetic therapy, the
compositions of the present invention will comprise one or more
nucleic acid molecules (preferably DNA molecule(s)) that encode
p21-inhibitor compound(s). Preferably, such nucleic acid Molecules
will be incorporated into a recombinant expression vector such as a
chimeric virus, a plasmid DNA, etc., and will optionally be
operatively linked to one or more regulatory elements (promoters,
translation initiation sites, etc.) so as to permit the
transcription of the nucleic acid molecule in a recipient cell. The
promoter may be a cell-specific promoter that directs substantial
transcription of the DNA only in predetermined cells. Highly
efficient transcription can be achieved with the early and late
promoters from SV40, the long terminal repeats (LTRS) from
retroviruses, e.g., RSV, HTLVI, HIVI, MPSV and the immediate early
promoter of the cytomegalovirus (CMV IEP). Alternatively, the
administered nucleic acid molecules will not contain such
regulatory elements, and will require cellular processes (such as
recombination, integration into nuclear or mitochondrial DNA, etc.)
in order to produce RNA transcripts.
[0054] Suitable DNA virus genomes include herpesvirus genomes,
adenovirus genomes, adeno-associated virus genomes, and poxvirus
genomes. However, cellular elements can also be used (e.g., the
human actin promoter, metallothionein promoter). In humans, CMV IEP
is preferred. Suitable expression vectors for use in practicing the
present invention include, for example, vectors such as PSVL and
PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr
(ATCC 37146) and pBC12MI (ATCC 67109), VR1051, VR1055, and pcDNA3
(Invitrogen, San Diego, Calif.). In a preferred embodiment, such
cytomegalovirus (CMV)-derived vectors, such as VRC8100, VRC 8103,
VRC8106, VRC 8107, or VRC8108 are employed. All forms of DNA,
whether replicating or non-replicating, preferably which do not
become integrated into the genome, and which are expressible, are
within the methods and compositions contemplated by the
invention.
[0055] In one embodiment, a polynucleotide or polynucleotide
construct of the present invention is RNA. Preferably in this
embodiment, the RNA is in the form of messenger RNA (mRNA). Methods
for introducing RNA sequences into vertebrate cells is described in
U.S. Pat. No. 5,580,859. Alternatively, the RNA may be in the form
of an RNA virus genome. Preferably an RNA virus genome of the
present invention is noninfectious, (i.e., does not result in the
production of infectious virus particles in vertebrate cells).
Suitable RNA virus genomes include, but are not limited to,
alphavirus genomes, picornavirus genomes, and retrovirus genomes.
Methods for the in vivo introduction of non-infectious viral
genomes to vertebrate tissues are well known to those of ordinary
skill in the art and are described, e.g., in Altman-Harnamdzic, S.,
et al. (1997) "EXPRESSION OF BETA-GALACTOSIDASE IN MOUSE BRAIN:
UTILIZATION OF A NOVEL NONREPLICATIVE SINDBIS VIRUS VECTOR AS A
NEURONAL GENE DELIVERY SYSTEM," Gene Therapy 4:815-822, in U.S.
Pat. No. 4,980,289, and in Miller, A. D., et al. 1993) "USE OF
RETROVIRAL VECTORS FOR GENE TRANSFER AND EXPRESSION," Meth.
Enzymol. 217:581-599. Viral replicons, i.e., non-infectious RNA
virus genomes packaged in a viral coat, e.g., a picornavirus coat
or an alphavirus coat, are also useful for efficient administration
of RNA. See, e.g., U.S. Pat. Nos. 5,766,602; 5,614,413, and PCT
Publication No. WO 95/07994.
[0056] The nucleic acid molecules of such compositions may be
single stranded or double-stranded, and may be circular or linear.
The term "nucleic acid" is intended to encompass a singular nucleic
acid molecule as well as plural nucleic acid molecules, and to
refer to an isolated molecule or construct, e.g., virus genomes
(preferably non-infectious), messenger RNA (mRNA), plasmid DNA
(pDNA), or derivatives of pDNA (e.g., minicircles as described in
(Darquet, A-M et al., (1997) "A NEW DNA VEHICLE FOR NONVIRAL GENE
DELIVERY: SUPERCOILED MINICIRCLE," Gene Therapy 4:1341-1349)
comprising a polynucleotide or polynucleotide construct. A nucleic
acid may be provided in linear (e.g., mRNA), circular (e.g.,
plasmid), or branched form as well as double-stranded or
single-stranded forms. A nucleic acid may comprise a conventional
phosphodiester bond or a non-conventional bond (e.g., an amide
bond, such as found in peptide nucleic acids (PNA)). Preferably, a
polynucleotide or polynucleotide construct of the present invention
is part of a circular or linearized plasmid which is preferably
non-infectious (i.e., does not result in the production of
infectious virus particles in vertebrate cells), and
noninitegrating (i.e., does not integrate into the genome of
vertebrate cells). A linearized plasmid is a plasmid that was
previously circular but has been linearized, for example, by
digestion with a restriction endonuclease.
[0057] Methods of gene therapy are reviewed by Relph, K. et al.
(2004) "RECENT DEVELOPMENTS AND CURRENT STATUS OF GENE THERAPY
USING VIRAL VECTORS IN THE UNITED KINGDOM," B M J.
329(7470):839-842; Takimoto, R. et al. (2004) "TUMOR SUPPRESSOR
GENE P53 AND MOLECULAR TARGETING THERAPY," Gan To Kagaku Ryoho
31(9):1309-1313; Hannon, G. J. et al. (2004) "UNLOCKING THE
POTENTIAL OF THE HUMAN GENOME WITH RNA INTERFERENCE," Nature
431(7006):371-378; Seufert, J. (2004) "CELL THERAPY AND GENE
THERAPY IN DIABETES MELLITUS," MMW Fortschr Med. 146(20):39-40;
Kawakami, Y. et al. (2004) "Cancer gene therapy and immunotherapy,"
Cancer Chemother Biol Response Modif. 21:327-337; Segota, E. et al.
(2004) "THE PROMISE OF TARGETED THERAPY: CANCER DRUGS BECOME MORE
SPECIFIC," Cleve Clin 3 Med. 71(7):551-560; Morral, N. (2004) "GENE
THERAPY FOR TYPE 1 DIABETES. NEW APPROACHES," Minerva Med.
95(2):93-104; Shen, W. G. (2004) "RNA INTERFERENCE AND ITS CURRENT
APPLICATION IN MAMMALS," Chin Med J (Engl). 117(7):1084-1091;
Nicklin, S. A. et al. (2003) "DEVELOPMENT OF TARGETED VIRAL VECTORS
FOR CARDIOVASCULAR GENE THERAPY," Genet Eng (N Y). 25:15-49;
Davidoff, A. M. et al. (2004) "ANTIANGIOGENIC GENE THERAPY FOR
CANCER TREATMENT," Curr Hematol Rep. 2004 3(4):267-273; Yonemitsu,
Y. et al. (2004) "GENE THERAPY: ITS HISTORY, CURRENT STATUS, AND
FUTURE PERSPECTIVES," Fukuoka Igalcu Zasshi. 95(4):81-88; Donahue,
J. K. et al. (2004) "GENE TRANSFER TECHNIQUES FOR CARDIAC
ARRHYTHMIAS," Ann Med. 36 Suppl 1:98-105; Guo, Z. S. at al. (2004)
"VACCINIA AS A VECTOR FOR GENE DELIVERY," Expert Opin Biol Ther.
4(6):901-917; Truckenmiller, M. E. at al. (2004) "VIRAL VECTORS FOR
INDUCING CD 8+ T CELL RESPONSES," Expert Opin Biol Ther.
4(6):861-868; Wang, S. at al. (2004) "THE P53 PATHWAY: TARGETS FOR
THE DEVELOPMENT OF NOVEL CANCER THERAPEUTICS," Cancer Treat Res.
119:175-187; Downward, J. (2004) "RNA INTERFERENCE," B M J.
328(7450):1245-1248; Scanlon, K. J. (2004) "CANCER GENE THERAPY:
CHALLENGES AND OPPORTUNITIES," Anticancer Res. 24(2A):501-504;
Ozawa, K. (2004) "AAV VECTOR-MEDIATED GENE TRANSFER AND ITS
APPLICATION TO THE NERVOUS SYSTEM," Rinsho Shinkeigaku.
43(11):835-838; and Kanerva, A. et al. (2004) "MODIFIED
ADENOVIRUSES FOR CANCER GENE THERAPY," Int J Cancer. 2004 Jul. 1;
110(4):475-480. Additional descriptions of gene therapy approaches
that can be modified to accomplish the goals of the present
invention are described in U.S. Pat. Nos. 6,806,080; 6,800,479;
6,797,703; 6,797,505; 6,784,162; 6,783,980; 6,780,639; 6,746,441;
6,743,620; 6,743,444; 6,740,331; 6,697,669; 6,692,966; 6,692,737;
6,689,758; 6,689,600; 6,677,313; 6,669,935; 6,667,294; 6,645,942;
6,632,670; 6,627,615; 6,592,864; 6,579,855; and 6,576,463.
[0058] If the p21 inhibitor compound(s) of the present invention
is/are administered as a pharmaceutical composition, such
pharmaceutical composition can be formulated according to known
methods for preparing pharmaceutical compositions, whereby the
substance to be delivered is combined with a pharmaceutically
acceptable carrier vehicle, Suitable vehicles and their preparation
are described, for example, in Remington's Pharmaceutical Sciences,
16.sup.th Edition, A. Osol, Ed., Mack Publishing Co., Easton, Pa.
(1980), and Remington's Pharmaceutical Sciences, 19.sup.th Edition,
A. R. Gennaro, Ed., Mack Publishing Co., Easton, Pa. (1995).
[0059] The amount of a polynucleotide or polynucleotide construct
or other p21 inhibitor included in such a composition depends on
factors including the age and weight of the subject, the delivery
method and route, the type of treatment desired, and the type of
polynucleotide or polynucleotide construct or other p21 inhibitor
being administered. In general, a composition of the present
invention that includes polynucleotide or polynucleotide constructs
will contain from about 1 ng to about 30 mg of such polynucleotide
or polynucleotide construct, more preferably, from about 100 ng to
about 10 mg of such polynucleotide or polynucleotide construct.
Certain preferred compositions of the present invention may include
about 1 ng of such polynucleotide or polynucleotide construct,
about 5 ng of such polynucleotide or polynucleotide construct,
about 10 ng of such polynucleotide or polynucleotide construct,
about 50 ng of such polynucleotide or polynucleotide construct,
about 100 ng of such polynucleotide or polynucleotide construct,
about 500 ng of such polynucleotide or polynucleotide construct,
about 1 .mu.g of such polynucleotide or polynucleotide construct,
about 5 ng of such polynucleotide or polynucleotide construct,
about 10 .mu.g of such polynucleotide or polynucleotide construct,
about 50 .mu.g of such polynucleotide or polynucleotide construct,
about 100 .mu.g of such polynucleotide or polynucleotide construct,
about 150 .mu.g of such polynucleotide or polynucleotide construct,
about 200 .mu.g of such polynucleotide or polynucleotide construct,
about 250 .mu.g of such polynucleotide or polynucleotide construct,
about 300 .mu.g of such polynucleotide or polynucleotide construct,
about 350 .mu.g of such polynucleotide or polynucleotide construct,
about 400 .mu.g of such polynucleotide or polynucleotide construct,
about 450 .mu.g of such polynucleotide or polynucleotide construct,
about 500 .mu.g of a polynucleotide, about 550 .mu.g of such
polynucleotide or polynucleotide construct, about 600 .mu.g of such
polynucleotide or polynucleotide construct, about 650 .mu.g of such
polynucleotide or polynucleotide construct, about 700 .mu.g of such
polynucleotide or polynucleotide construct, about 750 .mu.g of such
polynucleotide or polynucleotide construct, about 800 .mu.g of such
polynucleotide or polynucleotide construct, about 850 .mu.g of a
polynucleotide, about 900 .mu.g of such polynucleotide or
polynucleotide construct, about 950 .mu.g of such polynucleotide or
polynucleotide construct, about 1 mg of such polynucleotide or
polynucleotide construct, about 5 mg of such polynucleotide or
polynucleotide construct, about 10 mg of such polynucleotide or
polynucleotide construct, about 15 mg of such polynucleotide or
polynucleotide construct, about 20 mg of such polynucleotide or
polynucleotide construct, about 25 mg of such polynucleotide or
polynucleotide construct, or about 30 mg of such polynucleotide or
polynucleotide construct.
[0060] Nucleic acids and/or polynucleotides and/or polynucleotide
constructs of the present invention, e.g., plasmid DNA, derivatives
of plasmid DNA, mRNA, linear DNA, viral genomes, or polynucleotide
fragments contained therein may be formulated into any of the
various compositions and may be used in any of the methods
disclosed herein. As used herein, the term "polynucleotide
fragment" refers to a polynucleotide that is either a portion of a
gene, cDNA or RNA molecule, or a complement of such Molecules, and
which possesses a length of at least 10 nucleotide residues, at
least 15 nucleotide residues, at least 20 nucleotide residues, at
least 25 nucleotide residues, at least 35 nucleotide residues, at
least 50 nucleotide residues, at least 75 nucleotide residues, at
least at least 100 nucleotide residues, at least 150 nucleotide
residues, or at least 200 nucleotide residues.
[0061] For aqueous compositions used in vivo, use of sterile
pyrogen-free water is preferred. Such formulations will contain an
effective amount of such polynucleotide or polynucleotide construct
together with a suitable salt and/or auxiliary agent as disclosed
herein, in order to prepare pharmaceutically acceptable
compositions suitable for optimal administration to a vertebrate.
Insoluble polynucleotides or polynucleotide constructs may be
solubilized in a weak acid or weak base, and then diluted to the
desired volume, for example, with an aqueous solution of the
present invention. The pH of the solution may be adjusted as
appropriate. In addition, a pharmaceutically acceptable additive
can be used to provide an appropriate osmolarity.
[0062] As used herein a "salt" is a substance produced from the
reaction between acids and bases which comprises a metal (cation)
and a nonmetal (anion). Salt crystals may be "hydrated" i.e.,
contain one or more water molecules. Such hydrated salts, when
dissolved in an aqueous solution at a certain molar concentration,
are equivalent to the corresponding anhydrous salt dissolved in an
aqueous solution at the same molar concentration. For the present
invention, salts which are readily soluble in an aqueous solution
are preferred.
[0063] The terms "saline" or "normal saline" as used herein refer
to an aqueous solution of about 145 mM to about 155 mM sodium
chloride, preferably about 154 mM sodium chloride. The terms
"phosphate buffered saline" or PBS'' refer to an aqueous solution
of about 145 mM to about 155 mM sodium chloride, preferably about
154 mM sodium chloride, and about 10 mM sodium phosphate, at a pH
ranging from about 6.0 to 8.0, preferably at a pH ranging from
about 6.5 to about 7.5, most preferably at pH 7.2.
[0064] Such compositions of the present invention may include one
or more transfection facilitating materials that facilitate
delivery of polynucleotides or polynucleotide constructs to the
interior of a cell, and/or to a desired location within a cell.
Examples of the transfection facilitating materials include, but
are not limited to lipids, preferably cationic lipids; inorganic
materials such as calcium phosphate, and metal (e.g., gold or
tungsten) particles (e.g., "powder" type delivery solutions);
peptides, including cationic peptides, targeting peptides for
selective delivery to certain cells or intracellular organelles
such as the nucleus or nucleolus, and amphipathic peptides, i.e.
helix forming or pore forming peptides; basic proteins, such as
histories; asialoproteins; viral proteins (e.g., Sendai virus coat
protein); pore-forming proteins; and polymers, including
dendrimers, star-polymers, "homogenous" poly-amino acids (e.g.,
poly-lysine, poly-arginine), "heterogenous" poly-amino acids (e.g.,
mixtures of lysine & glycine), co-polymers,
polyvinylpyrrolidinone (PVP), and polyethylene glycol (PEG).
Furthermore, those auxiliary agents of the present invention which
facilitate and enhance the entry of a polynucleotide or
polynucleotide construct into vertebrate cells in vivo, may also be
considered "transfection facilitating materials."
[0065] Certain embodiments of the present invention may include
lipids as a transfection facilitating material, including cationic
lipids (e.g., DMRIE, DOSPA, DC-Chol, GAP-DLRIE), basic lipids
(e.g., steryl amine), neutral lipids (e.g., cholesterol), anionic
lipids (e.g., phosphatidyl serine), and zwitterionic lipids (e.g.,
DOPE, DOPC).
[0066] Examples of cationic lipids are 5-carboxyspermylglycine
dioctadecylamide (DOGS) and
dipalmitoyl-phophatidylethanolamine-5-carboxy-spermylamide (DPPES).
Cationic cholesterol derivatives are also useful, including {3
.beta.-[N-N',N'-dimethylamino)ethane]-carbomoyl}-cholesterol
(DC-Chol). Dimethyldioctdecyl-ammonium bromide (DDAB),
N-(3-aminopropyl)-N,N-(bis-(2-tetradecyloxyethyl))-N-methyl-ammonium
bromide (PADEMO),
N-(3-aminopropyl)-N,N-(bis-(2-dodecyloxyethyl))-N-methyl-1-ammonium
bromide (PADELO),
N,N,N-tris-(2-dodecyloxy)ethyl-N-(3-amino)pro-pyl-ammonium bromide
(PATELO), and
N.sub.1-(3-aminopropyl)((2-dodecyloxy)e-thyl)-N.sub.2-(2-dodecyloxy)ethyl-
-1-piperazinaminium bromide (GALOE-BP) can also be employed in the
present invention.
[0067] Non-diether cationic lipids, such as
DL-1,2-dioleoyl-3-dimethylaminopropyl-.beta.-hydroxyethylammonium
(DORI diester),
1-O-oleyl-2-oleoyl-3-dimethylaminopropyl-3-hydroxyethylammonium
(DORI ester/ether), and their salts promote in vivo gene delivery.
Preferred cationic lipids comprise groups attached via a heteroatom
attached to the quaternary ammonium moiety in the head group. A
glycyl spacer can connect the linker to the hydroxyl group.
[0068] Cationic lipids for use in certain embodiments of the
present invention include DMRIE
((.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2-,3-bis(tetradecyloxy)-1-propana-
minium bromide), and GAP-DMORIE
((+)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-pro-
-panaminium bromide), as well as
(.+-.)-N,N-dimethyl-N-[2-(sperminecarboxamido)et-hyl]-2,3-bis(dioleyloxy)-
-1-propaniminium pentahydrochloride (DOSPA),
(.+-.)-N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanimini-
-um bromide (.beta.-aminoethyl-DMRIE or .beta.AE-DMRIE) (Wheeler,
et al., Biochim. Biophys. Acta 1280:1-11 (1996)), and
(.+-.)-N-(3-aminopropyl)-N,-N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminiu-
m bromide (GAP-DLRIE) (Wheeler, et al., Proc. Natl. Acad. Sci. USA
93:11454-11459 (1996)), which have been developed from DMRIE. Other
examples of DMRIE-derived cationic lipids that are useful for the
present invention are
(.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-decyloxy)-1-propanaminium
bromide (GAP-DDRIE),
(.+-.)-N-(3-aminopropyl)-N,-N-dimethyl-2,3-(bis-tetradecyloxy)-1-propanam-
inium bromide (GAP-DMRIE),
(.+-.)-N-((N''-methyl)-N'-ureyl)propyl-N,N-dimethyl-2,3-bis(tetradecyloxy-
)-1-propanaminium bromide (GMU-DMRTE),
(.+-.)-N-(2-hydroxyethyl)-N,N-dimeth-yl-2,3-bis(dodecyloxy)-1-propanamini-
um bromide (DLRIE), and
(.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis-([Z]-9-octadecenyloxy)prop-
-yl-1-propaniminium bromide (HP-DORIE).
[0069] A cationic lipid that may be used in concert with the p21
inhibitor polynucleotide compositions of the present invention is a
"cytofectin." As used herein, a "cytofectin" refers to a subset of
cationic lipids which incorporate certain structural features
including, but not limited to, a quaternary ammonium group and/or a
hydrophobic region (usually with two or more alkyl chains), but
which do not require amine protonation to develop a positive
charge. Examples of cytofectins may be found, for example, in U.S.
Pat. No. 5,861,397. Cytofectins that may be used in the present
invention, include DMRIE
((.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-pr-opana-
minium bromide), GAP-DMORIE
(.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,-3-bis(syn-9-tetradeceneyloxy)-1--
propanaminium bromide), and GAP-DLRIE
((.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-1-propanamini-
um bromide).
[0070] The cationic lipid may be mixed with one or more co-lipids.
The term "co-lipid" refers to any hydrophobic material which may be
combined with the cationic lipid component and includes amphipathic
lipids, such as phospholipids, and neutral lipids, such as
cholesterol. Cationic lipids and co-lipids may be mixed or combined
in a number of ways to produce a variety of non-covalently bonded
macroscopic structures, including, for example, liposomes,
multilamellar vesicles, unilamellar vesicles, micelles, and simple
films. A preferred class of co-lipids are the zwitterionic
phospholipids, which include the phosphatidylethanolamines and the
phosphatidylcholines. Most preferably, the co-lipids are
phosphatidylethanolamines, such as, for example, DOPE, DMPE and
DPyPE. DOPE and DPyPE are particularly preferred. The most
preferred co-lipid is DPyPE, which comprises two phytanoyl
substituents incorporated into the diacylphosphatidylethanolamine
skeleton. The cationic lipid:co-lipid molar ratio may range from
about 9:1 to about 1:9, or from about 4:1 to about 1:4, or from
about 2:1 to about 1:2, or about 1:1. In order to maximize
homogeneity, such cationic lipid and co-lipid components may be
dissolved in a solvent such as chloroform, followed by evaporation
of the cationic lipid/co-lipid solution under vacuum to dryness as
a film on the inner surface of a glass vessel (e.g., a Rotovap
round-bottomed flask). Upon suspension in an aqueous solvent, the
amphipathic lipid component molecules self-assemble into homogenous
lipid vesicles. These lipid vesicles may subsequently be processed
to have a selected mean diameter of uniform size prior to
complexing with, for example, plasmid DNA according to methods
known to those skilled in the art. For example, the sonication of a
lipid solution is described in Feigner, P. L., et al. (1987)
"LIPOFECTION: A HIGHLY EFFICIENT, LIPID-MEDIATED DNA-TRANSFECTION
PROCEDURE," Proc. Natl. Acad. Sci. USA 84:7413-7417 and in U.S.
Pat. No. 5,264,618.
[0071] In some embodiments, such polynucleotide or polynucleotide
construct(s) are combined with lipids by mixing, for example, a
plasmid DNA solution and a solution of cationic lipid:co-lipid
liposomes. Preferably, the concentration of each of the constituent
solutions is adjusted prior to mixing such that the desired final
plasmid DNA/cationic lipid:co-lipid ratio and the desired plasmid
DNA final concentration will be obtained upon mixing the two
solutions. For example, if the desired final solution is to be 2.5
mM sodium phosphate, the various components of the composition,
e.g., plasmid DNA, cationic lipid:co-lipid liposomes, and any other
desired auxiliary agents, transfection facilitating materials, or
additives are each prepared in 2.5 mM sodium phosphate and then
simply mixed to afford the desired complex. Alternatively, if the
desired final solution is to be, e.g., 2.5 mM sodium phosphate,
certain components of the composition, e.g., the auxiliary agent
and/or cationic lipid:co-lipid liposomes, is prepared in a volume
of water which is less than that of the final volume of the
composition, and certain other components of the composition, e.g.,
the plasmid DNA, is prepared in a solution of sodium phosphate at a
higher concentration than 2.5 mM, in a volume such that when. the
components in water are added to the components in the sodium
phosphate solution, the final composition is in an aqueous solution
of 2.5 mM sodium phosphate. For example, the plasmid DNA could be
prepared in 5.0 mM sodium phosphate at one half the final volume,
the auxiliary agent and/or cationic lipid:co-lipid liposome is
prepared in water at one half the final volume, and then these two
elements are mixed together to produce the final composition. The
cationic lipid:co-lipid liposomes are preferably prepared by
hydrating a thin film of the mixed lipid materials in an
appropriate volume of aqueous solvent by vortex mixing at ambient
temperatures for about 1 minute. The thin films are prepared by
admixing chloroform solutions of the individual components to
afford a desired molar solute ratio followed by aliquoting the
desired volume of the solutions into a suitable container. The
solvent is removed by evaporation, first with a stream of dry,
inert gas (e.g. argon) followed by high vacuum treatment.
[0072] A transfection facilitating material can be used alone or in
combination with one or more other transfection facilitating
materials. Two or more transfection facilitating materials can be
combined by chemical bonding (e.g, covalent and ionic such as in
lipidated polylysine, PEGylated polylysine) (Toncheva, V., et al.
(1998) "NOVEL VECTORS FOR GENE DELIVERY FORMED BY SELF-ASSEMBLY OF
DNA WITH POLY(L-LYSINE) GRAFTED WITH HYDROPHILIC POLYMERS," Bio
shim. Biophys. Acta 1380(3):354-368), mechanical mixing (e.g., free
moving materials in liquid or solid phase such as
"polylysine+cationic lipids") (Gao, X. et al. (1996) "POTENTIATION
OF CATIONIC LIPOSOME-MEDIATED GENE DELIVERY BY POLYCATIONS,"
Biochemistry 35:1027-1036); Trubetskoy, V. S., et al. (1992)
"CATIONIC LIPOSOMES ENHANCE TARGETED DELIVERY AND EXPRESSION OF
EXOGENOUS DNA MEDIATED BY N-TERMINAL MODIFIED
POLY(L-LYSINE)-ANTIBODY CONJUGATE IN MOUSE LUNG ENDOTHELIAL CELLS,"
Biochem. Biophys. Acta 1131:311-313), and aggregation (e.g.,
co-precipitation, gel forming such as in cationic
lipids+poly-lactide co-galactide, and polylysine+gelatin).
[0073] Other hydrophobic and amphiphilic additives, such as, for
example, sterols, fatty acids, gangliosides, glycolipids,
lipopeptides, liposaccharides, neobees, niosomes, prostaglandins
and sphingolipids, may also be included in the compositions of the
present invention. In such compositions, these additives may be
included in an amount between about 0.1 mol % and about 99.9 mmol %
(relative to total lipid). Preferably, these additives comprise
about 1-50 mol % and, most preferably, about 2-25 mol %. Preferred
additives include lipopeptides, liposaccharides and steroids.
[0074] In embodiments of the present invention in which
non-polynucleotide p21 inhibitors are provided, such compounds can
be formulated according to known methods for preparing such
pharmaceutical compositions, whereby the substance to be delivered
is combined with a pharmaceutically acceptable carrier vehicle.
Suitable vehicles and their preparation are described, for example,
in Remington's Pharmaceutical Sciences, 16.sup.th Edition, A. Osol,
Ed., Mack Publishing Co., Easton, Pa. (1980), and Remington's
Pharmaceutical Sciences, 19.sup.th Edition, A. R. Gennaro, Ed.,
Mack Publishing Co., Easton, Pa. (1995). The amount of such
compounds included in such a composition depends on factors
including the age and weight of the subject, the delivery method
and route, the type of treatment desired, and the type of
polynucleotide or polynucleotide construct or other p21 inhibitor
being administered. In general, a composition of the present
invention that includes such inhibitors will contain from about 1
ng to about 30 mg, and more preferably, from about 100 ng to about
10 mg of such inhibitor. Certain preferred compositions of the
present invention may include about 1 ng of such inhibitor, about 5
ng of such inhibitor, about 10 ng of such inhibitor, about 50 ng of
such inhibitor, about 100 ng of such inhibitor, about 500 ng of
such inhibitor, about 1 .mu.g of such inhibitor, about 5 .mu.g of
such inhibitor, about 10 pg of such inhibitor, about 50 .mu.g of
such inhibitor, about 100 .mu.g of such inhibitor, about 150 .mu.g
of such inhibitor, about 200 .mu.g of such inhibitor, about 250
.mu.g of such inhibitor, about 300 .mu.g of such inhibitor, about
350 pg of such inhibitor, about 400 .mu.g of such inhibitor, about
450 pg of such inhibitor, about 500 .mu.g of a polynucleotide,
about 550 .mu.g of such inhibitor, about 600 pg of such inhibitor,
about 650 .mu.g of such inhibitor, about 700 pg of such inhibitor,
about 750 .mu.g of such inhibitor, about 800 pg of such inhibitor,
about 850 pg of a polynucleotide, about 900 .mu.g of such
inhibitor, about 950 pg of such inhibitor, about 1 mg of such
inhibitor, about 5 mg of such inhibitor, about 10 mg of such
inhibitor, about 15 mg of such inhibitor, about 20 mg of such
inhibitor, about 25 mg of such inhibitor, or about 30 mg of such
inhibitor.
[0075] Such compositions may be formulated into any of the various
compositions and may be used in any of the methods disclosed
herein. For aqueous compositions used in vivo, use of sterile
pyrogen-free water is preferred. Such formulations will contain an
effective amount of such inhibitor together with a suitable salt
and/or auxiliary agent as disclosed herein, in order to prepare
pharmaceutically acceptable compositions suitable for optimal
administration to a vertebrate. Insoluble inhibitors may be
solubilized in a weak acid or weak base, and then diluted to the
desired volume, for example, with an aqueous solution of the
present invention. The pH of the solution may be adjusted as
appropriate. In addition, a pharmaceutically acceptable additive
can be used to provide an appropriate osmolarity. Alternatively,
lipids and lipid vehicles (as discussed above) may be used to
facilitate the inhibitor administration. Other hydrophobic and
amphiphilic additives, such as, for example, sterols, fatty acids,
gangliosides, glycolipids, lipopeptides, liposaccharides, neobees,
niosomes, prostaglandins and sphingolipids, may also be included in
such compositions of the present invention. In such compositions,
these additives may be included in an amount between about 0.1 mol
% and about 99.9 mol % (relative to total lipid). Preferably, these
additives comprise about 1-50 mol % and, most preferably, about
2-25 mol %. Preferred additives include lipopeptides,
liposaccharides and steroids.
[0076] Pharmaceutical Compositions
[0077] The pharmaceutical composition of the present invention may
be in the form of an emulsion, gel, solution, suspension, etc. In
addition, the pharmaceutical composition can also contain
pharmaceutically acceptable additives including, for example,
diluents, binders, stabilizers, and preservatives. Administration
of pharmaceutically acceptable salts of the polynucleotides
described herein is preferred. Such salts can be prepared from
pharmaceutically acceptable non-toxic bases including organic bases
and inorganic bases. Salts derived from inorganic bases include
sodium, potassium, lithium, ammonium, calcium, magnesium, and the
like. Salts derived from pharmaceutically acceptable organic
non-toxic bases include salts of primary, secondary, and tertiary
amines, basic amino acids, and the like. Preferred salts include
but are not limited to sodium phosphate, sodium acetate, sodium
bicarbonate, sodium sulfate, sodium pyruvate, potassium phosphate,
potassium acetate, potassium bicarbonate, potassium sulfate,
potassium pyruvate, disodium DL-.alpha.-glycerol-phosphate, and
disodium glucose-6-phosphate. "Phosphate" salts of sodium or
potassium can be either the monobasic form, e.g., NaHPO.sub.4, or
the dibasic form, e.g., Na.sub.2HPO.sub.4, but a mixture of the
two, resulting in a desired pH, is most preferred. The most
preferred salts are sodium phosphate or potassium phosphate. As
used herein, the terms "sodium phosphate" or "potassium phosphate,"
refer to a mixture of the dibasic and monobasic forms of each salt
to present at a given pH.
[0078] Additional embodiments of the present invention are drawn to
pharmaceutical compositions comprising one or more p21 inhibitor
molecules and an auxiliary agent. The present invention is further
drawn to methods to use such compositions, methods of making such
compositions, and pharmaceutical kits. As used herein, an
"auxiliary agent" is a substance included in a composition for its
ability to enhance, relative to a composition which is identical
except for the inclusion of the auxiliary agent, the effectiveness
of a p21 inhibitor molecule. Auxiliary agents of the present
invention include nonionic, anionic, cationic, or, zwitterionic
surfactant or detergents, with nonionic, anionic, cationic, or
zwitterionic surfactant or detergents, with nonionic surfactant or
detergents being preferred, chelators, DNAse inhibitors, agents
that aggregate or condense nucleic acids, emulsifying or
solubilizing agents, wetting agents, gel-forming agents, and
buffers.
[0079] Suitable auxiliary agents include non-ionic detergents and
surfactant such as poloxaners. Poloxamers are a series of non-ionic
surfactant that are block copolymers of ethylene oxide and
propylene oxide. The poly(oxyethylene) segment is hydrophillic and
the poly(oxypropylene) segment is hydrophobic. The physical forms
are liquids, pastes or solids. The molecular weight ranges from
1000 to greater than 16000. The basic structure of a poloxaner is
HO--[CH.sub.2CH.sub.2O].sub.X--[CH.sub.2CHO(CH.sub.3)].sub.y--[CH.sub.2CH-
.sub.2O].sub.x--H, where x and y represent repeating units of
ethylene oxide and propylene oxide respectively. Thus, the
propylene oxide (PO) segment is sandwiched between two ethylene
oxide (EO) segments, (EO-PO-EO). The number of x's and y's
distinguishes individual poloxamers. If the ethylene oxide segment
is sandwiched between two propylene oxide segments, (PO-BO-PO),
then the resulting structure is a reverse poloxaner. The basic
structure of a reverse poloxamer is
HO--[CH(CH.sub.3)CH.sub.2O)].sub.x--[CH.sub.2CH.sub.2O].sub.y--[CH.sub.2C-
-HO(CH.sub.3)].sub.x--H.
[0080] Poloxamers that may be used in concert with the methods and
compositions of the present invention include, but are not limited
to commercially available poloxamers such as Pluronic.RTM. L121
(avg. MW:4400), Pluronic.RTM. L101 (avg. MW:3800), Pluronic.RTM.
L81 (avg. MW:2750), Pluronic.RTM. L61 (avg. MW:2000), Pluronic.RTM.
L31 (avg. MW: 1100), Pluronic.RTM. L122 (avg. MW:5000),
Pluronic.RTM. L92 (avg. MW:3650), Pluronic.RTM. L72 (avg. MW:2750),
Pluronic.RTM. L62 (avg. MW:2500), Pluronic.RTM. L42 (avg. MW:1630),
Pluronic.RTM. L63 (avg. MW:2650), Pluronic.RTM. L43 (avg. MW:
1850), Pluronic.RTM. L64 (avg. MW:2900), Pluronic.RTM. L44 (avg.
MW:2200), Pluronic.RTM. L35 (avg. MW:1900), Pluronic.RTM. P123
(avg. MW:5750), Pluronic.RTM. P103 (avg. MW:4950), Pluronic.RTM.
P104 (avg. MW:5900), Pluronic.RTM. P84 (avg. MW:4200) Pluronic.RTM.
P105 (avg. MW:6500), Pluronic.RTM. P85 (avg. MW:4600),
Pluronic.RTM. P75 (avg. MW:4150), Pluronic.RTM. P65 (avg. MW:3400),
Pluronic.RTM. F127 (avg. MW: 12600), Pluronic.RTM. F98 (avg. MW:
13000), Pluronic.RTM. F87 (avg. MW:7700), Pluronic.RTM. F77 (avg.
MW:6600), Pluronic.RTM. F 108 (avg. MW: 14600), Pluronic.RTM. F98
(avg. MW: 13000), Pluronic.RTM. F88 (avg. MW: 11400), Pluronic.RTM.
F68 (avg. MW:8400), and Pluronic.RTM. F38 (avg., MW:4700).
[0081] Reverse poloxamers of the present invention include, but are
not limited to Pluronic.RTM. R31R1 (avg. MW:3250), Pluronic.RTM.
R25R1 (avg. MW: 2700), Pluronic.RTM. R17R1 (avg. MW:1900),
Pluronic.RTM. R31R2 (avg. MW:3300), Pluronic.RTM. R25R2 (avg.
MW:3100), Pluronic.RTM. R17R2 (avg. MW:2150), Pluronic.RTM. R12R3
(avg. MW:1800) Pluronic.RTM. R31R4 (avg. MW:4150), Pluronic.RTM.
R25R4 (avg. MW:3600), Pluronic.RTM. R22R4 (avg. MW:3350),
Pluronic.RTM. R17R4 (avg. MW:3650), Pluronic.RTM. R25R5 (avg.
MW:4320), Pluronic.RTM. R10R5 (avg. MW:1950), Pluronic.RTM. R25R8
(avg. MW:8850), Pluronic.RTM. R17R8 (avg. MW:7000), Pluronic.RTM.
R10R8 (avg. MW:4550).
[0082] Other commercially available poloxamers include compounds
that are block copolymer of polyethylene and polypropylene glycol
such as Synperonic.RTM. L121, Synperonic.RTM. L122, Synperonic.RTM.
P104, Synperonic.RTM. P105, Synperonic.RTM. P123, Synperonic.RTM.
P85, and Synperonic.RTM. P94; and compounds that are nonylphenyl
polyethylene glycol such as Synperonic.RTM. NP10, Synperonic.RTM.
NP30, and Synperonic.RTM. NP5.
[0083] Suitable auxiliary agents include non-ionic detergents and
surfactants such as Pluronic.RTM. F68, Pluronic.RTM. F77,
Pluronic.RTM. F108, Pluronic.RTM. F127, Pluronic.RTM. P65,
Pluronic.RTM. P85, Pluronic.RTM. P103, Pluronic.RTM. P104,
Pluronic.RTM. P105, Pluronic.RTM. P123, Pluronic.RTM. L31,
Pluronic.RTM. L43; Pluronic.RTM. L44, Pluronic.RTM. L61,
Pluronic.RTM. L62, Pluronic.RTM. L64, Pluronic.RTM. L81,
Pluronic.RTM. L92, Pluronic.RTM. L101, Pluronic.RTM. L121,
Pluronic.RTM. R17R4, Pluronic.RTM. 125R4, Pluronic.RTM. R25R2,
IGEPAL CA 630.RTM., NONIDET NP-40, Nonidet.RTM. P40, Tween-20.RTM.,
Tween-80.RTM., Triton X-100.RTM., Triton X-114.TM., Thesit.RTM.;
the anionic detergent sodium dodecyl sulfate (SDS); the sugar
stachyose; the condensing agent DMSO; and the chelator/DNAse
inhibitor EDTA. Even more preferred are the auxiliary agents
Nonidet.RTM. P40, Triton X-100.RTM., Pluronic.RTM. F68,
Pluronic.RTM. F77, Pluronic.RTM. F108, Pluronic.RTM. P65,
Pluronic.RTM. P103, Pluronic.RTM. L31, Pluronic.RTM. L44,
Pluronic.RTM. L61, Pluronic.RTM. L64, Pluronic.RTM. L92,
Pluronic.RTM. R17R4, Pluronic.RTM. R25R4 and Pluronic.RTM. R25R2.
Most preferred auxiliary agent is Pluronic.RTM. R25R2.
[0084] Optimal concentrations of auxiliary agents of the present
invention are disclosed in U.S. Patent Application Publication No.
20020019358 and PCT Publication WO0180897A3. For example, in
certain embodiments, pharmaceutical compositions of the present
invention comprise about 5 ng to about 30 mg of a suitable
polynucleotide or a polynucleotide construct, and/or a
non-polynucleotide p21 inhibitor, and about 0.001% (w/v) to about
2.0% (w/v) of Pluronic.RTM. R 25R4, preferably about 0.002% (w/v)
to about 1.0% (w/v) of Pluronic.RTM. R 25R4, more preferably about
0.01% (w/v) to about 0.01% (w/v) of Pluronic.RTM. R 25R4, with
about 0.01% (w/v) of Pluronic.RTM. R 25R4 being the most preferred;
about 0.001% (w/v) to about 2.0% (w/v) of Pluronic.RTM. R 25R2,
preferably about 0.001% (w/v) to about 1.0% (w/v) of Pluronic.RTM.
R 25R2, more preferably about 0.001% (w/v) to about 0.1% (w/v) of
Pluronic.RTM. R 25R2, with about 0.01% (w/v) of Pluronic.RTM. R
25R2 being the most preferred.
[0085] Administration of the Pharmaceutical Compositions of the
Present Invention
[0086] The pharmaceutical compositions of the present invention may
be administered by any suitable means, for example, inhalation, or
intradermally, intracavity (e.g., oral, vaginal, rectal, nasal,
peritoneal, ventricular, or intestinal), intradermally,
intramuscularly, intranasally, intraocularly, intraperitoneally,
intrarectally, intratracheally, intravenously, orally,
subcutaneously, transdermally, or transmucosally (i.e., across a
mucous membrane) in a dose effective for the production of
neutralizing antibody and resulting in protection from infection or
disease. The present pharmaceutical compositions can generally be
administered in the form of a spray for intranasal administration,
or by nose drops, inhalants, swabs on tonsils, or a capsule,
liquid, suspension or elixirs for oral administration. The
pharmaceutical compositions may be in the form of single dose
preparations or in multi-dose flasks. Reference is made to
Remington's Pharmaceutical Sciences, Mack Publising Co., Easton,
Pa., Osol (ed.) (1980).
[0087] Administration can be into one or more tissues including but
not limited to muscle, skin, brain, lung, liver, spleen, bone
marrow, thymus, heart, e.g., myocardium, endocardium, and
pericardium; lymph nodes, blood, bone, cartilage, pancreas, kidney,
gall bladder, stomach, intestine, testis, ovary; uterus, rectum,
nervous system, eye, gland, or connective tissue. Furthermore, in
the methods of the present invention; the pharmaceutical
compositions may be administered to any internal cavity of a
mammal, including, but not limited to, the lungs, the mouth, the
nasal cavity, the stomach, the peritoneal cavity, the intestine,
any heart chamber, veins, arteries, capillaries, lymphatic
cavities, the uterine cavity, the vaginal cavity, the rectal
cavity, joint cavities, ventricles in brain, spinal canal in spinal
cord, and the ocular cavities. Any mode of administration can be
used so long as the mode results in prophylactic or therapeutic
efficacy. Methods to detect such a response include serological
methods, e.g., western blotting, molecular (transcriptional)
staining tissue sections by immunohistochemical methods, and
measuring the activity of the polypeptide. Pharmaceutical DNA
compositions and methods of their manufacture and delivery that may
be used in accordance with the present invention are disclosed in
U.S. Pat. Nos. 5,589,466; 5,620,896; 5,641,665; 5,703,055;
5,707,812; 5,846,946; 5,861,397; 5,891,718; 6,022,874; 6,147,055;
6,214,804; 6,228,844; 6,399,588; 6,413,942; 6,451,769, European
Patent Documents EP1165140A2; EP1006796A1 and EP0929536A1; and PCT
Patent Publications WO00/57917; WO00/73263; WO01/09303;
WO03/028632; WO94/29469; WO95/29703; and WO98/14439.
[0088] Administration may be by needle injection, catheter
infusion, biolistic injectors, particle accelerators (e.g., "gene
guns" or pneumatic "needleless" injectors) Med-E-Jet (Vahlsing, H.,
et al. (1994) "IMMUNIZATION WITH PLASMID DNA USING A PNEUMATIC
GUN," J. Immunol. Methods 171:11-22), Pigjet (Schrijver, R. S. et
al. (1997) "IMMUNIZATION OF CATTLE WITH A BHV 1 VECTOR VACCINE OR A
DNA VACCINE BOTH CODING FOR THE G PROTEIN OF BRSV," Vaccine
15:1908-1916), Biojector (Davis, H. L. et al. (1994) "DIRECT GENE
TRANSFER IN SKELETAL MUSCLE: PLASMID DNA-BASED IMMUNIZATION AGAINST
THE HEPATITIS B VIRUS SURFACE ANTIGEN," Vaccine 12:1503-1509;
Gramzinski, R., et al. (1998) "IMMUNE RESPONSE TO A HEPATITIS B DNA
VACCINE IN AOTUS MONKEYS: A COMPARISON OF VACCINE FORMULATION,
ROUTE, AND METHOD OF ADMINISTRATION," Mol Med 4:109-118),
AdvantaJet (Lindmayer, I., et al. (1986) "DEVELOPMENT OF NEW JET
INJECTOR FOR INSULIN THERAPY," Diabetes Care 9:294-297),
Medi-jector (Martins, J. K. et al. (1979) "MEDIJECTRO--A NEW METHOD
OF CORTICOSTEROID-ANESTHETIC DELIVERY," J. Occup. Med. 21:821-824),
gelfoam sponge depots, other commercially available depot materials
(e.g., hydrogels), osmotic pumps (e.g., Alza minipumps), oral or
suppositorial solid (tablet or pill) pharmaceutical formulations,
topical skin creams, and decanting, use of polynucleotide coated
suture (Qin, J. Y. at al. (1999) "GENE SUTURE--A NOVEL METHOD FOR
INTRAMUSCULAR GENE TRANSFER AND ITS APPLICATION IN HYPERTENSION
THERAPY," Life Sciences 65:2193-2203) or topical applications
during surgery.
[0089] Thus, in one embodiment, administration is into muscle
tissue, i.e., skeletal muscle, smooth muscle, or myocardium. Most
preferably, the muscle is skeletal muscle. For polynucleotide
constructs in which the polynucleotide or polynucleotide construct
is DNA, the DNA can be operably linked to a cell-specific promoter
that directs substantial transcription of the DNA only in
predetermined cells. In certain embodiments, a polynucleotide
construct, or composition comprising an polynucleotide or
polynucleotide construct, is delivered to any tissue including, but
not limited to those disclosed herein, such that the polynucleotide
or polynucleotide construct is free from association with liposomal
formulations and charged lipids. Alternatively, the polynucleotide,
polynucleotide construct, or composition is delivered to a tissue
other than brain or nervous system tissue, for example, to muscle,
skin, or blood, in any composition as described herein.
[0090] Preferably, the pharmaceutical composition is delivered to
the interstitial space of a tissue. "Interstitial space" comprises
the intercellular, fluid, mucopolysaccharide matrix among the
reticular fibers of organ tissues, elastic fibers in the walls of
vessels or chambers, collagen fibers of fibrous tissues, or that
same matrix within connective tissue ensheathing muscle cells or in
the lacunae of bone. It is similarly the space occupied by the
plasma of the circulation and the lymph fluid of the lymphatic
channels.
[0091] Nucleic acid pharmaceutical compositions, preferably in the
form of plasmid DNA, may be administered (especially by injection)
into tissue and voltage pulses applied between electrodes disposed
in the tissue, thus applying electric fields to cells of the
tissue. The electrically-mediated enhancement covers administration
using either iontophoresis or electroporation in vivo. Suitable
techniques of electroporation and iontophoresis are provided by
Singh, J. et al. (1989) "TRANSDERMAL DELIVERY OF DRUGS BY
IONTOPHORESIS: A REVIEW," Drug Des. Deliv. 4:1-12; Theiss, U. et
al. (1991) "IONTOPHORESIS--IS THERE A FUTURE FOR CLINICAL
APPLICATION?," Methods Find. Exp. Clin. Pharmacol. 13:353-359;
Singh and Maibach (1993) "TOPICAL IONTOPHORETIC DRUG DELIVERY IN
VIVO: HISTORICAL DEVELOPMENT, DEVICES AND FUTURE PERSPECTIVES,"
Dermatology. 187:235-238; Singh, P. et al. (1994) "IONTOPHORESIS IN
DRUG DELIVERY: BASIC PRINCIPLES AND APPLICATIONS," Crit. Rev. Ther.
Drug Carrier Syst. 11:161-213; Su, Y. at al. (1994) "SPHINGOSINE
1-PHOSPHATE, A NOVEL SIGNALING MOLECULE, STIMULATES DNA BINDING
ACTIVITY OF AP-1 IN QUIESCENT SWISS 3T3 FIBROBLASTS," 3. Pharm.
Sci. 83:12-17; Costello, C. T. at al. (1995) "IONTOPHORESIS:
APPLICATIONS IN TRANSDERMAL MEDICATION DELIVERY," Phys. Ther.
75:554-563; Howard, J. p. at al. (1995). "EFFECTS OF ALTERNATING
CURRENT IONTOPHORESIS ON DRUG DELIVERY," Arch. Phys. Med. Rehabil.
76:463-466; Kassan, D. G. at al. (1996) "PHYSICAL ENHANCEMENT OF
DERMATOLOGIC DRUG DELIVERY: IONTOPHORESIS AND PHONOPHORESIs," S.
Amer, Acad. Dermatol. 34:657-666; Riviere at al. (1997)
"ELECTRICALLY-ASSISTED TRANSDERMAL DRUG DELIVERY," Pharm. Res.
14:687-697; Zempslcy, W. T. et al. (1998) "IONTOPHORESIS:
NONINVASIVE DRUG DELIVERY," Amer. J. Anesthesiol. 25:158-162;
Muramatsu, T. et al. (1998) "IN VIVO ELECTROPORATION: A POWERFUL
AND CONVENIENT MEANS OF NONVIRAL GENE TRANSFER TO TISSUES OF LIVING
ANIMALS," Int. J. Mol. Med. 1:55-62; Garrison J. (1998).
"IONTOPHORESIS: AN ALTERNATIVE DRUG-DELIVERY SYSTEM," Med. Device
Technol. 9:32-36; Banga A. K. et al. (1998) "ASSESSING THE
POTENTIAL OF SKIN ELECTROPORATION FOR THE DELIVERY OF PROTEIN-AND
GENE-BASED DRUGS," Trends Biotechnol. 16; 408-412; Banga A. K. et
al. (1999) "IONTOPHORESIS AND ELECTROPORATION: COMPARISONS AND
CONTRASTS," Int. 3. Pharm. 179:1-19; Neumann E. et al. (1999)
"FUNDAMENTALS OF ELECTROPORATIVE DELIVERY OF DRUGS AND GENES,"
Bioelectrochem. Bioenerg. 48:3-16; and Heiser, W. C. (2000)
"OPTIMIZING ELECTROPORATION CONDITIONS FOR THE TRANSFORMATION OF
MAMMALIAN CELLS," Methods Mol. Biol. 130:117-134.
[0092] The nature of the electric field generated in accordance
with such methods is determined by the nature of the tissue, the
size of the selected tissue and its location. The use of
insufficient or excessive field strength is to be avoided. As used
herein, a field strength is excessive if it results in the lysing
of cells. A field strength is insufficient if it results in a
reduction of efficacy of 90% relative to the maximum efficacy
obtainable. The electrodes may be mounted and manipulated in many
ways known in the art. The waveform of the electrical signal
provided by the pulse generator can be an exponentially decaying
pulse, a square pulse, a unipolar oscillating pulse train or a
bipolar oscillating pulse train. The waveform, electric field
strength and pulse duration are dependent upon the type of cells
and the DNA that are to enter the cells via electrical-mediated
delivery and thus are determined by those skilled in the art in
consideration of these criteria. Any number of known devices may be
used for delivering polynucleotides and generating the desired
electric field. Examples of suitable devices include, but are not
limited to, a single needle probe, a bipolar probe and a
combination needle and plate probe. Alternatively, methods such as
continuous-flow electroporation may be employed (See, U.S. Pat.
Nos. 6,485,961; 6,090,617; 6,074,605; 5,720,921; 5,612,207; and
5,098,843).
[0093] The compositions of the present invention can be lyophilized
to produce pharmaceutical compositions in a dried form for ease in
transportation and storage. The pharmaceutical compositions of the
present invention may be stored in a sealed vial, ampule or the
like. In the case where the pharmaceutical composition is in a
dried form, the composition is dissolved or suspended (e.g., in
sterilized distilled water) before administration. An inert carrier
such as saline or phosphate buffered saline or any such carrier in
which the pharmaceutical compositions has suitable solubility, may
be used.
[0094] Further, the pharmaceutical compositions may be prepared in
the form of a mixed composition that contains one or more
additional constituents so long as such additional constituents do
not interfere with the effectiveness of the p2.1 inhibitor and the
side effects and adverse reactions are not increased additively or
synergistically. The pharmaceutical compositions of the present
invention can be associated with chemical moieties which may
improve the composition's solubility, absorption, biological half
life, etc. The moieties may alternatively decrease the toxicity of
the pharmaceutical compositions, eliminate or attenuate any
undesirable side effect of the pharmaceutical compositions, etc.
Moieties capable of mediating such effects are disclosed in
Remington's Pharmaceutical Sciences (1980). Procedures for coupling
such moieties to a molecule are well known in the art.
[0095] Determining an effective amount of a composition depends
upon a number of factors including, for example, the chemical
structure and biological activity of the substance, the age and
weight of the subject, the precise condition requiring treatment
and its severity, and the route of administration. Based on the
above factors, determining the precise amount, number of doses, and
timing of doses are within the ordinary skill in the art and will
be readily determined by the attending physician or
veterinarian.
[0096] In one embodiment, the pharmaceutical compositions of the
present invention are administered free from association with
liposomal formulations, charged lipids, or
transfection-facilitating viral particles. In another embodiment,
the compositions of the present invention are administered free
from association with any delivery vehicle now known in the art
that can facilitate entry into cells.
[0097] As used herein, "ex vivo" cells are cells into which the
pharmaceutical compositions are introduced, for example, by
transfection, lipofection, electroporation, bombardment, or
microinjection. The cells containing the pharmaceutical
compositions are then administered in vivo into mammalian tissue
(see, for example, see Belldegrun, A., et al. (1993) "HUMAN RENAL
CARCINOMA LINE TRANSFECTED WITH INTERLEUKIN-2 AND/OR INTERFERON
ALPHA GENE(S): IMPLICATIONS FOR LIVE CANCER VACCINES," J. Natl.
Cancer Inst. 85: 207-216; Ferrantini, M. et al. (1993) "ALPHA
1-INTERFERON GENE TRANSFER INTO METASTATIC FRIEND LEUKEMIA CELLS
ABROGATED TUMORIGENICITY IN IMMUNOCOMPETENT MICE: ANTITUMOR THERAPY
BY MEANS OF INTERFERON--PRODUCING CELLS," Cancer Research
53:1107-1112; Ferrantini, M. et al (1994) "IFN-ALPHA 1 GENE
EXPRESSION INTO A METASTATIC MURINE ADENOCARClNOMA (TS/A) RESULTS
IN CD8.sup.+ T CELL-MEDIATED TUMOR REJECTION AND DEVELOPMENT OF
ANTITUMOR IMMUNITY. COMPARATIVE STUDIES WITH IFN-GAMMA-PRODUCING
TS/A CELLS," I. Immunology 153:4604-4615; Kaido, T. et al. (1995)
"TN-ALPHA 1 GENE TRANSFECTION COMPLETELY ABOLISHES THE
TUMORIGENICITY OF MURINE B 16 MELANOMA CELLS IN ALLOGENEIC DBA/2
MICE AND DECREASES THEIR TUMORIGENICITY IN SYNGENEIC C57BL/6 MICE,"
Int. 3. Cancer 60: 221-229; Ogura, H. et al. (1990) "IMPLANTATION
OF GENETICALLY MANIPULATED FIBROBLASTS INTO MICE AS ANTITUMOR
ALPHA-INTERFERON THERAPY," Cancer Research 50:5102-5106;
Santodonato, L. et al. (1996) "CURE OF MICE WITH ESTABLISHED
METASTATIC FRIEND LEUKEMIA CELL TUMORS BY A COMBINED THERAPY WITH
TUMOR CELLS EXPRESSING BOTH INTERFERON-ALPHA 1 AND HERPES SIMPLEX
THYMIDINE KINASE FOLLOWED BY GANCICLOVIR," Human Gene Therapy
7:1-10; Santodonato, L., et al. (1997) "LOCAL AND SYSTEMIC
ANTITUMOR RESPONSE AFTER COMBINED THERAPY OF MOUSE METASTATIC
TUMORS WITH TUMOR CELLS EXPRESSING IFN-ALPHA AND HSVTK:
PERSPECTIVES FOR THE GENERATION OF CANCER VACCINES," Gene Therapy
4:1246-1255; and Zhang, J. F. et al. (1996) "GENE THERAPY WITH AN
ADENO-ASSOCIATED VIRUS CARRYING AN INTERFERON GENE RESULTS IN TUMOR
GROWTH SUPPRESSION AND REGRESSION," Cancer Gene Therapy
3:31-38.
[0098] In the "local delivery" embodiment of the present invention,
a pharmaceutical composition is administered in vivo, such that the
p21 inhibitor is incorporated into the local cells at the site of
administration. The pharmaceutical compositions can be administered
either within ex vivo cells or free of ex vivo cells or ex vivo
cellular material. Preferably, the polynucleotide construct is
administered free of ex vivo cells or ex vivo cellular
material.
[0099] The pharmaceutical compositions can be solubilized in a
buffer prior to administration. Suitable buffers include, for
example, phosphate buffered saline (PBS), normal saline, Tris
buffer, and sodium phosphate vehicle (100-150 mM preferred).
Insoluble polynucleotides can be solubilized in a weak acid or
base, and then diluted to the desired volume with a neutral buffer
such as PBS. The pH of the buffer is suitably adjusted, and
moreover, a pharmaceutically acceptable additive can be used in the
buffer to provide an appropriate osmolarity within the lipid
vesicle. Preferred salt solutions and auxiliary agents are
disclosed herein.
[0100] A systemic delivery embodiment is particularly preferred for
treating non-localized disease conditions. A local delivery
embodiment can be particularly useful for treating disease
conditions that might be responsive to high local concentration.
When advantageous, systemic and local delivery can be combined.
U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and PCT
publication WO94/29469 provide methods for delivering compositions
comprising naked DNA, or DNA cationic lipid complexes to
mammals.
[0101] Compositions used in the present invention can be formulated
according to blown methods. Suitable preparation methods are
described, for example, in Remington's Pharmaceutical Sciences,
16.sup.th Edition, A. Osol, ed., Mack Publishing Co., Easton, Pa.
(1980), and Remington's Pharmaceutical Sciences, 19.sup.th Edition,
A. R. Gennaro, ed., Mack Publishing Co., Easton, Pa. (1995), both
of which are incorporated herein by reference in their entireties.
Although the composition is preferably administered as an aqueous
solution, it can be formulated as an emulsion, gel, solution,
suspension, lyophilized form, or any other form known in the art.
According to the present invention, if the composition is
formulated other than as an aqueous solution, it will require
resuspension in an aqueous solution prior to administration. In
addition, the composition may contain pharmaceutically acceptable
additives including, for example, diluents, binders, stabilizers,
and preservatives.
[0102] The present invention also provides kits for use in treating
infection comprising an administration means and a container means
containing a pharmaceutical composition of the present invention.
Preferably, the container in which the composition is packaged
prior to use will comprise a hermetically sealed container
enclosing an amount of the lyophilized formulation or a solution
containing the formulation suitable for a pharmaceutically
effective dose thereof, or multiples of an effective dose. The
composition is packaged in a sterile container, and the
hermetically sealed container is designed to preserve sterility of
the pharmaceutical formulation until use. Optionally, the container
can be associated with administration means and/or instruction for
use.
[0103] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples, which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
Example 1
Materials and Methods for the Analysis Of Temporal Events
Associated with the Initial Virus-Macrophage Encounter
[0104] A. Purification of Human Monocytes by Counterflow
Centrifugal Elutriation
[0105] Human peripheral blood cells are obtained by leukapheresis
from normal volunteers in the Department of Transfusion Medicine at
the National Institutes of Health (Bethesda, Md.) and diluted in
endotoxin-free PBS without Ca.sup.2+ and Mg.sup.2+ (BioWhittaker,
Walkersville, Md.) for density sedimentation. The monocytes in the
mononuclear cell layer are purified by counterflow centrifugal
elutriation within 4 hr after leukapheresis (Wahl, S. M. et al.
(1984) "ISOLATION OF HUMAN MONONUCLEAR CELL SUBSETS BY COUNTERFLOW
CENTRIFUGAL ELUTRIATION(CCE). II. FUNCTIONAL PROPERTIES OF
B-LYMPHOCYTE-, T-LYMPHOCYTE-, AND MONOCYTE-ENRICHED FRACTIONS. Cell
Immunol 85:384-395).
[0106] B. HIV-1 Infection of Human Monocyte-Derived Macrophages
[0107] Monocytes are plated in 6 well plates (Corning Costar
Corporation, Cambridge, Mass.), at 6.times.10.sup.6 cells/well or
in Lab-Tek chamber slides (Naperville, Ill.) at
1.5.times.10.sup.6/chamber in Dulbecco's modified Eagle's medium
(DMEM) with 2 mM L-glutamine and 10 .mu.g/ml gentamicin
(BioWhittaker) or in other suitable vessels. After adherence (4-6
hr at 37.degree. C. and 5% CO.sub.2), 10% human AB-serum (Sigma,
St. Louis, Mo.) is added to the culture medium. Cells are cultured
5-10 days to allow differentiation into macrophages before being
infected with HIV-1.sub.BaL TCID.sub.50=1000-5000 (Advanced
Biotechnologies Inc., Columbia, Md.) or other M tropic. (R5) virus
for 90 minutes at 37.degree. C. The levels of endotoxin are below
the limit of detection in virus preparations. Unbound virus is
removed by washing the cells with media and refeeding with complete
DMEM containing 10% human serum. Control populations of adherent
macrophages are mock-infected and cultured in parallel. Every 3 to
4 days, half the medium is removed for virus assay and replaced
with fresh complete medium for two weeks. Supernatant p24 antigen
is assayed using the p24 core profile enzyme-linked immunosorbent
assay (ELISA) kit (Perkin-Elmer Life Sciences, Wilmington, Del.).
As described below, in certain experiments, macrophages are
pre-treated with 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid
(CDDO) or a CDDO analog (di-CDDO) for 45 minutes prior to exposure
to HIV-1. Additionally, macrophages are treated with p21 anti-sense
phosphorothioate oligonucleotides:
TABLE-US-00008 SEQ ID NO. 7: 5'-TGTCAGGCTGGTCTGCCTCC-3' (oligo 1),
and SEQ ID NO. 9: 5'-ACATCACCAGGATTGGACAT-3' (oligo 2)
or negative control oligonucleotide:
TABLE-US-00009 SEQ ID NO. 14: 5'-TGGATCCGACATGTCAGA-3' (oligo
3)
(sequence obtained from Dr. Argyrios N. Theofilopoulos, The Scripps
Research Institute, La Jolla, Calif.) after HIV-1 infection and at
the time of refeeding the cultures and tested for viral replication
at day 12. SEQ NOS. 7 and 9 are derived from the murine p21
antisense sequence, yet are effective in inhibiting human p21
expression and the replication and transmission of HIV.
[0108] C. Northern Blot Analysis and RNase Protection Assay
(RPA)
[0109] Total cellular RNA is extracted from adherent control or
infected monocytes with the RNeasy minikit (Qiagen, Valencia,
Calif.) and analyzed by northern blot (Wahl, S. M. et al. (1998)
"MYCOBACTERIUM AVIUM COMPLEX AUGMENTS MACROPHAGE HIV-1 PRODUCTION
AND INCREASES CCR5 EXPRESSION. Proc Natl Acad Sci USA
95:12574-12579) using an HIV-full length probe (NIH AIDS Research
and Reference Reagent Program) and GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) (Gibco BRL,
Gaithersburg, Md.). For the RPA 3 pg of RNA is evaluated using the
hStress tRiboquant Multi-Probe RPA system (BD Pharmingen, San
Diego, Calif.) and densities are normalized to the GAPDH gene using
ImageQuant (Molecular Dynamics, Sunnyvale, Calif.)
[0110] D. cDNA Expression Array
[0111] The Altlas human cDNA Expression Array 1.2 I (Clontech, Palo
Alto, Calif.) (catalog #7850-1) is performed using 5 .mu.g of DNase
digested total RNA as described by Greenwell-Wild, T. at al. (2002)
"MYCOBACTERIUM AVIUM INFECTION AND MODULATION OF HUMAN MACROPHAGE
GENE EXPRESSION." Immunol 169:6286-6297. Gene expression in
infected cells is compared with the corresponding control
population from the same donor expressed as a ratio (fold change)
after normalization to housekeeping genes. A total of seven
different donors are analyzed.
[0112] E. Transmission Electron Microscopy (TEM)
[0113] Gluteraldehyde-fixed uninfected and infected cells are
postfixed in OsO.sub.4, dehydrated through graded ethanol and
propylene oxide, embedded in Spurr's epoxy, and thick-and
thin-sectioned. Thin sections are placed on copper grids, stained
with uranyl acetate and lead citrate, and viewed in a Zeiss EM10
microscope (LEO Electron Microscope; Oberkochen, Germany).
[0114] F. Immunofluorescence Microscopy
[0115] Uninfected and HIV-1 infected macrophages that have been
cultured for twelve days are washed twice with PBS, fixed with 2%
paraformaldehyde in PBS, washed and incubated in 100 mM glycine in
PBS for 20 min, followed by 0.5% Triton-X-100 for 10 min and rinsed
with PBS. Cells are then labeled with rabbit anti-p21 antibody, at
5 .mu.g/ml (Santa Cruz Biotechnology, Santa Cruz, Calif.) in PBS
containing 5% donkey serum (Jackson ImmunoResearch Laboratories,
Inc., West Grove, Pa.) for 1 hr, washed extensively, then incubated
with Texas-Red conjugated secondary antibody at 3 .mu.g/ml at room
temperature (Molecular Probes, Eugene, Oreg.). Non-specific
background is determined using an irrelevant rabbit isotype control
antibody and secondary antibody alone at the same concentrations
above. Images are captured using a Leica TCS-4D confocal microscope
system with a Kr-Af laser on a DMR upright microscope using a
40.times., 1.0 numerical aperture objective. Fluorescence intensity
analysis, is performed using confocal microscopy and Metamorph
analysis (Universal Imaging, Downington, Pa.).
[0116] G. Immunoprecipitation and Western Blot Analysis
[0117] Whole cell lysates are generated using a lysis buffer that
consisted of 1% Nonidet P-40, 150 mM NaCl, 20 mM Tris-HCl (pH,
7.5), 10 mM NaF, 10 mM NaPPi, 2.5 nM EDTA, 1 mM Na.sub.3VO.sub.4,
0.2 mM 3, 4 dichloroisocoumarin, 1 mM phenylmethylsulfonyl
fluoride, 100 ug/ml chymostatin and 1.times. complete protease
inhibitor (BoehringerMannheim, Indianapolis, Ind.). CDKNIA is
immunoprecipitated from cell lysates using an anti-CDKN1A antibody
conjugated to agarose (Santa Cruz Biotechnology) and incubated with
constant rotation at 4.degree. C. for 2 hours. Immunoprecipitates
are electrophoresed onto Tricine gels (Invitrogen, Carlsbad,
Calif.), transferred to nitrocellulose membrane and immunoblotted
with anti-CDKN1A (BD Pharmingen). Immunoblots are developed using
enhanced chemiluminescence and the Super-Signal substrate according
to manufacturer's instructions (Pierce Chemical Co, Rockford,
Ill.).
Example 2
Results of the Analysis of Temporal Events Associated with the
Initial Virus-Macrophage Encounter
[0118] A. Kinetics of HIV-1 Replication in Adherent Macrophages
[0119] Elutriated monocytes are adhered for 7 days, exposed to an
R5 HIV.sub.BaL for 90 minutes, washed and the kinetics of cellular
and viral changes are monitored. HIV-1 RNA is typically detected on
day 5, becoming increasingly apparent by 10-16 days after initial
exposure to the virus (FIG. 1A). In parallel, detectable p24
antigen becomes evident within 5 days, then increases dramatically
(FIG. 1B). Ultrastructurally, viral particles are not be seen in
the adherent macrophages at day 1-5, although an increase in
binucleated cells and then an increasing frequency of
multinucleated cells can be observed (FIG. 1C). Consistent with
viral RNA and p24 antigen, virus is visibly detected by electron
microscopy around day 7 (FIG. 1C; FIG. 1D) with .gtoreq.70% of the
cells typically harboring large numbers of virions by day 10 (FIG.
1C; FIG. 1D). HIV-1 is being produced on the complex surfaces
between cells, on the free surfaces and in cytoplasmic vacuoles of
the Golgi apparatus. Nonetheless, once the majority of cells are
infected and large numbers of virions are present within and on the
surface of the macrophages, p24 levels plateau, likely dependent on
host factors.
[0120] B. Initial Gene Expression in HIV-1-Infected Macrophage
Populations
[0121] To examine the host factors underlying viral propagation,
transcriptional pathways that are activated downstream of the
CD4-HIV-1 co-receptor binding/signaling event are examined by cDNA
expression arrays. Compared with the control mock-infected
macrophage population, an early and transient gene expression
profile occurs, followed by a delayed pattern that emerges in
association with viral replication. Within 3-6 hours upregulated
genes defined as exhibiting a .gtoreq.2 fold increase above
baseline in .gtoreq.4 donors (134 of 1200 interrogated) are
associated predominantly with signal transduction pathways (24%)
and transcription (25%) (FIGS. 2A-2E), many consistent with
downstream effects of engaging the G protein signaling pathway.
Genes corresponding to the mitogen activated protein kinase (MAPK)
family are increased, including p38 MAPK and MAPKAP-K2.
[0122] In addition to genes involved in transcription and signal
transduction, multiple genes associated with cell cycle, apoptosis,
and cellular recruitment (Table 1), including chemokines (IL-8,
MCP-1, and MRP14) involve viral replication (Lane, B. R. et al.
(2001) "INTERLEUKIN-8 STIMULATES HUMAN IMMUNODEFICIENCY VIRUS TYPE
1 REPLICATION AND IS A POTENTIAL NEW TARGET FOR ANTIRETROVIRAL
THERAPY," J Virol 75:8195-8202; Cinque, P. et al. (1998) "ELEVATED
CEREBROSPINAL FLUID LEVELS OF MONOCYTE CHEMOTACTIC PROTEIN-1
CORRELATE WITH HIV-1 ENCEPHALITIS AND LOCAL VIRAL REPLICATION,"
Aids 12:1327-1332; Strasser, F. et al. (1997) "ELEVATED SERUM
MACROPHAGE INHIBITORY FACTOR-RELATED PROTEIN (MRP) 8/14 LEVELS IN
ADVANCED HIV INFECTION AND DURING DISEASE EXACERBATION," I Acquir
Immune Defic Syndr Hum Retrovirol 16:230-238) are upregulated.
Enhanced surface adhesion molecules VNRA, CD11c, ICAM1, FNRA, CD44,
ITGAE can influence HIV infection, virion interaction with the
target cell and syncytium formation (Shattock, R. J. et al. (1996)
"ENHANCED HIV REPLICATION IN MONOCYTIC CELLS FOLLOWING ENGAGEMENT
OF ADHESION MOLECULES AND CONTACT WITH STIMULATED T CELLS," Res
Virol 147:171-179) and also an incorporation into HIV virions (Guo,
M. M. et al. (1995) "HIV ACQUIRES FUNCTIONAL ADHESION RECEPTORS
FROM HOST CELLS," AIDS Res Hum Retroviruses 11: 1007-1013).
TABLE-US-00010 TABLE 1 Early HIV-1 Upregulated Macrophage Gene
Expression GenBank Accession Gene Average Fold Number Description
Increase Signal Transduction M36430 GNBI 5.0 X15014 Ral A 3.3
D26309 LIMK-1 3.1 M14631 GNAS 3.0 L35253 MAP kinase p38 3.0 L29511
GRB2 2.7 AF068920 SHOC 2 2.5 L25080 Ras homolog A (RhoA) 2.5
AF055581 LNK adaptor 2.4 X17576 NCK melanoma cytoplasmic 2.3
src/homolog M98343 Cortactin (ems-I) 2.3 M65066 PRKAR1B 2.3 U10550
Gem (ras family) 2.2 U78576 PI4P5 kinase alpha 2.2 U12779 MAPKAP
kinase 2 2.2 M19922 INT2 2.1 X15219 SnoN 2.1 M29870 Rac1 2.1 M28213
Rab2 2.1 U24166 EB1 2.1 L20321 Serine/threonine kinase NRK2 2.1
M34181 PKC beta 2.1 X60957 Tyrosine kinase receptor Tie-1 2.0
L05624 MAPKK1 2.0 X03484 Raf1 protooncogene 2.0 L22075 G13 2.0
X94991 Zyxin 2 2.0 M63960 PPlalpha 2.0 X06318 PKC beta 1 2.0 M77234
Fte-l 2.0 X08004 Raplb 2.0 Transcription U10323 NF45 3.6 L19871
Activating factor 3 (ATF3) 3.1 U12979 Activated RNA polymerase n
2.9 transcriptional coactivator p15 (PC4) M81601 Transcription
elongation factor SII 2.8 M29038 Stem cell protein 2.8 D90209
Activating factor 4 (ATF4) 2.8 M34079 TAT binding protein (TBP-1)
2.7 L34587 RNA polymerase II Elongation 2.6 factor SIII p15 subunit
L04282 CACCC-box DNA binding protein 2.6 U22431 Hypoxia-inducible
factor 1 alpha 2.5 L23959 E2F dimerization partner 1 (DP1) 2.5
M83234 NSEP 2.5 S40706 GADD153 2.4 M96824 nucleobindin precursor
(NUC) 2.4 M36717 Ribonuclease/angiogenin inhibitor 2.4 (RAI) D26156
SW1/SNF-related actin-dependent 2.3 regulator of chromatin X69391
60s ribosomal protein (RPL6) 2.3 X59738 Zinc finger x-chromosomal
protein 2.3 M59079 CBF-B 2.2 M96944 PAX5 2.2 AF084199 PRDI-BFl
(transcription repressor 2.2 protein) M97796 Inhibitor of DNA
binding 2 (ID2) 2.2 U07418 MutL protein homolog 1 (MLH1) 2.2
AF060222 DNase II 2.2 U58198 Interleukin enhancer binding factor
2.1 (ILF) Z36715 Elk-3 2.1 AF032119 CASK 2.1 M97935 STAT1
alpha/beta 2.1 Z30094 Basic transcription factor 2 2.1 (BTF2p44)
J04111 jun protooncogene, AP-1 2.1 D26155 Transcriptional activator
(hsnF2a) 2.0 M80397 DNA polymerase delta catalytic 2.0 subunit
AF076974 Transformation/transcription 2.0 domain associated protein
Cell Cycle/Apoptosis U13737 Caspase 3 2.2 L29222 CDC-like kinase
(CLK1) 2.1 AF071596 IEX-IL anti-death protein 2.1 M15796
Proliferating cyclic nuclear antigen 2.1 (PCNA) X96586 FAN protein
2.1 U28014 Caspase-4 2.1 Z23115 bcl-x 2.0 U09579 cyclin-dependent
kinase 2.0 inhibitor 1A (CDKNIA) Adhesion Molecules/Receptors
M14648 Vitronectin receptor alpha 3.3 (VNRA) J03132 Intercellular
adhesion molecule 1 3.1 (ICAM1) M81695 CDllc antigen 2.5 X06256
Fibronectin receptor alpha (FNRA) 2.5 D84657 Photolyase/blue-light
receptor 2.4 homolog X07979 Fibronectin receptor beta (FNRB) 2.7
D13866 Alpha 1 catenin 2.4 X72304 Corticotropin releasing factor
2.4 receptor 1 M59911 Integrin alpha 3 (ITGA3) 2.3 M37722
Fibroblast growth factor receptor 1 2.2 L25851 Integrin alpha E
(ITGAE) 2.1 M27492 IL-1 receptor type I 2.0 J04536 Leukosialin 2.0
X01057 IL2R alpha 2.0 M59040 CD44 antigen 2.0 Chemokines/Cytokines
Y00787 Interleukin-8 9.7 M65291 Interleukin-12 alpha 5.0 M24545
Monocyte chemotactic protein 1 4.5 (MCP-1) X06233 Migration
inhibitory factor-related 3.8 protein 14 (MRP14) M92381 Thymosin
beta 10 3.7 MI7733 Thymosin beta 4 3.6 X01394 Tumor necrosis factor
alpha (TNF 3.4 alpha) X53655 Neurotrophin-3 precursor 2.6 M21121
Small inducible protein A5 2.5 (SCYA5) M86492 Glia maturation
factor beta 2.2 M31145 Insulin-like growth factor binding 2.2
protein 1 M27288 Oncostatin M (OSM) 2.1 U13699 IL-1 beta converting
enzyme (ICE) 2.1 U16296 T-lymphoma-invasion & 2.0 metastasis
inducing (TIAM1) M25667 Neuromodulin 2.0 X02530 Interferon
gamma-induced protein 2.0 (IP-IO) Proteases/Protease Inhibitors
M11233 Cathepsin D 3.2 J05070 Matrix metalloproteinase 9 3.1 (MMP9)
X56692 C reactive protein 2.9 AF059244 Cystatin related protein 2.8
X05562 Procollagen alpha 2 2.5 L23808 Matrix metalloproteinase 12
2.2 D00762 Proteasome C8 2.1 Z81326 Protease inhibitor 12 2.0
L40377 Cytoplasmic antiprotease 2 2.0 (CAP2) M23254 Calpain 2 2.0
X04106 Calpain 2.0 Metabolism U03688 dioxin inducible cytochrome
p450 4.5 (CYP1A1) X06985 Heme oxygenase 1 (HO-1) 4.2 U34683
Glutathione synthetase 3.0 X07270 90-kDa heat-shock protein A 2.7
U29091 Selenium binding protein 2.7 L14595 Neural amino acid
transporter A 2.6 (SATT) DO0099 Na+/K+ transporting ATPase 2.4
alpha 1 M74524 Ubiquitin conjugating enzyme 2.3 (UBE2A) X91247
Thioredoxin reductase 2.3 X54079 27-kDa heat-shock protein 2.2
Ml1717 70-kDa heat shock protein 1 2.1 L20046 Xeroderma pigmentosum
group G 2.0 complementing protein Y00264 Alzheimer's disease
amyloid A4 2.0 protein
[0123] Although gene expression for caspases 3, 4 and 8 is
increased, genes encoding factors that contribute to cellular
resistance to apoptosis including IEX-1L and bcl-x (Wu, M. X. et
al. (1998) "IEX-1L, AN APOPTOSIS INHIBITOR INVOLVED IN
NF-KAPPAB-MEDIATED CELL SURVIVAL, Science 281:998-1001; Antonsson,
B. et al. (2000) "THE BCL-2 PROTEIN FAMILY," Exp Cell Res
256:50-57) are concurrently elevated (Table 1). As reported, IL-2
receptor mRNA is also enhanced in HIV-infected macrophages (Allen,
J. B. et al. (1990) "EXPRESSION OF INTERLEUKIN 2 RECEPTORS BY
MONOCYTES FROM PATIENTS WITH ACQUIRED IMMUNODEFICIENCY SYNDROME AND
INDUCTION OF MONOCYTE INTERLEUKIN 2 RECEPTORS BY HUMAN
IMMUNODEFICIENCY VIRUS IN VITRO," 3 Clin Invest 85:192-199).
Another gene upregulated in infected cells within hours and at day
1 is CYP1A1, previously associated with enhanced HIV-1 gene
expression in vitro and with acceleration in the progression of
AIDS mediated by an oxidative stress pathway Yao, Y. et al. (1995)
"DIOXIN ACTIVATES HIV-1 GENE EXPRESSION BY AN OXIDATIVE STRESS
PATHWAY REQUIRING A FUNCTIONAL CYTOCHROME P450 CYP1A1 ENZYME,"
Environ Health Perspect 103:366-71). However, the augmented
transcriptional activity for heme oxygenase-1 (HO-1), a
multifunctional protein that plays a role in the regulation of
cellular herne, could protect these viral host cells against
oxidative stress and is increased in PBMC of AIDS patients (Levere,
R. D. et al. (1993) "ELEVATED LEVELS OF HEME OXYGENASE-1 ACTIVITY
AND MRNA IN PERIPHERAL BLOOD ADHERENT CELLS OF ACQUIRED
IMMUNODEFICIENCY SYNDROME PATIENTS," Am 3 Hematol 43, 19-23).
[0124] HO-1, together with increased glutathione synthetase (Table
1) may help provide a balance and protect the macrophage from
oxidative stress generated by the virus (Mialocq, P. et al. (2001)
"OXIDATIVE METABOLISM OF HIV-INFECTED MACROPHAGES: THE ROLE OF
GLUTATHIONE AND A PHARMACOLOGIC APPROACH," Pathol Biol (Paris)
49:567-571; Toborek, M. et al. (2003) "HIV-TAT PROTEIN INDUCES
OXIDATIVE AND INFLAMMATORY PATHWAYS IN BRAIN ENDOTHELIUM," J
Neurochem 84:169-179). Furthermore, transcription for the host cell
Tat binding protein (TBP-1) that interacts with viral Tat is
rapidly elevated in HIV infected macrophages) Nelbock, P. et al,
(1990) "A CDNA FOR A PROTEIN THAT INTERACTS WITH THE HUMAN
IMMUNODEFICIENCY VIRUS TAT TRANSACTIVATOR," Science 248:1650-1653
(1990). In the early virus-induced transcriptional events, HIV-1
clearly enhances more genes than it suppresses since only
tripeptidyl peptidase I, a lysosomal serine protease with a minor
endoprotease activity responsible for cleaving tripeptides from the
N terminus of oligopeptides (Tomkinson, B. (1999) "TRIPEPTIDYL
PEPTIDASES: ENZYMES THAT COUNT," Trends Biochem Sci 24:355-359) is
reproducibly suppressed. Since this protein is involved in protein
turnover, control of its expression in the host cell could benefit
the virus to ensure that infection is established.
[0125] C. Kinetics of HIV-1 Induced Gene Expression
[0126] The initial pattern of expression observed following binding
of HIV-1 to macrophages in 4-7 donors, consistent with receptor
engagement, is transient and by 24 hr, a restricted number of genes
remain or are newly elevated (Table 2).
TABLE-US-00011 TABLE 2 Genes Upregulated in Macrophages from Day
1-14 after HIV-1 Infection GenBank Accession Fold Number Gene
Description Increase Day 1 M17733 thymosin beta 4 4.6 X15480
glutathione S-transferase pi 3.4 X12451 cathepsin L 3.0 M24545
monocyte chemotactic protein 1 (MCP-1) 2.7 L16785 nucleoside
diphosphate kinase B 2.7 X93499 ras-related protein RAB-7 2.5
U07418 mutL protein homolog1 (MLH1) 2.4 M15796 proliferating cyclic
nuclear antigen (PCNA) 2.4 M83234 nuclease sensitive element (NSEP)
2.3 D10495 protein kinase C delta 2.3 Z29678 microphthalmia-assoc.
transc. factor(MITF) 2.3 X04106 Calpain 2.3 X02920
alpha-1-antiproteinase 2.2 M19922 INT-2 proto-oncogene protein 2.2
L41816 calcium/calmodulin-dependent protein 2.2 kinase I(camki)
X79067 EGF response factor 1 2.1 U18840 myelin-oligodendrocyte
glycoprotein 2.1 U03688 CYP1B1 2.1 X69391 60S ribosomal protein L6
(RPL6) 2.1 M92381 thymosin beta-10 2.1 K02770 interleukin-1 beta
2.1 X06233 calgranulin B 2.0 M97796 inhibitor of DNA binding 2
protein 2.0 M59911 integrin alpha 3 (ITGA3) 2.0 AF055581 Ink
adaptor protein 2.0 X67951 thioredoxin peroxidase 2 (TDPX2) 2.0 Day
3 D88378 proteasome inhibitor HPI31 8.3 D28118 ZNF 161 2.6 M26708
prothymosin alpha 2.1 J05070 matrix metalloproteinase 9 (MMP9) 2.0
M29366 ERBB-3 receptor 2.0 U09579 CDKN1A 2.0 Day 5 M24545 monocyte
chemotactic protein 1 (MCP-1) 2.3 M14631 guanine nucleotide-binding
protein(GNAS) 2.2 X04106 Calpain 2.1 U07418 mutL protein homolog1
(MLH1) 2.1 Day 7 Y00796 integrin alpha L (ITGAL) 8.7 X15014 Ral A;
GTP-binding protein 3.5 U09579 CDKN1A 3.5 L16785 nucleoside
diphosphate kinase B 3.1 X01057 IL-2 receptor alpha 2.9 M29870 Rac1
2.5 M23619 high mobility group protein (HMG-I) 2.3 X93499 ras
related protein RAB-7 2.3 U07418 mutL protein homolog1 (MLH1) 2.2
D28118 zinc finger protein 161 (ZNF161) 2.2 L25080 Ras homolog A
(RhoA) 2.1 X08020 glutathione S-transferase mu1 2.0 Day 14 U09579
CDKN1A 7.9 U07418 mutL protein homolog1 (MLH1) 3.8 U48296 nuclear
tyrosine phosphatase (PRL1) 3.7 U12779 MAPKAP Kinase 2 3.3 D15057
defender against cell death 1(DAD1) 3.2 J04111 jun proto-oncogene
3.1 L07597 S6KII alpha 1 3.1 M23619 high mobility group protein
(HMG-I) 2.8 X69391 60s ribosomal protein L6 2.5 L25080 Ras homolog
A (RhoA) 2.5 U12979 PC4 2.5 X08804 ras-related protein RAP-1B 2.4
M74524 ubiquitin-conjugating enzyme E2 2.2 M17733 thymosin beta 4
2.2 U28014 caspase 4 2.2 U51004 PKC inhibitor 1 2.2 U08316 S6KII
alpha 3 2.0
[0127] Of considerable interest are the limited detectable
alterations in gene expression in the cells between 3-5 days after
infection, preceding evidence of viral replication. Concomitant
with evidence of the HIV replicative cycle (FIG. 1), a resurgence
of gene expression begins to manifest (Table 2, days 7-14). Albeit
the majority of cells are maximally producing HIV by day 10-16
(FIG. 1), only a limited repertoire of genes is upregulated
compared to control macrophages (Table 2). Among these are several
G-protein related molecules, which could reflect cell-cell
transmission and HIV-1 induced signaling. Mutt, protein homolog
1(MLH1), a component of the DNA mismatch repair gene (Modrich, P.
(1997) "STRAND-SPECIFIC MISMATCH REPAIR IN MAMMALIAN CELLS," J Biol
Chem 272:24727-30) is also increased consistently, not only within
a few hours of encountering the virus (Table 1), but during the
progression of infection (days 1, 5, 7 and 14) (Table 2). This
protein could be playing a role in aiding to repair DNA damage
after viral integration into the host and thus preserving genome
stability to continue the viral life, cycle. Furthermore,
transcription of the anti-apoptotic gene DAD1 (Hong, N. A. et al.
(2000) "MICE LACKING DAD1, THE DEFENDER AGAINST APOPTOTIC DEATH-1,
EXPRESS ABNORMAL N-LINKED GLYCOPROTEINS AND UNDERGO INCREASED
EMBRYONIC APOPTOSIS. Dev Biol 220:76-84) is enhanced at the peak of
viral replication. Although the proteasome inhibitor HPI31 subunit
(Table 2) is elevated at 3 days after infection, it is dramatically
decreased during replication, consistent with evidence that
proteasome activity is essential for viral maturation (Schubert, U.
et al. (2000) "PROTEASOME INHIBITION INTERFERES WITH GAG
POLYPROTEIN PROCESSING, RELEASE, AND MATURATION OF HIV-1 AND
HIV-2," Proc Natl Acad Sci USA 97:13057-13062). This may reflect a
tight control of the macrophage metabolism to regulate protein
turnover at this late stage of infection to preserve integrity of
viral protein or host cell factors necessary for efficient virion
maturation and release (Schubert, U. et al. (2000) "PROTEASOME
INHIBITION INTERFERES WITH GAG POLYPROTEIN PROCESSING, RELEASE, AND
MATURATION OF HIV-1 AND HIV-2," Proc Natl Acad Sci USA
97:13057-13062).
[0128] D. Increased CDKN1A Gene and Protein Expression In Virus
Infected Macrophages
[0129] Although all of the virus-induced genes discussed above can
be exploited as targets for drugs that would inhibit HIV-1
propagation, the unique biphasic expression pattern of CDKN1A makes
it a preferred gene for inhibiting HIV, but does not rule out the
additional genes as potential regulatory candidates. Because it is
rapidly upregulated following HIV-1 binding/entry and then again
during the emergence of viral replication (FIG. 3A), this gene is
selected for further study. RNAse protection assay (RPA) confirmed
the rapid induction of CDKN1A (FIG. 3B; FIG. 3C), followed by
maximum expression concomitant with viral infection with no
corresponding changes in another cell cycle related gene p53.
Macrophages infected with HIV-1.sub.BAL, a laboratory viral isolate
(ADA) or a primary clinical isolate (727) that are analyzed for p21
transcription using PCR reveal expression of p21 (FIG. 3D). To
further explore the relationship between viral infection and p21,
p21 protein expression is examined by immunofluorescence. Not only
increased nuclear, but also cytoplasmic p21 staining is observed in
infected cells (FIG. 4A; FIG. 4B), consistent with enhanced protein
expression in whole cell lysates detected by western blot (FIG.
4C).
[0130] To determine whether the increased p21 influenced viral life
cycle, the cells are treated with two distinct p21 anti-sense
oligonucleotides (SEQ ID NO 7 and SEQ ID NO. 9). Both
oligonucleotides reduce viral replication as assessed by p24
levels, most evident at day 12 when untreated cells show
substantial HIV-1 production. In contrast, a missense control
oligonucleotide (SEQ ID NO. 13) did not suppress HIV-1 p24 (FIG.
6A). The oligonucleotides have no negative effect on cell viability
in infected or uninfected macrophage cultures as determined by cell
number, morphology and ultrastuctural analysis. Moreover, the p21
oligos have no direct effect on macrophage CD4 nor CCR5.
[0131] E. Effect of CDDO on HIV-1 Replication
[0132] The ability of p21 oligonucleotides to block HIV-1
replication prompted the exploration of potential therapeutically
relevant mechanisms of modulating p21 to inhibit HIV-1. It has been
reported that peroxisome proliferator-activated receptor gamma
(PPAR.gamma.) ligands, one of which includes the synthetic
triterpenoid CDDO, modulate p21 activity (Wang, Y. et al. (2000) "A
SYNTHETIC TRITERPENOID, 2-CYANO-3,12-DIOXOOLEANA-1,9-DIEN-28-OIC
ACID (CDDO), IS A LIGAND FOR THE PEROXISOME PROLIFERATOR-ACTIVATED
RECEPTOR GAMMA," Mol Endocrinol 14:1550-1556); Wakino, S. et al.
(2001) "PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA LIGANDS
INHIBIT MITOGENIC INDUCTION OF P21 (CIP1) BY MODULATING THE PROTEIN
KINASE CDELTA PATHWAY IN VASCULAR SMOOTH MUSCLE CELLS," J Biol Chem
276, 47650-47657). To examine the effect of this synthetic
triterpenoid, CDDO (0.1 .mu.M) and its derivative di-CDDO are added
prior to, or at the time of, infection of macrophages with HIV-1,
resulting in a dose dependent suppression in the release of the
viral protein p24 Ag (FIG. 5A). Without reducing cell viability or
cell number, both CDDO and di-CDDO dramatically reduced the levels
of detectable virus (FIG. 5D; FIG. 5B). Paradoxically, CDDO and
di-CDDO variably decrease p21 protein levels concomitant with
reducing HIV infection (FIGS. 5C and 5E). Moreover, p21 prbtein
accumulates in uninfected cells, suggesting a post-translational
effect on the p21 pathway. FIG. 5E shows the result of an
investigation of macrophages infected with HIV-1.sub.BaL or ADA and
treated or not with CDDO (0.1 .mu.M) and analyzed by PCR for p21
and GAPDH. FIG. 5F shows the result of an investigation in which
supernatants (12 days) collected from HIV-1.sub.BaL, ADA or 727
infected cells that were treated or not with CDDO are analyzed for
viral replication by p24 ELISA.
Example 3
Analysis of Gene Silencing
[0133] Gene silencing was carried out using SMARTpool.TM. si RNA
duplexes (Dharmacon Research, Inc.; Smart Pool siRNA p21 waf1;
Catalog No. M-003471-00-05), which targets CDKN1A. A non-specific
si RNA pool (Dharmacon) was also utilized as a negative
control.
[0134] After preparing siRNA:Lipofectamine 2000 complexes cells
were transfected according to manufacture's instructions
(Invitrogen-Life
[0135] Technologies). Macrophages were infected after four days of
transfection. The results of siRNA for p21 and its effect on HIV
infection are shown in FIGS. 6B-6D.
[0136] P21 specific oligonucleotides (SEQ ID NO. 7 and SEQ NO. 9,
50 nM), but not control oligonucleotide (SEQ ID NO. 14) are found
to inhibit HIV-1 growth in replicate cultures as determined by p24
levels (day 12 shown) (% of positive HIV control, no oligo
treatment) (FIG. 6A).
[0137] Macrophages were also treated with p21 siRNA duplexes (5 nM)
five days prior to HIV infection and the effect on HIV-growth was
determined (% of positive HIV control, no siRNA treatment)
(representative experiment, n=3) (FIG. 6B). The percent of HIV-1
infection was determined comparing the p24 levels in untreated vs
oligo or siRNA treated macrophages. Cells treated with p21 and
negative control siRNA (5 days) were also analyzed by flow
cytometry for CD4 and CCR5 cell surface expression (FIG. 6C).
Nested PCR was employed in order to detect pro-viral DNA on days 1
and 3 after HIV-1.sub.BaL infection in macrophages treated with p21
or control siRNA or negative control si RNA. Control represents
uninfected cells (FIG. 6D).
[0138] The results obtained with the antisense oligonucleotides
were confirmed using gene silencing technology. Macrophages treated
with CDKN1A si RNA duplexes, show a reduction in HIV-1 replication
as determined by p24 ELISA on 14 day supernatants. This is not the
case if macrophages are treated with a non-specific si RNA duplex
control.
Example 4
Analysis of Effect of Vpr on p21 Expression
[0139] As indicated above, the HIV-1 Vpr gene product has been
found to prevent cell proliferation by activating p21 expression
(Chowdhury I. H. at al (2003) "HIV-1 VPR ACTIVATES CELL CYCLE
INHIBITOR P211WAF1/CIP1: A POTENTIAL MECHANISM OF G2/M CELL CYCLE
ARREST," Virol. 305:371-377). Macrophages were treated with Vpr (6
.mu.g/ml) for 3 hr and found to show increased gene transcription
and protein expression for p21 when treated with Vpr (FIG. 7A and
FIG. 7B).
[0140] The expression of p21 and GADPH in these cells was evaluated
using PCR. For such experiments, macrophages are infected with the
wild virus type clone pNLAD8, or pNLAD8 Vpr minus (#1) or
pNLAD8-delta R (#2) R5 macrophage tropic viruses and 12 day
supernatants analyzed by p24 ELISA (FIG. 7C). The total RNA from
cells infected with the indicated viruses was analyzed for p21 and
GAPDH by PCR (FIG. 7D; representative experiment, n=2),
Example 5
Analysis of Temporal Events Associated with the Initial
Virus-Macrophage Encounter
[0141] The present invention demonstrates that HIV-1 infection
promotes successful viral replication by modulating macrophage gene
transcription. In comparison to previous studies, using viral
envelope gp120 (Popik, W. et al. (2000) "EXPLOITATION OF CELLULAR
SIGNALING BY HIV-1: UNWELCOME GUESTS WITH MASTER KEYS THAT SIGNAL
THEIR ENTRY," Virology 276:1-6; Cicala, C. et al. (2002) "HIV
ENVELOPE INDUCES A CASCADE OF CELL SIGNALS IN NON-PROLIFERATING
TARGET CELLS THAT FAVOR VIRUS REPLICATION," Proc. Natl. Acad, Sci.
USA 99:9380-9385; Liu, Q. H. et al. (2000) "HIV-1 GP120 AND
CHEMOKINES ACTIVATE ION CHANNELS IN PRIMARY MACROPHAGES THROUGH
CCR5 AND CXCR4 STIMULATION," Proc. Natl. Acad. Sci. USA
97:4832-4837), one aspect of the invention relates to the finding
that intact, infectious R5 HIV-1 induces a cascade of events
associated with reproducible alteration of gene transcription in
primary macrophage hosts. Consistent with viral binding to CD4 and
CCR5 seven transmembrane G protein receptors, viral initiated
signal transduction induces transcriptional changes. While the
functional significance attributable to each of the 134 genes
upregulated within hours after viral binding is complex, the data
support an initial burst of transcriptional activity followed by a
quiescent phase and a resurgence of new genes associated with viral
replication. In addition to phosphorylation of p38 MAPK, HIV-1
enhanced gene expression of p38 MAPK and downstream mediators, such
as, MAPKAP-2, which may be critical in early post-entry and late
stages of HIV-1 infection (Del Corno, M. et al. (2001) "HIV-1 GP120
AND CHEMOKINE ACTIVATION OF PYK2 AND MITOGEN-ACTIVATED PROTEIN
KINASES IN PRIMARY MACROPHAGES MEDIATED BY CALCIUM-DEPENDENT,
PERTUSSIS TOXIN-INSENSITIVE CHEMOKINE RECEPTOR SIGNALING," Blood
98:2909-2916; Shapiro, L. et al. (1998) "ROLE OF P38
MITOGEN-ACTIVATED PROTEIN KINASE IN HIV TYPE 1 PRODUCTION IN
VITRO," Proc. Natl. Acad, Sci. USA 95:7422-7426 (1998), and p38
MAPK also plays an important role in multiple aspects of the immune
response (Dong, C. et al. (2002) "MAP KINASES IN THE IMMUNE
RESPONSE," Ann. Rev. Immunol 20, 55-72 (2002). MAPK also
contributes to chemokine expression and recruitment of leukocytes,
and inhibition of p38 MAPK reportedly also abrogates gp120-induced
MMP9 in T cells (Misse, D. et al. (2001) "HIV-1 GLYCOPROTEIN 120
INDUCES THE MMP-9 CYTOPATHOGENIC FACTOR PRODUCTION THAT IS
ABOLISHED BY INHIBITION OF THE P38 MITOGEN-ACTIVATED PROTEIN KINASE
SIGNALING PATHWAY," Blood 98:541-547). MMP9, a member of the matrix
metalloproteinase gene family is one of the genes expressed at day
3 after viral infection of macrophages that can facilitate the
migration of HIV-infected monocytes across the vascular endothelium
(Dhawan, S. et al. (1995) "HIV-1 INFECTION ALTERS MONOCYTE
INTERACTIONS WITH HUMAN MICROVASCULAR ENDOTHELIAL CELLS," J Immunol
154:422-432 (1995) and has been detected in the cerebrospinal
fluids of HIV-1 patients (Sporer, B. at al. (1998) "PRESENCE OF
MATRIX METALLOPROTEINASE-9 ACTIVITY IN THE CEREBROSPINAL FLUID OF
HUMAN IMMUNODEFICIENCY VIRUS-INFECTED PATIENTS," J. Infect. Dis.
178:854-857). Recruitment of viral host cells may also occur in
response to increased MCP-1 expression consistent with the results
obtained by other investigators (Mengozzi, M. et al. (1999) "HUMAN
IMMUNODEFICIENCY VIRUS REPLICATION INDUCES MONOCYTE CHEMOTACTIC
PROTEIN-1 IN HUMAN MACROPHAGES AND U937 PROMONOCYTIC CELLS," Blood
93:1851-1857). Enhanced gene transcription for other inflammatory
mediators associated with increased viral replication hi
macrophages and pathophysiology of HIV, including TNF.alpha.,
IP-10, MRP14, IL-8 and LIP (Lane, B. R. et al. (2001)
"INTERLEUKIN-8 STIMULATES HUMAN IMMUNODEFICIENCY VIRUS TYPE 1
REPLICATION AND IS A POTENTIAL NEW TARGET FOR ANTIRETROVIRAL
THERAPY," I Virol 75:8195-202; Strasser, F. at al. (1997) "ELEVATED
SERUM MACROPHAGE INHIBITORY FACTOR-RELATED PROTEIN (MRP) 8/14
LEVELS IN ADVANCED HIV INFECTION AND DURING DISEASE EXACERBATION,"
J Acquir Immune Defic Syndr Hum Retrovirol 16:230-23 8); Kinter, A.
et al. (2000) "CHEMOKINES, CYTOKINES AND HIV: A COMPLEX NETWORK OF
INTERACTIONS THAT INFLUENCE HIV PATHOGENESIS," Immunol Rev
177:88-98; Agostini, C. et al. (2000) "CXC CHEMOKINES IP-10 AND MIG
EXPRESSION AND DIRECT MIGRATION OF PULMONARY CD8+/CXCR3+ T CELLS IN
THE LUNGS OF PATIENTS WITH HIV INFECTION AND T-CELL ALVEOLITIS," Am
J Respir Crit. Care Med 162:1466-1473; Broor, S. et al. (1994)
"STIMULATION OF HIV REPLICATION IN MONONUCLEAR PHAGOCYTES BY
LEUKEMIA INHIBITORY FACTOR," J Acquir Immune Defic Syndr 7:647-654)
were also reproducibly detected. Cell homeostasis and genomic
stability may be aided by glutathione synthetase, heme oxygenase-1
and MLH1 ensuring the survival of the macrophage to allow viral
replication. Furthermore, reduction of lysosomal enzyme activity
can potentially enhance virus entry and infectivity in the host
cell (Fredericksen, B. L. et al. (2002) "INHIBITION OF
ENDOSOMAL/LYSOSOMAL DEGRADATION INCREASES THE INFECTIVITY OF HUMAN
IMMUNODEFICIENCY VIRUS," I Virol 76, 11440-11446).
[0142] Macrophages can co-exist with the virus for a prolonged
time, during which they contribute to the pathogenesis of AIDS,
acting as Viral reservoirs and transmitting HIV-1 to neighboring
cells. Although proapoptotic genes for caspase 3, 4 and g were
upregulated within 3 hours after infection, the antiapoptotic genes
bcl-x, DAD1 and IEX-1L (Wu, M. X. et al. (1998) "IEX-1L, AN
APOPTOSIS INHIBITOR INVOLVED IN NF-KAPPAB-MEDIATED CELL SURVIVAL,"
Science 281:998-1001; Antonsson, B. et al. (2000) "THE BCL-2
PROTEIN FAMILY," Exp Cell Res 256:50-57; Hong, N. A. et al. (2000)
"MICE LACKING DAD1, THE DEFENDER AGAINST APOPTOTIC DEATH-1, EXPRESS
ABNORMAL N-LINKED GLYCOPROTEINS AND UNDERGO INCREASED EMBRYONIC
APOPTOSIS," Dev Biol 220:76-84), were also increased by HIV-1. The
balance between pro and anti-apoptotic genes must favor the
survival of virus-infected macrophages in vitro and in vivo, as a
strategy developed by the virus to prolong the life of the host for
its uninterrupted cycle of replication.
[0143] As indicated above, following the initial HIV-1 induced
burst of gene expression (6-24 hr), little evidence of
transcriptional activity occurred until the onset of viral
replication, when an increase in host molecules was again detected
(day 7-14). The lack of induction of new host molecules during this
interim period may allow the infected cells to escape immune
surveillance while the virus initiates its life cycle to commence
replication. Once ready to replicate, new transcription may be
essential to facilitate the replicative process. For example the
data indicate CDKN1A/p21 as a host molecule critical to viral
replication in macrophages. CDKN1A is a cyclin-dependent kinase
inhibitor induced during G1 cell cycle arrest by p53-dependent
pathway following DNA damage, as well as p53-independent pathways
involving growth factors
[0144] Dotto, G. P. (2000) "p21(WAP1/CIP1): MORE THAN A BREAK TO
THE CELL CYCLE?," Biochim Biophys Acta 1471M43-56; Ogryzko, V. V.
et al. (1997) "WAF1 RETARDS S-PHASE PROGRESSION PRIMARILY BY
INHIBMON OF CYCLIN-DEPENDENT KINASES," Mol Cell Biol 17, 4877-4882
(1997); Zeng, Y. X. et al. (1996) "REGULATION OF P21WAF1/CIP1
EXPRESSION BY P53-INDEPENDENT PATHWAYS," Oncogene 12:1557-1564).
Progressive upregulation of p21 mRNA and protein have also been
associated with maturation of hematopoietic progenitor cells
(Steinman, R. A. et al. (1998) "REGULATION OF P21(WAF1) EXPRESSION
DURING NORMAL MYELOID DIFFERENTIATION," Blood 91:4531-4542), but
its connection with viral replication in macrophages has not been
demonstrated. Increased p21 in skin lesions of human papillomavirus
has been found to be further enhanced by HIV co-infection (Arany,
I. et al. (1997) "p53, WAF1/CIP1 AND MDM2 EXPRESSION IN SKIN
LESIONS ASSOCIATED WITH HUMAN PAPILLOMAVIRUS AND HUMAN
IMMUNODEFICIENCY VIRUS," Anticancer Res 17:1281-1285). An
upregulation in CDKN1A induced in macrophages infected with the
opportunistic bacteria, Mycobacterium avium (Greenwell-Wild, T. et
al. (2002) "MYCOBACTERIUM AVIUM INFECTION AND MODULATION OF HUMAN
MACROPHAGE GENE EXPRESSION," J Immunol 169:6286-6297, (2002) may
also be linked to the increased susceptibility for HIVreplication
reported in these cells (Wahl, S. M. et al. (1998) "MYCOBACTERIUM
AVIUM COMPLEX AUGMENTS MACROPHAGE HIV-1 PRODUCTION AND INCREASES
CCR5 EXPRESSION," Proc Natl Acad Sci USA 95:12574-12579). The
specific role played by CDKN1A in HIV-1 macrophage infection has
not been fully determined, however, it may either directly or
indirectly enhance viral replication. Although originally described
as a cell cycle inhibitor, CDKN1A has more recently been associated
with apoptosis, cytoplasmic regulation of nuclear import, and
transcriptional regulation by its capacity to act as a
transcriptional co-factor/adaptor molecule (Coqueret, O. (2003)
"NEW ROLES FOR P21 AND P27 CELL-CYCLE INHIBITORS: A FUNCTION FOR
EACH CELL COMPARTMENT?," Trends Cell Biol 13:65-70; LaBaer, J. et
al. (1997) "NEW FUNCTIONAL ACTIVITIES FOR THE P21 FAMILY OF CDK
INHIBITORS," Genes Dev 11:847-862). In this regard, HIV-1 Tat is
essential for efficient viral replication and interacts with cAMP
response element binding protein (CREB) and the transcriptional
coactivator p300 (Hottiger, M. O. et al. (1998) "INTERACTION OF
HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 TAT WITH THE TRANSCRIPTIONAL
COACTIVATORS P300 AND CREB BINDING PROTEIN," I Virol 72:8252-8256),
which can be stimulated by the coexpression of CDKN1A through a
novel transcriptional repression domain on p300 (Snowden, A. W. et
al. (2000) "A NOVEL, TRANSCRIPTIONAL REPRESSION DOMAIN MEDIATES
P21(WAF1/CIP1) INDUCTION OF P300 TRANSACTIVATION," Mol Cell Biol
20, 2676-2686), In addition, an increase in TBP-1 could be a
strategy of the virus to ensure efficient regulation of viral
transcription and replication. A causal relationship is thus
established between HIV and induced p21 expression, which appears
to support viral replication in macrophages.
[0145] While the initial enhancement of p21 gene expression likely
represents a downstream consequence of CCR5/G protein signaling,
the rise in gene transcription could be due to either intracellular
or extracellular viral signals. Definition of such factors may
provide a means of altering host involvement in viral infection and
replication kinetics, possibly in conjuction with antiviral
therapy. The presence of p21 in the nucleus has been related to its
cell cycle functions (Dotto, G. P. (2000) "P21(WAF1/CIP1): MORE
THAN A BREAK To THE CELL CYCLE?" Biochim Biophys Acta 1471:M43-56)
and the cytoplasmic localization of this protein has been
implicated in controlling/preventing apoptosis of alveolar
macrophages and during monocytic differentiation (Tomita, K. et al.
(2002) "INCREASED P21(CIP1/WAF1) AND B CELL LYMPHOMA LEUKEMIA-X(L)
EXPRESSION AND REDUCED APOPTOSIS IN ALVEOLAR MACROPHAGES FROM
SMOKERS," Am J Respir Crit. Care Med 166:724-731; Asada, M. et al.
(1999) "APOPTOSIS INHIBITORY ACTIVITY OF CYTOPLASMIC P21
(CIP1/WAF1) IN MONOCYTIC DIFFERENTIATION," Embo J 18:1223-1234).
Increased p21 protein in both nuclear and cytoplasmic compartments
of HIV-1 infected macrophages may both generate a permissive
environment for viral replication and prevent the death of the host
cells. The ability to dramatically suppress HIV-1 replication with
the p21 anti-sense oligonucleotides indicates that CDKN1A is
critical in promoting viral replication. CDDO a synthetic oleanane
triterpenoid with potent differentiating, anti-proliferative and
anti-inflammatory activities (Sub, N. et al. (1999) "A NOVEL
SYNTHETIC OLEANANE TRITERPENOID,
2-CYANO-3,12-DIOXOOLEAN-1,9-DIEN-28-OIC ACID, WITH POTENT
DIFFERENTIATING, ANTIPROLIFERATIVE, AND ANTI-INFLAMMATORY
ACTIVITY," Cancer Res 59:336-341) being developed as a
chemotherapeutic agent for cancer (Stadheim, T. A. et al. (2002)
"THE NOVEL TRITERPENOID 2-CYANO-3,12-DIOXOOLEANA-1,9-DIEN-28-OIC
ACID (CDDO) POTENTLY ENHANCES APOPTOSIS INDUCED BY TUMOR NECROSIS
FACTOR IN HUMAN LEUKEMIA CELLS," J Biol Chem 277:16448-16455
(2002)) may also inhibit HIV-1 via a p21-dependent pathway,
possibly by a post-translational mechanism. CDDO has been recently
identified as a member of a new class of nuclear PPAR.gamma.
ligands (Wang, Y. et al. (2000) "A SYNTHETIC TRITERPENOID,
2-CYANO-3,12-DIOXOOLEANA-1,9-DIEN-28-OIC ACID (CDDO), IS A LIGAND
FOR THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA," Mol
Endocrinol 14:1550-1556), which reportedly reduces p21 protein
expression (Walcino, S. et al. (2001) "PEROXISOME
PROLIFERATOR-ACTIVATED RECEPTOR GAMMA LIGANDS INHIBIT MITOGENIC
INDUCTION OF P21(CIP1) BY MODULATING THE PROTEIN KINASE C.delta.
PATHWAY IN VASCULAR SMOOTH MUSCLE CELLS," J Biol Chem
276:47650-47657). PPARy is a nuclear hormone receptor implicated in
the gene regulation of lipid and glucose metabolism, cellular
differentiation and control of macrophage inflammatory responses
(Bar-Tana, J. (2001) "PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR
GAMMA (PPARGAMMA) ACTIVATION AND ITS CONSEQUENCES IN HUMANS,"
Toxicol Lett 120:9-19; Delerive, P. (2001) "PEROXISOME
PROLIFERATOR-ACTIVATED RECEPTORS IN INFLAMMATION CONTROL," J
Endocrinol 169:453-459). Natural and synthetic agonists of
PPAR.gamma. have been recently shown to inhibit retroviral
replication (Hayes, M. M. et al. (2002) "PEROXISOME
PROLIFERATOR-ACTIVATED RECEPTOR GAMMA AGONISTS INHIBIT HIV-1
REPLICATION IN MACROPHAGES BY TRANSCRIPTIONAL AND
POST-TRANSCRIPTIONAL EFFECTS," J Biol Chem 277:16913-16919) and
although the target was not defined, some studies have implicated
TNF-.alpha. in the PPAR.gamma.-induced suppression of HIV (Skolnik,
P. R. at al. (2002) "STIMULATION OF PEROXISOME
PROLIFERATOR-ACTIVATED RECEPTORS ALPHA AND GAMMA BLOCKS HIV-1
REPLICATION AND TNFALPHA PRODUCTION IN ACUTELY INFECTED PRIMARY
BLOOD CELLS, CHRONICALLY INFECTED Ui CELLS, AND ALVEOLAR
MACROPHAGES FROM HIV-INFECTED SUBJECTS," J Acquir Immune Defic
Syndr 31:1-10). Still unresolved is whether the antiviral effect of
CDDO is mediated through this receptor entirely by its effect on
p21 function by post-translational modification (Scott, M. T. at
al. (2000) "REVERSIBLE PHOSPHORYLATION AT THE C-TERMINAL REGULATORY
DOMAIN OF P21(WAF1/CIP1) MODULATES PROLIFERATING CELL NUCLEAR
ANTIGEN BINDING," J Biol Chem 275:11529-11537), through inhibition
of NP.kappa.B (Straus, D. S. at al. (2000) "15-DEOXY-DELTA
12,14-PROSTAGLANDIN J2 INHIBITS MULTIPLE STEPS IN THE NE-KAPPA B
SIGNALING PATHWAY," Proc Natl Acad Sci USA 97:4844-4849, modulation
of p38 MAPK (Kim, J. Y. et al. (2002) "INVOLVEMENT OF P38
MITOGEN-ACTIVATED PROTEIN KINASE IN THE CELL GROWTH INHIBITION BY
SODIUM ARSENITE," J Cell Physiol 190:29-37 (2002) and/or the
production of cytokines that regulate cellular and viral
components, such as TGF-.beta. (Li, C. Y. et al. (1995) "POTENTIAL
ROLE OF WAF1/CIP1/P21 AS A MEDIATOR OF TOP-BETA CYTOINHIBITORY
EFFECT," J Biol Chem 270:4971-4974; Wahl, S. M, et al. (1991)
"MACROPHAGE-AND ASTROCYTE-DERIVED TRANSFORMING GROWTH FACTOR BETA
As A MEDIATOR OF CENTRAL NERVOUSSYSTEM DYSFUNCTION IN ACQUIRED
IMMUNE DEFICIENCY SYNDROME," J Exp Med 173:981-991). Comparison of
genes upregulated by HIV in T lymphocytes (Corbell, I. et al.
(2001) "TEMPORAL GENE REGULATION DURING HIV-1 INFECTION OF HUMAN
CD4+ T CELLS," Genome Res 11:1198-1204) with those identified in
macrophage hosts also revealed an early increase in genes
associated with cellular defense. However, increased expression of
proapoptotic transcripts, and inhibition of mitochondria and DNA
repair genes are also observed, which could explain the unavoidable
death pathway in HIV-1 infected T cells and survival of macrophage
hosts. The differential gene expression and cell specific
modulation of host protein function as a result of HIV-1 infection
in these cell populations may help better understand the reasons
leading to HIV-induced apoptosis in T cells (Corbeil, J. et al.
(2001) "TEMPORAL GENE REGULATION DURING HIV-1 INFECTION OF HUMAN
CD4+ T CELLS," Genome Res 11:1198-1204; Clark, E. et al. (2000)
"Loss OF G(1)/S CHECKPOINT IN HUMAN IMMUNODEFICIENCY VIRUS TYPE
1-INFECTED CELLS IS ASSOCIATED WITH A LACK OF CYCLIN-DEPENDENT
KINASE INHIBITOR P21/WAF I. 3 Virol 74, 5040-5052), while allowing
the macrophage to sustain a prolonged viral burden.
[0146] Since the macrophage represents a key target for HIV-1
infection and one of the major obstacles in eradicating the virus
even during HAART (Igarashi, T. et al. (2001) "MACROPHAGE ARE THE
PRINCIPAL RESERVOIR AND SUSTAIN HIGH VIRUS LOADS IN RHESUS MACAQUES
AFTER THE DEPLETION OF CD4+ T CELLS BY A HIGHLY PATHOGENIC SIMIAN
IMMUNODEFICIENCY VIRUS/HIV TYPE 1 CHIMERA (SHIV): IMPLICATIONS FOR
HIV-1 INFECTIONS OF HUMANS," Proc Natl Acad Sci USA 98:658-663;
Garbuglia, A. R. et al. (2001) "DYNAMICS OF VIRAL LOAD IN PLASMA
AND HIV DNA IN LYMPHOCYTES DURING HIGHLY ACTIVE ANTIRETROVIRAL
THERAPY (HAART): HIGH VIRAL BURDEN IN MACROPHAGES AFTER 1 YEAR OF
TREATMENT," J Chemother 13:188-194), the above-described data
analyzing the influence of HIV on the macrophage transcriptome
reveals important insights into the pattern of host cell gene
expression underlying viral success in this population. CDKN1A and
other virus-regulated macrophage genes critical for HIV-1
replication provide mechanisms by which to target the macrophage
reservoir and/or serve as prognostic markers of disease
progression.
[0147] Finally, since anti-HIV therapy is limited by the side
effects that have accompanied conventional anti-retroviral drugs,
and the constant emergence of drug-resistant HIV strains, CDDO
provides an important candidate drug to target HIV-1, particularly
in conjunction with additional anti-viral therapy, to prevent or
attenuate the infection of new viral hosts.
[0148] All publications and patent documents mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent document was
specifically and individually indicated to be incorporated by
reference.
[0149] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth.
Sequence CWU 1
1
14115RNAHomo sapiens 1uccgcgccca gcucc 15215RNAHomo sapiens
2uccgcccgca gcucc 1532265DNAHomo sapiens 3gctgccgaag tcagttcctt
gtggagccgg agctgggcgc ggattcgccg aggcaccgag 60gcactcagag gaggtgagag
agcggcggca gacaacaggg gaccccgggc cggcggccca 120gagccgagcc
aagcgtgccc gcgtgtgtcc ctgcgtgtcc gcgaggatgc gtgttcgcgg
180gtgtgtgctg cgttcacagg tgtttctgcg gcaggcgcca tgtcagaacc
ggctggggat 240gtccgtcaga acccatgcgg cagcaaggcc tgccgccgcc
tcttcggccc agtggacagc 300gagcagctga gccgcgactg tgatgcgcta
atggcgggct gcatccagga ggcccgtgag 360cgatggaact tcgactttgt
caccgagaca ccactggagg gtgacttcgc ctgggagcgt 420gtgcggggcc
ttggcctgcc caagctctac cttcccacgg ggccccggcg aggccgggat
480gaattgggag gaggcaggcg gcctggcacc tcacctgctc tgctgcaggg
gacagcagag 540gaagaccatg tggacctgtc actgtcttgt acccttgtgc
ctcgctcagg ggagcaggct 600gaagggtccc caggtggacc tggagactct
cagggtcgaa aacggcggca gaccagcatg 660acagatttct accactccaa
acgccggctg atcttctcca agaggaagcc ctaatccgcc 720cacaggaagc
ctgcagtcct ggaagcgcga gggcctcaaa ggcccgctct acatcttctg
780ccttagtctc agtttgtgtg tcttaattat tatttgtgtt ttaatttaaa
cacctcctca 840tgtacatacc ctggccgccc cctgcccccc agcctctggc
attagaatta tttaaacaaa 900aactaggcgg ttgaatgaga ggttcctaag
agtgctgggc atttttattt tatgaaatac 960tatttaaagc ctcctcatcc
cgtgttctcc ttttcctctc tcccggaggt tgggtgggcc 1020ggcttcatgc
cagctacttc ctcctcccca cttgtccgct gggtggtacc ctctggaggg
1080gtgtggctcc ttcccatcgc tgtcacaggc ggttatgaaa ttcaccccct
ttcctggaca 1140ctcagacctg aattcttttt catttgagaa gtaaacagat
ggcactttga aggggcctca 1200ccgagtgggg gcatcatcaa aaactttgga
gtcccctcac ctcctctaag gttgggcagg 1260gtgaccctga agtgagcaca
gcctagggct gagctgggga cctggtaccc tcctggctct 1320tgataccccc
ctctgtcttg tgaaggcagg gggaaggtgg ggtcctggag cagaccaccc
1380cgcctgccct catggcccct ctgacctgca ctggggagcc cgtctcagtg
ttgagccttt 1440tccctctttg gctcccctgt accttttgag gagccccagc
tacccttctt ctccagctgg 1500gctctgcaat tcccctctgc tgctgtccct
cccccttgtc ctttcccttc agtaccctct 1560cagctccagg tggctctgag
gtgcctgtcc cacccccacc cccagctcaa tggactggaa 1620ggggaaggga
cacacaagaa gaagggcacc ctagttctac ctcaggcagc tcaagcagcg
1680accgccccct cctctagctg tgggggtgag ggtcccatgt ggtggcacag
gcccccttga 1740gtggggttat ctctgtgtta ggggtatatg atgggggagt
agatctttct aggagggaga 1800cactggcccc tcaaatcgtc cagcgacctt
cctcatccac cccatccctc cccagttcat 1860tgcactttga ttagcagcgg
aacaaggagt cagacatttt aagatggtgg cagtagaggc 1920tatggacagg
gcatgccacg tgggctcata tggggctggg agtagttgtc tttcctggca
1980ctaacgttga gcccctggag gcactgaagt gcttagtgta cttggagtat
tggggtctga 2040ccccaaacac cttccagctc ctgtaacata ctggcctgga
ctgttttctc tcggctcccc 2100atgtgtcctg gttcccgttt ctccacctag
actgtaaacc tctcgagggc agggaccaca 2160ccctgtactg ttctgtgtct
ttcacagctc ctcccacaat gctgaatata cagcaggtgc 2220tcaataaatg
attcttagtg actttaaaaa aaaaaaaaaa aaaaa 226542265DNAHomo sapiens
4tttttttttt tttttttttt aaagtcacta agaatcattt attgagcacc tgctgtatat
60tcagcattgt gggaggagct gtgaaagaca cagaacagta cagggtgtgg tccctgccct
120cgagaggttt acagtctagg tggagaaacg ggaaccagga cacatgggga
gccgagagaa 180aacagtccag gccagtatgt tacaggagct ggaaggtgtt
tggggtcaga ccccaatact 240ccaagtacac taagcacttc agtgcctcca
ggggctcaac gttagtgcca ggaaagacaa 300ctactcccag ccccatatga
gcccacgtgg catgccctgt ccatagcctc tactgccacc 360atcttaaaat
gtctgactcc ttgttccgct gctaatcaaa gtgcaatgaa ctggggaggg
420atggggtgga tgaggaaggt cgctggacga tttgaggggc cagtgtctcc
ctcctagaaa 480gatctactcc cccatcatat acccctaaca cagagataac
cccactcaag ggggcctgtg 540ccaccacatg ggaccctcac ccccacagct
agaggagggg gcggtcgctg cttgagctgc 600ctgaggtaga actagggtgc
ccttcttctt gtgtgtccct tccccttcca gtccattgag 660ctgggggtgg
gggtgggaca ggcacctcag agccacctgg agctgagagg gtactgaagg
720gaaaggacaa gggggaggga cagcagcaga ggggaattgc agagcccagc
tggagaagaa 780gggtagctgg ggctcctcaa aaggtacagg ggagccaaag
agggaaaagg ctcaacactg 840agacgggctc cccagtgcag gtcagagggg
ccatgagggc aggcggggtg gtctgctcca 900ggaccccacc ttccccctgc
cttcacaaga cagagggggg tatcaagagc caggagggta 960ccaggtcccc
agctcagccc taggctgtgc tcacttcagg gtcaccctgc ccaaccttag
1020aggaggtgag gggactccaa agtttttgat gatgccccca ctcggtgagg
ccccttcaaa 1080gtgccatctg tttacttctc aaatgaaaaa gaattcaggt
ctgagtgtcc aggaaagggg 1140gtgaatttca taaccgcctg tgacagcgat
gggaaggagc cacacccctc cagagggtac 1200cacccagcgg acaagtgggg
aggaggaagt agctggcatg aagccggccc acccaacctc 1260cgggagagag
gaaaaggaga acacgggatg aggaggcttt aaatagtatt tcataaaata
1320aaaatgccca gcactcttag gaacctctca ttcaaccgcc tagtttttgt
ttaaataatt 1380ctaatgccag aggctggggg gcagggggcg gccagggtat
gtacatgagg aggtgtttaa 1440attaaaacac aaataataat taagacacac
aaactgagac taaggcagaa gatgtagagc 1500gggcctttga ggccctcgcg
cttccaggac tgcaggcttc ctgtgggcgg attagggctt 1560cctcttggag
aagatcagcc ggcgtttgga gtggtagaaa tctgtcatgc tggtctgccg
1620ccgttttcga ccctgagagt ctccaggtcc acctggggac ccttcagcct
gctcccctga 1680gcgaggcaca agggtacaag acagtgacag gtccacatgg
tcttcctctg ctgtcccctg 1740cagcagagca ggtgaggtgc caggccgcct
gcctcctccc aactcatccc ggcctcgccg 1800gggccccgtg ggaaggtaga
gcttgggcag gccaaggccc cgcacacgct cccaggcgaa 1860gtcaccctcc
agtggtgtct cggtgacaaa gtcgaagttc catcgctcac gggcctcctg
1920gatgcagccc gccattagcg catcacagtc gcggctcagc tgctcgctgt
ccactgggcc 1980gaagaggcgg cggcaggcct tgctgccgca tgggttctga
cggacatccc cagccggttc 2040tgacatggcg cctgccgcag aaacacctgt
gaacgcagca cacacccgcg aacacgcatc 2100ctcgcggaca cgcagggaca
cacgcgggca cgcttggctc ggctctgggc cgccggcccg 2160gggtcccctg
ttgtctgccg ccgctctctc acctcctctg agtgcctcgg tgcctcggcg
2220aatccgcgcc cagctccggc tccacaagga actgacttcg gcagc
226551909DNAMus musculus 5gagccgagag gtgtgagccg ccgcggtgtc
agagtctagg ggaattggag tcaggcgcag 60atccacagcg atatccagac attcagagcc
acaggcacca tgtccaatcc tggtgatgtc 120cgacctgttc cgcacaggag
caaagtgtgc cgttgtctct tcggtcccgt ggacagtgag 180cagttgcgcc
gtgattgcga tgcgctcatg gcgggctgtc tccaggaggc ccgagaacgg
240tggaactttg acttcgtcac ggagacgccg ctggagggca acttcgtctg
ggagcgcgtt 300cggagcctag ggctgcccaa ggtctacctg agccctgggt
cccgcagccg tgacgacctg 360ggaggggaca agaggcccag tacttcctct
gccctgctgc aggggccagc tccggaggac 420cacgtggcct tgtcgctgtc
ttgcactctg gtgtctgagc ggcctgaaga ttccccgggt 480gggcccggaa
catctcaggg ccgaaaacgg aggcagacca gcctgacaga tttctatcac
540tccaagcgca gattggtctt ctgcaagaga aaaccctgaa gtgcccacgg
gagccccgcc 600ctcttctgct gtgggtcagg aggcctcttc cccatcttcg
gccttagccc tcactctgtg 660tgtcttaatt attatttgtg ttttaattta
aacgtctcct gtatatacgc tgcctgccct 720ctcccagtct ccaaacttaa
agttatttaa aaaaagaaca aaacaaaaca aaaaaaaacc 780aaaacaaaac
aaacctaaat tagtaggacg gtagggccct tagtgtgggg gatttctatt
840atgtagatta ttattattta agcccctccc aacccaagct ctgtgtttcc
tataccggag 900gaacagtcct actgatatca acccatctgc atccgtttca
cccaaccccc ctccccccat 960tccctgcctg gttccttgcc acttcttacc
tgggggtgat cctcagacct gaatagcact 1020ttggaaaaat gagtaggact
ttggggtctc cttgtcacct ctaaggccag ctaggatgac 1080agtgaagcag
tcacagccta gaacagggat ggcagttagg actcaaccgt aatatcccga
1140ctcttgacat tgctcagacc tgtgaagaca ggaatggtcc ccactctgga
tcccctttgc 1200cactcctggg gagcccacct ctcctgtggg tctctgccag
ctgcccctct attttggagg 1260gttaatctgg tgatctgctg ctcttttccc
ccaccccata cttccccttc tgcaggtcgg 1320caggaggcat atctaggcac
ttgccccaca gctcagtgga ctggaaggga atgtatatgc 1380agggtacact
aagtgggatt ccctggtctt accttaggca gctccagtgg caaccccctg
1440cattgtgggt ctagggtggg tccttggtgg tgagacaggc ctcccagagc
attctatggt 1500gtgtggtggt gggggtgggc ttatctggga tggggacccc
agttggggtt ctcagtgact 1560tctcccattt cttagtagca gttgtacaag
gagccaggcc aagatggtgt cttgggggct 1620aagggagctc acaggacact
gagcaatggc tgatcctttc tcagtgttga ataccgtggg 1680tgtcaaagca
cttagtgggt ctgactccag ccccaaacat ccctgtttct gtaacatcct
1740ggtctggact gtctaccctt agcccgcacc ccaagaacat gtattgtggc
tccctccctg 1800tctccactca gattgtaagc gtctcacgag aagggacagc
accctgcatt gtcccgagtc 1860ctcacacccg accccaaagc tggtgctcaa
taaatacttc tcgatgatt 190961909DNAMus musculus 6aatcatcgag
aagtatttat tgagcaccag ctttggggtc gggtgtgagg actcgggaca 60atgcagggtg
ctgtcccttc tcgtgagacg cttacaatct gagtggagac agggagggag
120ccacaataca tgttcttggg gtgcgggcta agggtagaca gtccagacca
ggatgttaca 180gaaacaggga tgtttggggc tggagtcaga cccactaagt
gctttgacac ccacggtatt 240caacactgag aaaggatcag ccattgctca
gtgtcctgtg agctccctta gcccccaaga 300caccatcttg gcctggctcc
ttgtacaact gctactaaga aatgggagaa gtcactgaga 360accccaactg
gggtccccat cccagataag cccaccccca ccaccacaca ccatagaatg
420ctctgggagg cctgtctcac caccaaggac ccaccctaga cccacaatgc
agggggttgc 480cactggagct gcctaaggta agaccaggga atcccactta
gtgtaccctg catatacatt 540cccttccagt ccactgagct gtggggcaag
tgcctagata tgcctcctgc cgacctgcag 600aaggggaagt atggggtggg
ggaaaagagc agcagatcac cagattaacc ctccaaaata 660gaggggcagc
tggcagagac ccacaggaga ggtgggctcc ccaggagtgg caaaggggat
720ccagagtggg gaccattcct gtcttcacag gtctgagcaa tgtcaagagt
cgggatatta 780cggttgagtc ctaactgcca tccctgttct aggctgtgac
tgcttcactg tcatcctagc 840tggccttaga ggtgacaagg agaccccaaa
gtcctactca tttttccaaa gtgctattca 900ggtctgagga tcacccccag
gtaagaagtg gcaaggaacc aggcagggaa tggggggagg 960ggggttgggt
gaaacggatg cagatgggtt gatatcagta ggactgttcc tccggtatag
1020gaaacacaga gcttgggttg ggaggggctt aaataataat aatctacata
atagaaatcc 1080cccacactaa gggccctacc gtcctactaa tttaggtttg
ttttgttttg gttttttttt 1140gttttgtttt gttctttttt taaataactt
taagtttgga gactgggaga gggcaggcag 1200cgtatataca ggagacgttt
aaattaaaac acaaataata attaagacac acagagtgag 1260ggctaaggcc
gaagatgggg aagaggcctc ctgacccaca gcagaagagg gcggggctcc
1320cgtgggcact tcagggtttt ctcttgcaga agaccaatct gcgcttggag
tgatagaaat 1380ctgtcaggct ggtctgcctc cgttttcggc cctgagatgt
tccgggccca cccggggaat 1440cttcaggccg ctcagacacc agagtgcaag
acagcgacaa ggccacgtgg tcctccggag 1500ctggcccctg cagcagggca
gaggaagtac tgggcctctt gtcccctccc aggtcgtcac 1560ggctgcggga
cccagggctc aggtagacct tgggcagccc taggctccga acgcgctccc
1620agacgaagtt gccctccagc ggcgtctccg tgacgaagtc aaagttccac
cgttctcggg 1680cctcctggag acagcccgcc atgagcgcat cgcaatcacg
gcgcaactgc tcactgtcca 1740cgggaccgaa gagacaacgg cacactttgc
tcctgtgcgg aacaggtcgg acatcaccac 1800gattggtcat ggtgcctgtg
gctctgaatg tctggatatc gctgtggatc tgcgcctgac 1860tccaattccc
ctagactctg acaccgcggc ggctcacacc tctcggctc 1909720DNAMus musculus
7tgtcaggctg gtctgcctcc 20820DNAHomo sapiens 8tgtcatgctg gtctgccgcc
20920DNAMus musculus 9acatcaccag gattggacat 201023DNAHomo sapiens
10acatccccag ccggttctga cat 2311202DNAHomo sapiens 11accatcccct
tcctcacctg aaaacaggca gcccaaggac aaaatagcca ccagcctctt 60ctatgccaga
gctcaacatg ttgggacatg ttcctgacgg ccagaaagcc aatcagagcc
120acagcctgct gcccaagcat gttcctggga agcaggcagc atagggatgg
agggaggctc 180agcctggggg aacaagagtg cc 20212202DNAHomo sapiens
12ggcactcttg ttcccccagg ctgagcctcc ctccatccct atgctgcctg cttcccagga
60acatgcttgg gcagcaggct gtggctctga ttggctttct ggccgtcagg aacatgtccc
120aacatgttga gctctggcat agaagaggct ggtggctatt ttgtccttgg
gctgcctgtt 180ttcaggtgag gaaggggatg gt 20213160PRTHomo sapiens
13Met Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro Cys Gly Ser Lys 1
5 10 15 Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gln Leu Ser
Arg 20 25 30 Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gln Glu Ala
Arg Glu Arg 35 40 45 Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu
Glu Gly Asp Phe Ala 50 55 60 Trp Glu Arg Val Arg Gly Leu Gly Leu
Pro Lys Leu Tyr Leu Pro Thr 65 70 75 80Gly Pro Arg Arg Gly Arg Asp
Glu Leu Gly Gly Gly Arg Arg Pro Gly 85 90 95 Thr Ser Pro Ala Leu
Leu Gln Gly Thr Ala Glu Glu Asp His Val Asp 100 105 110 Leu Ser Leu
Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gln Ala Glu 115 120 125 Gly
Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln 130 135
140 Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser
145 150 155 1601418DNAMus musculus 14tggatccgac atgtcaga 18
* * * * *
References