U.S. patent application number 10/166372 was filed with the patent office on 2003-01-09 for methods and compositions for modulating the interaction between the apj receptor and the hiv virus.
This patent application is currently assigned to Trustees of the University of Pennsylvania. Invention is credited to Doms, Robert W., Faulds, Daryl, Hesselgesser, Joseph E., Horuk, Richard, Mitrovic, Branislava, Zhou, Yiqing.
Application Number | 20030008391 10/166372 |
Document ID | / |
Family ID | 22528559 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030008391 |
Kind Code |
A1 |
Doms, Robert W. ; et
al. |
January 9, 2003 |
Methods and compositions for modulating the interaction between the
APJ receptor and the HIV virus
Abstract
The orphan seven transmembrane domain receptor, APJ, can
function as a coreceptor for cellular infection by the HIV virus.
The establishment of cell lines that coexpress CD4 and APJ provide
valuable tools for continuing research on HIV infection and the
development of anti-HIV therapeutics.
Inventors: |
Doms, Robert W.; (Berwyn,
PA) ; Faulds, Daryl; (Mill Valley, CA) ;
Hesselgesser, Joseph E.; (San Francisco, CA) ; Horuk,
Richard; (Belmont, CA) ; Mitrovic, Branislava;
(Walnut Creek, CA) ; Zhou, Yiqing; (El Sobrante,
CA) |
Correspondence
Address: |
MILLEN, WHITE, ZELANO & BRANIGAN, P.C.
2200 CLARENDON BLVD.
SUITE 1400
ARLINGTON
VA
22201
US
|
Assignee: |
Trustees of the University of
Pennsylvania
Philadelphia
PA
|
Family ID: |
22528559 |
Appl. No.: |
10/166372 |
Filed: |
June 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10166372 |
Jun 11, 2002 |
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09149045 |
Sep 8, 1998 |
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6475718 |
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Current U.S.
Class: |
435/345 ;
435/325; 435/5; 530/300 |
Current CPC
Class: |
A01K 2217/05 20130101;
A61P 31/18 20180101; C07K 14/7158 20130101; A61K 38/00
20130101 |
Class at
Publication: |
435/345 ;
435/325; 435/5; 530/300 |
International
Class: |
C12Q 001/70; C07K
002/00; C07K 004/00; C07K 005/00; C07K 007/00; C07K 014/00; C07K
016/00; C07K 017/00; A61K 038/00; C12N 005/00; C12N 005/02; C12N
005/06; C12N 005/16 |
Claims
What is claimed is:
1. A recombinant eukaryotic cell transformed with a polynucleotide
encoding an APJ polypeptide or a polynucleotide encoding a CD4
polypeptide, wherein the cell coexpresses APJ and CD4
polypeptides.
2. A recombinant eukaryotic cell transformed with a polynucleotide
encoding an APJ polypeptide and a polynucleotide encoding a CD4
polypeptide, wherein the cell coexpresses APJ and CD4
polypeptides.
3. A recombinant eukaryotic cell according to claim 1, wherein the
cell is stably transformed.
4. A recombinant eukaryotic cell according to claim 2, wherein the
cell is stably transformed both polynucleotides.
5. The cell as in any of claims 1-4, wherein the cell is a human
cell.
6. The cell as in any of claims 1-4, wherein the cell is a
non-human cell.
7. An antibody which specifically binds to an extracellular domain
of APJ, wherein the antibody inhibits HIV infection of a target
cell that coexpresses APJ and CD4 polypeptides.
8. An antibody which specifically binds to an extracellular domain
of APJ, wherein the antibody inhibits membrane fusion between a
first cell coexpressing APJ and CD4 polypeptides and second cell
expressing an HIV env protein.
9. An antibody according to claim 7 or 8, wherein the antibody is a
monoclonal antibody.
10. An antibody according to claim 9, wherein the antibody
recognizes an epitope comprising an amino acid sequence
corresponding to a portion of the first extracellular domain of
APJ.
11. An antibody according to claim 10, wherein the amino acid
sequence corresponding to a portion of the first extracellular
domain of APJ comprises the amino acid sequence Asn-Tyr-Tyr-Gly
(SEQ ID NO: 3).
12. An antibody according to claim 9, wherein the antibody
recognizes an epitope comprising an amino acid sequence
corresponding to a portion of the second extracellular domain of
APJ.
13. A substantially purified peptide fragment of APJ, wherein the
peptide inhibits HIV infection of a target cell that coexpresses
APJ and CD4 polypeptides.
14. A substantially purified peptide fragment of APJ, wherein the
peptide inhibits cell fusion between a first cell coexpressing APJ
and CD4 polypeptides and a second cell expressing an HIV env
protein.
15. A substantially purified peptide fragment of APJ according to
claim 13 or 14, wherein the peptide fragment comprises an amino
acid sequence corresponding to a portion of the first extracellular
domain of APJ.
16. A substantially purified peptide fragment of APJ according to
claim 15, wherein the amino acid sequence corresponding to a
portion of the first extracellular domain of APJ comprises the
amino acid sequence Asn-Tyr-Tyr-Gly (SEQ ID NO: 3).
17. A substantially purified peptide fragment of APJ according to
claim 13 or 14, wherein the peptide fragment comprises an amino
acid sequence corresponding to a portion of the second
extracellular domain of APJ.
18. A method for identifying a compound that modulates interaction
between an HIV virus and an APJ receptor comprising incubating a
first cell line which coexpresses CD4 and APJ polypeptides with a
second cell line which expresses an env protein under conditions
which promote cell fusion, in the presence and absence of a test
compound, and determining whether the presence of the test compound
inhibits cell fusion between the first cell line and the second
cell line.
19. A method according to claim 18, wherein cell fusion is
determined by detection of a reporter molecule.
20. A method according to claim 19, wherein the reporter molecule
is selected from the group consisting of a radioisotope, a
fluorescent compound, a bioluminescent compound, a chemiluminescent
compound, a metal chelator, or an enzyme.
21. A method according to claim 19 wherein the reporter molecule is
B-galactosidase or luciferase.
22. A method for identifying a compound that modulates interaction
between an HIV virus and an APJ receptor comprising incubating a
cell line which expresses CD4 and APJ polypeptides with a test
virus carrying an env protein, in the presence and absence of a
test compound, and determining whether the presence of the test
compound inhibits infection of the cell line by the test virus.
23. A method according to claim 22, wherein infection is determined
by detection of a reporter molecule.
24. A method according to claim 23, wherein the reporter molecule
is selected from the group consisting of a radioisotope, a
fluorescent compound, a bioluminescent compound, a chemiluminescent
compound, a metal chelator, or an enzyme.
25. A method according to claim 23, wherein the reporter molecule
is B-galactosidase or luciferase.
26. A method of inhibiting HIV infection of a target cell
expressing an APJ and CD4 polypeptides comprising contacting the
target cell with an effective amount of a APJ binding or blocking
agent.
27. The method of claim 26, wherein the agent is an anti-APJ
antibody or epitope binding fragment thereof.
28. The method of claim 27, wherein the antibody is a monoclonal
antibody or a polyclonal antibody.
29. The method of claim 26, wherein said contacting is accomplished
by in vivo administration to a subject.
30. The method of claim 26, wherein the agent is a peptide fragment
of APJ.
31. A method of treating a subject having an HIV-related disorder
associated with expression of APJ comprising administering an agent
that suppresses APJ to the subject.
32. The method of claim 31, wherein the agent is an anti-APJ
antibody.
33. The method of claim 31, wherein the agent is an antisense
polynucleotide that hybridizes to an APJ polynucleotide.
34. The method of claim 31, wherein the agent is introduced into a
cell using a carrier.
35. The method of claim 34, wherein the carrier is a vector.
36. A method of treating a subject having or at risk of having an
HIV infection or related disorder, comprising administering a
therapeutically effective amount of an anti-APJ antibody or a
peptide fragment to the subject.
37. A method according to claim 36, wherein the subject is a
fetus.
38. A transgenic non-human animal having a phenotype characterized
by expression of APJ polypeptide and CD4 polypeptide otherwise not
naturally occurring in the animal, wherein the phenotype is
conferred by a transgene contained in the somatic cells and germ
cells of the animal, and wherein the transgene comprises a
polynucleotide encoding an APJ polypeptide and a polynucleotide
encoding a CD4 polypeptide.
Description
BACKGROUND OF THE INVENTION
[0001] The entry of HIV-1 into cells involves binding of the viral
envelope (env) protein to CD4 followed by interaction with one of
several coreceptors (reviewed in E. A. Berger, 1997, AIDS,
11:S3-S16; Broder et al., 1997, J. Leukocyte Biol., 62:20-29; Doms
et al., 1997, Virology, 235:279-190; and Moore et al., 1997, Curr.
Opinion Immunol., 9:551-562). Binding of the env protein to the
appropriate coreceptor is thought to trigger conformational changes
in env that mediate fusion between the viral membrane and the host
cell membrane. The CCR5 and CXCR4 chemokine receptors have been
identified as major HIV-1 coreceptors in that all HIV-1 strains
examined to date use one or both of these molecules as second
receptors. CCR5 supports infection by R5 (M-tropic) virus strains,
while CXCR4 supports infection by X4 (T-tropic) virus isolates
(Alkhatib et al., 1996, Science, 272:1955-1958; Berger et al.,
1998, Nature, 391:240; Choe et al., 1996, Cell, 85:1135-1148; Deng
et al., 1996, Nature, 381:661-666; Doranz et al., 1996, Cell,
85:1149-1158; Dragic et al., 1996, Nature, 381:667-673; and Feng et
al., 1996, Science, 272:872-877). R5-X4 (dual-tropic) viral env
proteins can, in conjunction with CD4, use either CCR5 or CXCR4 for
cellular entry. The differential utilization of CCR5 and CXCR4 by
HIV strains, coupled with their expression patterns in CD4 positive
cells largely explains viral tropism at the level of entry.
[0002] In addition to CCR5 and CXCR4, a number of other chemokine
and orphan seven transmembrane domain receptors have been shown to
function as coreceptors for one or more virus strains in vitro,
including CCR2b, CCR3, CCR8, CX3CR1, GPR1, GPR 15, STRL33, US28,
and ChemR23 (Choe et al., 1996, Cell 85:1135-1148; Deng et al.,
1997, Nature 388:296-300; Doranz et al., 1996, Cell 85:1149-1158;
Farzan et al., 1997, J. Exp. Med. 186:405-411; Liao et al., 1997,
J. Exp. Med. 195:2015-2023; Pleskoffet al., 1997, Science
276:1874-1878; Reeves et al., 1997, Virology 231:120-134; Rucker et
al., 1997, J. Virol. 71:8999-9007). In general, these alternative
coreceptors support virus infection less efficiently than either
CCR5 or CXCR4. However, use of alternative coreceptors may help
explain certain facets of HIV-1 tropism and pathogenesis in vivo.
For example, neurologic disease is a serious and relatively
frequent consequence of HIV-1 infection, with microglia being the
primary targets of virus infection in the central nervous system
(Bagasra et al., 1996, AIDS, 10:573-585; Sharer et al., 1992, J.
Neuropath. Exp. Neurol., 51:3-11; Wiley et al., 1986, Proc. Natl.
Acad. Sci., USA, 83:7089-7093). Microglia express both CCR3 and
CCR5 and it has been suggested that utilization of CCR3 by a virus
strain may correlate with neurotropism (He et al., 1997, Nature,
385:645-649).
[0003] The identification of additional coreceptors for the HIV
virus would provide an important tool for investigating and
controlling HIV infection.
SUMMARY OF THE INVENTION
[0004] In one aspect of the invention, the invention relates to a
recombinant eukaryotic cell transformed with a polynucleotide
encoding an APJ polypeptide and/or a polynucleotide encoding a CD4
polypeptide, wherein the cell coexpresses APJ and CD4
polypeptides.
[0005] The invention also relates to an antibody which specifically
binds to an extracellular domain of APJ, wherein the antibody
inhibits HIV infection of a target cell that coexpresses APJ and
CD4 polypeptides or wherein the antibody inhibits membrane fusion
between a first cell coexpressing APJ and CD4 polypeptides and a
second cell expressing an HIV env protein.
[0006] The invention also relates to a substantially purified
peptide fragment of APJ, wherein the peptide inhibits HIV infection
of a target cell that coexpresses APJ and CD4 polypeptides or
wherein the peptide inhibits cell fusion between a first cell
coexpressing APJ and CD4 polypeptides and a second cell expressing
an HIV env protein.
[0007] In another aspect of the invention the invention relates to
methods for identifying compounds that modulate the interaction
between an HIV virus and an APJ receptor.
[0008] The invention also relates to a method of inhibiting HIV
infection of a target cell expressing an APJ and CD4 polypeptides
comprising contacting the target cell with an effective amount of
an APJ binding or blocking agent.
[0009] The invention also relates to a method of treating a subject
having or at risk of having an HIV infection or related disorder,
comprising administering a therapeutically effective amount of an
anti-APJ antibody to the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a graph showing cell-cell fusion mediated by HIV-1
or HIV-2 env proteins. All of the indicated env proteins are
derived from HIV-1, with the exception of HIV-2/ST which was
derived from HIV-2. QT6 cells expressing CD4, the indicated
coreceptor, and luciferase under the control of the T7 promoter
were mixed with cells expressing T7 polymerase and the indicated
HIV-1 or HIV-2 env protein. The degree of cell-cell fusion was
determined 8 hours post-mixing by measuring luciferase activity.
The results were normalized by setting the extent of fusion
obtained when CD4 and either CCR5 (for R5 env proteins) or CXCR4
(for X4 env proteins) were coexpressed to 100%. The extent of
fusion obtained with the major HIV-1 coreceptors was generally 40
to 100 times above background levels. Error bars here and in
subsequent figures represent the standard error of the mean derived
from multiple independent experiments.
[0011] FIG. 2 is a graph showing cell-cell fusion mediated by SIV
env proteins. Cell-cell fusion was determined as in FIG. 1, but
using SIV rather than HIV env proteins. The results were normalized
by setting the extent of fusion obtained when CD4 and CCR5 were
coexpressed to 100%. The extent of fusion obtained with the
different env proteins was generally 40 to 100 times above
background levels (defined as CD4 alone).
[0012] FIG. 3 is a graph showing pseudotype virus infection. HEK
293 cells expressing CD4 and the indicated coreceptor were infected
with luciferase virus pseudotypes bearing the indicated HIV or SIV
env protein, and luciferase activity was determined 2-3 days after
infection.
[0013] FIG. 4 is a graph showing viral infection of HEK 293 cells
that express CD4 and the indicated coreceptor and that also contain
a plasmid encoding luciferase under the control of the HIV-1 LTR.
The cells were infected with live HIV-1 IIIB (HxB) or HIV-1 89.6.
Values were normalized by setting the extent of infection obtained
with either CCR5 (R5 env proteins) or CXCR4 (X4 env proteins) to
100%.
[0014] FIG. 5 is an image of a Southern blot showing the entry of
virus into cells expressing CD4 and APJ. QT6 cells stably
expressing CD4 and transiently expressing the desired coreceptor
were infected with DNAase-treated, cell-free virus. Viral specific
LTR DNA sequences were detected 2 days after infection by PCR
amplification, followed by resolution of the products on a 2%
agarose gel and detection of sequences using a labeled probe.
[0015] FIG. 6 is an image of a Northern blot showing the expression
of APJ in human brain tissue. Membranes containing poly A.sup.+ RNA
from various human brain regions were obtained from Clontech and
were incubated with a labeled cDNA probe specific for APJ
overnight. The membranes were then exposed to a Fuji Imaging plate
for 4 hours. The following tissues were examined: Panel (A): (1)
Amygdala; (2) Caudate nucleus; (3) Corpus callosum; (4)
Hippocampus; (5) Whole brain; (6) Substantia nigra; (7) Subthalamic
nucleus; (8) Thalamus; Panel (B): (1) Cerebellum; (2) Cerebral
cortex; (3) Medulla; (4) Spinal cord; (5) Occipital lobe; (6)
Frontal lobe; (7) Temporal lobe; (8) Putamen.
[0016] FIG. 7 is an image of a Northern blot showing the expression
of APJ in human peripheral tissue. Membranes containing poly
A.sup.+ RNA from various human tissues were obtained from Clontech
and were incubated with a labeled cDNA probe specific for APJ
overnight. The membranes were then exposed to a Fuji Imaging plate
for 4 hours. The following tissues were examined: (1) Spleen; (2)
Thymus; (3) Prostate; (4) Testis; (5) Ovary; (6) Small intestine;
(7) Colonic mucosa; (8) Total peripheral blood lymphocytes.
[0017] FIG. 8 is an image of a stained agarose gel showing the
expression of APJ in primary cells and in T-cell lines. RNA from
the indicated cells was used in one-tube RT-PCR reactions and 10
.mu.l of each 25 .mu.l reaction was run out on a 2% agarose gel.
The size of the predicted APJ band is 481 base pairs. Both plasmid
DNA and RNA isolated from U87-APJ stably transfected cells are
included as positive controls and water was used as template for a
negative control.
[0018] FIG. 9 is the nucleotide and deduced amino acid sequence for
human APJ (O'Dowd et al., 1993, Gene 136:355-360) (SEQ ID NO: 1).
The positions of the seven trans-membrane regions are as follows:
transmembrane region 1 (TM 1) corresponds to amino acids 26-54;
transmembrane region 2 (TM2) corresponds to amino acids 66-90;
transmembrane region 3 (TM3) corresponds to amino acids 104-125;
transmembrane region 4 (TM4) corresponds to amino acids 144-167;
transmembrane region 5 (TM5) corresponds to amino acids 199-225;
transmembrane region 6 (TM6) corresponds to amino acids 246-271;
transmembrane region 7 (TM7) corresponds to amino acids 285-312.
Extracellular portions of the APJ polypeptide are located in the
amino terminal segment before transmembrane domain 1 (e.g. amino
acids 1-25), between transmembrane domains 2 and 3 (e.g. amino
acids 91-103), between transmembrane domains 4 and 5 (e.g. amino
acids 168-198), and between transmembrane domains 6 and 7 (e.g.
amino acids 272-284).
DETAILED DESCRIPTION OF THE INVENTION
[0019] In accordance with this invention, it has been discovered
that the orphan seven transmembrane domain receptor, APJ (O'Dowd et
al., 1993, Gene 136:355-360), functions as an efficient coreceptor
for a number of HIV-1 and SIV strains. APJ serves as a very
efficient coreceptor for some X4 (T-tropic) and R5-X4 (dual tropic)
virus strains, while two R5 (M-tropic) isolates use APJ less
efficiently. APJ also served as a coreceptor for several SIV
strains.
[0020] Also in accordance with this invention, the widespread
expression of APJ in the human central nervous system has been
discovered. The efficient use of APJ by a number of virus strains,
coupled with the expression of APJ in the central nervous system,
indicates that utilization of this receptor may be important in HIV
neuropathogenesis.
[0021] Also in accordance with this invention, the expression of
APJ in a CD4 positive T-cell line, C8166, has been discovered.
[0022] In one embodiment, the present invention relates to
recombinant cell lines, the cells of which co-express APJ and CD4
polypeptides, and which contain an exogenous polynucleotide that
encodes either an APJ polypeptide or a CD4 polypeptide. The present
invention also relates to recombinant cell lines, the cells of
which co-express APJ and CD4 polypeptides, and which contain an
exogenous polynucleotide that encodes an APJ polypeptide and an
exogenous polynucleotide that encodes a CD4 polypeptide. As used
herein, a "CD4 polypeptide" means a mammalian CD4 polypeptide,
preferably a human or a simian CD4 polypeptide, or a biologically
active fragment thereof. As used herein, an "APJ polypeptide" means
a mammalian APJ polypeptide, preferably a human or a simian APJ
polypeptide, or a biologically active fragment thereof.
Biologically active, as used herein, refers to polypeptides having
an ability to specifically interact with an HIV or SIV virus and to
polypeptides having at least one epitope for an antibody
immunoreactive with an APJ or a CD4 polypeptide.
[0023] The invention relates not only to naturally-occurring APJ
and CD4 polypeptides, but also to mutant APJ and CD4 polypeptides.
For example, changes in the amino acid sequence of APJ are
contemplated in the present invention. APJ can be altered by
changing the DNA encoding the protein. Preferably, only
conservative amino acid sequence alterations are undertaken, using
amino acids that have the same or similar properties. Illustrative
amino acid substitutions include the following changes: alanine to
serine; arginine to lysine; asparagine to glutamine or histidine;
aspartate to glutamate; cysteine to serine; glutamine to
asparagine; glutamate to aspartate; glycine to proline; histidine
to asparagine or glutamine; isoleucine to leucine or valine;
leucine to valine or isoleucine; lysine to arginine, glutamine, or
glutamate; methionine to leucine or isoleucine; phenylalanine to
tyrosine, leucine or methionine; serine to threonine; threonine to
serine; tryptophan to tyrosine; tyrosine to tryptophan or
phenylalanine; valine to isoleucine or leucine.
[0024] Polynucleotide sequences of the invention include DNA, cDNA
and RNA sequences which encode APJ or CD4 polypeptides. Such
polynucleotides include naturally occurring, synthetic, and
intentionally manipulated polynucleotides. For example, portions of
the mRNA sequence may be altered due to alternate RNA splicing
patterns or the use of alternate promoters for RNA transcription.
As another example, APJ and CD4 polynucleotides may be subjected to
site-directed mutagenesis. The polynucleotide sequence for APJ and
CD4 also includes antisense sequences. The invention also includes
a polynucleotide encoding an APJ polypeptide having biological
activity or a CD4 polypeptide having biological activity.
[0025] Suitable cell types include, but are not limited to, cells
of the following types: NIH 3T3 (Murine), Mv 1 lu (mink), BS-C-1
(African Green Monkey), human embryonic kidney (HEK) 293 cells
(ATCC CRL 1573), and quail QT6 cells. Such cells are described, for
example, in the Cell Line Catalog of the American Type Culture
Collection (ATCC). These cells can be stably transformed or
transiently transformed by a method known to the skilled artisan.
See, for example, Ausubel, et al., Introduction of DNA Into
Mammalian Cells, in Current Protocols in Molecular Biology,
sections 9.5.1-9.5.6 (John Wiley & Sons, Inc. 1995). "Stable"
transformation in the context of the invention means that the cells
are immortal to the extent of having gone through at least 50
divisions.
[0026] Exogenous APJ or CD4 polynucleotides can be expressed using
inducible or constitutive regulatory elements for expression.
Commonly used constitutive or inducible promoters, for example, are
known in the art. For example promoters derived from the genome of
mammalian cells (e.g. metallothionein promoter) or from mammalian
viruses (e.g., the retrovirus long terminal repeat, the adenovirus
late promoter, the vaccinia virus 7.5K promoter) may be used.
Promoters produced by recombinant DNA or synthetic techniques may
also be used to provide for transcription of the exogenous APJ or
CD4 polynucleotides.
[0027] The desired protein encoding sequence and operably linked
promoter may be introduced into a recipient cell either as a
non-replicating DNA (or RNA) molecule, which may either be a linear
molecule or, more preferably, a closed covalent circular molecule.
Since such molecules are incapable of autonomous replication, the
expression of the desired molecule may occur through the transient
expression of the introduced sequence. Alternatively, permanent
expression may occur through the integration of the introduced
sequence into the host chromosome.
[0028] In a preferred embodiment, the introduced sequence will be
incorporated into a plasmid or viral vector capable of autonomous
replication in the recipient host. A wide variety of vectors may be
employed for this purpose. Factors of importance in selecting a
particular plasmid or viral vector include the following: the ease
with which recipient cells that contain the vector may be
recognized and selected from those recipient cells which do not
contain the vector; the number of copies of the vector which are
desired in a particular host; and whether it is desirable to
"shuttle" the vector between host cells of different species.
[0029] Several possible vector systems are available for
expression. One class of vectors utilize DNA elements which provide
autonomously replicating extra-chromosomal plasmids, derived from
animal viruses such as bovine papilloma virus, polyoma virus,
adenovirus, or SV40 virus. A second class of vectors include
vaccinia virus expression vectors. A third class of vectors relies
upon the integration of the desired gene sequences into the host
chromosome. Cells which have stably integrated the introduced DNA
into their chromosomes may be selected by also introducing one or
more markers (e.g., an exogenous gene) which allow selection of
host cells which contain the expression vector. The marker may
provide, for example, prototrophy to an auxotrophic host or biocide
resistance, e.g., antibiotic resistance or heavy metal resistance,
such as copper resistance. The selectable marker gene can either be
directly linked to the DNA sequences to be expressed, or introduced
into the same cell by co-transformation. Additional elements may
also be needed for optimal synthesis of mRNA. These elements may
include splice signals, as well as transcription promoters,
enhancers, and termination signals. The cDNA expression vectors
incorporating such elements include those described by Okayama, H.,
Mol. Cell. Biol., 3:280 (1983), and others.
[0030] Once the vector or DNA sequence containing the construct has
been prepared for expression, the DNA construct may be introduced
(transformed) into an appropriate host. Various techniques which
may be employed include, for example, protoplast fusion, calcium
phosphate precipitation, electroporation, microinjection, delivery
via liposomes, viral infection, or other conventional
techniques.
[0031] In another embodiment, the present invention relates to
transgenic animals having cells that coexpress human CD4 and APJ
polypeptides. Such transgenic animals represent a model system for
the study of HIV infection and the development of more effective
anti-HIV therapeutics.
[0032] The term "animal", as used herein, denotes all mammalian
species except human. It also includes an individual animal in all
stages of development, including embryonic and fetal stages. Farm
animals (pigs, goats, sheep, cows, horses, rabbits, etc.), rodents
(such as mice), and domestic pets (for example, cats and dogs) are
included within the scope of the present invention.
[0033] A "transgenic" animal is any animal containing cells that
bear genetic information received, directly or indirectly, by
deliberate genetic manipulation at the subcellular level, such as
by microinjection or by infection with a recombinant virus.
"Transgenic" in the present context does not encompass classical
crossbreeding or in vitro fertilization, but rather denotes animals
in which one or more cells receive a recombinant DNA molecule.
Although it is highly preferred that this molecule be integrated
within the animal's chromosomes, the present invention also
contemplates the use of extrachromosomally replicating DNA
sequences, such as might be engineered into yeast artificial
chromosomes.
[0034] The term "transgenic animal" also includes a "germ cell
line" transgenic animal. A germ cell line transgenic animal is a
transgenic animal in which the genetic information has been taken
up and incorporated into a germ cell line, therefore conferring the
ability to transfer the information to offspring. If such offspring
in fact possess some or all of that information they are also
considered transgenic animals.
[0035] It is highly preferred that the transgenic animals of the
present invention be produced by introducing into single cell
embryos a polynucleotide encoding an APJ polypeptide and or a
polynucleotide encoding a CD4 polypeptide, in a manner such that
these polynucleotides are stably integrated into the DNA of germ
line cells of the mature animal and inherited in normal mendelian
fashion. Advances in technologies for embryo micromanipulation now
permit introduction of exogenous polynucleotides into fertilized
mammalian ova. For instance, totipotent or pluripotent stem cells
can be transformed by microinjection, calcium phosphate mediated
precipitation, liposome fusion, retroviral infection or other
means, the transformed cells are then introduced into the embryo,
and the embryo then develops into a transgenic animal. In a
preferred-method, developing embryos are infected with a retrovirus
containing the desired polynucleotide, and transgenic animals
produced from the infected embryo.
[0036] In a most preferred method, the appropriate polynucleotides
are coinjected into the pronucleus or cytoplasm of embryos,
preferably at the single cell stage, and the embryos allowed to
develop into mature transgenic animals. These techniques are well
known. For example, reviews of standard laboratory procedures for
microinjection of exogenous DNAs into mammalian (mouse, pig,
rabbit, sheep, goat, cow) fertilized ova include: Hogan et al.,
Manipulating The Mouse Embryo (Cold Spring Harbor Press 1986);
Krimpenfort et al., Bio/Technology 9:86 (1991); Palmiter et al.,
Cell 41:343 (1985); Kraemer et al., Genetic Manipulation of The
Early Mammalian Embryo (Cold Spring Harbor Laboratory Press 1985);
Hammer et al., Nature 315:680 (1985); Purcel et al., Science
244:1281 (1986); Wagner et al., U.S. Pat. No. 5,175,385;
Krimpenfort et al., U.S. Pat. No. 5,175,384, the respective
contents of which are herein incorporated by reference.
[0037] The polynucleotide that encodes APJ or CD4 can be fused in
proper reading frame under the transcriptional and translational
control of a vector to produce a genetic construct that is then
amplified, for example, by preparation in a bacterial vector,
according to conventional methods. See, for example, the standard
work: Sambrook et al., Molecular Cloning: a Laboratory Manual (Cold
Spring Harbor Press 1989), the contents of which are herein
incorporated by reference. The amplified construct is thereafter
excised from the vector and purified for use in producing
transgenic animals.
[0038] Production of transgenic animals containing the gene for
human CD4 have been described. See Snyder et al., Mol. Reprod.
& Devel. 40:419-428 (1995); Dunn et al., J. Gen. Virology
76:1327-1336 (1995), the contents of which are incorporated by
reference.
[0039] In another embodiment, the present invention relates to
antibodies that bind APJ and that inhibit HIV entry into a
CD4-positive target cell or that inhibit cell-cell fusion between a
first cell type that expresses CD4 and APJ polypeptides and a
second cell type that expresses the env protein. As used herein, an
env protein means any env protein derived from an HIV virus, either
HIV-1 or HIV-2, or derived from an SIV virus. Expression of an env
protein by a cell will typically result in the expression of the
gp120 moiety of the env protein on the cell surface. Antibodies of
the invention may also inhibit gp120 binding to APJ. Such
antibodies could represent research and diagnostic tools in the
study of HIV infection and the development of more effective
anti-HIV therapeutics. In addition, pharmaceutical compositions
comprising antibodies against APJ may represent effective anti-HIV
therapeutics.
[0040] An antibody suitable for blocking env-mediated cell-cell
fusion, HIV entry into a CD4 positive cell, or gp120 binding to APJ
is specific for at least one portion of an extracellular region of
the APJ polypeptide, e.g. the first extracellular region (amino
acids 1-25), the second extracellular region (amino acids 91-103),
the third extracellular region (amino acids 168-198), or the fourth
extracellular region (amino acids 272-284), as shown in FIG. 9.
Preferred antibodies are those which recognize an epitope
comprising a portion of either the first extracellular region or
the second extracellular region. Particularly preferred antibodies
are those which recognize an epitope comprising the Asn-Tyr-Tyr-Gly
(SEQ ID NO: 3) amino acid sequence contained within the first
extracellular region.
[0041] Anti-APJ antibodies of the invention include polyclonal
antibodies, monoclonal antibodies, and fragments of polyclonal and
monoclonal antibodies. The preparation of polyclonal antibodies is
well-known to those skilled in the art. See, for example, Green et
al., Production of Polyclonal Antisera, in Immunochemical Protocols
(Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al.,
Production of Polyclonal Antisera in Rabbits, Rats, Mice and
Hamsters, in Current Protocols in Immunology, section 2.4.1 (1992),
which are hereby incorporated by reference.
[0042] The preparation of monoclonal antibodies likewise is
conventional. See, for example, Kohler & Milstein, Nature
256:495 (1975); Coligan et al., Current Protocols in Immunology,
sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory
Manual, page 726 (Cold Spring Harbor Pub. 1988), which are hereby
incorporated by reference. Briefly, monoclonal antibodies can be
obtained by injecting mice with a composition comprising an
antigen, verifying the presence of antibody production by removing
a serum sample, removing the spleen to obtain B lymphocytes, fusing
the B lymphocytes with myeloma cells to produce hybridomas, cloning
the hybridomas, selecting positive clones that produce antibodies
to the antigen, and isolating the antibodies from the hybridoma
cultures. Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatograhy, antigen affinity
purification and ion-exchange chromatography. See, e.g., Coligan et
al., Current Protocols in Immunology, sections 2.7.1-2.7.12 and
sections 2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin
G (IgG), in Methods in Molecular Biology, Vol. 10, pages 79-104
(Humana Press 1992).
[0043] Methods of in vitro and in vivo multiplication of monoclonal
antibodies are well-known to those skilled in the art.
Multiplication in vitro may be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium,
optionally replenished by a mammalian serum such as fetal calf
serum or trace elements and growth-sustaining supplements such as
normal mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages. Production in vitro provides relatively pure antibody
preparations and allows scale-up to yield large amounts of the
desired antibodies. Large scale hybridoma cultivation can be
carried out by homogenous suspension culture in an airlift reactor,
in a continuous stirrer reactor, or in immobilized or entrapped
cell culture. Multiplication in vivo may be carried out by
injecting cell clones into mammals histocompatible with the parent
cells, e.g., osyngeneic mice, to cause growth of antibody-producing
tumors. Optionally, the animals are primed with a hydrocarbon,
especially oils such as pristane (tetramethylpentadecane) prior to
injection. After one to three weeks, the desired monoclonal
antibody is recovered from the body fluid of the animal.
[0044] Therapeutic applications for anti-APJ antibodies disclosed
herein are also part of the present invention. For example,
antibodies of the present invention may also be derived from
subhuman primate antibody. General techniques for raising
therapeutically useful antibodies in baboons can be found, for
example, in Goldenberg et al., International Patent Publication WO
91/11465 (1991) and Losman et al., Int. J. Cancer 46:310 (1990),
which are hereby incorporated by reference.
[0045] Alternatively, a therapeutically or diagnostically useful
anti-APJ antibody may be derived from a "humanized" monoclonal
antibody. Humanized monoclonal antibodies are produced by
transferring mouse complementary determining regions from heavy and
light variable chains of the mouse immunoglobulin into a human
variable domain, and then substituting human residues in the
framework regions of the murine counterparts. The use of antibody
components derived from humanized monoclonal antibodies obviates
potential problems associated with the immunogenicity of murine
constant regions. General techniques for cloning murine
immunoglobulin variable domains are described, for example, by
Orlandi et al., Proc. Natl. Acad. Sci. USA 86:3833 (1989), which is
hereby incorporated in its entirety by reference. Techniques for
producing humanized monoclonal antibodies are described, for
example, by Jones et al., Nature 321:522 (1986); Riechmann et al.,
Nature 332:323 (1988); Verhoyen et al., Science 239:1534 (1988);
Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Sandhu,
Crit. Rev. Biotech. 12:437 (1992); and Singer et al., J. Immunol.
150:2844 (1933), which are hereby incorporated by reference.
[0046] Antibodies of the invention may also be derived from human
antibody fragments isolated from a combinatorial immunoglobulin
library. See, for example, Barbas et al., Methods: A Companion to
Methods in Enzymology, Vol. 2, page 119 (1991); Winter et al., Ann.
Rev. Immunol. 12:433 (1994), which are hereby incorporated by
reference. Cloning and expression vectors that are useful for
producing a human immunoglobulin phage library can be obtained, for
example, from Stratagene Cloning Systems (La Jolla, Calif.).
[0047] In addition, antibodies of the present invention may be
derived from a human monoclonal antibody. Such antibodies are
obtained from transgenic mice that have been "engineered" to
produce specific human antibodies in response to an antigenic
challenge. In this technique, elements of the human heavy and light
chain loci are introduced into strains of mice derived from
embryonic cell lines that contain targeted disruptions of the
endogenous heavy and light chain loci. The transgenic mice can
synthesize human antibodies specific for human antigens, and the
mice can be used to produce human antibody-secreting hybridomas.
Methods for obtaining human antibodies from transgenic mice are
described by Green et al., Nature Genet. 7:13 (1994); Lonberg et
al., Nature 368:856 (1994); and Taylor et al., Int. Immunol. 6:579
(1994), which are hereby incorporated by reference.
[0048] Antibody fragments of the present invention can be prepared
by proteolytic hydrolysis of the antibody or by expression in E.
coli of DNA encoding the fragment. Antibody fragments can be
obtained by pepsin or papain digestion of whole antibodies by
conventional methods. For example, antibody fragments can be
produced by enzymatic cleavage of antibodies with pepsin to provide
a 5S fragment denoted F(ab').sub.2. This fragment can be further
cleaved using a thiol reducing agent, and optionally a blocking
group for the sulfhydryl groups resulting from cleavage of
disulfide linkages, to produce 3.5S Fab' monovalent fragments and
an Fc fragment directly. These methods are described, for example,
by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and
references contained therein. These patents are hereby incorporated
in their entireties by reference. See also Nisonhoff et al., Arch.
Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959);
Edelman et al., Methods in Enzymology, Vol. 1, page 422 (Academic
Press 1967); and Coligan et al., Current Protocols in Immunology,
sections 2.8.1-2.8.10 and 2.10.1-2.10.4.
[0049] Other methods of cleaving antibodies, such as separation of
heavy and light chains to form monovalent light-heavy chain
fragments, further cleavage of fragments, or other enzymatic,
chemical, or genetic techniques may also be used, so long as the
fragments bind to the antigen that is recognized by the intact
antibody.
[0050] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association may be noncovalent, as
described in Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659
(1972). Alternatively, the variable-chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. See, e.g., Sandhu, Crit. Rev. Biotech. 12:437
(1992). Preferably, the Fv fragments comrpise V.sub.H and V.sub.L
chains connected by a peptide linker. These single-chain antigen
binding proteins (sFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains connected by an oligonucleotide. The structural gene is
inserted into an expression vector, which is subsequently
introduced into a host cell such as E. coli. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing sFvs are
described, for example, by Whitlow et al., Methods: A Companion to
Methods in Enzymology, Vol. 2, page 97 (1991); Bird et al., Science
242:423-426 (1988); Ladner et al., U.S. Pat. No. 4,946,778; Pack et
al., Bio/Technology 11:1271-77 (1993); and Sandhu, Crit. Rev.
Biotech. 12:437 (1992).
[0051] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
(`minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick et al., Methods: A Companion to
Methods in Enzymology, Vol. 2, page 106 (1991).
[0052] Antibodies that bind to the CXCR4 chemokine receptor,
another HIV coreceptor, have been shown to block fusion of HTV
strains that use CXCR4 receptor for infection (Feng, et al.,
Science 272:872 (1996); Endres, et al., Cell 87:745 (1996)).
[0053] In another embodiment, the present invention relates to "APJ
variants". An APJ variant, as used herein, means a molecule that
simulates at least part of the structure of APJ and that inhibits
HIV entry into a target cell expressing CD4 and APJ polypeptides or
that inhibits cell-cell fusion between a first cell type that
expresses CD4 and APJ polypeptides and a second cell type that
expresses the env protein. The env protein of certain HIV isolates
may participate in HIV infectivity by binding to APJ at the cell
surface. While not wishing to be bound by a particular theory of
the invention, the inventors believe that APJ variants may
interfere in HIV infectivity by competing with APJ in binding to
env.
[0054] In one embodiment, the present invention relates to APJ
variants that are peptides and peptide derivatives that have fewer
amino acid residues than APJ. Such peptides and peptide derivatives
could represent research and diagnostic tools in the study of HIV
infection and the development of more effective anti-HIV
therapeutics. Peptides and peptide derivatives of APJ, according to
the present invention, include those which correspond to the
extracellular regions of APJ, e.g. the first extracellular region
(amino acids 1-25), the second extracellular region (amino acids
91-103), the third extracellular region (amino acids 168-198), or
the fourth extracellular region (amino acids 272-284), as shown in
FIG. 9. Peptides that correspond to the extracellular loops of
another HIV coreceptor, the CCR5 coreceptor, have previously been
shown to inhibit fusion between cells expressing the HIV-1 env and
murine cells co-expressing CD4 and CCR5 (Combadiere et al.,
PCT/US97/09586, publication number WO 97/45543). Preferred peptides
and peptide derivatives are those which correspond to a portion of
either the first extracellular region or the second extracellular
region. Particularly preferred peptides or peptide derivatives are
those which comprise the Asn-Tyr-Tyr-Gly (SEQ ID NO: 3) amino acid
sequence contained within the first extracellular region.
[0055] APJ variants useful for the present invention comprise
analogs, homologs, muteins and mimetics of APJ. The variants can be
generated directly from APJ itself by chemical modification, by
proteolytic enzyme digestion, or by combinations thereof.
Additionally, genetic engineering techniques, as well as methods of
synthesizing polypeptides directly from amino acid sequences, can
also be employed.
[0056] Peptides of the invention can be synthesized by such
commonly used methods as t-BOC or FMOC protection of alpha-amino
groups. Both methods involve stepwise syntheses whereby a single
amino acid is added at each step starting from the C terminus of
the peptide (See, Coligan, et al., Current Protocols in Immunology,
Wiley Interscience, 1991, Unit 9). Peptides of the invention can
also be synthesized by the well known solid phase peptide synthesis
methods (described in Merrifield, J. Am. Chem. Soc., 85:2149 (1962)
and Stewart and Young, Solid Phase Peptide Synthesis (Freeman, San
Francisco, 1969) pp. 27-62), using a copoly(styrene-divinylbenzene)
containing 0.1-1.0 mMol amines/g polymer. On completion of chemical
synthesis, the peptides can be deprotected and cleaved from the
polymer by treatment with liquid HF-10% anisole for about 1/4-1
hours at 0.degree. C. After evaporation of the reagents, the
peptides are extracted from the polymer with 1% acetic acid
solution which is then lyophilized to yield the crude material. The
crude material can normally be purified by standard techniques such
as, for example, by gel filtration on Sephadex G-15 using 5% acetic
acid as a solvent. Lyophilization of appropriate fractions of the
column will yield the homogeneous peptide or peptide derivatives,
which can then be characterized by such standard techniques as
amino acid analysis, thin layer chromatography, high performance
liquid chromatography, ultraviolet absorption spectroscopy, molar
rotation, solubility, and quantitated by solid phase Edman
degradation analysis.
[0057] Alternatively, peptides can be produced by recombinant
methods which are well known to those of skill in the art.
[0058] The term "substantially purified", as used herein, refers to
a molecule, such as a peptide that is substantially free of other
proteins, lipids, carbohydrates, nucleic acids, and other
biological materials with which it is naturally associated. For
example, a substantially pure molecule, such as a polypeptide, can
be at least 60%, by dry weight, the molecule of interest. One
skilled in the art can purify APJ peptides using standard protein
purification methods and the purity of the polypeptides can be
determined using standard methods including, e.g., polyacrylamide
gel electrophoresis (e.g., SDS-PAGE), column chromatography (e.g.,
high performance liquid chromatography (HPLC), and amino-terminal
amino acid sequence analysis.
[0059] Non-peptide compounds that mimic the binding and function of
APJ ("mimetics") can be produced by the approach outlined in
Saragovi et al., Science 253:792-95 (1991). Mimetics are molecules
which mimic elements of protein secondary structure. See, for
example, Johnson et al., Peptide Turn Mimetics, in Biotechnology
and Pharmacy, Pezzuto et al., Eds., (Chapman and Hall, New York
1993). The underlying rationale behind the use of peptide mimetics
is that the peptide backbone of proteins exists chiefly to orient
amino acid side chains in such a way as to facilitate molecular
interactions. For the purposes of the present invention,
appropriate mimetics can be considered to be the equivalent of APJ
itself.
[0060] Longer peptides can be produced by the "native chemical"
ligation technique which links together peptides (Dawson, et al.,
Science 266:776 (1994)). Variants can be created by recombinant
techniques employing genomic or cDNA cloning methods. Site-specific
and region-directed mutagenesis techniques can be employed. See
Current Protocols in Molecular Biology, Vol. 1, ch. 8 (Ausubel et
al. eds., J. Wiley & Sons 1989 & Supp. 1990-93); Protein
Engineering (Oxender & Fox eds., A. Liss, Inc. 1987). In
addition, linker-scanning and PCR-mediated techniques can be
employed for mutagenesis. See PCR Technology (Erlich ed., Stockton
Press 1989); Current Protocols in Molecular Biology, Vols. 1 &
2, supra. Protein sequencing, structure and modeling approaches for
use with any of the above techniques are disclosed in Protein
Engineering, loc. cit., and Current Protocols in Molecular Biology,
Vol. 1 & 2, supra.
[0061] If the compounds described above are employed, the skilled
artisan can routinely ensure that such compounds are amenable for
use with the present invention in view of the cell-cell fusion
assay systems and the infectivity assay systems described
herein.
[0062] The invention also includes various pharmaceutical
compositions that inhibit HIV entry into a target cell expressing
CD4 and APJ polypeptides. The pharmaceutical compositions according
to the invention are prepared by bringing an APJ variant or an
antibody against APJ, according to the present invention, into a
form suitable for administration to a subject using carriers,
excipients and additives or auxiliaries. Frequently used carriers
or auxiliaries include magnesium carbonate, titanium dioxide,
lactose, mannitol and other sugars, talc, milk, protein, gelatin,
starch, vitamins, cellulose and its derivatives, animal and
vegetable oils, polyethylene glycols and solvents, such as sterile
water, alcohols, glycerol and polyhydric alcohols. Intravenous
vehicles include fluid and nutrient replenishers. Preservatives
include antimicrobial agents, anti-oxidants, chelating agents and
inert gases. Other pharmaceutically acceptable carriers include
aqueous solutions, non toxic excipients, including salts,
preservatives, buffers and the like, as described for instance, in
Remington's Pharmaceutical Sciences, 15th ed. Easton: Mack
Publishing Co., 1405-1412, 1461-1487 (1975), the contents of which
are hereby incorporated by reference. The pH and exact
concentration of the various components of the pharmaceutical
composition are adjusted according to routine skills in the art.
See Goodman and Gilman's The Pharmacological Basis for Therapeutics
(7th ed.).
[0063] In another embodiment, the invention relates to a method of
inhibiting HIV entry into a target cell. This method involves
administering to a subject a therapeutically effective dose of a
pharmaceutical composition containing the compounds of the present
invention and a pharmaceutically acceptable carrier.
"Administering" the pharmaceutical composition of the present
invention may be accomplished by any means known to the skilled
artisan. By "subject" is meant any mammal, preferably a human. For
example, neuropathy has been observed in the brains of newborn
infants that are born to HIV-1 seropositive mothers (Kolson et al.,
1998, Adv. Virus Res. 50:1-47). Therefore, a particularly preferred
method is a method of treating a fetal subject having or at risk of
having an HIV infection by the administration of an anti-APJ
antibody or an APJ peptide fragment. The anti-APJ antibody and the
APJ peptide fragment are preferably administered to the fetal
subject via administration to the mother.
[0064] The pharmaceutical compositions are preferably prepared and
administered in dose units. Solid dose units are tablets, capsules
and suppositories. For treatment of a subject, depending on
activity of the compound, manner of administration, nature and
severity of the disorder, age and body weight of the patient,
different daily doses are necessary. Under certain circumstances,
however, higher or lower daily doses may be appropriate. The
administration of the daily dose can be carried out both by single
administration in the form of an individual dose unit or else
several smaller dose units and also by multiple administration of
subdivided doses at specific intervals.
[0065] The pharmaceutical compositions according to the invention
are in general administered topically, intravenously, orally, or
parenterally or as implants, but even rectal use is possible in
principle. Suitable solid or liquid pharmaceutical preparation
forms are, for example, granules, powders, tablets, coated tablets,
(micro)capsules, suppositories, syrups, emulsions, suspensions,
creams, aerosols, drops or injectable solution in ampule form and
also preparations with protracted release of active compounds, in
whose preparation excipients and additives and/or auxiliaries such
as disintegrants, binders, coating agents, swelling agents,
lubricants, flavorings, sweeteners or solubilizers are customarily
used as described above. The pharmaceutical compositions are
suitable for use in a variety of drug delivery systems. For a brief
review of present methods for drug delivery, see Langer, Science
249:1527-1533 (1990), which is incorporated herein by
reference.
[0066] The pharmaceutical compositions according to the invention
may be administered locally or systemically. By "therapeutically
effective dose" is meant the quantity of a compound according to
the invention necessary to prevent, to cure or at least partially
arrest the symptoms of the disease and its complications. Amounts
effective for this use will, of course, depend on the severity of
the disease and the weight and general state of the subject.
Typically, dosages used in vitro may provide useful guidance in the
amounts useful for in situ administration of the pharmaceutical
composition, and animal models may be used to determine effective
dosages for treatment of particular disorders. Various
considerations are described, e.g., in Gilman et al. (eds.) (1990)
Goodman and Gilman's: The Pharmacological Bases of Therapeutics,
8th ed., Pergamon Press; and Remington's Pharmaceutical Sciences,
17th ed. (1990), Mack Publishing Co., Easton, Pa., each of which is
herein incorporated by reference.
[0067] Another preferred embodiment of this invention is in the
diagnosis of susceptibility to HIV infection. Nucleotide sequences
encoding the APJ receptor and antibodies to the APJ receptor can be
particularly useful for diagnosis of susceptibility to infection
where higher levels of the receptors indicate an increased risk for
HIV infection. For example, higher levels of the APJ receptor in
tissues of the central nervous system may indicate an increased
risk of neuropathogenesis associated with HIV infection.
[0068] Using any suitable technique known in the art, such as
Northern blotting, quantitative PCR, etc., the nucleotide sequences
of the receptor or fragments thereof can be used to measure levels
of APJ RNA expression.
[0069] Alternatively, antibodies to APJ can be used in standard
techniques such as Western blotting to detect the presence of cells
expressing the APJ receptor and in standard techniques, e.g. FACS
or ELISA, to quantify the level of expression. For any biological
tissue sample, a level of APJ expression that is greater than a
reference level is indicative of increased susceptibility to HIV
infection. A reference level may be established by surveying a
large population of individuals.
[0070] In a preferred embodiments, the invention relates to methods
for screening a compound ("test compound") for anti-HIV
pharmacological activity.
[0071] In one embodiment, a cell fusion assay is used to screen for
a compound with anti-HIV pharmacological activity. In the cell
fusion assay,. one type of eukaryotic cell that coexpresses APJ and
CD4 polypeptides is incubated with a second type of eukaryotic cell
that expresses an HIV envelope protein ("env"). Fusion between the
two different cell types is then monitored. The test compound is
added to the incubation solution before or after mixing of the
cells and its effect on the fusion rate of cells is determined by
any number of means, including through morphological observation or
through the use of an indicator system in conjunction with the cell
fusion assay. Indicator systems useful in conjunction with a cell
fusion assay can be any combination of elements wherein a
detectable signal is produced when a first component in a first
cell is brought into contact with a second component in a second
cell by cell-cell fusion. For example, the first component may be a
gene encoding a polymerase such as T7 polymerase and the second
component may be a gene encoding a reporter molecule which is under
the control of the T7 promoter, such as a luciferase gene. For
example, a system that results in the production of an active
.beta.-galactosidase reporter molecule upon cell fusion is also
contemplated.
[0072] In another embodiment, an infectivity assay is used to
screen for a compound with anti-HIV pharmacological activity. In an
infectivity assay, a target eukaryotic cell that expresses APJ and
CD4 polypeptides is incubated with a test virus expressing an HIV
env protein. Infection of the target cell with the test virus is
then monitored. The test compound is added to the incubation
solution before or after mixing of the target cells with the test
virus, and the effect of the compound on the infection rate of
target cells is determined by any number of means. The test virus
may be a reporter virus in which the env protein is pseudotyped
onto a reporter virus background. Alternatively, the test virus may
be an intact HIV virus. Infectivity is generally monitored by use
of an indicator system in conjunction with the infectivity assay.
Any number of indicator systems may be used, and indicator systems
which produce a detectable signal upon infection are preferred. For
example, the reporter virus may be constructed with a gene encoding
a reporter molecule such as luciferase and .beta.-galactosidase,
which is expressed when the reporter virus infects the target cell.
As another example, the target cell may contain a gene encoding a
reporter molecule under the control of the LTR promoter, thereby
resulting in expression of the reporter molecule upon infection of
the target cell with the HIV virus. Alternatively, viral infection
may be monitored through the use of a PCR to detect viral sequences
within the infected cell.
[0073] The cell fusion assay and the HIV infectivity assay can be
used to determine the functional ability of APJ to confer
env-mediated fusion competence to a diverse range of CD4 positive
cell types (either recombinantly produced or naturally occurring),
including but not limited to NIH 3T3 (murine), BS-C-1 (African
green monkey), HEK 293 (human), Mv1Lu (mink), U-87 MG glioblastoma,
SCL1, and QT6 (quail). HIV strains that may be used in conjunction
with the assays, or as sources for env protein genes to be used in
conjunction with the assays, include M-tropic, T-tropic and dual
tropic strains. For example, the 89.6 dual tropic strain, the JRFL
M-tropic strain, and the IIIB T-tropic HIV-1 strains may be
employed (Matthews et al., 1986, Proc. Natl. Acad. Sci. USA
83:9709-9713; Collman et al., 1992, J. Virol. 66:7517-7521; Gartner
et al., 1986, Science 233:215-219). Additionally, selected primary
isolates may also be employed.
[0074] Variations of drug screening methods are known to the
artisan of average skill in this field. Consequently, the cell
fusion assay and the HIV infectivity assay can be used in a wide
variety of formats to exploit the properties of the APJ receptor to
screen for drugs that are effective against HIV.
[0075] Another embodiment of the invention employs the use of
antisense technology as a specific and potent means of inhibiting
HIV infection of cells that contain APJ, for example, by decreasing
the amount of APJ expression in a cell. Antisense polynucleotides
in context of the present invention includes both short sequences
of DNA known as oligonucleotides of usually 10-50 bases in length
as well as longer sequences of DNA that may exceed the length of
the APJ gene sequence itself. Antisense polynucleotides useful for
the present invention are complementary to specific regions of a
corresponding target mRNA. Hybridization of antisense
polynucleotides to their target transcripts can be highly specific
as a result of complementary base pairing. The capability of
antisense polynucleotides to hybridize is affected by such
parameters as length, chemical modification and secondary structure
of the transcript which can influence polynucleotide access to the
target site. See Stein et al., Cancer Research 48:2659 (1988). An
antisense polynucleotide can be introduced to a cell by introducing
a DNA segment that codes for the polynucleotide into the cell such
that the polynucleotide is made inside the cell. An antisense
polynucleotide can also be introduced to a cell by adding the
polynucleotide to the environment of the cell such that the cell
can take up the polynucleotide directly. The latter route is
preferred for the shorter polynucleotides of up to about 20 bases
in length.
[0076] In selecting the preferred length for a given
polynucleotide, a balance must be struck to gain the most favorable
characteristics. Shorter polynucleotides such as 10-mers to 1
5-mers, while offering higher cell penetration, have lower gene
specificity. In contrast, while longer polynucleotides of 20-30
bases offer better specificity, they show decreased uptake kinetics
into cells. See Stein et al., Phosphorothioate Oligodeoxynucleotide
Analogues in Oligodeoxynucleotides-Antisense Inhibitors of Gene
Expression (Cohen, ed., McMillan Press, London 1988). Accessibility
to mRNA target sequences also is of importance and, therefore,
loop-forming regions in targeted mRNAs offer promising targets.
[0077] In this disclosure, the term "polynucleotide" encompasses
both oligomeric nucleic acid moieties of the type found in nature,
such as the deoxyribonucleotide and ribonucleotide structures of
DNA and RNA, and man-made analogues which are capable of binding to
nucleic acids found in nature. The polynucleotides of the present
invention can be based upon ribonucleotide or deoxyribonucleotide
monomers linked by phosphodiester bonds, or by analogues linked by
methyl phosphonate, phosphorothioate, or other bonds. They may also
comprise monomer moieties which have altered base structures or
other modifications, but which still retain the ability to bind
naturally occurring DNA and RNA structures. Such polynucleotides
may be prespared by methods well well-known in the art, for
instance using commercially available machines and reagents
available from Perkin-Elmer/Applied Biosystems (Foster City,
Calif.).
[0078] Phosphodiester-linked polynucleotides are particularly
susceptible to the action of nucleases in serum or inside cells,
and therefore in a preferred embodiment the polynucleotides of the
present invention are phosphorothioate or methyl phosphonate-linked
analogues, which have been shown to be nuclease resistant. Persons
of ordinary skill in this art will be able to select other linkages
for use in the invention. These modifications also may be designed
to improve the cellular uptake and stability of the
polynucleotides.
[0079] In another embodiment of the invention, the antisense
polynucleotide is an RNA molecule produced by introducing an
expression construct into the target cell. The RNA molecule thus
produced is chosen to have the capability to hybridize to APJ mRNA.
Such molecules that have this capability can inhibit translation of
the APJ mRNA and thereby inhibit the ability of HIV to infect cells
that contain the RNA molecule.
[0080] The polynucleotides which have the capability to hybridize
with mRNA targets can inhibit expression of corresponding gene
products by multiple mechanisms. In "translation arrest", the
interaction of polynucleotides with target mRNA blocks the action
of the ribosomal complex and, hence, prevents translation of the
messenger RNA into protein. Haeuptle et al., Nucl. Acids. Res.
14:1427 (1986). In the case of phosphodiester or phosphorothioate
DNA polynucleotides, intracellular RNase H can digest the targeted
RNA sequence once it has hybridized to the DNA oligomer. Walder and
Walder, Proc. Natl. Acad. Sci. USA 85:5011 (1988). As a further
mechanism of action, in "transcription arrest" it appears that some
polynucleotides can form "triplex," or triple-helical structures
with double stranded genomic DNA containing the gene of interest,
thus interfering with transcription by RNA polymerase.
Giovannangeli et al., Proc. Natl. Acad. Sci. 90:10013 (1993);
Ebbinghaus et al., J. Clin. Invest. 92:2433 (1993).
[0081] In one preferred embodiment, APJ polynucleotides are
synthesized according to standard methodology. Phosphorothioate
modified DNA polynucleotides typically are synthesized on automated
DNA synthesizers available from a variety of manufacturers. These
instruments are capable of synthesizing nanomole amounts of
polynucleotides as long as 100 nucleotides. Shorter polynucleotides
synthesized by modem instruments may be purified by polyacrylamide
gel electrophoresis or reverse phase chromatography. See Sambrook
et al., Molecular Cloning: A Laboratory Manual, Vol. 2, Chapter 11,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989).
[0082] Alternatively, a APJ polynucleotide in the form of antisense
RNA may be introduced to a cell by its expression within the cell
from a standard DNA expression vector. APJ DNA antisense sequences
can be cloned from standard plasmids into expression vectors, which
expression vectors have characteristics permitting higher levels
of, or more efficient expression of the resident polynucleotides.
At a minimum, these constructs require a prokaryotic or eukaryotie
promoter sequence which initiates transcription of the inserted DNA
sequences. A preferred expression vector is one where the
expression is inducible to high levels. This is accomplished by the
additionof a regulatory region which provides increased
transcription of downstream sequences in the appropriate host cell.
See Sambrook et al., Molecular Cloning: A Laboratory Manual Vol. 3,
Chapter 16, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989).
[0083] For example, APJ antisense expression vectors can be
constructed using the polymerase chain reaction (PCR) to amplify
appropriate fragments from single-stranded cDNA of a plasmid such
as pRc in which APJ cDNA has been incorporated. Fang et al., J.
Biol. Chem. 267:25889-25897 (1992). Polynucleotide synthesis and
purification techniques are described in Sambrook et al. and
Ausubel et al. (eds.), Current Protocols in Molecular Biology
(Wiley Interscience 1987) (hereafter "Ausubel"), respectively. The
PCR procedure is performed via well-known methodology. See, for
example, Ausubel, and Bangham, The Polymerase Chain Reaction:
Getting Started, in Protocols in Human Molecular Genetics (Humana
Press 1991). Moreover, PCR kits can be purchased from companies
such as Stratagene Cloning Systems (La Jolla, Calif.) and
Invitrogen (San Diego, Calif.).
[0084] The products of PCR are subcloned into cloning vectors. In
this context, a "cloning vector" is a DNA molecule, such as a
plasmid, cosmid or bacteriophage, that can replicate autonomously
in a host prokaryotic cell. Cloning vectors typically contain one
or a small number of restriction endonuclease recognition sites at
which foreign DNA sequences can be inserted in a determinable
fashion without loss of an essential biological function of the
vector, as well as a marker gene that is suitable for use in the
identification and selection of cells transformed with the cloning
vector. Suitable cloning vectors are described by Sambrook et al.,
Ausubel, and Brown (ed.), Molecular Biology Labfax (Academic Press
1991). Cloning vectors can be obtained, for example, from GIBCO/BRL
(Gaitherburg, Md.), Clontech Laboratories, Inc. (Palo Alto,
Calif.), Promega Corporation (Madison, Wis.), Stratagene Cloning
Systems (La Jolla, Calif.), Invitrogen (San Diego, Calif.), and the
American Type Culture Collection (Rockville, Md.).
[0085] Preferably, the PCR products are ligated into a "TA" cloning
vector. Methods for generating PCR products with a thymidine or
adenine overhang are well-known to those of skill in the art. See,
for example, Ausubel at pages 15.7.1-15.7.6. Moreover, kits for
performing TA cloning can be purchased from companies such as
Invitrogen (San Diego, Calif.).
[0086] Cloned antisense fragments are amplified by transforming
competent bacterial cells with a cloning vector and growing the
bacterial host cells in the presence of the appropriate antibiotic.
See, for example, Sambrook et al., and Ausubel. PCR is then used to
screen bacterial host cells for APJ antisense orientation clones.
The use of PCR for bacterial host cells is described, for example,
by Hoffman et al., Sequencing DNA Amplified Directly from a
Bacterial Colony, in PCR Protocols: Methods And Applications, White
(ed.), pages 205-210 (Humana Press 1993), and by Cooper et al.,
PCR-Based Full-Length cDNA Cloning Utilizing the
Universal-Adaptor/Specific DOS Primer-Pair Strategy, Id. at pages
305-316.
[0087] Cloned antisense fragments are cleaved from the cloning
vector and inserted into an expression vector. For example, HindIII
and XbaI can be used to cleave the antisense fragment from TA
cloning vector pCR.TM.-II (Invitrogen; San Diego, Calif.). Suitable
expression vectors typically contain (1) prokaryotic DNA elements
coding for a bacterial origin of replication and an antibiotic
resistance marker to provide for the amplification and selection of
the expression vector in a bacterial host; (2) DNA elements that
control initiation of transcription, such as a promoter; and (3)
DNA elements that control the processing of transcripts, such as a
transcription termination/polyadenylation sequence.
[0088] For a mammalian host, the transcriptional and translational
regulatory signals preferably are derived from viral sources, such
as adenovirus, bovine papilloma virus, simian virus, or the like,
in which the regulatory signals are associated with a particular
gene which has a high level of expression. Suitable transcriptional
and translational regulatory sequences also can be obtained from
mammalian genes, such as actin, collagen, myosin, and
metallothionein genes.
[0089] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene (Hamer et al., J. Mol. Appl. Genes. 1:273
(1982)); the TK promoter of Herpes virus (McKnight, Cell 31:355
(1982)); the SV40 early promoter (Benoist et al., Nature 290:304
(1981); the Rous sarcoma virus promoter (Gorman et al., Proc.
Nat'l. Acad. Sci. USA, 79:6777 (1982)); and the cytomegalovirus
promoter (Foecking et al., Gene 45:101 (1980)).
[0090] Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA polymerase promoter, can be used to control
fusion gene expression if the prokaryotic promoter is regulated by
a eukaryotic promoter. Zhou et al., Mol. Cell. Biol. 10:4529
(1990); Kaufman et al., Nucl. Acids Res. 19:44-85 (1991).
[0091] A vector for introducing at least one antisense
polynucleotide into a cell by expression from a DNA is the vector
pRc/CMV (Invitrogen, San Diego, Calif.), which provides a high
level of constitutive transcription from mammalian
enhancer-promoter sequences. Cloned APJ antisense vectors are
amplified in bacterial host cells, isolated from the cells, and
analyzed as described above.
[0092] Another possible method by which antisense sequences may be
exploited is via gene therapy. Virus-like vectors, usually derived
from retroviruses, may prove useful as vehicles for the importation
and expression of antisense constructs in human cells. Generally,
such vectors are non-replicative in vivo, precluding any unintended
infection of non-target cells. In such cases, helper cell lines are
provided which supply the missing replicative functions in vivo,
thereby permitting amplification and packaging of the antisense
vector. A further precaution against accidental infection of
non-target calls involves the use of target cell-specific
regulatory sequences. When under the control of such sequences,
antisense constructs would not be expressed in normal tissues.
[0093] Two prior studies have explored the feasibility of using
antisense polynucleotides to inhibit the expression of a heparin
binding growth factor. Kouhara et al., Oncogene 9:455-462 (1994);
Morrision, J. Biol. Chem. 266:728 (1991). Kouhara et al. showed
that androgen-dependent growth of mouse mammary carcinoma cells
(SC-3) is mediated through induction of androgen-induced, heparin
binding growth factor (AIGF). An antisense 15-mer corresponding to
the translation initiation site of AIGF was measured for its
ability to interfere with androgen-induction of SC-3 cells. At
concentrations of 5 .mu.M, the antisense polynucleotide-effectively
inhibited DNA synthesis. Morrision showed that antisense
polynucleotides targeted against basic fibroblast growth factor can
inhibit growth of astrocytes in culture. Thus, the general
feasibility of targeting an individual gene product in a mammalian
cell has been established.
[0094] Antisense polynucleotides according to the present invention
are derived from any portion of the open reading frame of the APJ
cDNA. Preferably, mRNA sequences (i) surrounding the translation
initiation site and (ii) forming loop structures are targeted.
Based upon the size of the human genome, statistical studies show
that a DNA segment approximately 14-15 base pairs long will have a
unique sequence in the genome. To ensure specificity of targeting
APJ RNA, therefore, it is preferred that the antisense
polynucleotides are at least 15 nucleotides in length. Thus, the
shortest polynucleotides contemplated by the present invention
encompass nucleotides corresponding to positions 1-14, 1-15, 1-16,
1-17, 1-18, 1-19, 2-16, 3-17, etc. of the APJ cDNA sequence.
Position 1 refers to the first nucleotide of the APJ coding
region.
[0095] Not every antisense polynucleotide will provide a sufficient
degree of inhibition or a sufficient level of specificity for the
APJ target. Thus, it will be necessary to screen polynucleotides to
determine which have the proper antisense characteristics. A
preferred method to assay for a useful antisense polynucleotide is
the inhibition of cell fusion between (1) cells that contain CD4
and APJ; and (2) cells that contain env.
[0096] Administration of an antisense polynucleotide to a subject,
either as a naked, synthetic polynucleotide or as part of an
expression vector, can be effected via any common route (oral,
nasal, buccal, rectal, vaginal, or topical), or by subcutaneous,
intramuscular, interperitoneal, or intravenous injection.
Pharmaceutical compositions of the present invention, however, are
advantageously administered in the form of injectable compositions.
A typical composition for such purpose comprises a pharmaceutically
acceptable solvent or diluent and other suitable, physiologic
compounds. For instance, the composition may contain polynucleotide
and about 10 mg of human serum albumin per milliliter of a
phosphate buffer containing NaCl.
[0097] As much as 700 milligrams of antisense polynucleotide has
been administered intravenously to a patient over a course of 10
days (i.e., 0.05 mg/kg/hour) without signs of toxicity. Sterling,
"Systemic Antisense Treatment Reported," Genetic Engineering News
12:1, 28 (1992).
[0098] Other pharmaceutically acceptable excipients include
non-aqueous or aqueous solutions and non-toxic compositions
including salts, preservatives, buffers and the like. Examples of
non-aqueous solutions are propylene glycol, polyethylene glycol,
vegetable oil and injectable organic esters such as ethyloleate.
Aqueous solutions include water, alcoholic/aqueous solutions,
saline solutions, parenteral vehicles such as sodium chloride,
Ringer's dextrose, etc. Intravenous vehicles include fluid and
nutrient replenishers. Preservatives include antimicrobial,
anti-oxidants, chelating agents and inert gases. The pH and exact
concentration of the various components the pharmaceutical
composition are adjusted according to routine skills in the art. A
preferred pharmaceutical composition for topical administration is
a dermal cream or transdermal patch.
[0099] Antisense polynucleotides or their expression vectors may be
administered by injection as an oily suspension. Suitable
lipophilic solvents or vehicles include fatty oils, such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides. Moreover, antisense polynucleotides or vectors may
be combined with a lipophilic carrier such as any one of a number
of sterols including cholesterol, cholate and deoxycholic acid. A
preferred sterol is cholesterol. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension
include, for example, sodium carboxymethyl cellulose, sorbitol,
and/or dextran. Optionally, the suspension also contains
stabilizers.
[0100] An alternative formulation for the administration of
antisense APJ polynucleotides involves liposomes. Lipsome
encapsulation provides an alternative formulation for the
administration of antisense APJ polynucleotides and expression
vectors. Liposomes are microscopic vesicles that consist of one or
more lipid bilayers surrounding aqueous compartments. See,
generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.
Infect. Dis. 12 (Suppl. 1): S61 (1993), and Kim, Drugs 46:618
(1993). Lipsomes are similar in composition to cellular membranes
and as a result, liposomes can be administered safety and are
biodegradable. Depending on the method of preparation, liposomes
may be unilamellar or multilamellar, and liposomes can vary in size
with diameters ranging from 0.01 .mu.m to greater than 10 .mu.m. A
variety of agents can be encapsulated in liposomes: hydrophobic
agents partition in the bilayers and hydrophilic agents partition
within the inner aqueous space(s). See, for example, Machy et al.,
Liposomes in Cell Biology And Pharmacology (John Libby 1987), and
Ostro et al., American J. Hosp. Pharm. 46:1576 (1989). Moreover, it
is possible to control the therapeutic availability of the
encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
[0101] Liposomes can adsorb to virtually any type of cell then
slowly release the encapsulated agent. Alternatively, an absorbed
liposome may be endocytosed by cells that are phagocytic.
Endocytosis is followed by intralysosomal degradation of liposomal
lipids and release of the encapsulated agents. Scherphof et al.,
Ann. N.Y. Acad. Sci. 446:368 (1985).
[0102] After intravenous administration, conventional liposomes are
preferentially phagocytosed into the reticuloendothelial system.
However, the reticuloendothelial system can be circumvented by
several methods including saturation with large doses of liposome
particles, or selective macrophage inactivation by pharmacological
means. Claassen et al., Biochim. Biophys. Acta 802:428 (1984). In
addition, incorporation of glycolipid- or polyethelene
glycol-derivatised phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the
reticuloendothelial system. Allen et al., Biochim. Biophys. Acta.
1068:133 (1991); Allen et al., Biochim. Biophys. Acta 1150:9
(1993). These Stealth.RTM. liposomes have an increased circulation
time and an improved targeting to tumors in animals. Woodle et al.,
Proc. Ameri. Assoc. Cancer Res. 33:2672 (1992); Gregoriadis et al.,
Drugs 45:15 (1993).
[0103] Antisense polynucleotides and expression vectors can be
encapsulated within liposomes using standard techniques. A variety
of different liposome compositions and methods for synthesis are
known to those of skill in the art. See, for example, U.S. Pat. No.
4,844,904, U.S. Pat. No. 5,000,959, U.S. Pat. No. 4,863,740, and
U.S. Pat. No. 4,975,282, all of which are hereby incorporated by
reference.
[0104] Liposomes can be prepared for targeting to particular cells
or organs by varying phospholipid composition or by inserting
receptors or ligands into the liposomes. For instance, antibodies
specific to tumors associated with antigens may be incorporated
into liposomes, together with antisense polynucleotides or
expression vectors, to target the liposome more effectively to the
tumor cells. See, for example, Zelphati et al., Antisense Research
and Development 3:323-338 (1993), describing the use
"immunoliposomes" containing antisense polynucleotides for human
therapy.
[0105] In general, the dosage of administered liposome-encapsulated
antisense polynucleotides and vectors will vary depending upon such
factors as the patient's age, weight, height, sex, general medical
condition and previous medical history. Dose ranges for particular
formulations can be determined by using a suitable animal
model.
[0106] The above approaches can also be used not only with
antisense nucleic acids, but also with ribozymes, or triplex agents
to block transcription or translation of a specific APJ mRNA,
either by masking that mRNA with an antisense nucleic acid or
triplex agent, or by cleaving it with a ribozyme.
[0107] Use of an oligonucleotide to stall transcription is known as
the triplex strategy since the oligomer winds around double-helical
DNA, forming a three-strand helix. Therefore, these triplex
compounds can be designed to recognize a unique site on a chosen
gene (Maher, et al., Antisense Res. and Dev., 1(3):227, 1991;
Helene, C., Anticancer Drug Design, 6(6):569, 1991).
[0108] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases. Through the modification of
nucleotide sequences which encode these RNAs, it is possible to
engineer molecules that recognize specific nucleotide sequences in
an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030,
1988). A major advantage of this approach is that, because they are
sequence-specific, only mRNAs with particular sequences are
inactivated.
[0109] There are two basic types of ribozymes namely,
tetrahymena-type (Hassellhoff, Nature, 334:585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
which are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18-base
recognition sequences are preferable to shorter recognition
sequences.
[0110] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples are
to be considered illustrative and thus are limiting to the
remainder of the disclosure.
EXAMPLES
Example 1
A Cell-Cell Fusion Assay to Determine Whether APJ Could Function as
a Coreceptor for HIV-1 or SIV.
[0111] A cell-cell fusion assay was employed to determine whether
APJ could function as a coreceptor for HIV-1 or SIV. This assay has
been described in detail in Nussbaum et al., 1994, J. Virol.,
68:5411-5422 and in Rucker et al., 1997, Meth. Enzymol.,
288:118-133. Effector cells were prepared by infecting quail QT6
cells with a recombinant vaccinia virus encoding T7 polymerase (vTF
1.1) and then either transfecting the cells with a plasmid bearing
the envelope gene of interest under the control of the T7 promoter
or introducing the env constructs via recombinant vaccinia virus.
Env constructs SIVmac251, SIVmac239, SIVmac316, SIVmac316mut, DH12,
RF, BK132, ADA, JR-FL, IIIB and HIV-2 ST were introduced into
effector cells via recombinant vaccinia virus rather than by
transfection. QT6 target cells were prepared by transient
transfection with plasmids encoding CD4, the coreceptor of interest
under the control of the CMV promoter, and luciferase under the
control of the T7 promoter. Effector and target cells were mixed
the day after transfection and cell-cell fusion was quantified by
measuring luciferase activity in cell lysates 7-8 hours following
mixing.
[0112] In this assay, cell-cell fusion results in cytoplasmic
mixing and luciferase production, which can be easily quantified.
As shown in FIG. 1, co-expression of either the CCR5 or the CXCR4
coreceptor with CD4 resulted in efficient fusion by R5 and X4 Env
proteins, respectively. R5-X4 env proteins such as HIV-1 89.6
mediated fusion with cells bearing either the CCR5 or the CXCR4
coreceptor. Fusion was not observed when CD4 was expressed alone.
When APJ was co-expressed with CD4 in QT6 cells, cell-cell fusion
was mediated by the R5-X4 Env protein 89.6 and by several X4 env
proteins at levels .gtoreq.70% of that observed with CXCR4 (FIG.
1). For one primary X4 env protein, ZR0001.3, fusion with cells
expressing APJ was more efficient than with CXCR4. A majority of R5
env proteins mediated fusion with APJ expressing cells, but only at
very low levels relative to that observed with CCR5 (FIG. 1 and
Table 1). However, ADA and the primary isolate TH 22-4 exhibited
fusion mediated by APJ at roughly half the level observed when CCR5
served as the viral coreceptor. The HIV-2 ST env protein also
mediated very inefficient fusion with cells expressing both CD4 and
APJ. The ability of APJ to support fusion for some X4 and R5-X4
viral env proteins nearly as efficiently as the major coreceptors
is notable, since most other alternative HIV-1 coreceptors
typically support cell-cell fusion much less efficiently than CCR5
or CXCR4.
[0113] The ability of APJ to support fusion by a panel of SIV
envelope proteins was also examined. Unlike HIV-1, both M- and
T-tropic SIV strains utilize CCR5 as a coreceptor, while CXCR4 is
either not used or rarely used by SIV (Chen et al., 1997, J.
Virol., 71:2705-2714; Edinger et al., 1997, Proceedings of the
National Academy of Sciences, USA, 94:4005-4010; and Marcon et al.,
1997, J. Virol., 71:2522-2527). In addition, the orphan receptors
STRL33, GPR15, and GPR1 can be used as coreceptors by both T- and
M-tropic SIV strains (Deng et al., 1997, Nature, 388:296-300 and
Farzan et al., 1997, J. Exp. Med., 186:405-411). In the instant
experiments, it was determined that APJ supported fusion by several
M- and T-tropic SIV env proteins at levels that were less efficient
than those observed with CCR5, with the exception of the M-tropic
SIVmac316 and a variant of this env protein (316mut) which
efficiently used APJ as a coreceptor in cell-cell fusion assays
(FIG. 2 and Table 1). Additionally, APJ typically supported fusion
less efficiently than the orphan receptors GPR1, GPR15/BOB, and
STRL33/Bonzo. Finally, because it was previously determined that
many SIV strains can infect cells in a CD4-independent,
CCR5-dependent manner (Edinger et al., 1997, Proc. Natl. Acad.
Sci., USA, 94:14742-14747), the ability of HIV-1, HIV-2, and SIV
env proteins to mediate fusion with cells expressing APJ alone was
tested. The results showed that APJ coreceptor activity was
strictly CD4 dependent, as cells expressing APJ alone did not
support cell-cell fusion with any of the env proteins tested.
1 TABLE 1 ENY Tropism CCR5 CXCR4 APJ DH12 D +++ +++ + RF (D) +++
+++ - YU2 M +++ - + JR-FL M +++ - - SF162 M +++ - + 91US005.11 +++
- + 93BR019.10 +++ - + 92UG031.7 +++ - - 93BR029.2 +++ - - UG37-8
+++ - + TH 22-4 +++ - ++ RW20-5 +++ - + SIVmacBK28 +++ - +
SIV/17E-C1 M +++ - + SIVmac1A11 M +++ - + SIVagmSab1.4 +++ - +
SIVsm62A T +++ - + SIVsm62D T +++ - + SIVsm543-3 M +++ - ++
SIVsm543-B10 +++ - + SIVsmPBj6 +++ - +
Example 2
Determination of APJ Ability to Support Virus Infection
[0114] The ability of APJ to support virus infection was determined
in order to more rigorously assess the ability of APJ to function
as a coreceptor. A first assay system employed a luciferase
reporter virus assay in which various env proteins were pseudotyped
onto the luciferase reporter virus backbone. Luciferase reporter
viruses were prepared by transfecting human HEK 293 cells with a
plasmid that expresses env under the control of the CMV or SV40
promoter, and with a plasmid containing a proviral genome with an
inactive env gene and the luciferase gene in place of nef (e.g. the
NL4-3 luciferase virus backbone (pNL-Luc-E.sup.-R.sup.-)) (Chen et
al., 1994, J. Virol., 68:654-660 and Connor et al., 1995, Virology,
206:935-944). Target cells for infection were HEK 293 or CCCS cells
with CD4 and coreceptors introduced by calcium phosphate
transfection. Infections were performed in media containing 8
.mu.g/ml DEAE dextran. Cells were lysed 3-4 days post-infection by
resuspension in 0.5% NP-40 in PBS and assayed for luciferase
activity.
[0115] Unfortunately, most env proteins which efficiently catalyzed
fusion with cells expressing CD4 and APJ (such as HIV-1 89.6) could
not be successfully pseudotyped. Viral env proteins that could be
pseudotyped, as judged by infection of CCR5 or CXCR4 positive
cells, either failed to infect cells expressing CD4 and the APJ
coreceptor or did so inefficiently (FIG. 3). In some cases, env
proteins that mediated fusion with APJ expressing cells at
intermediate levels failed to support virus infection. For example,
the virus pseudotype with the ADA env protein did not infect
APJ-positive cells even though cells expressing the ADA env protein
mediated fusion with APJ-positive cells half as efficiently as with
CCR5-positive cells. The reasons for these assay dependent
discrepancies are not clear, but may reflect the efficiencies with
which various env proteins can be pseudotyped.
[0116] Another assay system was employed in order to test envelope
proteins which could not be pseudotyped but which were able to
mediate cell-cell fusion with APJ expressing cells in an efficient
manner. Target HEK 293 cells that had been transfected with
plasmids expressing CD4, the desired coreceptor, and luciferase
under control of the viral LTR were infected with intact HIV-1 89.6
or HIV-1 IIIB (FIG. 4). Luciferase activity was measured 2
days-post infection. The results showed that HIV-1 89.6 infected
APJ positive cells nearly as efficiently as cells expressing CXCR4.
HIV-1 IIIB (HxB3) also infected APJ positive cells a levels well
above background.
[0117] Finally, a PCR based entry assay was also employed to
determine if APJ could support infection by HIV-1 89.6 and IIIB.
QT6 cells stably expressing human CD4 and transiently expressing
the desired coreceptor were infected with 50 ng p24 of
DNAase-treated, cell-free virus. After two days, the cells were
washed and lysed, and HIV-1 specific LTR DNA sequences were
detected by PCR using primers LTR-plus/LTR-minus
(5'-ACAAGCTAGTACCCAGTTGAGCC-3' (SEQ ID NO: 4),
5'-CACACACTACTTGAAGCACTCA-- 3' (SEQ ID NO: 5)). Products were
resolved by electrophoresis on 2% agarose gels, transferred to
Hybond N+ (Amersham), and detected by using the 3'-End Labeling
Biotin Kit (DuPont; probe 5'-ATCTACAAGGGACTTTCCCGC-3' (SEQ ID
NO:6), followed by exposure. As shown in FIG. 5, both HIV-1 IIIB
and 89.6 could enter QT6 cells expressing both CD4 and APJ,
although entry was less efficient than with the major HIV-1
coreceptors.
Example 3
Examination of the of the Distribution of APJ in Human Brain by
Northern Blot Analysis
[0118] APJ was originally cloned from human genomic DNA, and
analysis of rat tissues using a probe based on the rat homolog
revealed that APJ is expressed widely in brain (O'Dowd et al.,
1993, Gene, 136:355-360). APJ has also been shown to be expressed
in some areas of the human brain (Matsumoto et al., 1996, Neurosci.
Lett., 219:119-122). Because of the efficient use of APJ as a
coreceptor by some virus strains, APJ distribution in the human
brain has been further examined by Northern blot analysis.
[0119] Membranes containing poly A.sup.+ RNA from various human
brain regions were obtained from Clontech. The Prime-It II Random
Primer Labeling Kit (Stratagene, La Jolla, Calif.) was used to
label the cDNA probe with .alpha.-.sup.32P-dATP (3,000 Ci/mmol)
using the Klenow enzyme. The .alpha.-.sup.32P-labeled cDNA probe
was purified using Quick Spin columns (Boehringer Mannheim,
Indianapolis, Ind.). The membranes were hybridized overnight with
10.sup.7 cpms of the labeled probe in hybridization buffer
containing 25 mM Na/Na.sub.2PO.sub.4, 50 mM Tris pH 7.4, 6.times.
SSPE, 0.1% SDS, 100 .mu.g/ml single stranded DNA and 1.times.
Denhardt's solution. The membranes were washed twice in 1.times.
SSPE, 0.1% SDS at 42.degree. C. for 10 minutes and changed to a
high stringency wash solution of 0.2.times. SSPE, 0.1% SDS at
42.degree. C. for 10 minutes. The membrane was then exposed to a
Fuji Imaging plate for 4 hours. Images of the plate were captured
on a BAS1000Mac Bio-Imaging Analyzer (Fuji) and processed with Mac
BAS software. Images were printed on a Pictography 3000 (Fuji)
digital printer.
[0120] The results showed that high levels of APJ transcripts were
present in the corpus callosum, spinal cord, and medulla. Lower
levels of APJ transcripts were detected in other regions of the
human brain (FIG. 6). In peripheral tissues, the APJ transcript was
readily detected in spleen but absent in PBLs (FIG. 7). Lower
levels of transcript were detected in other peripheral tissues.
[0121] To investigate the distribution of APJ in cells commonly
used to propagate HIV-1, RT-PCR analysis was performed on a large
number of cell lines and some primary cell types. A U87 cell line
that stably expressed APJ was generated and used as a positive
control.
[0122] Primary cells were isolated as follows. Human blood
mononuclear cells (PBMC) were isolated from blood of normal
volunteers using Ficoll-Hypaque, depleted of monocytes by serial
adherence to plastic, stimulated with phytohemagglutinin (PHA-L, 5
.mu.g/ml; Sigma) for 3 days and then resuspended with interleukin 2
(20 U/ml, Boehringer Mannheim Biochemicals). RNA was extracted
after 3 days of PHA stimulation and also following 1 week in IL-2.
Monocytes were purified from PBMC by selective adherence to gelatin
followed by plastic, and then maintained in culture to allow
differentiation into monocyte-derived macrophages (MDM) as
previously described (Collman et al., 1989, J. Exp. Med.,
170:1149-1163). RNA was extracted from undifferentiated monocytes
immediately after purification and from MDM after 1 week in
culture.
[0123] For the isolation of total cellular RNA for RT-PCR,
5-10.times.10.sup.6 cells were resuspended in 1 ml Trizol
(GIBCO-BRL) and processed as recommended by the manufacturer. Total
RNA was then treated with 1 .mu.l (10-50 units) DNAse (RNAse-free)
(Boehringer Mannheim) per 10 .mu.g RNA for 30 min at 37.degree. C.
in the presence of 5 mM MgCl.sub.2 with subsequent inactivation at
65.degree. C. for 10 minutes in the presence of 5 mM EDTA; RNA
concentration was calculated based on the OD.sub.260. The Titan
RT-PCR system (Boehringer Mannheim) was used to evaluate RNA
expression patterns. Specific, internal upstream and downstream
primers were used which resulted in an amplified product of 481
base pairs. The primers used were the following: forward
5'-TACACAGACTGGAAATCCTCG-3' (SEQ ID NO: 7) and reverse
5'-TGCACCTTAGTGGTGTTCTCC-3' (SEQ ID NO: 8). In order to control for
contamination of the RNA sample with genomic DNA despite treatment
with DNAse, all RNA samples were also amplified with Titan enzyme
mix in which the RT but not PCR activity had been destroyed by
treatment at 95.degree. C. for 10 minutes (this inactivation
protocol was found to eliminate the ability to amplify a RNA but
not a DNA template). In each RT-PCR reaction, RNA isolated from
U87-APJ stably transfected cells were included as a positive RNA
control and plasmid DNA was included as a second positive
control.
[0124] The results of the investigation of the distribution of APJ
in cell lines and cell types showed that APJ was expressed in C8166
cells, but APJ-specific reaction products could not be detected in
the other cell lines examined, including Jurkat, Hut78, CEMx174,
and PM1 cells. Additionally, expression of APJ was not detected in
PHA, PHA with IL-2, or anti-CD3 and IL-2 stimulated PBMC or in
monocytes or monocyte derived macrophages (FIG. 8).
[0125] For other aspects of the nucleic acids, polypeptides,
antibodies, etc., reference is made to standard textbooks of
molecular biology, protein science, and immunology. See, e.g.,
Davis et al. (1986), Basic Methods in Molecular Biology, Elsevir
Sciences Publishing, Inc., New York; Haames et al. (1985), Nucleic
Acid Hybridization, IL Press, Molecular Cloning, Sambrook et al.;
Current Protocols in Molecular Biology, Edited by F. M. Ausubel et
al., John Wiley & Sons, Inc; Current Protocols in Human
Genetics, Edited by Nicholas C. Dracopoli et al., John Wiley &
Sons, Inc.; Current Protocols in Protein Science; Edited by John E.
Coligan et al., John Wiley & Sons, Inc.; Current Protocols in
Immunology; Edited by John E. Coligan et al., John Wiley &
Sons, Inc.
[0126] The entire disclosure of all patent applications, patents,
and publications cited herein are hereby incorporated by
reference.
[0127] From the foregoing description, on skilled in the art can
easily ascertain the essential characteristics of this invention,
and without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions.
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