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Is Antisense An Appropriate Nomenclature or Design For Oligodeoxynucleotides
Is Antisense An Appropriate Nomenclature or Design For Oligodeoxynucleotides
In this work, we studied the antiviral activity of various Cells and Virus
ODN sequences in different HIV infection cell culture
models. We compared the activity of free and DLS- The human lymphoid cell line MOLT-3 was kindly
associated PS-ODNs with that of DLS-delivered provided by Dr R.P. Sekaly (Clinical Research Institute
unmodified ODNs (PO-ODNs) and PS-ODNs, in both of Montreal, Quebec, Canada). H9 cells chronically
acute- and chronic-infection models. We looked at infected with HIV-1 (IIIB) (H9/human T-lymphotropic
several factors that have been proposed to account for virus [HTLV]-IIIB [NIH] 1983)21,22 were obtained from the
discrepancies in the literature on antisense technology NIH Acquired Immunodeficiency Syndrome (AIDS)
such as dose-response range, number and choice of Research and Reference Reagent Program (Rockville,
experimental controls, backbone modifications of the MD). Uninfected and infected cells were cultivated in
ODNs, and type of cell infection (acute or chronic), along RPMI 1640 culture medium (Invitrogen / Life
with length of assays and delivery approach, in order to Technologies, Burlington, Ontario, Canada)
improve further protocol design in this field area. supplemented with 10% heat-inactivated fetal calf
Although this paper describes observations already serum, L-glutamine (4 mM), and gentamycin (50 μg/mL)
made independently elsewhere, it constitutes a unique at 37°C in a 5% CO2 atmosphere. Peripheral blood
comprehensive work, as no previous report has mononuclear cells (PBMCs) from healthy HIV-1-
addressed the effect of all these parameters influencing seronegative donors were isolated by Ficoll-Hypaque
ODN's anti-HIV activity. The overall goal and objective of (Amersham Biosciences, Piscataway, NJ) gradient
this report are to present a thorough analysis of the centrifugation of heparinized venous blood. The cells
question of sequence specificity and, consequently, to were collected, washed, and stimulated with
evaluate antisense ODN design. 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
MATERIALS AND METHODS (10-20 U/mL; ZeptoMetrix Corporation, Buffalo, NY).
HIV-1 laboratory strain IIIB was obtained from Advanced
Antisense and Control ODNs BioScience Laboratories Inc (Kensington, MD) and was
used to infect MOLT-3 cells and primary cells.
PO-ODNs and PS-ODNs (ODN with a sulfur atom
introduced at each phosphodiester linkage) were
synthesized by using an automated DNA synthesizer Preparation of ODN-liposome Complexes
(BioServe Biotechnologies, Laurel, MD) and purified by
DLS liposomes are a mixture of equal amounts of
polyacrylamide gel electrophoresis. In this study, we
dioctadecylamidoglycylspermidine (Promega, Madison,
used 4 antisense sequences known to have anti-HIV-1
WI) and dioleoyl phosphatidylethanolamine (Sigma-
activity in either phosphorothioate (PS-ODN) or
Aldrich Corp. St-Louis, MO) and consist of small
unmodified (PO-ODN) form: antisenses DIS and Pac,
unilamellar vesicles, which can complex with ODNs in an
which are complementary to a portion of the 5'-long
interactive molecular manner. Liposomes were prepared
terminal repeat of the HIV-1 genome and are considered
as previously described.23,24 Oligonucleotides were first
to be essential for HIV-1 RNA encapsidation16,17;
complexed to DLS preparation in sterile deionized water
antisense GEM 91 (gene expression modulator 91), a
as described earlier,15 and the preparation was
25-mer complement to the gag initiation site of HIV-118;
incubated at room temperature for at least 30 minutes
and antisense rev, a 28-mer complementary to the 5'-
just prior to addition to the cells. Dilution in deionized
end sequence of HIV-1 rev mRNA.19 The ODN
water was made to obtain appropriate concentrations.
sequences are shown in Table 1. As a control, a 28-mer
The lipoplexes used here are specifically formulated to
random phosphorothioate (RS) or phosphodiester (RD)
obtain highly stable and reproducible preparations.
sequence was made from a mixture of all 4 nucleotides
These preparations showed high homogeneity in size
at each synthesis step, in order to verify the sequence
(polydispersity ~ 0.20) as determined by dynamic light
specificity of the antisense ODNs. As controls for PS-
scattering. The coefficient of variation of the mean size
ODNs, we also used 2 sense sequences named SSDIS
(~120 nm) was found to be 19.5%. ODN-liposome
2
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
complexes were stored at 4°C up to the next treatment 3 were plaed into 96-well microtiter plates at a
or 4 days later. Fresh ODN-liposome complexes were concentration of 4 x 105 cells/mL, and antisense ODNs
prepared every week (every 2 treatments). 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
Short-term Evaluation of Anti-HIV Activity in kept in culture for 3 to 4 days, and HIV-1 replication was
Acutely Infected Cells determined by the p24 antigen assay.
Antisense compounds are single-stranded nucleic acids Cell Culture Models and Antiviral Activity
that, in principle, disrupt the synthesis of a targeted
protein by hybridizing in a sequence-dependent manner Use of various cell culture models in this work allows for
to the mRNAs that encode it. This mechanism is the quantitative comparison of ODN activity, as
conventionally termed in the literature and in this report significant differences (P < .05) were obtained for
as "antisense." It is well established that26-32 antisense numerous experimental groups.
ODNs might have, in addition, activity that is related to a
"non-antisense" effect (ie, not related to an antisense All free ODNs showed significant inhibition (> 80%) at
effect) as we have referred to it here. This non-antisense the highest dose tested. Nevertheless, viral replication
effect can be due to multiple mechanisms comprising appears to be affected differently depending on the cell
those originating from the activity of a specific ODN culture model used, with the following growing order
sequence (termed here as a "sequence-specific" effect) upon estimated IC50: chronically infected H9 cells <
and those originating from the activity of an ODN that is acutely infected PBMCs = acutely infected MOLT-3 cells
not specific to a particular sequence (termed here as a in a short-term assay < acutely infected MOLT-3 cells in
"non-sequence-specific" effect). Alternatively, we can a long-term assay (Tables 2, 3, 4, 5). All free PS-ODNs
distinguish these effects as being either the sequence- tested showed the highest activity in the long-term assay
specific effect (comprising antisense and non-antisense for MOLT-3 acutely infected cells with >99% inhibition at
sequence-specific effects) or the non-sequence-specific 1 μM; in addition, SPac and SDIS showed >99%
effect. inhibition at 0.1 μM.
The experimental design of this work aimed at dissecting Use of a Delivery Approach
the contributions of both sequence-specific and non-
sequence-specific interactions to the HIV-inhibitory Use of DLS to deliver ODN does not improve the optimal
properties of ODNs. The results are expressed as viral inhibition of ODN when compared with ODN in a
percent inhibition of p24 production in culture cell free form. Substantial antiviral activity with DLS-
medium. We, arbitrarily, defined the sequence-specific associated PS-ODNs was obtained with up to 83%
activity as the percent of the difference between the inhibition, although we never observed the very high
4
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
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
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
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).
7
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
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
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%
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 compared to MOLT-3 cells (Tables 2, 4). This difference
ODN binding to either the CD4 receptor or the gp120 suggests that the cellular uptake of DLS- PS-ODNs
might be higher in PBMCs than in MOLT-3 cells or that
viral protein. We found in our short-term acute-infection
model using MOLT-3 cell line that approximately 25% of the intracellular distribution of DLS- PS-ODNs might be
the activity of PS-ODNs can be attributed to extracellular more diffuse in PBMCs than in MOLT-3 cells, as
previously observed.43 The highest IC50 of DLS- ODNs
and/or membranar effects (P = .03). It is interesting that
in our PBMC assay and in our chronic-infection model, was observed in chronically infected cells in which a high
no significant difference between the level of the level of inhibition was observed at the very low
concentration of 1 pM. This result might be explained, in
inhibition of free PS-ODNs and that of DLS-delivered
PS-ODNs could be observed, suggesting that the portion part, by a higher level of cellular uptake and/or a higher
of the activity that can be attributed to non-specific intranuclear localization compared to acutely infected
extracellular and/or membranar effects may be less cells. In this chronic-infection model, an increased
important in primary cells and chronically infected cells intranuclear localization may be advantageous
compared to an acute-infection model and may result in
than it is in acutely infected established cell lines. With
regard to this hypothesis, the higher level of inhibition increased inhibitory activity, since in chronically infected
observed in MOLT-3 cells treated with free PS-ODNs cells, the site of action of ODNs is expected to be
predominantly in the nucleus where the viral nucleic acid
compared to that in primary cells (Table 7) might be
explained, in part, by the addition of extracellular and/or is integrated into the cellular genome. Taken together,
membranar effects on overall antiviral activity in our results suggest that most of the PS-ODNs' activity
should be due to intracellular activity. Assays on ODN's
immortalized cell lines. In chronically infected cells, PS-
ODNs cannot interact with virus entry or reverse activity using primary cells should be promoted over
transcription steps that occur only in acute infections assays using established cell lines, since primary cells
are more representative of in vivo infections and since
and, thus, can only interfere with post-integration steps.
As such, free PS-ODNs and DLS- PS-ODNs might both cellular uptake and intracellular distribution may vary
exert their activity only in the cytoplasm and/or the among cell models employed.
nucleus, explaining in part why free PS-ODNs and DLS-
PS-ODNs showed the same maximum level of activity in Antisense-Dependent vs Non-Specific Activities
this model. Furthermore, the difference in the levels of
the inhibition between DLS- PS-ODNs and DLS- PO- In the literature, most of the studies that report
ODNs was more important in our assays using MOLT-3 antisense-dependent activity of antisense molecules
cells than in our assays using either primary or have been done in short-term acute-infection
chronically infected cells, suggesting that non-specific assays.31,37,38,44 In the present study, the highest level of
intracellular effects due to backbone modification are sequence-specific activity of our antisense PS-ODNs
more apparent in immortalized cell lines. and PO-ODNs, either free or DLS-delivered, was
observed in our short-term assay using acutely infected
In addition, intracellular uptake and bioavailability of MOLT-3 cells with HIV-1 (IIIB) (Table 7). In agreement
ODNs may vary, on the one hand, between immortalized with our data, other investigators also observed, in a
and primary cells43 and, on the other hand, between long-term acute-infection assay using the MOLT-3 cell
acutely and chronically infected cells.15 The cellular line, a high level of antiviral activity in HIV-infected cell
uptake and the intracellular distribution of the free PS- cultures treated with 28-mer random and mismatch
ODN GEM 91 was evaluated in MOLT-3 cells and sequences for up to 21 days postinfection.29 However, in
PBMCs by flow cytofluorometry and laser-assisted contrast to our results, antisense-dependent activity of
confocal microscopy for uptake and biodistribution, GEM 91 was observed in another study43 in which the
respectively.43 The cellular uptake was slow and similar activity of GEM 91 was compared to a random control in
in both MOLT-3 cells and PBMCs. However, in MOLT-3 a long-term assay using acutely infected MOLT-3 cells
cells, the intracellular distribution of GEM 91 was mainly with HIV-1 (IIIB).
concentrated in cytoplasmic vesicles, in contrast to
PBMCs, in which the intracellular distribution of GEM 91
was more diffuse and, thus, more available for In another experiment in which the efficacy of the 3
cytoplasmic and nuclear activity. Complexation with DLS antisense PS-ODNs, Srev, SDIS, and Spac, to block the
formulation may protect ODNs from degradative replication of HIV-1 clinical isolate VR2846A was
enzymes in endosomal vesicles and allow bypassing evaluated in infected PBMCs, SDIS and SPac showed a
vesicular trafficking, resulting in a higher intracellular and significant sequence-specific activity for up to 14 days
intranuclear localization compared to free ODNs.24 In when compared to their sense control, but no antisense-
this study, when PS-ODNs were delivered by DLS, the dependent activity could be found when compared to the
level of activity achieved with the DLS-delivered PS- random control at 0.1 μM (Lavigne et al, unpublished
ODNs was similar in both MOLT-3 cells and PBMCs in observations, 2001). Non-antisense-dependent antiviral
our short-term assays (Table 7), but in PBMCs, high activity was also observed in PBMCs infected with the
antiviral activity was observed at lower concentrations 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 immunostimulation approaches.35,36 This might result in
in cells treated with the random control (74% inhibition at better adjustment to the universal pharmacological
day 11 and 0% at day 14) compared to cells treated with paradigm of the structure/function rationale and,
GEM 91 (>99% inhibition up to day 14). consequently, in development of more effective ODN
therapeutics.
In our chronic-infection model, we did not observe
significant sequence-specific activity of our free ACKNOWLEDGMENTS
antisense PS-ODNs. Non-sequence-specific inhibition
observed in our chronic-infection model might be This work was supported by grants from the Medical
explained, in part, by direct interactions with proteins or Research Council of Canada. C. Lavigne benefited from
by hybridization to other mRNA targets.4,28 In a recent postgraduate scholarships from the Fonds pour la
study, inhibition of virus production in chronically infected formation de chercheurs et l'aide à la recherche (FCAR).
H9 cells was evaluated at about 60% (as measured by We thank Mrs M. Fauvel for giving access to the
p24 determination) with either the antisense molecule Laboratoire de Sante Publique du Quebec's P3 facilities
GEM 91 or the mismatched oligo control at 1 μM, that are used for virus manipulation.
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 REFERENCES
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