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BACKGROUND
Human T-cell leukemia-lymphotropic virus (HTLV) refers to a family of T
cell tropic retroviruses. Such viruses, which have a role in causing
certain T cell neoplasms, are presently divided into three main types or
subgroups: (1) HTLV-type I (HTLV-I), which appears to cause adult T-cell
leukemia-lymphoma (ATLL); (2) HTLV-type II (HTLV-II), which has been
isolated from an individual having a T-cell variant of hairy cell
leukemia; and (3) HTLV-type III (HTLV-III), which has been identified as
the etiologic agent of acquired immune deficiency syndrome (AIDS).
HTLV-III is also known as lymphadenopathy-associated virus (LAV), AIDS
related virus (ARV) and human immunodeficiency virus (HIV). Popovic, M. et
al., Science, 224: 497-500 (1984); Gallo, R. C. et al., Science, 224:
500-503 (1984); Wong-Staal, F. and Gallo, R. C., Nature, 317: 395-403
(1985); and Curran, J. W. et al., Science, 229: 1352-1357 (1985).
AIDS was first recognized in 1981 and since that time, the disease has come
to be recognized as a new epidemic. RNA Tumor Viruses (2d edition), Volume
2, pp 437-443, Cold Spring Harbor Laboratory (1985).
Patients with AIDS exhibit clinical manifestations which include severe
immunodeficiency which generally involves a depletion of helper T
lymphocytes; malignancies; and opportunistic infections. The disease at
this time is incurable and the mortality rate among AIDS patients is high.
Because the disease has severe, generally life threatening effects, there
is great interest in finding means of protecting the population from it
and of treating those who contract it. At the present time, much effort is
being put into developing methods of detecting the presence of HTLV-III in
body tissues and fluids (e.g., blood, saliva) and into developing vaccines
which will protect recipients from HTLV-III. However, there is no known
method which is satisfactory either for preventing the disease or for
treating those who become infected with the virus. In fact, current
efforts to develop a broad spectrum anti-HTLV-III vaccine may be seriously
compromised, in light of the variation in envelope proteins (which are the
principal antigenic determinants of the virus) observed among various
strains of HTLV-III. Hahn, G. H. et al., Proceedings of the National
Academy of Sciences, USA, 82: 4813-4817 (1985); Benn, S. et al., Sciences,
230: 949-951 (1985). Other methods of blocking the effects of the virus
are clearly needed.
SUMMARY OF THE INVENTION
This invention relates to exogenous oligonucleotides which are
complementary to regions of the HTLV-III genome and inhibit HTLV-III
replication or gene expression; methods of inhibiting HTLV-III replication
and HTLV-III gene expression in cultured human cells; methods of detecting
the presence of HTLV-III in biological samples; and methods of
administering the oligonucleotides to individuals for the purpose of
inhibiting HTLV-III replication or gene expression.
The oligonucleotides of this invention, which can be
oligodeoxyribonucleotides or oligoribonucleotides, are complementary to
regions on the HTLV-III genome which are highly conserved, and whose
function is necessary for normal replication or gene expression by
HTLV-III. The oligonucleotides can be used to block HTLV-III replication,
gene expression or both and thus can be used as chemotherapeutic agents in
inhibiting replication and gene expression by the virus. In addition, they
can be used to detect the presence of HTLV-III in samples such as blood,
urine and saliva.
Oligonucleotides of the present invention are complementary to target sites
which are highly conserved regions of the HTLV-III genome. These include
the cap site; the primer binding site; nucleotide sequences vicinal to the
primer binding site in the 5' direction; mRNA donor splice and acceptor
splice sites; the HTLV-III initiator codons, including those for the gag,
the sor, the tat, the env, and the 3'ORF sequences; the art gene or a
portion thereof; and the region of the genome responsible for the
frameshift known to occur during transcription. These
oligodeoxynucleotides can be used to inhibit HTLV-III replication and/or
gene expression in the HTLV-III infected cells. They can be administered
to individuals to block HTLV-III replication and/or gene expression as a
means of chemotherapeutic treatment of acquired immune deficiency syndrome
(AIDS) and of AIDS related complex (ARC).
Use of such oligonucleotides has at least two important advantages. First,
the antiviral effects observed are very specific. For example, a specific
sequence of 20 nucleotides would not be expected to occur at random more
often than about one time in 10.sup.12. There are about 4.times.10.sup.9
nucleotide pairs in the human genome and thus, the specificity of a
20-nucleotide sequence chosen from a conserved region of HTLV-III is
predicted to be great. Second, the cellular toxicity of the
oligonucleotides is also low, in comparison with most nucleoside analogues
(e.g., those used in cancer chemotherapy, graft-host immunology and viral
inhibition); such analogues are converted into nucleotides, which are
subsequently incorporated into cellular DNA.
Oligonucleotides complementary to the same regions of the HTLV-III genome
can be used to determine whether HTLV-III is present or absent in a sample
such as blood, saliva or urine by determining whether cell death occurs in
cells which are normally killed by HTLV-III virus (such as T lymphocytes)
when they are cultured with the sample to be tested and whether cell death
can be inhibited by the oligonucleotide.
BRIEF DESCRIPTION OF THE FIGURE
The FIGURE is a schematic representation of the primary nucleotide sequence
of the HTLV-III genome and of the location on the genome of
oligonucleotide competitive inhibition targets.
DETAILED DESCRIPTION OF THE INVENTION
The primary nucleotide sequence of the HTLV-III/LAV genome has been
determined by several groups of investigators. Ratner, L. et al., Nature
313: 277-284 (1985); Wain-Hobson, S. et al., Cell 40: 9-17 (1985);
Sanchez-Pescador, R. et al., Science 227: 484-492 (1985); Muesing, M. A.
et al., Nature 313: 450-458 (1985).
The genome of HTLV-III is shown in the FIGURE. The HTLV-III genome has been
shown to be considerably more variable than the genomes of most
retroviruses. RNA Tumor Viruses (2d edition) Volume 2, p 446, Cold Spring
Harbor Laboratory (1985). Like other retroviruses, HTLV-III has in its
genome three genes which encode viral proteins: (1) the gag gene, which
encodes nucleocapsid or internal structural proteins of the virus; (2) the
pol gene, which encodes reverse transcriptase (an RNA-directed DNA
polymerase responsible for transcribing RNA into DNA); and (3) the env
gene, which encodes the envelope glycoproteins of the virion. In addition,
two other open reading frames are known; one (sor) overlaps with the 3'
end of the pol gene and the other (3'ORF), located at the extreme 3' end
of the genome, slightly overlaps the env gene and continues through most
of the U.sub.3 region. The genome has also been shown to contain tat-III
and art. Tat-III is the trans-activation gene of HTLV-III; it gene encodes
for trans-activator protein, which greatly accelerates viral protein
synthesis in infected cells. Art (antirepression of the
translation-transactivator gene) has only recently been found in the
HTLV-III genome and appears to work cooperatively with tat in producing
viral core and envelope proteins.
Other regions of the RNA of HTLV-III are a cap nucleotide, which occurs at
the extreme 5' end of the genome; a short sequence (R) which is repeated
at both ends of the RNA; a short sequence unique to the 5' end (U.sub.5);
and a sequence unique to the 3' end (U.sub.3). Each of the last three
components is present twice in viral DNA; each forms part of the long
terminal repeat (LTR) sequence found at both ends of the unintegrated
linear DNA product of reverse transcription. The HTLV-III genome also
contains a primer binding site (PBS) adjacent to U.sub.5 (at its 3' end);
the PBS is complementary to the 3' end of tRNA lysine and functions as
primer for synthesis of the minus strand of viral DNA. Donor splice (S.D.)
and acceptor splice (S.A.) sites are also located on the viral RNA. Donor
splice sites are sequences at which a 5' portion of the viral genome is
joined to a portion of the 3' end of viral RNA, forming a spliced,
subgenomic messenger RNA. Acceptor splice sites are sequences at which
portions of the 3' end of viral RNA join donor splice sites to form
subgenomic messenger RNA.
As mentioned above, different HTLV-III strains have been reported to have
variations in envelope proteins. These variations may compromise the
development of a broad spectrum anti-HTLV-III vaccine. In contrast, the
primary nucleotide sequence of the primer area and certain other parts of
the HTLV-III genome are highly conserved.
It has been now shown that complementary oligodeoxynucleotides directed
toward such highly conserved regions of the HTLV-III genome inhibit virus
replication and/or gene expression in cultured HTLV-III-transformed human
lymphocytes.
TARGETED REGIONS OF THE HTLV-III GENOME
As mentioned, several regions of the HTLV-III genome are highly conserved;
these regions or parts thereof can be targeted for inhibition by
complementary oligonucleotide sequences. These regions, referred to as
oligonucleotide competitive inhibition targets, include: (1) the cap site;
(2) sequences of nucleotides 5' to the primer tRNA.sup.lys binding site;
(3) the primer binding site or a segment thereof; (4) a combination of
sequences 5' to the primer tRNA.sup.lys binding site and the primer
binding site; (5) sequences of the mRNA donor or acceptor splice sites;
(6) the initiator codons for the gag, the sor, the tat, the env and the
3'ORF sequences; (7) the art gene or portions thereof; and (8) the region
of the genome responsible for the frameshift known to occur during
transcription. The location of these regions (except the art gene) is
indicated in the FIGURE; the art gene is located close to the tat gene.
It has been demonstrated that oligodeoxynucleotides complementary to four
of the above mentioned highly conserved regions inhibit virus replication
or gene expression in cultured HTLV-III-transformed human lymphocytes.
That is, oligodeoxynucleotides complementary to (1) sequences 5' to the
primary tRNA.sup.lys binding site; (2) the primer binding site; (3)
sequences of a mRNA donor splice site; or (4) sequences of a mRNA acceptor
splice site have been shown to cause inhibition. In general, any highly
conserved region of the HTLV-III genome which encodes information
necessary for viral replication or gene expression (e.g., protein
synthesis) is a potential target for complementary oligodeoxynucleotides.
COMPLEMENTARY OLIGONUCLEOTIDE SEQUENCES
The oligonucleotide sequences complementary to the competitive inhibition
targets can be oligoribonucleotide sequences or oligodeoxyribonucleotide
sequences. Both types are referred to herein as oligonucleotides. As
described here, the oligonucleotides were synthesized on an automated DNA
synthesizer. It is possible, however, to produce the desired sequences by
using genetically engineered organisms, such as bacteria or viruses.
Oligodeoxynucleotide sequences of varying lengths were used to assess their
inhibitory effect on viral replication and gene expression. For example,
several nucleotide sequences complementary either to nucleotide sequences
of the HTLV-III genome which are 5' to the primer tRNA.sup.lys binding
site or to nucleotide sequences which straddle the primer binding site
and the adjacent region (in the 5' direction) were synthesized and their
inhibitory effects tested. As described in greater detail in Example 3, a
12-nucleotide sequence (mer), a 20-nucleotide sequence and a 26-nucleotide
sequence have been made and their inhibitory effects on viral replication
and gene expression measured. The 12-nucleotide and the 20-nucleotide
sequences are complementary to portions of the HTLV-III genome close to
the primer binding site, in the 5' direction. The 26-nucleotide sequence
is complementary to the primer binding site.
In addition, oligodeoxynucleotide sequences complementary to splice donor
or splice acceptor sites of HTLV-III mRNA have been made and their
inhibitory effects assessed. In particular, a 20-nucleotide sequence
complementary to a splice donor site from the 3'-open reading frame region
(FIG. 1) and two 20-nucleotide sequence complementary to the a-1 and a-1'
splice acceptor sites, the former necessary for the production of
transactivating factor, have been synthesized and their inhibitory effects
measured.
Viral replication was assayed as reverse transcriptase activity level and
gene expression as production of viral proteins p15 and p24. Inhibition of
viral replication is reflected in reduced reverse transcriptase activity
levels; inhibition of viral gene expression is indicated by reduction in
viral protein production. As shown in Table 1, HTLV-III replication and
protein expression were inhibited in almost every instance. The greatest
inhibitory effect was evident when the 20-nucleotide sequence
complementary to the splice acceptor site was tested on cultures of
HTLV-III infected cells.
Other complementary oligonucleotide sequences which can be used are
determined by the competitive inhibition target(s) selected.
Oligonucleotide sequences can be complementary to a single competitive
inhibition target or can be complementary to more than one such target.
For example, sequences can be produced which are complementary to the
HTLV-III primer binding site and the region of the genome immediately
adjacent to that site in the 5' direction; to two splice donor sites; to
two splice acceptor sites; or to any combination of competitive inhibition
targets.
Other characteristics of the oligonucleotides used to inhibit viral
processes include their length; their modification and the location of
groups used to modify them. For example, the length of the
oligonucleotides to be used will be determined by factors such as the
desired specificity of inhibition, size necessary to block viral function,
and effect on transmembrane passage. For example, the work described
herein has made use of complementary oligodeoxynucleotides ranging in
length from 14 to 26 nucleotides. However, there is potentially no limit
to the length of the oligonucleotides to be used and length must be
determined carefully, in light of the fact it plays a role in viral
inhibition. Generally, oligonucleotides used to inhibit HTLV-III will be
8-50 nucleotides in length.
Oligonucleotides to be used can be modified at a variety of locations along
their length. For example, they can be modified by the addition of groups
at the 5' end, the 3' end or both, as well as on the internal phosphate
groups or on the bases. Whether oligonucleotides to be used are modified
and, if so, the location of the modification(s) will be determined, for
example, by the desired effect on viral activity (e.g., inhibition of
viral replication, gene expression or both), uptake into infected cells,
inhibition of degradation of the oligonucleotides once they are inside
cells, and prevention of their use as a primer by reverse transcriptase.
For example, if inhibition of reverse transcriptase activity (and thus of
viral replication) is desired, it may be necessary to block the 3' end of
a sequence complementary to the primer binding site and/or sequences
vicinal to the primer binding site in the 5' direction (for example by a
2'3' dideoxynucleotide). In this way, the oligonucleotide complementary to
either or both of those regions cannot itself serve as a template for
transcriptase activity. If the desired effect is increased uptake of the
oligonucleotide into infected cells, modification of the oligonucleotide
by addition of a lipophillic group at the 5' end would be beneficial.
Modification of oligonucleotides can also be carried out by the addition
of an intercalating agent (e.g., acridine dye) at 5' or 3' termini, on
bases, or on internucleophosphate groups. Modification in this manner may
result in stronger binding between the oligonucleotides and the HTLV-III
nucleic acids. Asseline, U. et al., C.R. Acad. Sc. Paris, 369-372 (1983).
As shown in Table 1, the 12 nucleotide sequences complementary to the
region of the HTLV-III genome 5' to the primer binding site were blocked
at the 3' end by ddT. Early work on Rous sarcoma virus inhibition
indicates that the 3' end blocked hybridon was a more effective inhibitor
than an unblocked hybridon. A hybridon is defined as an oligonucleotide
complementary to single-stranded DNA or RNA, which modulates the function
of the DNA or RNA by competitive hybridization. Zamecnik, P. and M. L.
Stephenson, Proceedings of the National Academy of Sciences, USA, 75:
280284 (1978).
Chain terminator(s) to be used in modifying oligonucleotides for use in
inhibiting viral replication and gene expression can be, for example, ddT
(as described above and in Example 3), the isourea group, the
dimethoxytrityl group, or, in fact, any 3' modified function. Selection of
the chain terminator is based, for example, on the absence of a 3' OH
group (which can act as a substrate for reverse transcriptase); lack of or
low cellular toxicity; lipophilicity; and lack of impairment of hydrogen
bonding properties of the oligonucleotide.
INHIBITION OF HTLV-III-INFECTED CELLS
Using the oligodeoxynucleotide sequences described above and in Example 3,
it was possible to inhibit HTLV-III replication and gene expression in
HTLV-III-infected cells in tissue culture. The oligodeoxynucleotides
described were added to peripheral human blood cells (PB) infected with
HTLV-III and to transformed T-lymphocyte (H9) cells infected with
HTLV-III. The oligodeoxynucleotide was usually added at time zero only and
observation of inhibitory effects was made at 96 hours. In one case, the
oligonucleotide was added to fresh culture medium daily for 3 days.
Reverse transcriptase activity and viral p15 and p24 protein production
were used as indicators of inhibition of HTLV-III replication and gene
expression, respectively. As shown in Table 1 and described in detail in
Example 3, inhibition was greatest when a 20-nucleotide sequence
complementary to a splice acceptor site was added to HTLV-III-infected
transformed T-lymphocytes. Inhibition was observed under essentially all
experimental conditions (see Table 1).
Important considerations in this context are the concentration at which the
complementary oligodeoxynucleotides are applied and the timing
(scheduling) of their administration. As shown in Table 1, the
oligodeoxynucleotides were added at concentrations ranging from 5
.mu.g/ml. to 50 .mu.g/ml. culture medium. These concentrations were
generally effective in producing an inhibitory effect but this range is by
no means to be considered limiting. As described, the oligodeoxynucleotide
was usually added at one time only; it seems, however, that daily addition
(or more frequent addition) is more effective than a single dose.
INHIBITION OF HTLV-III IN HUMANS
Based on the information gained from inhibition of HTLV-III-infected cells
in tissue culture, it is possible to formulate a strategy for similar
inhibition of HTLV-III in AIDS patients, as well as in individuals
carrying the AIDS virus but not manifesting symptoms of the disease.
The strategy used in treating a particular individual depends on the status
of the individual and the objective of the treatment. That is, an
individual who has been found to be carrying the HTLV-III virus but shows
no symptoms of AIDS might be treated differently, in terms of both the
type of oligonucleotide(s) administered and the dose given, than an
individual who does, in fact, have AIDS. In addition, treatment might well
differ if its objective is to protect uninfected cells or to have an
effect on cells which are already infected.
For example, an individual known to be harboring the virus but yet
manifesting no sign of AIDS could be given a long-term or lifetime
maintenance dose of oligonucleotides whose inhibitory effects stop reverse
transcription (e.g., oligonucleotides complementary to the primer binding
site and/or sequences close to the primer binding site in the 5'
direction). In this way, the first step in viral life or replication is
inhibited because viral DNA cannot be made and the virus is unable to
proliferate. However, in an AIDS patient, cells are already infected and
treatment must inhibit expression of genes (viral DNA) already present in
the infected cells. In this case, oligonucleotides complementary to, for
example, initiator codons for genes encoding viral proteins, are required
to prevent viral construction. In an AIDS patient, uninfected cells can
also be protected by administration of oligonucleotides capable of
blocking reverse transcription.
In any treatment situation, however, oligonucleotides must be administered
to individuals in a manner capable of getting the oligonucleotides
initially into the blood stream and subsequently into cells. As a result,
the oligonucleotides can have the desired effects: getting into HTLV-III
infected cells to slow down or prevent viral replication and/or into as
yet uninfected cells to provide protection.
Oligonucleotides whose presence in cells can stop reverse transcription and
oligonucleotides whose presence in cells can inhibit protein synthesis can
be administered by intravenous injection, intravenous drip or orally. The
dose to be administered varies with such factors as the size and age of
the patient, stage of the disease and the type of oligonucleotide to be
given.
DETECTION OF THE HTLV-III VIRUS IN SAMPLES
The oligonucleotide sequences of the present invention can also be used in
determining whether the HTLV-III virus is present or absent in samples
such as blood, urine, saliva and tears. An aliquot of the sample to be
analyzed is added to a culture of cells which are normally killed by the
HTLV-III virus (e.g., T lymphocytes); this is the control. A second
aliquot is added to a separate culture of T lymphocytes, along with
oligonucleotides complementary to one or more of the regions of the
HTLV-III genome describe above; this is the test sample. Both cultures are
maintained under conditions appropriate for growth and subsequently
analyzed (e.g., visually/microscopically) for growth of the T lymphocytes.
If the HTLV-III virus is present, the T lymphocytes in the control sample
will be killed; if not, the T lymphocytes survive. T lymphocytes in the
test sample, however, will continue to be viable because of the protection
provided by the complementary oligonucleotides included in the culture.
Visual comparison of the two samples makes it possible to determine
whether HTLV-III virus is present or absent in each.
The present invention will now be further illustrated by the following
examples, which are not intended to be limiting in any way.
EXAMPLE 1
Synthesis and Characterization of Oligodeoxynucleotides
Unmodified oligodeoxynucleotides were synthesized on an automated DNA
synthesizer (Biosearch SAM I), using either standard triester or
phosphoramidite chemistry. Gait, M. J. (Ed.), Oligonucleotide Synthesis,
I.R.L. Press (1984). After deblocking, the products were purified first on
Merck silica gel 60 thin layer chromatographic plates in i-propanol:
concentrated ammonia:water (55:35:10) and eluted with ethanol:water (1:3).
Where necessary, further purification was performed by high pressure
liquid chromatography, using a Waters SAX Radial-Pak catridge or by
polyacrylamide gel electrophoresis (PAGE). The synthetic, preparative and
analytical procedures have been described in detail. See Gait, M. J.,
above. The oligonucleotide with terminal 3'-deoxythymidine (ddT) was made
by the solution phase triester method. This method is described in detail
by Narang, S. A. et al., in: Methods in Enzymology, L. Grossman and K.
Moldave (Ed.), 65: 610-620, Academic Press (1980), the teachings of which
are incorporated herein by reference. ddT (Sigma) was used directly in the
coupling reaction without protecting groups. The final product was
purified first on 2 mm thick silica gel plates (Analtech) as above and
subsequently by column chromatography on DEAE cellulose in a gradient of
0.02-0.8M triethylammonium bicarbonate.
Oligonucleotides were 5'-end-labeled by T.sub.4 polynucleotide kinase,
purified by polyacrylamide gel electrophoresis (PAGE) and sequenced by
either the Maxam-Gilbert or wandering spot methods. Maxam, A. M. and W.
Gilbert, in: Methods in Enzymology, L. Grossman and K. Moldave (ed.) pp
499-560, Academic Press (1980); Jay, E. et al., Nucleic Acids Research, 1:
331-353 (1974). For Maxam-Gilbert sequencing of fragments of this size, it
was found necessary to increase reaction times up to 30 minutes at
37.degree.. The presence of ddT at the 3' end of oligodeoxynucleotide did
not seem to hinder the action of the exonuclease snake venom
phosphodiesterase.
EXAMPLE 2
Oligodeoxynucleotide uptake studies
HeLa cells were grown in suspension culture, concentrated by centrifugation
at 600.times.g for 5 min. and resuspended at a concentration of
5.times.10.sup.7 to 5.times.10.sup.8 cells/ml of Dulbecco's modified
Eagle's medium (DME) without serum and kept on ice. Synthetic
oligodeoxynucleotides to be tested (10-30 nucleotides in length), were
labeled with .sup.32 P at the 5'-end by polynucleotide kinase at
2.times.10.sup.5 cpm/nmol, dissolved in DME without serum, and added to
the HeLa cell suspension (40 .mu.l oligodeoxynucleotide solution to 0.7 ml
ice cold HeLa cell suspension.). Alternatively, to generate an internally
labelled oligonucleotide, two decamers, one of them 5'.sup.32 P labelled,
were joined by T4 DNA ligase in the presence of an oligodeoxynucleotide
(12 nucleotides long) part of which was complementary to the 5' end of one
of the decamers and part of which was complementary to the 3' end of the
other decamer. The concentration of labelled oligodeoxynucleotide in the
HeLa cell suspension was usually 1.times.10.sup.-5 to 1.times.10.sup.-7 M.
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