AAPS PharmSci 2002; 4 (2) article 9 (http://www.aapspharmsci.org).

Is Antisense an Appropriate Nomenclature or Design for Oligodeoxynucleotides

Aimed at the Inhibition of HIV-1 Replication?
Submitted: December 21, 2002; Accepted: March 23, 2002; Published: April 30, 2002
Carole Lavigne1,2, Jocelyn Yelle2,Gilles Sauvé2, and Alain R. Thierry 3,4
1
Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec,
Canada H3C 3J7
2
Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada H7N 4Z3
3
Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255
4
MedinCell Project, Laboratoire des Défenses Antivirales et Antitumorales, UMR 5124, 1919 route de Mende, 34293
Montpellier Cedex 5, France

ABSTRACT We have evaluated the specificity and the

variation in activity against human immunodeficiency
virus (HIV) infection of antisense oligodeoxynucleotides
(ODNs) with regard to factors such as dose-response
range, number and choice of experimental controls,
backbone modifications of the ODNs, type of cell
infection, length of assays, and delivery approach. The
highest level of inhibition was achieved in our long-term
assay with MOLT-3 cells acutely infected with HIV-1
(IIIB) and treated with free phosphorothioate-modified
ODNs (PS-ODNs). The highest level of specificity was
observed in our short-term assay with MOLT-3 cells
acutely infected with HIV-1 (IIIB) and treated with free
PS-ODNs. The highest potency (IC50 level) was
observed in our short-term chronic-infection model with
(DLS)-delivered ODNs in which the DLS delivery
improved the ODN activity up to 106 times compared to
the activity of free ODNs. Thus, the near blocking of HIV
replication obtained when using PS-ODNs appears
because of the addition of extracellular and/or
membranar effects. The higher efficacy of PS-ODNs
compared to unmodified ODNs, when both are delivered
with the DLS system, was demonstrated solely in our
short-term assay with MOLT-3 cells. Important variations
in the level of sequence specificity were observed and
depended on the type of control used and the type of cell
assay employed. It seems that all 3 groups of control-
tested, random, sense sequence, and non-antisense
T30177 ODNs might have distinct activity and,
consequently, different modes of action in inhibiting HIV
replication. Our data buttress the notion that the
contribution of the sequence-specific mediated mode of
action is minor compared to the other mechanisms
involved in ODN antiviral activity.
KEY WORDS: fibroblast growth factor, keratinocyte growth
factor, pharmacology, pharmacokinetics clinical trial.
*Corresponding author: Alain R. Thierry, Laboratoire des
Défenses Antivirales et Antitumorales, UMR 5124, Case
courrier 37, 1919, route de Mende, 34293 Montpellier Cedex 5,
France; E-mail: thierry1@micronet.fr
1
INTRODUCTION
Antisense oligodeoxynucleotides (ODNs) are a novel
class of therapeutic agents that offer an attractive
approach for the treatment of viral infections, cancer,
and genetic disorders by controlling cellular or viral gene
expression at the mRNA and possibly gene levels.
These molecules are designed to block the action of
specific genes by binding to their RNA transcripts by a
phenomenon called hybridization arrest through Watson-
Crick base-pairing. Typically, antisense ODNs can
selectively interfere with RNA splicing or processing,
prevent initiation of translation, block progression of
ribosomes along the mRNA, and cause RNA cleavage
through activation of RNAse H.1 Paul Zamecnik's
laboratory published the first report on application of
antisense ODNs in 1978,2 along with a report on their
application against HIV replication in 1986.3 The authors
showed that a 13-mer synthetic ODN complementary to
the 3'-end of the Rous sarcoma virus genome inhibited
the formation of new virus and prevented transformation
of chick fibroblasts into sarcoma cells when added
exogenously to the infected cell cultures. Since then,
many oligonucleotide analogs have been synthesized
and studied as antisense agents, and antisense effects
of ODNs in mammalian cells have been reported in
numerous tissue culture experiments4 and in several
recent in vivo studies.5,6
The most widely used analogs of ODNs are
phosphorothioate-modified ODNs (PS-ODNs), which
have a sulfur-for-oxygen substitution for 1 of the
nonbridged oxygen atoms of internucleotide linkages.
PS-ODNs have good biological activity, pharmacology,
pharmacokinetics, and safety.7,8 PS-antisense ODNs are
now evaluated in clinical trials for their therapeutic
potential against several human diseases, including
cancer, viral infections, and connective tissue disorders,
and initial results are encouraging.9 Although results
from recent clinical trials of first-generation PS-ODN
drugs seem promising, the mechanism of action of these
compounds remains somewhat speculative and may
include antisense and non-antisense effects.10 PS-ODNs
have been shown to inhibit the replication of human
immunodeficiency virus (HIV-1) in vitro by both
sequence-specific and non-sequence-specific activity,
 AAPS PharmSci 2002; 4 (2) article 9 (http://www.aapspharmsci.org).
depending on the cell culture models used, the
concentrations, and the assay's design.10,11 Several
studies have demonstrated that the potency and
specificity of antisense ODNs can be greatly enhanced
by the use of lipid-based carrier system to deliver the
ODNs into cells.12,13 Lipid-based carriers, such as the
DLS system, increase cellular uptake and help antisense
compounds accumulate in the nucleus, allowing activity
at much lower concentrations.14,15
In this work, we studied the antiviral activity of various
ODN sequences in different HIV infection cell culture
models. We compared the activity of free and DLS-
associated PS-ODNs with that of DLS-delivered
unmodified ODNs (PO-ODNs) and PS-ODNs, in both
acute- and chronic-infection models. We looked at
several factors that have been proposed to account for
discrepancies in the literature on antisense technology
such as dose-response range, number and choice of
experimental controls, backbone modifications of the
ODNs, and type of cell infection (acute or chronic), along
with length of assays and delivery approach, in order to
improve further protocol design in this field area.
Although this paper describes observations already
made independently elsewhere, it constitutes a unique
comprehensive work, as no previous report has
addressed the effect of all these parameters influencing
ODN's anti-HIV activity. The overall goal and objective of
this report are to present a thorough analysis of the
question of sequence specificity and, consequently, to
evaluate antisense ODN design.
MATERIALS AND METHODS
Antisense and Control ODNs
PO-ODNs and PS-ODNs (ODN with a sulfur atom
introduced at each phosphodiester linkage) were
synthesized by using an automated DNA synthesizer
(BioServe Biotechnologies, Laurel, MD) and purified by
polyacrylamide gel electrophoresis. In this study, we
used 4 antisense sequences known to have anti-HIV-1
activity in either phosphorothioate (PS-ODN) or
unmodified (PO-ODN) form: antisenses DIS and Pac,
which are complementary to a portion of the 5'-long
terminal repeat of the HIV-1 genome and are considered
to be essential for HIV-1 RNA encapsidation16,17;
antisense GEM 91 (gene expression modulator 91), a
25-mer complement to the gag initiation site of HIV-118;
and antisense rev, a 28-mer complementary to the 5'-
end sequence of HIV-1 rev mRNA.19 The ODN
sequences are shown in Table 1. As a control, a 28-mer
random phosphorothioate (RS) or phosphodiester (RD)
sequence was made from a mixture of all 4 nucleotides
at each synthesis step, in order to verify the sequence
specificity of the antisense ODNs. As controls for PS-
ODNs, we also used 2 sense sequences named SSDIS
2
and SSPac for sense SDIS and sense SPac,
respectively (Table 1). In addition, an ODN sequence
named T30177 was used here as a non-antisense
control. T30177 is a 17-mer ODN that comprises only
deoxyguanosine and thymidine residues stabilized by an
intramolecular guanosine octet.20 T30177 contains single
phosphorothioate internucleoside linkages at its 5' and 3'
ends, and has shown strong anti-HIV activity in infected
cells on the basis of its 3-dimensional structure.
Cells and Virus
The human lymphoid cell line MOLT-3 was kindly
provided by Dr R.P. Sekaly (Clinical Research Institute
of Montreal, Quebec, Canada). H9 cells chronically
infected with HIV-1 (IIIB) (H9/human T-lymphotropic
virus [HTLV]-IIIB [NIH] 1983)21,22 were obtained from the
NIH Acquired Immunodeficiency Syndrome (AIDS)
Research and Reference Reagent Program (Rockville,
MD). Uninfected and infected cells were cultivated in
RPMI 1640 culture medium (Invitrogen / Life
Technologies, Burlington, Ontario, Canada)
supplemented with 10% heat-inactivated fetal calf
serum, L-glutamine (4 mM), and gentamycin (50 μg/mL)
at 37°C in a 5% CO2 atmosphere. Peripheral blood
mononuclear cells (PBMCs) from healthy HIV-1-
seronegative donors were isolated by Ficoll-Hypaque
(Amersham Biosciences, Piscataway, NJ) gradient
centrifugation of heparinized venous blood. The cells
were collected, washed, and stimulated with
phytohemagglutinin-P (PHA-P) (1 μg/mL; Pharmacia
Biotech) for 24 hours. The cells were then washed and
maintained in the same culture medium as above,
supplemented with recombinant human interleukin-2
(10-20 U/mL; ZeptoMetrix Corporation, Buffalo, NY).
HIV-1 laboratory strain IIIB was obtained from Advanced
BioScience Laboratories Inc (Kensington, MD) and was
used to infect MOLT-3 cells and primary cells.
Preparation of ODN-liposome Complexes
DLS liposomes are a mixture of equal amounts of
dioctadecylamidoglycylspermidine (Promega, Madison,
WI) and dioleoyl phosphatidylethanolamine (Sigma-
Aldrich Corp. St-Louis, MO) and consist of small
unilamellar vesicles, which can complex with ODNs in an
interactive molecular manner. Liposomes were prepared
as previously described.23,24 Oligonucleotides were first
complexed to DLS preparation in sterile deionized water
as described earlier,15 and the preparation was
incubated at room temperature for at least 30 minutes
just prior to addition to the cells. Dilution in deionized
water was made to obtain appropriate concentrations.
The lipoplexes used here are specifically formulated to
obtain highly stable and reproducible preparations.
These preparations showed high homogeneity in size
(polydispersity ~ 0.20) as determined by dynamic light
scattering. The coefficient of variation of the mean size
(~120 nm) was found to be 19.5%. ODN-liposome
 AAPS PharmSci 2002; 4 (2) article 8 (http://www.aapspharmsci.org).

Table 1. Sequences of the ODNs Used for the Treatment of HIV-1 Infection In Vitro
ODNs Nucleotide Sequence Targeted Sequence

GEM 91 (antisense) 5'-CTCTCGCACCCATCTCTCTCCTTCT-3' gag gene

Srev or rev (antisense) 5'-TCGTCGCTGTCTCCGCTTCTTCCTGCCA-3' rev gene
SDIS (antisense) 5'-CTCTTGCCGTGCGCGCTTCAGCAAGC-3' dimerization site
DIS (antisense) 5'-CTCTTGCCGTGCGCGCTTCAGCAAGCCG-3' dimerization site
SPac (antisense) 5'-TCTAGCCTCCGCTAGTCAAAATTTTTGGCG-3' packaging signal (\)
SSDIS (sense) 5'-CGGCTTGCTGAAGCGCGCACGGCAAGAG-3' dimerization site
SSPac (sense) 5'-CGCCAAAAATTTTGACTAGCGGAGGCTAGA-3' packaging signal (\)
T30177 5'-GTGGTGGGTGGGTGGGT-3' non-antisense control
RS (random) 28-mer sequence made from a mixture of all 4 nucleotides
RD (random) 28-mer sequence made from a mixture of all 4 nucleotides
ODNs GEM 91, Srev, SDIS, SPac, and RS (random) were synthesized with a phosphorothioate backbone. ODN T30177
was synthesized with a partial phosphorothioate backbone in which only the terminal 3' and 5' internucleoside linkages were
phosphorothioate. ODNs rev, DIS, and RD (random) were synthesized with a phosphodiester linkage.

complexes were stored at 4°C up to the next treatment 3
or 4 days later. Fresh ODN-liposome complexes were
prepared every week (every 2 treatments).
Short-term Evaluation of Anti-HIV Activity in
Acutely Infected Cells
were plaed into 96-well microtiter plates at a
concentration of 4 x 105 cells/mL, and antisense ODNs
were either added free or complexed with DLS
liposomes at 1.5 and 18.5 μM concentrations or at 0.001
and 0.01 nM concentrations, respectively. The cells were
kept in culture for 3 to 4 days, and HIV-1 replication was
determined by the p24 antigen assay.

Antiviral activity of ODNs was evaluated in 2 different

cell systems. MOLT-3 cells or PBMCs were infected with
HIV-1 (IIIB) at a multiplicity of infection of 0.1. Cells were
cultured with various concentrations of ODNs either
added free in culture medium or complexed with DLS
liposomes for 7 days. The supernatants were then
harvested and examined for virus production.
Long-term Evaluation of Anti-HIV Activity in
Acutely Infected Cell Lines
To test the antiviral activity of the antisense ODNs in a
long-term model treatment in acutely infected cells,
MOLT-3 cells were infected at a multiplicity of infection
of 0.01 with HIV-1 laboratory strain IIIB as previously
described.15 Cells were treated for up to 21 days with
PS-ODNs added free to the cultures at 0.01, 0.1, or 1
μM concentrations, or for up to 28 days with either PS-
ODNs or PO-ODNs complexed with DLS liposomes at
0.001, 0.01, 0.1, and 5 nM concentrations. Every 3 or 4
days, cells were split to 4 x 105 cells/mL and
supernatants were collected to determine the HIV-1 titer.
Except for the initial 2-hour period of virus adsorption,
the oligonucleotides were always present in the culture
medium during the course of the experiments.
Antiviral Assay in Chronically Infected Cell Lines
To study the inhibition of HIV-1 by antisense ODNs and
their efficiency in chronically infected cells, we used the
H9 cell line chronically infected with HIV-1 (IIIB). Cells
3
Detection of HIV-1 p24 Antigen
Virus replication was determined by detection of p24
HIV-1 viral core antigen in cell-free supernatants by a
p24 antigen-capture assay (Beckman Coulter
Immunology, Fullerton, CA or SAIC Frederick, Frederick,
MD). Cell viability and cytotoxicity were monitored by the
tetrazolium-based colorimetric cell proliferation assay
(MTT).25
Statistical Analysis
Data for experimental groups were expressed as mean ±
SD and compared with those of control groups or
different treatment groups using the single-factor
analysis of variance. When statistical significance (P <
.05) was reached with the F test, comparisons of the
means were then performed using either the Tukey-HSD
test or the Student t test. A P value lower than 0.05 was
considered significant.
RESULTS AND DISCUSSION
Experimental Protocol Design
The remarkable ability of ODNs to inhibit genetic
expression in a sequence-specific manner has become,
 AAPS PharmSci 2002; 4 (2) article 9 (http://www.aapspharmsci.org).
in recent years, the subject of increased scrutiny in the
field of antisense technology. This issue arises because
the mechanism of action of these compounds remains
unclear and can apparently include antisense and a
variety of non-antisense mechanisms simultaneously.11,
26,27,28
Here, we have investigated the influence of
several factors - such as backbone modification, cell
culture model, dose, sequence of the ODN, type of
control used, and delivery approach - on the sequence
specificity of activity of antisense ODNs against HIV-1.
Antiviral activity of antisense ODNs could be evaluated
by measuring reverse transcriptase activity as well. The
inhibitory activity can be more precisely determined by
quantifying the specific protein targeted by the antisense
molecule and could be best suited to estimate the true
antisense mechanism. Non-antisense mechanisms may
be determined by evaluating ODNs' capacity to hybridize
with viral and/or host sequences of a proteic or nucleic
nature. ODN activity, especially non-antisense activity,
could furthermore be explored by investigating biological
mechanisms such as measuring cellular, metabolically
active proteins to appreciate health of treated cells. This
study focused only on the contribution of the sequence-
specific anti-HIV activity of antisense ODNs. Inhibition of
HIV replication was conceived as the strategic end point
for treating infection with antisense ODNs.
Consequently, p24 assay was considered solely for
sensitive monitoring of ODN activity. In addition, this
study did not attempt to investigate why several ODN
sequences tested were more active than others.
Antisense compounds are single-stranded nucleic acids
that, in principle, disrupt the synthesis of a targeted
protein by hybridizing in a sequence-dependent manner
to the mRNAs that encode it. This mechanism is
conventionally termed in the literature and in this report
as "antisense." It is well established that26-32 antisense
ODNs might have, in addition, activity that is related to a
"non-antisense" effect (ie, not related to an antisense
effect) as we have referred to it here. This non-antisense
effect can be due to multiple mechanisms comprising
those originating from the activity of a specific ODN
sequence (termed here as a "sequence-specific" effect)
and those originating from the activity of an ODN that is
not specific to a particular sequence (termed here as a
"non-sequence-specific" effect). Alternatively, we can
distinguish these effects as being either the sequence-
specific effect (comprising antisense and non-antisense
sequence-specific effects) or the non-sequence-specific
effect.
The experimental design of this work aimed at dissecting
the contributions of both sequence-specific and non-
sequence-specific interactions to the HIV-inhibitory
properties of ODNs. The results are expressed as
percent inhibition of p24 production in culture cell
medium. We, arbitrarily, defined the sequence-specific
activity as the percent of the difference between the
4
inhibition level of the most active antisense ODNs and
the random control ODNs; and the non-sequence-
specific activity as the percent of the inhibition level of
the random control ODN to the inhibition level of the
most active ODN.
In our study, part of the activity was dependent on the
sequence of antisense sequences and of some control
sequences. Non-sequence-specific activity was
observed with the random control sequences, and this
activity can be attributed to all ODNs. The concentration
of a single sequence in a preparation of random 28-mer
ODNs is negligible, as it is more than 7 x 1016 times
lower than the concentration of total ODNs in the
preparation. To study the effect of PO-ODNs and to
avoid extracellular biodegradation and cell surface non-
specific interference of ODNs with virus entry, we used a
carrier system. We used the DLS delivery system, which
efficiently protected PO-ODNs from nuclease
degradation and increased intracellular availability,
allowing them to exert their activity against HIV-1 at low
concentration.33 In order to understand the importance of
specific inhibition by antisense molecules, we compared
the antiviral activity of either free or DLS-associated PS-
ODNs or PO-ODNs in 4 different cell assays, including 3
acute infection models (short-term and long-term assays
with HIV-1 [IIIB]-infected MOLT-3 cells and a short-term
assay with HIV-1 [IIIB]-infected PBMCs), with a chronic
infection model (with H9/HTLV-III cell line).
Cell Culture Models and Antiviral Activity
Use of various cell culture models in this work allows for
the quantitative comparison of ODN activity, as
significant differences (P < .05) were obtained for
numerous experimental groups.
All free ODNs showed significant inhibition (> 80%) at
the highest dose tested. Nevertheless, viral replication
appears to be affected differently depending on the cell
culture model used, with the following growing order
upon estimated IC50: chronically infected H9 cells <
acutely infected PBMCs = acutely infected MOLT-3 cells
in a short-term assay < acutely infected MOLT-3 cells in
a long-term assay (Tables 2, 3, 4, 5). All free PS-ODNs
tested showed the highest activity in the long-term assay
for MOLT-3 acutely infected cells with >99% inhibition at
1 μM; in addition, SPac and SDIS showed >99%
inhibition at 0.1 μM.
Use of a Delivery Approach
Use of DLS to deliver ODN does not improve the optimal
viral inhibition of ODN when compared with ODN in a
free form. Substantial antiviral activity with DLS-
associated PS-ODNs was obtained with up to 83%
inhibition, although we never observed the very high
 AAPS PharmSci 2002; 4 (2) article 8 (http://www.aapspharmsci.org).

Table 2. Antiviral Activity of PS-ODNs and PO-ODNs Either Added Free in Solution or Delivered by DLS Liposomes
in a Short-term Assay Using MOLT-3 Cells Acutely Infected with HIV-1 (IIIB)
Sequence % Inhibition of p24 Production

Free 0.01PM 0.1 PM 1 PM

GEM 91 ND 84 r 13* 98 r 2
Srev ND 76 r 11* 81 r 4
SDIS 0r0 91 r 6* 92 r 1
Spac 53 r 2 92 r 3* 92 r 2
RS 0r0 0r0 96 r 1

With DLS liposomes 0.001 nM 0.01 nM 0.1 nM 5 nM

DLS-Srev 12 r 2 31 r 6 57 r 6 48 r 7
DLS-SDIS 0r0 21 r 13 69 r 13 78 r 11
DLS-Spac 64 r19 47 r 10 64 r 20 47 r 6
DLS-RS 56 r19 74 r 6 71 r 5 52 r 0
DLS-SSDIS 55 r 3 72 r 8 41 r 7* ND
DLS-SSPac 57 r 5 ND 52 r 9 ND
DLS-T30177 12 r 6* 36 r 17 27 r 11* ND
DLS-rev ND 32 r 17* 54 r 05 24 r 0
DLS-DIS ND 29 r 0.4* 53 r 18 26 r 4
DLS-RD ND 2r2 60 r 21 51 r 5
*Values statistically different from the random control sequence (P < .05). ND indicates not determined. MOLT-3 cells were
acutely infected with HIV-1 (IIIB) and incubated with various concentrations of free ODNs or DLS-ODN complexes during 7
days. Data are given as percent inhibition of p24 production in the supernatants compared to infected, untreated control cell
cultures. The results represent the means (r SD) of triplicate determinations for free ODNs experiments and the means (r SD)
of at least 2 assays for DLS delivery experiments.

Table 3. Comparison of Antiviral Activity of PS-ODNs Either Added Free or Complexed with DLS Liposomes on
Acutely Infected MOLT-3 Cells with HIV-1 (IIIB) in a Long-term Assay
Sequence % Inhibition of p24 Production

Free With DLS

0.01 PM 0.1 PM 1 PM 0.001 nM 0.01 nM 0.1 nM 5 nM

GEM 91 ND > 99* > 99 ND ND ND ND

Srev 66 r 7 99 r 2* > 99 32 r 17 52 r 18 73 r 6 68 r 10
SDIS 73 r 18 > 99 > 99 44 r 16 52 r 17 77 r 9 48 r 8
SPac 33 r 18 > 99 > 99 36 r 11 46 r 6 71 r 6 81 r 23
RS 54 r 12 90 r 0.1 > 99 77 r 17 62 r 12 68 r 7 50 r 15
T30177 ND ND ND 73 r 10 63 r 22 55 r 4 ND
SSDIS ND ND ND 44 r 22 53 r 10 61 r 3 ND
SSPac ND ND ND 39 r 4 82 r 22 77 r 15 ND
rev ND ND ND ND 60 r 13 73 r 13 80 r 0.4
DIS ND ND ND ND 44 r 49 60 r 16 65 r 9
RD ND ND ND ND 48 r 19 61 r 5 92 r 9
*Values statistically different from the random control sequence (P < .05). ND indicates not determined. MOLT-3 cells were
acutely infected with HIV-1 (IIIB) and treated for up to 21 days with free PS-ODNs or treated for up to 28 days with DLS-
delivered PS-ODNs. Percent inhibition of p24 production in the supernatants was calculated at the peak of the infection for
each assay and compared to infected, untreated control cell cultures. Results represent the mean (r SD) of at least 3 assays.

5
 AAPS PharmSci 2002; 4 (2) article 9 (http://www.aapspharmsci.org).
Table 4. Antiviral activity of PS-ODNs or PO-ODNs Delivered by DLS Liposomes in a Short-term Assay on PBMCs
Acutely Infected with HIV-1 (IIIB)
Sequence % Inhibition of p24 Production

0.05 nM 0.1 nM 5 nM 10 nM

DLS-Srev 70 r 7 74 r 7 66 r 9 52 r 6
DLS-RS 74 r 3 68 r 1 76 r 3 20 r 8
DLS-rev 64 r 8 71 r 6 71 r 2 66 r 7
DLS-RD 57 r 11 79 r 3 72 r 3 29 r 7
Srev free (100 nM) 79 r 19
PBMCs were acutely infected with HIV-1 (IIIB) and incubated with various concentrations of liposome-encapsulated ODNs
during 7 days. Data are given as percent inhibition of p24 production in the supernatants compared to infected, untreated
control cell cultures. The results represent the means (r SD) of triplicate determinations.

Table 5. Comparison of Antiviral Activity of PS-ODNs or PO-ODNs Either Added Free in Solution or Complexed with
DLS Liposomes on Chronically Infected H9/HTLV-IIIB Cells
Sequence % Inhibition of p24 Production

Free With DLS

0.1 PM 1.5 PM 18.5 PM 0.001 nM 0.01 nM

Srev 28 r 1 83 r 1 87 r 2 77 r 4 83 r 2*
SDIS 25 r 1 75 r 2 79 r 4 86 r 13 ND
Spac 25 r 4 76 r 0.2 84 r 1 79 r 16 ND
RS ND 77 r 1 75 r 1 74 r 3 73 r 3
SSDIS ND 86 r 3 85 r 4 72 r 4 ND
SSPac 26 r 3 83 r 4 87 r 2 65 r 13 ND
T30177 ND ND ND 86 r 24 52 r 22
Rev ND ND ND 83 r 2* 84 r 4*
RD ND ND ND 68 r 2 70 r 4
DLS 19 r 11 19 r 8
*Values statistically different from the random control sequence (P < .05). ND indicates not determined. Chronically infected
H9/HTLV-IIIB cells were treated with free or DLS-complexed PS-ODNs during 4 days. Percent inhibition of p24 production in
the supernatants was calculated 4 days post-infection and compared to infected, untreated control cell cultures. The results
represent averages of at least 3 experiments (r SD).

equivalent concentrations used for DLS-ODNs at

level of inhibition of viral replication seen when using
relatively large amounts of free PS-ODNs. In contrast,
when using free ODNs, DLS-delivered ODNs affected
viral replication (on the basis of estimated IC50) in an
inverse manner: acutely infected MOLT-3 cells in a long-
term assay = acutely infected PBMCs < acutely infected
MOLT-3 cells in a short-term assay < chronically
infected H9 cells. Overall, the results did not indicate any
statistical difference between the activities of the ODNs
tested, and there was no significant difference between
PS and PO activities when they were delivered with DLS
to cells. DLS liposomes used as controls at the
6
effective doses did not show any effect on HIV
replication in MOLT-3 cells, and no major cytotoxicity
could be observed with either free or DLS-complexed
ODNs at effective doses in MOLT-3 and chronically
infected H9 cells (Table 6).
In all in vitro systems tested, DLS delivery did not
improve the level of antiviral activity of PS-ODNs
compared to that of free PS-ODNs, and DLS-unmodified
ODNs were not more potent than free PS-ODNs when
maximal inhibition is considered. On the basis of our
 AAPS PharmSci 2002; 4 (2) article 9 (http://www.aapspharmsci.org).
results, the use of free PS-ODNs seems to be the best
approach to block HIV replication in vitro. However, at
IC50, DLS delivery greatly improved cellular uptake since
inhibition of viral replication with DLS-delivered ODNs
could be achieved at subnanomolar concentrations
compared to micromolar concentrations with free ODNs
(Table 7). This confirms that the DLS carrier system
appears to be an efficient approach for ODN delivery. An
IC50 in the nanomolar range is clearly of high
pharmacological interest. IC50 around 0.001 nM was
never observed for any in vitro experimental protocol
using either free ODN or ODN complexed with a carrier.
The DLS formulation has also been tested for its in vivo
efficiency for systemic gene delivery in mice. After a
single injection of DLS-complexed plasmid DNA, the
transgene was detected in lung, liver, spleen, and heart,
and the transgene mRNA was detected in lung and
spleen for as long as 3 months.23 In addition, DLS- DNA
showed a rapid cellular uptake, a high cytoplasmic and
nuclear distribution of DNA in cell cultures, and a
relatively low plasma clearance following intravenous
24
administration in mice. Therefore, the DLS system may
represent a powerful tool for in vivo application of gene
therapeutics.
Sequence-Specific Effect
In this study, the level of antiviral activity of ODNs varied
depending on the cell assay used, and the level of the
activity that was due to a sequence-specific effect varied
depending on the type of control used to calculate the
specificity of the inhibition. We found that the portion of
the activity of the antisense molecules that could be
attributed to a sequence-dependent mechanism was
more considerable in our short-term assays with acutely
infected cells than in our long-term assay with acutely
infected MOLT-3 cells and our short-term assay with
chronically infected cells (Table 7). When using the
acutely infected MOLT-3 cell culture assay, specific
inhibition was dose-dependent (Table 2). At 0.01 μM,
SPac displayed antiviral activity (53% ± 2% inhibition of
viral replication), whereas SDIS and random control
completely failed to inhibit HIV-1 replication (0%
inhibition for each). At 0.1 μM, all ODNs exhibited
substantial antiviral activity varying from 76% ± 11% to
92% ± 3% inhibition. In contrast, no inhibition could be
achieved with the random control at this same
concentration. Therefore, at 0.1 μM, all the antiviral
activity (100% of the activity) observed with antisense
sequences could be attributed to a sequence-specific
mechanism (P = .05). When oligonucleotide doses were
increased to 1 μM, 81% ± 4% to 98% ± 2% inhibition
was obtained with ODN sequences, compared to 96% ±
1% inhibition with the random control. Thus, at higher
concentration, 100% of the inhibitory activity was due to
a non-antisense mechanism. Moreover, at optimal viral
inhibition, antiviral activity appeared essentially to result
from non-sequence-specific effects in all other cell
7
culture models used in this work (Tables 2, 3, 4, 5, 7).
Consequently, a sequence-specific effect was observed
for free antisense ODN in the acutely infected MOLT-3
short-term and long-term assays at concentrations lower
than 1 μM and 0.1 μM, respectively, and was observed
for DLS-delivered antisense ODN in the acutely infected
PBMCs and in the chronically infected H9 cells at
concentrations of 10 nM and less than 0.01 nM,
respectively.
Previously Described Non-Specific Effects
Non-specific effects of PS-ODNs have been reported in
many studies. PS-ODNs are polyanions and, as a
consequence, they are able to bind to various cellular
proteins on the basis of charge interaction and base
sequence (aptamer approach).27,34 The presence of a G-
quartet, along with particular sequences flanking the G-
quartet, may also enhance non-specific effects.28
Furthermore, CpG motifs have been found to be highly
immunostimulatory in mice.35,36 PS-ODNs containing the
dinucleotide motif CpG can increase immunoglobulin
secretion and expression of B-cell activation markers
such as major histocompatibility complex class II, induce
interferons (INFs), augment natural killer cell activity,
and stimulate the release of several interleukins (ILs)
from T cells. It is possible that release of ILs, such as
INF-gamma, tumor necrosis factor Į, or IL-12, may be
involved in anti-HIV activity in the assays used in this
study, thus contributing to the random ODNs' high
activity. PS-ODNs have been shown to inhibit the
replication of HIV-1 in vitro by both sequence-
specific19,29,37,38 and non-sequence-specific29-32
processes depending on the cell culture model
employed. In acute-infection models, non-antisense
inhibition of PS-ODNs has been well documented1 and
might occur, because of interference with virus
adsorption, by binding to the CD4 receptor or the V3
loop of viral gp120,39-41 or with reverse transcription.42 In
chronic-infection models, sequence-specific inhibition of
HIV production has been reported,19,32 but the precise
mechanism of action of PS-ODNs still remains to be
elucidated. Furthermore, it might be possible that non-
antisense sequence-specific activity could be partly
attributed to ODN interaction with cellular life proteins
such as proteins interfering with transcription,
phosphorylation, and cell cycle.
Cellular Compartmentalization of ODN Non-
Specific Effect
In comparing the activity of free PS-ODNs with DLS-
delivered PS-ODNs, it is possible to evaluate the amount
of activity that can be attributed to extracellular and/or
membranar effects such as interaction with virus by
binding to the gp120 viral protein and interaction with cell
surface molecules. In fact, DLS complexation prevents
ODN interaction with virus and/or cellular membrane and
 AAPS PharmSci 2002; 4 (2) article 8 (http://www.aapspharmsci.org).

Table 6. Cytotoxicity of PS-ODNs or PO-ODNs Either Added Free in Solution or Delivered by DLS Liposomes in MOLT-3
and H9/HTLV-IIIB Cell Lines
Sequence % Cell Viability

MOLT-3 Cell Line H9/HTLV-IIIB Cell Line

Free in Solution With DLS Liposomes With DLS Liposomes

1 PM (21-day)b 18.5 PM (3-day)a 0.005 PM (21-day)b 1 PM (7-day)a 0.1 nM (7-day) 0.1 PM (7-day)
GEM 91 96 r 0.3 ND ND ND ND ND
Srev 100 r 0 85 r 6 94 r 11 ND 90 r 8 70 r 15
SDIS 100 r 0 100 r 0.2 96 r 6 ND 89 r 8 89 r 11
SPac 100 r 0 79 r 14 77 r 2 ND 91 r 3 ND
RS 100 r 0 71 r 10 88 r 20 9r 7 91 r 9 84 r 5
SSDIS ND 91 r 14 ND ND 89 r 9 89 r 12
SSPac ND 92 r 6 ND ND 91 r 5 ND
rev ND ND 100 r 0 11 r 2 92 r 9 ND
DIS ND ND 89 r 19 19 r 1 ND ND
RD ND ND 93 r 12 4r 2 97 r 4 ND

ND indicates not determined.Values represent the number of cell viability calculated either as a percentage of the a) uninfected or b)
infected, unexposed control cultures in MOLT-3 cell cultures or as a percentage of the unexposed control cultures in chronically
infected H9/HTLV-III cells. Cells were exposed to various concentrations of ODNs either added free or DLS-complexed during variable
time of exposure. Viability was determined by the colorimetric (MTT) assay. The data represent averages of triplicate determinations
(MOLT-3) or averages of three experiments done in duplicate (H9/HTLV-III).

Table 7. Contributions of Sequence-Specific and Non–Sequence-Specific Effects of ODNs in Inhibiting HIV Replication
Cell Culture Model Delivery Optimal Sequence-Specific Activity Optimal Non–Sequence-Specific Activity Optimal Viral
Inhibition

Acute Infection Concentration % Total Inhibition Concentration % Total Inhibition

MOLT-3/HIV-1 (IIIB)
Short-term assay Free PS 0.01-0.1 PM 100%* 1 μM 100% 96%
DLS-PS 0.001-5 nM 0% 0.001-5 nM 100% 74%
DLS-PO 0.01 nM 94%* 0.1-5 nM 100% 60%

Long-term assay Free PS 0.1 PM 9%* 1 μM 100% >99%

DLS-PS 0.001-5 nM 0% 0.001-5 nM 100% 77%
DLS-PO 0.001-5 nM 0% 0.01-5 nM 100% 92%
PBMC/HIV-1 (IIIB)
Short-term assay Free PS ND ND ND ND ND
DLS-PS 10 nM 62% 0.05-5 nM 100% 74%
DLS-PO 10 nM 56% 0.05-5 nM 100% 79%

Chronic infection

H9/HTLV-1 IIIB
Short-term assay Free PS 0.1-18.5 PM 0% 18.5 μM 100% 77%
DLS-PS 0.01 nM 12%* 0.01 nM 88% 74%
DLS-PO 0.001 nM 18%* 0.01 nM 82% 70%

ND indicates not determined. Sequence-specific activity expressed as the percentage of the difference between the inhibition levels of the
most active antisense ODNs and the random control sequence; and non–sequence-specific activity expressed as the percentage of the
inhibition level of the random control ODN. Optimal viral inhibition expressed as percent inhibition of p24 production in cell culture
supernatant of the most active ODN. Values higher than 10% or *significant (P < .05) are presented.

8
 AAPS PharmSci 2002; 4 (2) article 8 (http://www.aapspharmsci.org).
then bypasses the non-specific effects that result from
ODN binding to either the CD4 receptor or the gp120
viral protein. We found in our short-term acute-infection
model using MOLT-3 cell line that approximately 25% of
the activity of PS-ODNs can be attributed to extracellular
and/or membranar effects (P = .03). It is interesting that
in our PBMC assay and in our chronic-infection model,
no significant difference between the level of the
inhibition of free PS-ODNs and that of DLS-delivered
PS-ODNs could be observed, suggesting that the portion
of the activity that can be attributed to non-specific
extracellular and/or membranar effects may be less
important in primary cells and chronically infected cells
than it is in acutely infected established cell lines. With
regard to this hypothesis, the higher level of inhibition
observed in MOLT-3 cells treated with free PS-ODNs
compared to that in primary cells (Table 7) might be
explained, in part, by the addition of extracellular and/or
membranar effects on overall antiviral activity in
immortalized cell lines. In chronically infected cells, PS-
ODNs cannot interact with virus entry or reverse
transcription steps that occur only in acute infections
and, thus, can only interfere with post-integration steps.
As such, free PS-ODNs and DLS- PS-ODNs might both
exert their activity only in the cytoplasm and/or the
nucleus, explaining in part why free PS-ODNs and DLS-
PS-ODNs showed the same maximum level of activity in
this model. Furthermore, the difference in the levels of
the inhibition between DLS- PS-ODNs and DLS- PO-
ODNs was more important in our assays using MOLT-3
cells than in our assays using either primary or
chronically infected cells, suggesting that non-specific
intracellular effects due to backbone modification are
more apparent in immortalized cell lines.
In addition, intracellular uptake and bioavailability of
ODNs may vary, on the one hand, between immortalized
and primary cells43 and, on the other hand, between
acutely and chronically infected cells.15 The cellular
uptake and the intracellular distribution of the free PS-
ODN GEM 91 was evaluated in MOLT-3 cells and
PBMCs by flow cytofluorometry and laser-assisted
confocal microscopy for uptake and biodistribution,
respectively.43 The cellular uptake was slow and similar
in both MOLT-3 cells and PBMCs. However, in MOLT-3
cells, the intracellular distribution of GEM 91 was mainly
concentrated in cytoplasmic vesicles, in contrast to
PBMCs, in which the intracellular distribution of GEM 91
was more diffuse and, thus, more available for
cytoplasmic and nuclear activity. Complexation with DLS
formulation may protect ODNs from degradative
enzymes in endosomal vesicles and allow bypassing
vesicular trafficking, resulting in a higher intracellular and
intranuclear localization compared to free ODNs.24 In
this study, when PS-ODNs were delivered by DLS, the
level of activity achieved with the DLS-delivered PS-
ODNs was similar in both MOLT-3 cells and PBMCs in
our short-term assays (Table 7), but in PBMCs, high
antiviral activity was observed at lower concentrations
compared to MOLT-3 cells (Tables 2, 4). This difference
suggests that the cellular uptake of DLS- PS-ODNs
might be higher in PBMCs than in MOLT-3 cells or that
the intracellular distribution of DLS- PS-ODNs might be
more diffuse in PBMCs than in MOLT-3 cells, as
previously observed.43 The highest IC50 of DLS- ODNs
was observed in chronically infected cells in which a high
level of inhibition was observed at the very low
concentration of 1 pM. This result might be explained, in
part, by a higher level of cellular uptake and/or a higher
intranuclear localization compared to acutely infected
cells. In this chronic-infection model, an increased
intranuclear localization may be advantageous
compared to an acute-infection model and may result in
increased inhibitory activity, since in chronically infected
cells, the site of action of ODNs is expected to be
predominantly in the nucleus where the viral nucleic acid
is integrated into the cellular genome. Taken together,
our results suggest that most of the PS-ODNs' activity
should be due to intracellular activity. Assays on ODN's
activity using primary cells should be promoted over
assays using established cell lines, since primary cells
are more representative of in vivo infections and since
cellular uptake and intracellular distribution may vary
among cell models employed.
Antisense-Dependent vs Non-Specific Activities
In the literature, most of the studies that report
antisense-dependent activity of antisense molecules
have been done in short-term acute-infection
assays.31,37,38,44 In the present study, the highest level of
sequence-specific activity of our antisense PS-ODNs
and PO-ODNs, either free or DLS-delivered, was
observed in our short-term assay using acutely infected
MOLT-3 cells with HIV-1 (IIIB) (Table 7). In agreement
with our data, other investigators also observed, in a
long-term acute-infection assay using the MOLT-3 cell
line, a high level of antiviral activity in HIV-infected cell
cultures treated with 28-mer random and mismatch
sequences for up to 21 days postinfection.29 However, in
contrast to our results, antisense-dependent activity of
GEM 91 was observed in another study43 in which the
activity of GEM 91 was compared to a random control in
a long-term assay using acutely infected MOLT-3 cells
with HIV-1 (IIIB).
In another experiment in which the efficacy of the 3
antisense PS-ODNs, Srev, SDIS, and Spac, to block the
replication of HIV-1 clinical isolate VR2846A was
evaluated in infected PBMCs, SDIS and SPac showed a
significant sequence-specific activity for up to 14 days
when compared to their sense control, but no antisense-
dependent activity could be found when compared to the
random control at 0.1 μM (Lavigne et al, unpublished
observations, 2001). Non-antisense-dependent antiviral
activity was also observed in PBMCs infected with the
HIV-1 clinical isolate 571 with 1 μM GEM 91 compared
to a random sequence 7 days after infection.43 However,
9
 AAPS PharmSci 2002; 4 (2) article 9 (http://www.aapspharmsci.org).
after 11 days, the level of inhibition started to decrease
in cells treated with the random control (74% inhibition at
day 11 and 0% at day 14) compared to cells treated with
GEM 91 (>99% inhibition up to day 14).
In our chronic-infection model, we did not observe
significant sequence-specific activity of our free
antisense PS-ODNs. Non-sequence-specific inhibition
observed in our chronic-infection model might be
explained, in part, by direct interactions with proteins or
by hybridization to other mRNA targets.4,28 In a recent
study, inhibition of virus production in chronically infected
H9 cells was evaluated at about 60% (as measured by
p24 determination) with either the antisense molecule
GEM 91 or the mismatched oligo control at 1 μM,
suggesting that inhibition of HIV-1 production in this
chronic-infection model was also a non-sequence-
specific phenomenon.41 However, these authors found
that treatment of the chronically infected H9 cells with
GEM 91 caused a significant decrease in gag mRNA
expression (60%-70% inhibition), while mismatched
oligo treatment resulted in gag mRNA expression similar
to the control, suggesting that GEM 91 had a sequence-
specific effect at this stage of the viral-replicative cycle.
Nevertheless, a reduction in mRNA level does not
necessarily result in biological activity since a lower
mRNA level might be sufficient for protein function.
Taken together, the above considerations indicate that
the nature of the activity of ODNs (antisense-dependent
vs non-specific activity) may vary depending on the type
of infection and length of the assay, and conclusions
regarding ODNs' activity may differ depending on the
stage of HIV replication investigated.
CONCLUSION
Inhibition of HIV-1 replication by antisense ODNs
depends on various factors in vitro that should be taken
into consideration for the design of any study on
antisense ODNs. For instance, the use of an efficient
delivery system has made it possible to point out
extracellular and/or membranar activity of PS-ODNs that
appears as the most effective presentation among those
tested for the antisense ODN-mediated inhibition of HIV
replication. In light of our results and previous
observations, the contribution of sequence-specific effect
and, consequently, antisense-mediated mode of action
appears minor compared to the other mechanisms
involved in ODN antiviral activity. As long as the
inhibition of HIV replication is concerned, the term
"antisense" does not appear appropriate to identify this
class of compound. As the antisense sequences used in
this study, and in others, were specifically designed for
eliciting the true antisense mechanism, it appears more
promising to shift the oligonucleotide compound design
to a design adapted for other modes of action such as
the aptamer27,45 or the sequence-specific
10
immunostimulation approaches.35,36 This might result in
better adjustment to the universal pharmacological
paradigm of the structure/function rationale and,
consequently, in development of more effective ODN
therapeutics.
ACKNOWLEDGMENTS
This work was supported by grants from the Medical
Research Council of Canada. C. Lavigne benefited from
postgraduate scholarships from the Fonds pour la
formation de chercheurs et l'aide à la recherche (FCAR).
We thank Mrs M. Fauvel for giving access to the
Laboratoire de Sante Publique du Quebec's P3 facilities
that are used for virus manipulation.
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