Inhibition of HTLV-III by exogenous oligonucleotides

Inhibition of HTLV-III by adminstration of an oligonucleotide complementary to highly conserved regions of the HTLV-III genome necessary for HTLV-III replication and/or gene expression is described, as are oligodeoxynucleotide sequences which are complementary to those regions, methods of inhibiting HTLV-III replication and gene expression and methods of determining the presence or absence of HTLV-III virus in samples such as blood, saliva, urine and tears.

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.

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. 
Cells were incubated under sterile conditions at 37.degree. for up to 20 
hours. Samples were cooled at 0.degree. , diluted to 10 ml with DME, and 
centrifuged lightly to pellet the cells. The supernatant fluid was poured 
off and saved and the centrifugation tube drained on filter paper. The 
cell pellets were then washed six times, each time in 9 ml of ice-cold 
DME. The supernatants were saved and monitored for .sup.32 P 
radioactivity. By the sixth wash, virtually no radioactivity was detected 
in the wash fluid. The cell pellets were then resuspended on 0.7 ml of ice 
cold DME and transferred to an electroporation cell. Electroporation was 
carried out at 0.degree. by a variation of the technique described by 
Potter and co-workers in Potter et al., Proceedings of the National 
Academy of Sciences, USA, 81: 7161-7165 (1984), the teachings of which are 
incorporated herein by reference. During electroporation, a short high 
voltage pulse was applied across the electroporation cuvette containing 
the cell pellets; in this way, the cell membranes were made temporarily 
leaky or porous, allowing oligonucleotides to pass out of the cells. The 
electroporation cuvette was kept in an ice bath for 15 min. following 
electroporation. The contents were transferred to a 1.5 ml Eppendorf 
microfuge tube, and centrifuged 5 min. at 12,000.times.g. The supernatant 
solution (i.e., oligomer which has entered the cell) was removed, and 
radioactivity of both the supernatant and the pellet (which contained the 
nuclear and cell membrane component) was determined by scintillation 
counting. 
Two other variants of this method were also used to determine whether 
externally added labeled oligodeoxynucleotides enter CEF and HeLa cells. 
In the case of CEF cells, which had been grown in monolayers in 75 
cm.sup.2 Falcon flasks, the DME medium containing serum was removed, the 
cells were washed once with serum-lacking DME; 2 ml of DME containing 
.sup.32 P-labeled oligodeoxynucleotide were added; and the resulting 
combination was incubated at 37.degree. C. for 15 minutes. The cells were 
next washed six times at 37.degree. C. (ambient), each time with 10 ml of 
DME. 2 ml of 1N formic acid was then added, and the cells were kept on ice 
for 15 min. The same procedure was carried out with CEF or HeLa cells 
except that instead of 1N formic acid, distilled water was added after 
incubation to lyse the cells. Results were similar with both procedures; 
approximately half as much radioactivity was associated with the nuclear 
and cell membrane fraction (sedimented by centrifugation 5 min. at 
12,000.times.g) as was associated with the non-sedementing fraction of the 
cell. 
The possibility that treatment of labeled cells with either 1N formic acid 
or distilled water caused dissociation of radiolabeled oligomer (which had 
never entered the interior of the cell) from the cell membrane fraction 
was tested by using the above modified electroporation technique as 
described. Results using electroporation agree with those where cells were 
ruptured by hypotonicity or 1N formic acid. 
These tests made it possible to assess uptake of .sup.32 P-oligonucleotides 
by the cultured cells described. Inhibition of viral replication by 
exogenous oligodeoxynucleotides depends upon their uptake in sufficient 
amounts by the cells; this is not the case when endogenously transcribed 
or microinjected anti-sense RNAs are used. The permeability of cultured 
mammalian cells to the oligodeoxynucleotides has been demonstrated by 
these tests to be as follows: 
(1) Under the experimental methods described, cellular uptake of 
20-nucleotide sequences, labeled with .sup.32 P either internally or 
terminally, increased during the initial few hours of incubation. At an 
external concentration of 1.times.10.sup.-7 M, after 4 hours of incubation 
at 37.degree. C., the internally labeled 20 -nucleotide sequence 
TAGTCTCAAT-.sup.32 P-GGGCTGATAA reached a concentration inside the HeLa 
cell of approximately 2.times.10.sup.-9 M. In another experiment conducted 
using the same conditions described, at an external concentration of 
2.times.10.sup.-5 M, after 15 minutes of incubation at 37.degree. C., the 
internally labelled 20-nucleotide sequence TAGTCTCAAT-.sup.32 P-GGGCTGATAA 
reached an apparent concentration inside the CEF cell of about 
1.5.times.10.sup.-6 M. 
(2) At 15 min. and 4 hour time periods labeled oligodeoxynucleotides 
released from the cells by electroporation were largely intact, as judged 
both by migration on thin layer DEAE plates, in Homo V Jay, E. et al., 
Nucleic Acids Research, 1: 331-353 (1974), and by PAGE using 
oligodeoxynucleotide markers. However, degradation of 
oligodeoxynucleotides increased with incubation time. By 20 hours, a large 
fraction of oligodeoxynucleotide was degraded intracellularly and 
extracellularly, but undergraded oligomer was still detected, and thus 
endured long enough to have the desired inhibiting effect. 
(3) Terminally labeled oligodeoxynucleotides disappeared more rapidly than 
those labeled internally. This indicates that phosphomonoesterase activity 
is more rapid than endonuclease activity. 
EXAMPLE 3 
Inhibition of HTLV-III replication by complementary oligodeoxynucleotides 
The primary nucleotide sequence of the HTLV-III/LAV genome has been 
determined during the past year by several groups of investigators, as 
indicated above. The following regions of the genome were selected as 
oligonucleotide competitive inhibition targets: (a) a sequence of 
nucleotides 5' to the primer tRNA.sup.lys binding (association) site; (b) 
a sequence straddling the primer binding site and the adjacent region, in 
the 5' direction; (c) a sequence at the primer binding site and (d) 
sequences from the splice sites (i.e., splice donor site, splice acceptor 
site) of the pre-mRNA that expresses the 3'-open reading frame regions. 
Sodroski, J. et al., Journal of Virology, 55: 831-835 (1985); Wong-Staal, 
F. and Gallo, R. C., Nature, 317: 395-403 (1985). Their locations on the 
HTLV-III/LAV genome are indicated in FIG. 1. 
(A) Sequences complementary to the primer binding site and sequences 
vicinal to the primer binding site in a 5' direction. 
Several sequences complementary to regions immediately adjacent, in a 
5'-direction, to the tRNA.sup.lys primer binding site in HTLV-III, or 
complementary to the primer binding site were synthesized. These are a 
12-nucleotide sequence (5'CTGCTAGAGATddT) a 20-nucleotide sequence 
(5'-CTGCTAGAGATTTTCCACAC), and a 26-nucleotide sequence with a 3' terminal 
non-complementary tail of (pA).sub.3 (5'-TTCAAGTCCCTGTTCGGGCGCCAAAA). As 
shown in Table 1, the 12-nucleotide sequence is complementary to a 
sequence of nucleotides close to the primer binding site (in the 5' 
direction); the 20-nucleotide sequence is also complementary to a sequence 
close to the primer binding site in the 5' direction, and includes the 
first 11 nucleotides of the 12-nucleotide sequence, as well as nine 
additional nucleotides. The 26-nucleotide sequence is complementary to the 
primer binding site. These oligodeoxynucleotides were tested on tissue 
cultures of HTLV-III-infected cells; they were added to the cultures at 
the concentrations shown in Table 1 (column 3). Both reverse transcriptase 
activity and production of viral-encoded p15 and p24 proteins were 
measured to determine inhibition of viral replication and inhibition of 
gene expression, respectively. 
TABLE I 
__________________________________________________________________________ 
Inhibition of HTLV-III Replication and Protein Expression 
by Complementary Oligodeoxynucleotides 
Oligomer 
Conc. 
HTLV-III Cell 
HTLV-III 
Percent Inhibition 
Sequence Length 
.mu.g/ml 
Binding Site 
Line 
Added RT p15 
p24 
__________________________________________________________________________ 
0 H9 - 0 0 0 
0 H9 + 0 0 0 
CCCCAACTGTGTACT 15 5 none H9 + 0 0 0 
" " 10 none H9 + 0 0 0 
CTGCTAGAGATddT 12 5 5'-vicinal to PBS 
PB + 30 0 17 
" " 10 " " + 36 0 50 
" " 20 " " + 40 35 36 
" " 5 " H9 + 10 15 35 
" " 10 " " + 17 15 50 
" " 10 " H9 + 0 10 12 
" " 20 " " + 0 28 38 
CTGCTAGAGATTTTCCACAC 
20 50 " PB + 50 50 50 
" " 10 .times. 3.sup.+ 
" H9 + 50 75 75 
" " 50 " " + 23 27 30 
TTCAAGTCCCTGTTC- 26 50 at PBS.sup..noteq. 
H9 + 80 4 8 
GGGCGCCAAAA 
GCGTACTCACCAGTCGCCGC 
20 50 splice donor 
H9 + 85 40 60 
site 
CTGCTAGAGATTAA 14 50 5'-vincinal to 
H9 + 75 8 11 
PBS.sup..sctn. 
ACACCCAATTCTGAAAATGG 
20 50 splice H9 + 67 95 88 
acceptor 
site 
__________________________________________________________________________ 
.sup.+ 10 ug/ml. on days 1,2 and 3 from time of infection 
.sup..noteq. Has 3 noncomplementary bases at 3' end 
.sup..sctn. Has 2 noncomplementary bases at 3' end 
A+ PBS indicates directly competing at primer binding site 
PB: peripheral human blood cells 
H9: transformed immortalized human T cell line 
Reverse transcriptase activity was measured by the method described by 
Sarin and co-workers, which is a modification of an earlier method 
described by Baltimore and Smoller. The modified method is described in 
Sarin, P. S. et al., Biochemica Biophysica Acta, 470: 198-206 (1977) and 
the earlier method in Baltimore, D. and Smoller, D., Proceedings of the 
National Academy of Sciences, U.S.A., 68: 1507-1511 (1971); the teachings 
of both references are incorporated herein by reference. 
HTLV-III-protein expression was measured by immunofluorescence using 
monoclonal antibodies to HTLV-III p15 and p24 as described in Sarin et 
al., Biochemistry and Pharmacology, 34: 4075-4078 (1985), the teachings of 
which are incorporated herein by reference. 
In separate experiments, peripheral human blood cells and transformed 
T-lymphocyte (H9) cells were infected with HTLV-III; the 
oligodeoxynucleotides were added just once (at time zero), unless 
otherwise indicated. Assays for inhibition were carried out at 96 hours. 
(B) Sequences complementary to splice sites of pre-mRNA 
A 20-nucleotide sequence complementary to a splice donor site from the 
3'-open reading frame region, and a 20-nucleotide sequence complementary 
to a splice acceptor site were produced. These oligodeoxynucleotides were 
tested as described in part A (above); their effects were also measured 
through determintion of reverse transcriptase activity and production of 
viral-encoded proteins. 
(C) Results of inhibition tests 
The results of testing using the oligodeoxynucleotides described in (A) and 
(B) of this example are shown in Table 1. The greatest inhibition occurred 
when an oligonucleotide having the sequence ACACCCAATTCTGAAAATGG, which is 
complementary to the splice acceptor site in H9 cells, was added at 50 
.mu.g/ml (9.times.10.sup.-6 M). Percent inhibition as shown in the table 
is based on comparison with control values obtained for HTLV-III-infected 
cells incubated without oligodeoxynucleotide. As indicated in Table 1 
(columns 7-9), reverse transcriptase activity was inhibited by 67%, p15 
protein production by 95% and p27 protein production by 88% when this 
sequence was used. The oligodeoxynucleotide was given just once (at time 
zero), and inhibitory effects were observed at 96 hours. Marked inhibition 
was also found with the other oligodeoxynucleotides, as shown in Table 1. 
For example, when the 12-mer sequence complementary to the region of the 
HTLV-III/LAV genome adjacent, in the 5' direction, the tRNA.sup.lys primer 
binding site was added to HTLV-III-infected cells at the concentrations 
shown in Table 1, reverse transcriptase activity was inhibited from 10-17% 
in H9 cells and 30-40% in PB cells. Viral p15 and p24 protein production 
was inhibited by 15% and by 35-50%, respectively, in H9 cells; in PB 
cells, inhibition of p15 protein production ranged from 0-35% and of p24 
protein production, from 17-50%. When the 20-nucleotide sequence was used, 
reverse transcriptase activity was inhibited in H9 cells by 23-50% and 
viral protein production by 27-75%. Fifty percent inhibition of all three 
activities was observed in PB cells as a result of addition of the 
20-nucleotide sequence. Based on work on inhibition of Rous sarcoma virus 
in tissue culture, it seems likely that daily addition of competitor 
oligodeoxynucleotide is more effective than a single dose at time zero. 
Zamecnik, P. C. and M. L. Stephenson, Proceedings of the National Academy 
of Sciences, USA, 75: 280-284 (1978). This is also consistent with 
time-related intracellular and extracellular degradation of added 
oligodeoxynucleotide, since measurement of efficacy occurs at 96 hours. 
Although overall variation in assays of other chemotherapeutic agents for 
HTLV-III is in the vicinity of .+-.5 percent, it is considerably higher 
where oligodeoxynucleotides are being tested (cf. Table I). This may be 
related to variable nuclease activity, both intracellular and 
extracellular, in tissue cultures of H9 and PBS cells. Such an effect 
would be more marked at lower concentration of oligodeoxynucleotides. 
Oligodeoxynucleotides blocked at the 3' end by ddT, the isourea group, or 
other chain terminators may prove to be more effective inhibitors than 
those described above. For example, work on inhibition of Rous sarcoma 
virus has shown that the 3' end blocked hybridon was a more effective 
inhibitor than a hybridon having an unblocked 3' end. This is particularly 
pertinent to prevention of initiation of replication at loci close to the 
primer binding site.