Antisense inhibitors of the human immunodeficiency virus phosphorothioate oligonucleotides

Methods for modulating the expression of the HIV tat gene are disclosed comprising contacting tat RNA with oligonucleotide or oligonucleotide analog which can bind with at least a portions of the RNA. In accordance with the preferred embodiments, oligonucleotides or oligonucleotide analogs are designed to bind to portions of the tat RNA which are of significance to the expression of the gene coding for said RNA. In accordance with a preferred embodiment, methods of treatment of human immunodeficiency virus are disclosed.

FIELD OF THE INVENTION 
This invention relates to the field of therapeutics, particularly the 
treatment of infections of the human immunodeficiency virus (HIV). It 
relates to the design, synthesis and application of oligonucleotides and 
oligonucleotide analogs which inhibit the activity of the HIV and other 
retroviruses. 
BACKGROUND OF THE INVENTION 
This invention relates to materials and methods for modulating the activity 
of HIV RNA. The invention generally relates to the field of "antisense" 
compounds, compounds which are capable of specific hybridization with a 
nucleotide sequence of an RNA. In accordance with preferred embodiments, 
this invention is directed to methods for achieving therapeutic treatment 
of disease and regulating gene expression in experimental systems. 
It is well known that most of the bodily states in mammals including 
infectious disease states, are effected by proteins. Such proteins, either 
acting directly or through their enzymatic functions, contribute in major 
proportion to many diseases in animals and man. Classical therapeutics has 
generally focused upon interactions with such proteins in efforts to 
moderate their disease causing or disease potentiating functions. 
Recently, however, attempts have been made to moderate the actual 
production of such proteins by interactions with molecules that direct 
their synthesis, intracellular RNA. By interfering with the production of 
proteins, it has been hoped to effect therapeutic results with maximum 
effect and minimal side effects. It is the general object of such 
therapeutic approaches to interfere with or otherwise modulate gene 
expression leading to undesired protein formation. 
One method for inhibiting specific gene expression which has been adopted 
to some degree is the "antisense" approach, where oligonucleotide analogs 
complimentary to a specific, target, messenger RNA, mRNA sequence are 
used. A number of workers have reported such attempts. Pertinent reviews 
include C. A. Stein & J. S. Cohen, Cancer Research, vol. 48, pp. 2659-2668 
(1988); J. Walder, Genes & Development, vol. 2, pp. 502-504 (1988); C. J. 
Marcus-Sekura, Anal. Biochemistry, vol 172, 289-295 (1988); G. Zon, 
Journal of Protein Chemistry, vol. 6, pp-131-145 (1987); G. Zon, 
Pharmaceutical Research, vol. 5, pp. 539-549 (1988); A. R. Van der Krol, 
J. N. Mol, & A. R. Stuitje, BioTechniques., vol. 6, pp. 958-973 (1988) and 
D. S. Loose-Mitchell, TIPS, vol. 9, pp. 45-47 (1988). Each of the 
foregoing provide background concerning general antisense theory and prior 
techniques. 
Prior attempts to inhibit HIV by various antisense approaches have been 
made by a number of researchers. Zamecnic and coworkers have used 
phosphodiester oligonucleotides targeted to the reverse transcriptase 
primer site and to splice donor/acceptor sites P. C. Zamecnik, J. Goodchil 
Taguchi, P. S. Sarin, Proc. Natl. Acad. Sci. USA 83, 4143 (1986). 
Goodchild and coworkers have made phosphodiester compounds targeted to the 
initiation sites for translation, the cap site, the polyadenylation 
signal, the 5' repeat region and a site between the gag and pol genes. J. 
Goodchild, S. Agrawal, M. P. Civeira, P. S. Sarin, D. Sun, P. C. Zamecnik, 
Proc. Natl. Acad. Sci. U.S.A. 85, 5507 (1988). In the Goodchild study, the 
greatest activity was achieved by targeting the polyadenylation signal. 
Agrawal and coworkers have extended the studies of Goodchild by using 
chemically modified oligonucleotide analogs which were also targeted to 
the cap and splice donor/acceptor sites. S. Agarwal, J. Goodchild, M.P. 
Civeira, A. H. Thornton, P. S. Sarin, P. C. Zamecnik, Proc. Nat'l. Acad. 
Sci. USA 85, 7079 (1988). A portion of one of these overlapped a portion 
of the HIV TAR region but was not found to have exemplary effect. Neither 
was this oligonucleotide analog designed to interfere with the HIV TAR 
region. Agrawal and coworkers have used oligo-nucleotide analogs targeted 
to the splice donor/acceptor site inhibit HIV infection in early infected 
and chronically infected cells. S. Agrawal, T. Ikeuchi, D. Sun, P. S. 
Sarin, A. Konopka, J. Maizel, Proc. Natl. Acad. Sci. U. S. A. 86, 7790 
(1989). 
Sarin and coworkers have also used chemically modified oligonucleotide 
analogs targeted to the cap and splice donor/acceptor sites. P. S. Sarin, 
S. Agrawal, M. P. Civeira, J. Goodchild, T. Ikeuchi, P. C. Zamecnik, Proc. 
Natl. Acad. Sci. U. S. A. 85, 7448 (1988). Zia and coworkers have also 
used an oligonucleotide analog targeted to a splice acceptor site to 
inhibit HIV. J. A. Zaia, J. J. Rossi, G. J. Murakawa, P. A. Spallone, D. 
A. Stephens, B. E. Kaplan, J. Virol. 62, 3914 (1988). Matsukura and 
coworkers have synthesized oligonucleotide analogs targeted to the 
initiation of translation of the rev gene mRNA. M. Matsukura, K. 
Shinozuka, G. Zon, et al, Proc. Natl. Acad. Sci. USA 84, 7706 (1987); R. 
L. Letsinger, G. R. Zhang, D. K. Sun, T. Ikeuchi, P. S. Sarin, Proc. Natl. 
Acad. Sci. U. S. A. 86, 6553 (1989). Mori and coworkers have used a 
different oligonucleotide analog targeted to the same region as Matsukura. 
K. Mori, C. Boiziau, C. Cazenave, et al, Nucleic Acads Res. 17, 8207 
(1989). Shibahara and coworkers have used oligonucleotide analogs targeted 
to a splice acceptor site as well as the reverse transcriptase primer 
binding site. S. Shibahara, S. Mukai, H. Morisawa, H. Nakashima, S. 
Kobayashi, N. Yamamoto, Nucl. Acids Res. 17, 239 (1989). Letsinger and 
coworkers have synthesized and tested a oligonucleotide analogs with 
conjugated cholesterol targeted to a splice site. K. Mori, C. Boiziau, C. 
Cazenave, et al, Nucleic Acids Res. 17, 8207 (1989). Stevenson and Iversen 
have conjugated polylysine to oligonucleotide analogs targeted to the 
splice donor and the 5'-end of the first exon of the tat gene. M. 
Stevenson, P. L. Iversen, J. Gen. Virol. 70, 2673 (1989). Buck and 
coworkers have recently described the use of phosphate-methylated DNA 
oligonucleotides targeted to HIV mRNA and DNA. H. M. Buck, L. H. Koole, M. 
H. P. van Gendersen, L. Smith, J. L. M. C. Green, S. Jurriaans and J. 
Goudsmit. Science 248, 208-212 (1990). 
These prior attempts at targeting HIV have largely focused on the nature of 
the chemical modification used in the oligonucleotide analog. Although 
each of the above publications have reported some degree of success in 
inhibiting some function of the virus, a general therapeutic scheme to 
target HIV and other retroviruses has not been found. Accordingly, there 
has been and continues to be a long-felt need for the design of 
oligonucleotides and oligonucleotide analogs which are capable of 
effective, therapeutic antisense use. 
This long-felt need has not been satisfied by prior work in the field of 
antisense oligonucleotide therapy for HIV and other retroviruses and 
viruses. Others have failed to identify target sites in which antisense 
oligonucleotides or oligonucleotide analogs are therapeutically effective 
at reasonable rates of application. 
OBJECTS OF THE INVENTION 
It is a principal object of the invention to provide therapies for human 
diseases, particularly the human immunodeficiency virus and other human 
retroviruses. 
It is a further object of the invention to provide molecules, especially 
oligonucleotides and oligonucleotide analogs which perturb the structure 
of mRNA. 
Yet another object of this invention is to modulate gene expression in 
cells. 
A further object is to interfere with the secondary structure of RNAs 
through interaction of those structures with oligonucleotides or 
oligonucleotide analogs. 
These and other objects of this invention will become apparent from a 
review of the instant specifications. 
SUMMARY OF THE INVENTION 
Prior attempts at antisense targeting to HIV have been focused on 
inhibition of the synthesis of some particular viral protein thought to be 
essential to the success of the infection. In the present invention, the 
same goal (inhibition of viral gene expression) is achieved, but greater, 
therapeutically significant activity is obtained by targeting particular 
sites on the HIV or other retrovirus RNA. In the present invention, target 
RNAs which have important biological function have been found to be the 
key target sites. It has been determined that targeting these RNA regions 
is a key to effective antisense therapy with oligonucleotides and 
oligonucleotide analogs. 
It has now been discovered that compounds which specifically bind the tat 
RNA structure and interfere with tat trans-activation have activity as 
therapeutic agents for HIV infection. It is intended that all strains of 
HIV fall within the spirit and scope of this invention. While different 
strains of HIV may have different tat RNA sequences, this invention can be 
practiced on alternative strains of HIV by changing the sequence of the 
oligonucleotide or oligonucleotide analog to complement the structure of 
the alternative strain in accordance with the present teachings. 
In accordance with the present invention, methods of modulating the 
expression of genes are provided. The targeted RNA, or cells containing 
it, is contacted with oligonucleotide or oligonucleotide analog which can 
bind with at least a portion of the RNA. The gene is generally one which 
is believed to give rise to a disease state in an organism and is 
typically a virus or, preferably, a retrovirus such as HIV, although other 
infectious organisms can be so attacked leading to therapeutic methods for 
treating diseased states by administering oligonucleotides or 
oligonucleotide analogs to animals suspected of having viral or retroviral 
infections. 
It has now been found that oligonucleotide or oligonucleotide analogs can 
be designed, especially for retroviruses such as HIV, which are effective 
in diminishing the infection. For HIV, a number of sequences have been 
found which are effective and persons of ordinary skill in the art will 
likely identify others. These sequences are the first, practically 
effective antisense sequences which have been shown to be effective in 
inhibiting the tat region of HIV and to lead to HIV therapeutics. 
Accordingly, this invention provides oligonucleotides and oligonucleotide 
analogs capable of binding with at least a portion of tat mRNA of HIV. 
Such oligonucleotides and analogs have been found corresponding to the 
nucleotide sequences G G C T C C A T T T C T T G C T C T C , 
CCATTTCTTGCTCTCCTCTGT, GCTATGTCGACACCCAATTC, CCGCCCCTCGCCTCTTGCCG, 
CGGGTCCCCTCGGGATTGGG, and CACCTTCTTCTTCTATTCCT. It is preferred that the 
oligonucleotides and analogs have at least about 6 contiguous subunits of 
such sequences, with at least about 10 being preferred and at least about 
15 being still more preferred. It is preferred for some embodiments that 
the oligonucleotide or oligonucleotide analog substantially correspond to 
a given sequence. By this is meant that the oligonucleotide or analog have 
every subunit of the sequence or an effective substitute. Thus, 
substitutions such as U for T and the like may be made in wild type 
oligonucleotides. Additionally, of course, other chemical modifications to 
form oligonucleotide analogs may be made without departing from the spirit 
of this invention. 
The sequence portions may fall anywhere within the given sequences to 
likely have effect. Oligonucleotides and analogs, such as the preferred 
phosphorothioate analogs may be presented in a pharmaceutically acceptable 
carrier. 
Methods of inhibiting gene expression, especially tat gene expression have 
also been discovered. Such inhibition may be employed for therapeutics, 
for diagnosis or for research. Thus, methods of treating an animal 
suspected of having a disease characterized by tat gene expression, 
especially AIDS, have been discovered comprising contacting the animal 
with oligonucleotides or oligonucleotide analogs in accordance with this 
invention. 
It is preferred that the oligonucleotide or oligonucleotide analog be 
capable of binding with at least about six subunits of the RNA portion. It 
is more preferred that from eight to fifty units be capable of being 
bound, with from about 10 to about 20 subunits being even more preferred. 
In accordance with preferred embodiments, the oligonucleotide of 
oligonucleotide analog is capable of forming a duplex structure with the 
portion of RNA. While the mechanism of the interaction is not known with 
certainty, it is possible that it may effect modulation of gene expression 
through a number of ways. 
In accordance with preferred embodiments, the RNA portion which is 
interfered with comprises at least a part of the tat mRNA of HIV. The 
oligonucleotides and oligonucleotide analogs in accordance with this 
invention are, themselves believed to be novel. Thus, oligonucleotides 
which are capable of interacting with portions of tat RNA are 
comprehended. Thus, animals suspected of having the disease are contacted 
with oligonucleotide or oligonucleotide analog which can bind with the tat 
RNA. In particular, the present invention is believed to be effective in 
the treatment of HIV infections in mammals, especially man.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
It has been discovered to be possible to regulate the activity of HIV tat 
RNA in cells by introducing oligonucleotides or oligonucleotide analogs 
which bind to the tat mRNA. The oligonucleotides or oligonucleotide 
analogs interfere with the normal function of the mRNA and these methods 
can be used to treat diseases, particularly HIV. 
In the context of this invention, the term oligonucleotide refers to a 
plurality of joined nucleotide units formed from naturally-occurring bases 
and cyclofuranosyl groups joined by native phosphodiester bonds. This term 
effectively refers to naturally-occurring species or synthetic species 
formed from naturally-occurring subunits. 
"Oligonucleotide analog," as that term is used in connection with this 
invention, refers to moieties which function similarly to oligonucleotides 
but which have non naturally-occurring portions. Thus, oligonucleotide 
analogs may have altered sugar moieties or inter-sugar linkages. Exemplary 
among these are the phosphorothioate and other sulfur containing species 
which are known for use in the art. They may also comprise altered base 
units or other modifications consistent with the spirit of this invention. 
In accordance with certain preferred embodiments, at least some of the 
phosphodiester bonds of the oligonucleotide have been substituted with a 
structure which functions to enhance the ability of the compositions to 
penetrate into the region of cells where the RNA whose activity is to be 
modulated is located. It is preferred that such linkages be 
sulfur-containing. It is presently preferred that such substitutions 
comprise phosphorothioate bonds. Others such as alkyl phosphothioate 
bonds, N-alkyl phosphoramidates, phosphorodithioates, alkyl phosphonates, 
and short chain alkyl or cycloalkyl structures may also be useful. In 
accordance with other preferred embodiments, the phosphodiester bonds are 
substituted with structures which are, at once, substantially non-ionic 
and non-chiral. Persons of ordinary skill in the art will be able to 
select other linkages for use in the practice of the invention. 
It is generally preferred for use in some embodiments of this invention 
that the 2' position of the linking sugar moieties in at least some of the 
subunits of the oligonucleotides or oligonucleotide analogs be 
substituted. Thus, 2' substituients such as OH, SH, F, OCH.sub.3, OCN, 
OCH.sub..epsilon. CH.sub.3 : where n is from 1 to about 20 and other 
substituients having similar properties may be useful in some embodiments. 
Oligonucleotide analogs may also include species which include at least 
some modified base forms. Thus, purines and pyrimidines other than those 
normally found in nature may be so employed. Similarly, modifications on 
the cyclofuranose portions of the nucleotide subunits may also occur as 
long as the essential tenets of this invention are adhered to. 
Such analogs are best described as being functionally interchangeable with 
natural oligonucleotides (or synthesized oligonucleotides along natural 
lines), but which have one or more differences from natural structure. All 
such analogs are comprehended by this invention so long as they function 
effectively to bind to selected portions of tat RNA. 
The oligonucleotides and oligonucleotide analogs in accordance with this 
invention preferably comprise from about 3 to about 100 subunits. It is 
preferred that such oligonucleotides and analogs comprise at least about 6 
subunits with from about 8 to about 50 subunits being more preferred, and 
still more preferred to have from about 10 to about 20 subunits. As will 
be appreciated, a subunit is a base and sugar combination suitably bound 
to adjacent subunits through phosphodiester or other bonds. 
The oligonucleotides and oligonucleotide analogs of this invention can be 
used in diagnostics, therapeutics and as research reagents and kits. For 
therapeutic use, the oligonucleotide or oligonucleotide analog is 
administered to an animal, especially a human, such as are suffering from 
a virus or retrovirus infection such as AIDS. 
It is generally preferred to apply the therapeutic agents in accordance 
with this invention internally such as orally, intravenously or 
intramuscularly. Other forms of administration, such as transdermally, 
topically or intralesionally may also be useful. Inclusion in 
suppositories may also be useful. Use of the oligonucleotides and 
oligonucleotide analogs of this invention in prophylaxis is also likely to 
be useful. Use of pharmacologically acceptable carriers is also preferred 
for some embodiments. 
In accordance with the present invention, it will be understood that the 
term "to bind" as it refers to the interaction between an oligonucleotide 
or oligonucleotide analog and an RNA portion or subportion may have any of 
several, related meanings. Thus, the present invention comprehends binding 
of an oligonucleotide or analog with at least one portion of tat RNA. It 
will be understood that the oligonucleotide or analog will bind with at 
least one portion of the RNA portion in a Watson-Crick fashion so as to 
form, locally, a heteroduplex between the RNA portion and the 
oligonucleotide or analog. This heteroduplex formation is believed to 
result in alteration of the structure or function of the RNA portion. The 
exact mechanism and the result of this effect is not known with certainty, 
yet it is believed that the normal structure or function of the RNA 
portion is gradually replaced by the binding of the oligonucleotide with 
one or more portions of the RNA. Since the electronic and steric factors 
which attend the new heteroduplex are different from those of the 
natural-occurring RNA portion, the effectiveness and nature of the 
function to generate protein from the RNA is interfered with. The 
resulting formation of defective or missing protein manifests itself 
overall as a modulation in the expression of the gene coding for the RNA. 
In short, any interaction or binding of oligonucleotide or oligonucleotide 
analog with tat RNA is believed to have the potential for interference 
with RNA function and, hence, for modulation the expression of the gene 
from which the RNA derives. Tat RNA targets have been found which exhibit 
overall diminution of activity of HIV when oligonucleotides or 
oligonucleotide analogs corresponding to portions of the target are 
administered to infected cells. 
While a wide variety of oligonucleotides and oligonucleotide analogs are 
believed to be useful in practice of the present invention, it has been 
found to be preferred to design such oligonucleotides and analogs so as to 
bind with at least about six subunits of a portion of RNA. In accordance 
with other preferred embodiments, oligonucleotides which combine with from 
about six to about 30 and even more preferably with about 10 to about 20 
subunits are preferred. As discussed above, it is presently believed that 
the tat RNA of HIV comprises an excellent target for employment of the 
present invention. Accordingly, preparation of oligonucleotide or 
oligonucleotide analog for binding with one or more portions of the tat 
RNA region of HIV are preferred. 
Therapeutics are particular objects of the present invention. Thus, 
presenting oligonucleotides and oligonucleotide analogs in accordance with 
the present invention in pharmaceutically acceptable carriers may be 
highly useful. This is especially true for treatment of the disease AIDS. 
Overall, it is preferred to administer to patients suspected of suffering 
from the foregoing disease states with amounts of oligonucleotide or 
analog, in either native form or suspended in a carrier medium in amounts 
and upon treatment schedules which are effective to reduce the 
symptomology of that disease. It is within the scale of a person's skill 
in the art to determine optimum dosages and treatment schedules for such 
treatment regimens. 
An elaborate set of control elements in the HIV genome determine whether 
the virus replicates or remains dormant. Of the nine genes identified in 
the HIV genome, only three are from the core and envelope. W. A. 
Haseltine, F. Wong-Staal, Scientific American Oct., 52 (1988). The other 
six genes are involved in regulation of the production of viral proteins. 
Regulatory genes work by encoding a protein that interacts with a 
responsive element somewhere else on the viral genome. The major 
regulatory gene responsible for initiating the burst of replication is the 
tat (transactivator) gene. FIGS. 1A and 1B is a sequence for the cDNA of 
the tat region in HIV. The product of the tat gene, tat protein, works by 
interaction with a short sequence element known as TAR (trans-acting 
responsive element). The TAR sequence is encoded in the viral long 
terminal repeats (LTR's), and therefore is included in the mRNA from every 
HIV gene. 
Expression of the tat protein results in increased expression of other HIV 
genes up to 1,000 fold, including the tat gene itself. Because of this 
autoregulatory positive feedback, and the fact that the TAR sequence in 
included in the mRNA from every HIV transcript, a immense amount of viral 
gene expression is triggered when the tat gene is activated. The 
interaction between the tat gene and the TAR element is therefore crucial 
to the life cycle of the HIV, and specific disruption of this interaction 
is likely to interrupt the propagation of the virus. 
The mechanism of trans-activation of TAR-containing genes by the tat 
protein has recently been studied intensely. Philip,A. Sharp, Robert,A. 
Marciniak, Cell 59, 229 (1989). Although much remains to be learned, two 
important points have become clear; that tat increases the expression of 
TAR-containing genes by increasing both the amount of viral mRNA and the 
efficiency of its translation, and that TAR functions as an RNA structure, 
rather than a DNA structure. 
The unusual conclusion that tat increases the transcription of 
TAR-containing genes, but does so by interacting with the TAR element in 
RNA was derived from a number of observations. Philip,A. Sharp, Robert,A. 
Marciniak, Cell 59, 229 (1989). In order to achieve trans-activation, the 
TAR element must be located immediately downstream from the site of 
initiation of transcription. Moreover, TAR is orientation dependent; if 
inserted in the inverse orientation, it fails to function. 
Some of the strongest evidence that tat interacts with TAR as an RNA 
structure has come from mutagenesis experiments. Efforts to study the TAR 
element were stimulated by the observation that the tat protein from HIV-1 
was capable of trans-activating vectors containing the TAR region of 
HIV-2, a different strain of virus, even though there is very little 
primary sequence homology in the TAR region between the two strains. S. 
Feng, E. C. Holland, Nature 334, 165 (1988). However, examination of the 
TAR sequence from HIV-1 and HIV-2 with computer programs that predict RNA 
secondary revealed the potential of RNA stem-loop structures, with a 
single stemloop in the TAR region of HIV-1 and three stem-loop structures 
in HIV-2. Although the compositions and lengths of the stems were 
divergent, all four loops contained the pentanucleotide CUGGG as shown in 
FIGS. 1A and 1B. Mutagenesis experiments revealed that each of the 
nucleotides present in the loop are absolutely essential for 
trans-activation by tat, but that base substitutions in the stem were 
tolerated to some extent so long as the stem structure was maintained. S. 
Feng, E. C. Holland, Nature 334, 165 (1988). 
Further evidence for the TAR structure functioning as RNA was obtained from 
experiments in which the sequences flanking the stem-loop structure were 
altered creating competing secondary structures in the RNA that were more 
stable than the natural TAR stem-loop. B. Berkhout, Cell 59, 273 (1989). 
This was accomplished by introducing additional sequences into the 
TAR-containing RNA that were antisense to the 5' side of the stem-loop 
structure. Trans-activation of the modified TAR structure was lost, 
suggesting that the TAR sequences alone are not sufficient for 
trans-activation, but that these sequences must fold up in the proper 
secondary structure to be active. It also suggests that antisense 
sequences to the TAR stem-loop are capable of disrupting the natural RNA 
structure. 
Direct biochemical evidence for TAR stem-loop structure has also been 
obtained. The TAR RNA has been enzymatically synthesized in vitro and 
probed with enzymes selectively cleave single stranded regions of RNA, but 
not duplex structures. The results of the enzyme cleavage patterns were 
consistent with the computer predicted RNA secondary structure. B. 
Berkhout, Cell 59, 273 (1989). 
In summary, there is strong and direct evidence from a number of studies 
that the HIV tat protein is responsible for triggering an enormous amount 
of viral gene expression, that this occurs by interaction with the TAR 
sequence which is incorporated into every HIV mRNA transcript, that the 
HIV TAR sequence functions as an RNA structure and that the correct TAR 
RNA structure is essential for tat transactivation. 
TAR and tat function has been studied by removing the genes from the HIV 
genome and studying them in cell lines in isolation. Vectors have been 
constructed to study the interactions between the tat protein and TAR 
element. The tat gene is expressed under the SV40 promoter. The TAR region 
is expressed from a separate plasmid fused to an easily assayed reporter 
gene, the placental alkaline phosphatase gene (PAP). P. Henthorn, P. 
Zervos, M. Raducha, H. Harris, T. Kadesch, Proc. Natl. Acad. Sci. USA 85, 
6342 (1988) Enzymatic activity in cell culture models has been shown to be 
dependent upon both the presence of the essential elements of the TAR 
region and the presence of the tat protein. P. Sharp, R. Marciniak, Cell 
59, 229 (1989); S. Feng, E. C. Holland, Nature 334, 165 (1988); Michael,F. 
Laspia, Andrew,P. Rice, Michael,B. Mathews, Cell 59, 283 (1989); J. A. 
Garcia, D. Harrich, E. Soultanakis, F. Wu, R. Mitsuyasu, R. B. Gaynor, 
EMBO J. 8, 765 (1989); and B. Berkhout, Cell 59, 273 (1989). In essence, 
the vector system reconstitutes the events of tatmediated TAR 
transactivation in which occurs in HIV infected cells. 
TAT/TAR trans activation can be conveniently assayed by placing the human 
placental alkaline phosphatase gene (PAP) under the regulatory control of 
the HIV-1 LTR sequences, which contain enhancer, promoter, and tar 
elements. A plasmid containing the HIV-1 LTR, pHIVCAT-0 (S. Feng, E. C. 
Holland, Nature 334, 165 (1988)), contains HIV U3 in its entirety and R up 
through position +78 (a HindIII site). Digestion of this plasmid with a 
combination of HindIII and AatII releases the CAT cassette along with the 
SV40 sequences responsible for the processing of the RNA. A second 
plasmid, pSV2Apap, contains the PAP cassette with eukaryotic processing 
signals, under the transcriptional control of an SV40 promoter. P. 
Henthorn, P. Zervos, M. Raducha, H. Harris, T. Kadesch, Proc. Natl. Acad. 
Sci. USA 85, 6342 (1988). The PAP cassette and processing sequences were 
released from the plasmid by digestion with HindIII and AatII. A new 
plasmid, pHIVPAP, was created by ligating the HindIII/AatII fragment 
containing the HIV-1 LTR and vector sequences from pHIVCAT-0, to the 
HindIII/AatII PAP cassette from pSV2Apap. 
To test the activity of oligonucleotide analogs, pcDEBtat and pHIVPAP were 
co-transfected into HeLa cells by calcium/phosphate precipitation. The 
effects of the selected oligonucleotide analogs was determined as follows. 
HeLa cells were split 1:8 into 6-well dishes the day prior to the 
transfections. For each dish, lug of pHIVPAP and 12ug of pcDEBtat were 
precipitated in 500 .mu.l of HBS and 32 .mu.l of 2.5 M CaCl.sub.2. The 
CaPO.sub.4 precipitate was divided evenly between the 6 wells. 
Oligonucleotides or oligonucleotide analogs were precepitated in the same 
manner and added to the cells at the concentrations indicated in the 
figures. The precipitate was allowed to sit on the cells for 20 minutes 
then complete media was added and the cells were incubated for an 
additional 4 hours. The cells were then shocked with 10% glycerol in HBS. 
After 48 hours, cells were harvested and protein and PAP assays performed 
as described by Henthorn et al. P. Henthorn, P. Zervos, M. Raducha, H. 
Harris, T. Kadesch, Proc. Natl. Acad. Sci. USA 85, 6342 (1988) with the 
following modifications. The cells were harvested in 0.5 ml of TBS, of 
which 0.1 mls were used for use in the protein assay. The remaining 0.4 
mls of cell suspension was pelleted then resuspended in 50 .mu.l TBS. 
Endogenous phosphatases were inactivated by heating the cells at 
65.degree. C. for 30 min. The heat stable human placental alkaline 
phosphatase activity was assayed by the addition of PNPP (0.5 ml, 5 mM 
PNPP) to the cell suspension, which was then incubated at 37.degree. C. 
Activity was determined at 30 minute intervals using 150 .mu.l aliquots of 
the reaction mixture and measuring absorbance at 405 nm with a Titertek 
Multiscan MCC 340 ELISA plate reader. The PAP activity was normalized 
total protein in each well as determined by Bio-Rad protein assay, in 
which 1 5 of the harvested cells in TBS(0.1 .mu.l) were added to 30 of 
BioRad Protein Reagent, then incubated for 10 minutes at room temperature, 
followed by measurement of absorbance at 595 nm using the Titertek plate 
reader. 
Cells were treated with the following oligonucleotide analogs having 
phosphorothioate backbones: 
##STR1## 
The data are displayed graphically in FIG. 2. Significant diminution in PAP 
activity, which is a direct measure of gene expression from the HIV LTR, 
was exhibited by the phosphorothioate oligonucleotide analogs bearing code 
numbers 461, 462, 463, 464 and 465. Oligonucleotide 466 and the control 
oligonucleotide, which was not designed to be complementary to the tat 
mRNA did not exhibit significant activities in this assay. The apparent 
increase in PAP activity at the 1 uM dose for the control oligonucleotide 
and compound number 466 probably resulted from a carrier effect in which 
the oligonucleotides facilitated uptake of the plasmids at the time of 
transfection. This effect may have been present in experiments with the 
active oligonucleotides, but was masked by the specific inhibitory 
activities of these compounds.