Gene Therapy (2017) 24, 453–461

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0969-7128/17
www.nature.com/gt

ORIGINAL ARTICLE
Silencing of HIV-1 by AgoshRNA molecules
E Herrera-Carrillo, A Harwig and B Berkhout

RNA interference (RNAi) is a sequence-specific gene silencing mechanism that is triggered by the expression of a short hairpin RNA
(shRNA). shRNA molecules enter the RNAi pathway at the Dicer processing step. Recent studies indicated that the cellular microRNA
miR-451 is not recognized by Dicer, but that it is processed instead by the Argonaute 2 (Ago2) protein. Subsequently, Dicer-
independent shRNAs were described that rely on Ago2 for processing, as well as the subsequent silencing step. We called
these AgoshRNA molecules because they depend on Ago2 both for maturation and activation. Processing of an AgoshRNA yields
only a single active RNA strand, thus reducing the chance of adverse off-target effects induced by the passenger strand of regular
shRNAs. In this study, we converted several anti-HIV-1 shRNAs into AgoshRNAs. Seven of the 21 designed AgoshRNAs were potent
anti-HIV molecules, although their RNAi activity is generally somewhat reduced compared with the matching shRNAs. The
AgoshRNA candidates revealed no cellular toxicity. This may relate to the absence of passenger strand expression, which was
verified for these AgoshRNA candidates. Furthermore, we demonstrate that a toxic shRNA can be converted into a non-toxic
AgoshRNA.

Gene Therapy (2017) 24, 453–461; doi:10.1038/gt.2017.44

INTRODUCTION shift to Ago2-mediated processing (bottom panel). Dicer

The cellular RNA interference (RNAi) mechanism uses small, non-
coding RNA molecules to regulate gene expression at the
posttranscriptional level.1–4 The canonical microRNA (miRNA) path-
way employs the Drosha endonuclease to liberate the pre-miRNA
hairpin from the primary transcript. The pre-miRNA is transported
by Exportin-5 to the cytoplasm, where the Dicer endonuclease
removes the loop of the hairpin to generate an active miRNA
duplex, which subsequently associates with the Argonaute 2 (Ago2)
protein forming the RNA-induced silencing complex to induce
degradation of complementary target mRNAs.5–7 Designed short
hairpin RNA (shRNA) molecules enter the RNAi pathway at the Dicer
processing step, thus avoiding the Drosha processing step.
Recent studies identified Dicer-independent RNAi pathways,
in which the unusually short miR-451 hairpin of just 17 base
pairs (bp) is processed by Ago2 instead of Dicer.8–10 Ago2 binds
the duplex and cleaves on the 3′ side between bp 10 and 11 to
generate a single, extended ~ 30 nucleotides (nt) guide strand.
In contrast, regular miRNAs generate a duplex of guide and
passenger strand, of which the latter molecule may cause
unwanted side effects. Subsequent 3′-end processing of
miR-451 by poly(A)-specific ribonuclease creates the ~ 22–26
nt mature miRNA, but this modification appears non-essential
for activity.11 Short shRNAs that resemble the unique miR-451
molecule are also recognized by Ago2 instead of Dicer.12–18 We
named these molecules AgoshRNA because Ago2 is involved in
both the processing and silencing steps.15 Compared with a
regular shRNA, the silencing activity switches from a 3′ side-
encoded guide to a single, extended 5′ side-encoded guide
strand. Deep sequencing confirmed 3′ side cleavage between
bp 10 and 11.14 The regular shRNA and the alternative
AgoshRNA processing routes are depicted in Figure 1a. Regular
shRNAs are processed by Dicer (top panel), but shorter
AgoshRNA hairpins (⩽18 bp) escape from Dicer cleavage and
generates the duplex small interfering RNA (siRNA) consisting
of the guide and passenger strands, both with potential
silencing activity (marked white-5′ and black-3′). In contrast,
Ago2 generates a single-guide RNA strand that upon poly(A)-
specific ribonuclease processing yields the AgoshTRIM molecule
(marked gray).
There is accumulating evidence that the AgoshRNA pathway is
very similar to that of miR-451. One of the hallmarks of miR-451 is
the 5′-terminal A residue instead of a more common U, and this A
remains unpaired at the bottom of the hairpin stem. A recent
study indicated that the identity of the bottom bp is also
important for AgoshRNA activity.13 We subsequently performed a
more in-depth analysis of the bottom bp, which includes variation
of the 5′-end nt and thus the transcription start site, to facilitate
the design of optimized AgoshRNA molecules.19 We concluded
that AgoshRNA optimization is possible by introduction of a
bottom mismatch with 5′-terminal A or G. The latter purine
requirement also has a role in optimized transcription by the H1
polymerase III promoter.19,20
A clear advantage of the AgoshRNA design over shRNAs is the
lack of possible off-target effects that are caused by a passenger
strand. We listed additional advantages of AgoshRNA
inhibitors,21 for example, the activity in monocytes that express
very little or no Dicer.22 We recently also addressed the
important mechanistic question whether AgoshRNAs trigger
cleavage or translational suppression of a perfect complemen-
tary mRNA.19 Precise cleavage at the predicted site in the target
mRNA was observed. In fact, the position of AgoshRNA-mediated
mRNA cleavage was more accurate than cleavage by a regular
shRNA, which is likely due to imprecise Dicer-mediated proces-
sing of the shRNA, a step that is circumvented by the AgoshRNA
design. We now converted previously validated and potent anti-
HIV-1 shRNA molecules into AgoshRNAs. We demonstrate that

Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of
Amsterdam, Amsterdam, The Netherlands. Correspondence: Professor B Berkhout, Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection
and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam Meibergdreef 15, Amsterdam, 1105 AZ Noord-Holland, The Netherlands.
E-mail: b.berkhout@amc.uva.nl
Received 13 January 2017; revised 13 April 2017; accepted 12 May 2017; accepted article preview online 29 May 2017; advance online publication, 29 June 2017
 Antiviral AgoshRNA design
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Figure 1. Anti-HIV shRNAs and AgoshRNAs. (a) In the canonical pathway (top) the shRNA stem is cleaved by Dicer into an siRNA duplex of
~ 21 bp with 3′ UU overhang that is loaded into RNA-induced silencing complex. The 5′ passenger (white arrow) is cleaved and degraded and
the 3′ guide strand (black arrow) acts in RNAi silencing. In the non-canonical pathway (bottom), the AgoshRNA is cleaved by Ago2 on the
3′ side between base pairs 10 and 11 into an extended guide of ~ 33 nt (gray arrow). AgoshRNA subsequently may instruct Ago2 for RNAi
silencing or may be trimmed by poly(A)-specific ribonuclease (PARN) to create a ~ 24 nt guide named AgoshTRIM. The predicted Dicer and
Ago2 cleavage sites are marked with black and gray arrows, respectively. (b) Secondary structure of the R/T5 shRNA and AgoshRNA as
predicted by Mfold, with the guide boxed in gray. The 5′-end nucleotide of AgoshRNA constructs and the basepairing partner were replaced
by A C. (c) The HIV-1 genome showing the position of all target sites. (d) HIV-1 target sequences. We designed two or even three AgoshRNAs
for targets with sequences that are highly conserved among HIV-1 isolates.

an active, but toxic shRNA antiviral can be reconfigured into an RESULTS

active and non-toxic AgoshRNA that targets the same HIV-1 RNA
sequence. We also show that these AgoshRNA inhibitors, unlike
shRNA inhibitors, remain active in Dicer-minus monocytic cells
that are host cells for HIV-1 infection. These findings will aid in
the future design of an active and safe AgoshRNA-based gene
therapy against HIV-1 infection.
Design of anti-HIV AgoshRNA molecules
Figure 1b depicts the secondary RNA structure of a potent antiviral
shRNA (shR/T5) and the newly designed AgoshRNA (AgoshR/T5) as
predicted by Mfold. Both molecules encode an identical anti-HIV
guide on their 3′ and 5′ side, respectively (marked by a gray box).
The predicted Dicer and Ago2 cleavage sites are indicated,

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respectively. We replaced the bottom bp in the AgoshRNA
molecule by an unpaired A C for optimal AgoshRNA activity.19
These small RNAs were synthesized from the H1 polymerase III
promoter, which requires a purine at the +1 position for optimal
transcription. We selected A over G because the MID domain of
the human Ago2 protein prefers to load small RNAs with a 5′-end
U or A in the RNA-induced silencing complex.23,24 We avoided
stretches of four or more Us in the AgoshRNA sequence because a
polyU tract acts as a termination signal for RNA polymerase III
transcription. As a favorite delivery vehicle for an AIDS gene
therapy is the HIV-based lentiviral vector, we avoided anti-HIV-1
guide sequences that trigger ‘self’-targeting of the JS1 lentiviral
vector.25,26
Over the years, we designed several sets of anti-HIV shRNAs
based on high conservation of the viral target sequence among
different virus isolates.27,28 Nearly 100 shRNA candidates were
tested for in vitro antiviral activity against the primary HIV-1 LAI
isolate,29 which is the prototype virus strain used in our
laboratory and a few shRNAs have progressed to in vivo tests.30
Four shRNAs (shPol1, shPol47, shR/T5 and shGag5) were
selected because of robust and durable inhibition of HIV-1
replication. However, shGag5 was subsequently reported to
exhibit a negative effect on T cell physiology in a competitive
cell growth assay in vitro and in vivo in the humanized immune
system mouse.30 In this study, we designed a set of AgoshRNA
mimics of this pre-selected set of potent shRNAs. The
conversion of shRNA into AgoshRNA molecules is not likely to
be easy because of the different processing routes of these
molecules. We thus wondered whether potent shRNAs can
indeed be converted into AgoshRNAs without losing antiviral Figure 2. Inhibition of HIV-1 production by AgoshRNA constructs.
potency. We also addressed whether the toxicity of shGag5 is (a) HEK293T cells were co-transfected with 250 ng of the HIV-1 pLAI,
removed in the AgoshRNA design that does not generate a 1 ng of Renilla luciferase plasmid (pRL) and 25 ng of the AgoshRNA
passenger strand. constructs. Two days post-transfection, inhibition of HIV-1 produc-
tion was determined by measuring CA-p24 levels in the culture
In addition, we evaluated AgoshRNAs against critically impor- supernatant. CA-p24 values were normalized to the Renilla luciferase
tant and highly conserved viral sequences that are perhaps not activities. The ratio between the CA-p24 level and the Renilla
allowed to mutate: the primer-binding site and the polypurine luciferase activity in the presence of 25 ng pBS control was set at
tract (PPT) and domains in the untranslated leader region (UTR-A 100%. Bars represent the average values from eight independent
and UTR-B). Early attempts to raise potent shRNAs against such transfections and error bars shows the s.d. (b) Dose-dependent
theoretically ideal targets failed, in part because of repressive RNA inhibition of HIV-1 production by the most potent AgoshRNA
structure.28,31 Figures 1c and d depict all target sites in the HIV-1 constructs. HEK293T cells were co-transfected with HIV-1 pLAI, pRL
and increasing amount of AgoshRNA constructs (1, 5 and 25 ng).
genome and the actual nt sequences, respectively. In total, we pBS control was set at 100%. The mean values and s.d. are based on
designed and tested 21 anti-HIV AgoshRNAs (Supplementary six independent transfections. Statistical analyses (one-way analysis
information). When conservation of the HIV-1 target was extended of variance followed by Tukey’s post-hoc tests) were performed and
into flanking sequences, we designed multiple overlapping differences among groups were considered significant when the
AgoshRNAs. For instance, the Gag target starting at position correspondent P-value was below 0.05.
1364 in HIV-1 LAI allowed the design of three overlapping
AgoshRNAs, AgoshGag4–6 (Figure 1d).
We selected the most potent AgoshRNAs (Gag4-6, Pol1,
Antiviral activity of the AgoshRNA inhibitors in transient assays Pol8, Pol45 and R/T5) for a titration in human embryonic
kidney 293T (HEK293T) cells co-transfected with the
Each AgoshRNA expression construct (25 ng) was co-transfected HIV-1 pLAI molecular clone, the Renilla plasmid and an
in HEK293 T cells with the HIV-1 molecular clone pLAI (250 ng) to increasing amount of the AgoshRNA constructs (1, 5 and
score inhibition of virus production. A fixed amount of Renilla 25 ng, Figure 2b). The results confirm the inhibitory potency of
luciferase plasmid was included to control for variation in the these seven AgoshRNA molecules, which act in a dose-
transfection efficiency. Two days post-transfection, HIV-1 produc- dependent manner.
tion was measured by the CA-p24 level in the culture supernatant, We next compared the AgoshRNA versus shRNA activity on
which was corrected for the Renilla luciferase activity (Figure 2a). matching luciferase reporters with the sense HIV-1 target
Virus production in the presence of 25 ng pBluescript (pBS) control sequence and a control encoding the antisense HIV-1 sequence
plasmid was set at 100%. Seven of the 21 AgoshRNAs showed a (Figure 3a). The HIV-sense reporter detects the activity of the
significant inhibitory activity (Gag4-6, Pol1, Pol8, Pol45 and R/T5) shRNA guide strand (black arrow) and the extended guide
with virus production levels dropping to o 50% versus the pBS strand from the 5′ side of AgoshRNA (gray arrow), whereas the
control (0.0001 ⩽ P o 0.01). The other 14 AgoshRNA candidates HIV-antisense reporter will score any shRNA passenger strand
showed little inhibitory activity (P40.05). Among the inactive activity (white arrow). HEK293T cells were co-transfected using
AgoshRNAs, 10 mimic shRNAs that are potent (Pol2, Pol6, Pol7, 25 ng of inhibitor and the luciferase activity was measured after
Pol9, Pol41, Pol42, Pol44, Pol47, R/T4 and Nef). Four inactive two days in cell lysates. We set the activity measured with the
AgoshRNAs target ideally conserved HIV-1 sequences (PBS, PPT, unrelated shNef construct at 100% (Figure 3b). All seven
UTR-A and UTR-B) that also resisted shRNA attack.28 AgoshRNAs produce 5′ guide strands that target the

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Figure 3. Luciferase knockdown assays by AgoshRNAs. (a) Luciferase reporter constructs with the sense and antisense HIV-derived sequences.
The Luc-sense reporter scores canonical shRNA guide activity and AgoshRNA activity. The Luc-antisense reporter can theoretically detect both
shRNA (21 nt) and AgoshRNA passenger strand (10 nt) activity. (b) Luciferase knockdown was determined by co-transfection of the reporters
with the AgoshRNA constructs. HEK293T cells were co-transfected with 100 ng of the respective firefly luciferase reporter plasmid, 1 ng of
Renilla luciferase plasmid and 25 ng of the AgoshRNA constructs, which were compared with the original shRNA constructs with identical
HIV-1 target sequence. An irrelevant shRNA (shNef ) served as negative control, for which the activity was set at 100% luciferase expression.
The Luc-sense results are shown in black bars and the Luc-antisense results in white bars scores shRNA passenger strand activity. The mean
values and s.d. are based on six independent transfections. Statistical analysis (one-way analysis of variance followed by Tukey’s post-hoc tests)
demonstrated that luciferase expression in the presence of antiviral AgoshRNAs differed significantly from luciferase expression measured
with the shNef control (ns P40.05, *P ⩽ 0.05, **P ⩽ 0.01, ***P ⩽ 0.001 and ****P ⩽ 0.0001).

corresponding HIV-sense reporter. No passenger strand activity
was measured as predicted for the AgoshRNA design. However,
passenger strand activity was measured for all regular shRNAs
on the respective HIV-antisense reporters, with relative lucifer-
ase expression levels dropping to between 20 and 80% of the
uninhibited value. The AgoshGag4–6 set reduced the relative
luciferase expression level to o 30%. The shRNA-AgoshRNA
comparison yielded a mixed outcome. The inhibitory potency
was increased for AgoshGag4, AgoshGag6 and AgoshPol8
versus the corresponding shRNAs, but AgoshGag5 and Agosh-
Pol45 were less potent than the matching shRNAs. AgoshPol1
and AgoshR/T5 showed similar inhibitory potential as the
corresponding shRNAs, with luciferase expression levels of the
HIV-sense reporter dropping to o 40%.

Gene Therapy (2017) 453 – 461 © 2017 Macmillan Publishers Limited, part of Springer Nature.

Figure 4. Northern blotting of the AgoshRNA processing products.
Processing of the AgoshGag4–6 variants and the corresponding
shRNA set was analyzed by northern blot analysis with (a) the ‘guide’
probe and (b) the ‘passenger’ probe. Ethidium bromide staining of
small rRNAs and tRNAs are shown as loading controls below
the blot.
Intracellular processing of shRNA/AgoshRNA inhibitors
The range of silencing potencies observed for the anti-HIV
AgoshRNAs might be due to altered RNAi activity, but could also
reflect differences in RNA processing by Ago2 or intracellular RNA
stability. Northern blot analysis revealed intracellular processing of
the overlapping AgoshRNA triplet (AgoshGag4-6) and the
corresponding shRNAs set (shGag4-6). Processing of the 3′ guide
strand of regular shRNAs and the 5′ guide strand of AgoshRNAs
variants was analyzed by northern blot analysis with a ‘guide’
probe (Figure 4a), while processing of the 5′ passenger strand of
regular shRNAs was detected by the ‘passenger’ probe (Figure 4b).
The most prominent ~ 21 nt fragment was observed for shGag5,
followed by shGag4 and shGag6, consistent with the ~ 2-fold
improved luciferase knockdown activity of shGag5. The guide
probe also detected the ~ 30 nt guide strand for the AgoshRNA
constructs and the trimmed products (~24 nt fragments). The
differential luciferase activity scored for the AgoshGag4-6 variants
correlated with the amount of guide RNA produced. The
AgoshGag6 guide is ~ 2-fold more abundant and consequently
more active than the guide strand of AgoshGag4 and AgoshGag5.
The passenger probe detected a prominent Dicer product of
~ 21 nt fragment exclusively for shGag5, which may relate to the
toxicity induced by this construct.30 Some striking exceptions
were also apparent, for example, shGag4 produces much more
guide RNA than AgoshGag4 (Figure 4a), although the order is
reversed in the luciferase assay (Figure 3b). We currently do not
fully understand these intricacies.
As predicted, none of the AgoshRNAs generated a passenger
strand. The 10 nt product of processed AgoshRNAs is never
detected on northern blottings, possibly because it is rapidly
degraded.15
Antiviral activity in stably transduced T cells and virus escape
options
The anti-HIV-1 AgoshRNAs (Gag4-6, Pol1, Pol8, Pol45 and R/T5)
were stably expressed in SupT1 T cells upon transduction with the
corresponding lentiviral vectors (LV-GFP) to allow HIV-1 replication
studies. GFP-positive cells were selected and challenged with the
HIV-1 LAI isolate (0.01 ng CA-p24). Six parallel infections were
performed for each AgoshRNA to monitor virus evolution, which is
a chance process. CA-p24 antigen was measured starting at day 3
post infection and we followed the spreading infection for
56 days. The replication curves were grouped by target gene in
three graphs, with JS1—the empty LV—as negative control
(Figure 5). Virus replication was delayed in all AgoshRNA-
expressing cells compared with JS1 cells, with differential
inhibitory activity among the AgoshRNA constructs.
© 2017 Macmillan Publishers Limited, part of Springer Nature.
Antiviral AgoshRNA design
E Herrera-Carrillo et al
457
Figure 5. Kinetics of HIV-1 LAI replication in AgoshRNA-expressing
cells. Six parallel infections per AgoshRNA-expressing SupT1 cells
were performed. The empty lentiviral vector JS1 served as the
negative control in all three panels. CA-p24 antigen was measured
starting at day three post-infection up to day 56.
We will first describe the results for AgoshGag4. It took around
21–56 days for viral escape to occur in five of six AgoshGag4
cultures (Figure 5, upper panel). We screened for the selection of
truly AgoshRNA-resistant HIV-1 variants by passage of cell-free
virus on AgoshRNA-expressing SupT1 cells and control cells
(untransduced). For all five escape cultures, the passaged virus
replicated equally well on these cells (data not shown), confirming
that a resistant virus was selected. Next, the 18 nt HIV-1 targets
and flanking regions were analyzed by population-based sequen-
cing. Point mutations within the target sequence (A3G, 2 × A6G
and C14U) were detected in four of the five cultures, confirming
the pressure imposed by the AgoshGag4 inhibitor. It may take
longer for resistance mutations to become visible in the
population-based sequence of the fifth culture.
AgoshGag5 and AgoshGag6 challenged cultures yielded a CA-
p24-positive supernatant around day 12–28. The phenotype test
indicated that the restricted cells were in fact partially permissive
to replication of the LAI isolate and we scored only wild-type (WT)
target sequences. We previously described similar breakthrough
replication of the WT virus in experiments with poorly active
shRNA inhibitors.32 The same phenomenon was observed for
AgoshPol8 cells (peak infection around day 18–39) and Agosh-
Pol1/AgoshPol45/AgoshR/T5 cells (peak infection around day 12).
Although some delay of virus replication was observed compared
to the JS1 control, the viral phenotype test indicated that
restricted cells were permissive to replication of the passaged
virus and only WT target sequences were detected by sequencing
(Figure 5, intermediate and bottom panel). We think that no
resistant virus was selected because the AgoshRNAs do not put
sufficient selective pressure on WT HIV-1.
Many effects were consistently observed among the different
assays, but we also want to mention some discrepancies in
AgoshRNA activity. For instance, only AgoshGag4 was potent in
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Figure 6. Anti-HIV activity of AgoshGag4 in monocyte-derived cells. Six parallel infections were performed for AgoshGag4 and the
corresponding shGag4 inhibitor in Dicer- minus (THP-1) and Dicer-positive (SupT1) cells. The empty lentiviral vector JS1 served as the negative
control. CA-p24 antigen was measured starting at day 3 post-infection up to day 25.

Figure 7. Competitive cell growth assay. SupT1 T cells were transduced with the lentiviral vector expressing shRNA or AgoshRNA at a
multiplicities of infection of 0.15 or 1.5, yielding a cell population with ~ 20 and 80% GFP-positive cells, respectively. The cells were passaged
and analyzed by fluorescence-activated cell sorting (FACS) measurement twice a week up to 50 days. JS1 represents the empty lentiviral
vector.

the assay of Figure 5, but AgoshGag6 was about twofold better in
the initial tests in Figures 2 and 3. Likewise, AgoshGag6 is more
efficient than AgoshGag4 in transient assays, but this advantage
disappears in long-term assays. We cannot provide a full
explanation for these differences, but the most likely explanation
is differential RNA processing or activity in different cell types.
AgoshRNAs maintain their activity in Dicer-minus cells
We next wanted to probe sustained antiviral activity of
AgoshGag4 versus shGag4 in cells that do not express Dicer.
The THP-1 cell line originated from a heterogeneous population of
tumor cells that were cultured from the blood of a patient with
acute monocytic leukaemia.33 These cells maintain monocyte
characteristics and do not express Dicer. Differentiation into
mature macrophage-like cells can be induced by phorbol
12-myristate 13-acetate, resulting in Dicer expression.33 This
would be the ideal Dicer-positive control, but this phenotype is
not stable and cells will undergo dedifferentiation.34 Furthermore,
although undifferentiated THP-1 cells can be infected with CXCR4-
using virus strains, phorbol 12-myristate 13-acetate-differentiated
cells are resistant to these viruses.34–38 We therefore used SupT1
T cells as Dicer-positive control cells.
THP-1 cells were transduced with the respective LV and GFP-
positive cells were selected for subsequent virus challenge with
5 ng HIV-1 LAI by means of spinoculation in six parallel cultures.
SupT1 T cells were transduced and GFP-positive cells were
selected for subsequent virus challenge with 0.01 ng HIV-1 LAI.
Cells with the empty LV JS1 served as negative control. CA-p24
antigen was measured starting at day 3 post-infection and we
followed the spreading infections for 25 days, after which reduced
cell surface expression of the CD4 receptor occurs in THP-1 cells.37
shGag4 showed antiviral activity in Dicer-positive cells, but lost its
HIV-1 inhibitory potency in Dicer-minus THP-1 cells (Figure 6).
AgoshGag4 demonstrated equal antiviral activity in Dicer-minus
THP-1 cells versus Dicer-positive SupT1 T cells, confirming this
AgoshRNA advantage.
AgoshRNA toxicity tests in a human T-cell line
The main theoretical advantage of the AgoshRNA design over a
regular shRNA is the absence of a passenger strand that can
induce adverse effects. This was tested by AgoshRNA-
transformation of the shGag5 antiviral, which was previously
shown to trigger reduced cell growth in vitro and in vivo.30 We
transduced SupT1 cells with the respective LV and the empty JS1
vector as negative control. To determine any negative effects on
growth of the transduced cells, we monitored the percentage
of GFP-positive (and RNA-expressing) cells in the transduced
culture for 50 days (Figure 7). Untransduced cells provide

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boost the silencing activity and to develop better design
Table 1. Competitive cell growth (CCG) assay
algorithms.
Lentiviral vector construct Reduction in GFP+ cells (%)a Recent evidence indicates that one can design shRNAs with a
small loop and short duplex length that avoid Dicer processing
moi:0.15 moi:1.5
and instead are processed by Ago2.12,15,17,18,47 This new
AgoshRNA design has a major hypothetical advantage that only
a single active guide RNA is generated, without a passenger strand
Mean s.d. Mean s.d. that can induce unwanted side effects. We previously listed
JS1 2.3 0.5 0.4 1.6 additional advantages of the AgoshRNA design.21 In this study, we
AgoshGag4 1.7 2.7 1.7 1.6 confirmed the safety advantage by converting a ‘toxic’ shRNA into
AgoshGag5 2.2 1.3 0.0 3.1 a ‘non-toxic’ AgoshRNA. An additional safety aspect relates to the
AgoshGag6 − 0.3 1.6 0.8 3.2 shorter duplex of AgoshRNAs versus shRNAs, which may activate
AgoshPol1 − 0.1 1.0 0.7 1.5 innate immunity sensors such as interferon less efficiently.48
AgoshPol8 2.1 2.1 0.7 1.6 Furthermore, AgoshRNAs like miR-451 are loaded exclusively in
AgoshPol45 − 0.8 1.0 − 0.8 1.0
AgoshRT5 0.3 1.5 − 1.4 1.1
Ago2-containing RNA-induced silencing complex complexes49
shGag5 14.1 1.1 22.2 1.7 (Harwig et al., unpublished results). Avoiding AgoshRNA loading
shRT5 1.4 1.7 0.4 2.0 in Ago1, 3 and 4 will restrict unwanted off-targeting,50 although
the contribution of the individual Ago proteins to siRNA off-target
Abbreviations: GFP, green fluorescent protein; moi, multiplicities of
activity is still unclear.50–53 The AgoshRNA design is ideally suited
infection. aGFP expression was measured up to 50 days.
for specific therapeutic strategies. A restriction for massive
AgoshRNA (and shRNA) use is that siRNA design algorithms
the internal control. The transduction was performed at two cannot be applied, which calls for a difficult trial-and-error design
different multiplicities of infection, 0.15 and 1.5, which obviously process.39–42
yielded a different starting level of GFP-positive cells. We
confirmed the selective loss of GFP-positive cells for shGag5, but
MATERIALS AND METHODS
no cell growth impairment was apparent in the AgoshRNA
cultures (included AgoshGag5) (Figure 7). Similar results were Plasmid construction
obtained in five independent experiments. A significant decrease For the AgoshRNA and shRNA constructs, complementary DNA oligonu-
of GFP-positive cells was observed exclusively for shGag5 cleotides encoding the corresponding sequences were annealed to create
(P o 0.0001) compared with the other shRNAs, all AgoshRNAs sticky BamHI and HindIII sites, and subsequently inserted into the
corresponding restriction sites of the pSUPER vector.54 Firefly luciferase
and the JS1 control (Table 1).
reporter constructs (pGL3; Promega, Madison, WI, USA) were made by
insertion of a 50–70 nt HIV-1 sequence, with the 19 nt target region in the
center, in the EcoRI and PstI sites of the pGL3 plasmid.31 The precise
DISCUSSION
positions of the inserted HIV-1 fragments are as follows: Gag (1341–1399),
We tested 21 novel anti-HIV AgoshRNA candidates by incorporat- Pol1 (1889–1948), Pol8 (4017–4155), Pol45 (4531–4589) and R/T5 (5535–
ing previously proven shRNA-derived guide sequences into the 5583). The luciferase reporters with the sense and antisense target
AgoshRNA design.27,28 Seven of these 21 AgoshRNAs were highly sequences were described previously.15 All constructs were verified by
active in reporter silencing. However, only AgoshPol8 and sequence analysis.
AgoshGag4 exhibited profound HIV-1 inhibition in spreading
infections on a T-cell line. Resistant HIV-1 variants were selected in Cell culture
the AgoshGag4 cultures, but breakthrough replication of the WT HEK293T adherent cells (ATCC CRL-11268, Manassas, VA, USA) were grown
virus was apparent for the other AgoshRNAs. The lack of selection as monolayer in Dulbecco’s modified Eagle’s medium (Life Technologies,
of AgoshRNA-resistant virus variants indicates that these antivirals Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum,
do not exert strong selective pressure on the HIV-1 RNA minimal essential medium non-essential amino acids, streptomycin
genome.27,32 Although this initial result of a single potent (100 μg ml − 1) and penicillin (100 U ml − 1) in a humidified chamber
AgoshRNA is not encouraging, we stress that only four potent at 37 °C and 5% CO2. SupT1 T cells (ATCC CRL-1942) were grown in
shRNAs were previously selected by us in a large screen of nearly Advanced RPMI (Gibco BRL, Carlsbad, CA, USA) supplemented with
100 shRNA candidates.27 RNAi tools are popular, but it is difficult L-glutamine, 1% fetal calf serum, streptomycin (30 μg ml − 1) and penicillin
to design active shRNAs and AgoshRNAs, as these molecules do (30 U ml − 1) in a humidified chamber at 37 °C and 5% CO2. THP-1 cells
not adopt the known siRNA design rules.39–42 Furthermore, HIV-1 (ATCC TIB-202) were grown in RPMI (Gibco BRL) supplemented with 10%
represents a difficult target mRNA because it adopts stable RNA fetal calf serum, streptomycin (1000 μg ml − 1) and penicillin (100 U ml − 1)
in a humidified chamber at 37 °C and 5% CO2. Regular testing for
structures that inhibit an RNAi attack.43–46 On the positive side, the
mycoplasma contamination was performed.
AgoshRNAs exclusively induced guide-mediated luciferase knock-
down, whereas the shRNA triggered guide and passenger strand
activity. We checked the absence of a passenger strand for the Transient HIV-1 inhibition assays
AgoshRNA candidates by northern blotting, which indeed seems a HEK293T cells were seeded 1 day before transfection in 24-well plates at a
major bonus over regular shRNAs. Furthermore, the AgoshRNA density of 1.2 × 105 cells per well. Co-transfection of 250 ng of the full-
candidates revealed no toxicity in the ultra-sensitive competitive length HIV-1 molecular clone pLAI,29 1 ng of pRL-CMV and 25 ng of
cell growth assay. Most strikingly, AgoshGag5 revealed no toxicity, AgoshRNA construct was performed using Lipofectamine 2000 reagent
(Invitrogen). We added pBS to have an equal DNA concentration per
whereas the corresponding shGag5 inhibitor showed a profound
transfection. Two days post transfection, samples were taken for virus
impact on T-cell growth. We demonstrated another advantage of production, which was determined by CA-p24 enzyme-linked immunosor-
AgoshRNAs: their ability to maintain antiviral activity in a Dicer- bent assay and cells were lysed for Renilla luciferase activity measurement
deficient monocyte cell line that lack Dicer expression. Thus, the with the Renilla Luciferase Assay System (Promega). Relative HIV-1
AgoshRNA design may constitute a new platform for gene production was calculated as the ratio between the CA-p24 level and
silencing that seems competitive with current miRNA and shRNA the Renilla luciferase activity. We performed three independent transfec-
technology. These data indicate that the future for AgoshRNA tions, each in duplicate. Values were corrected for between-session
therapeutics is promising, but further refinements are essential to variation as described previously.55

© 2017 Macmillan Publishers Limited, part of Springer Nature. Gene Therapy (2017) 453 – 461
 Antiviral AgoshRNA design
E Herrera-Carrillo et al
460
Luciferase assays
Luciferase tests were performed as previously described.56 In brief,
HEK293T cells were seeded one day before transfection in 24-well plates
at a density of 1.2 × 105 cells per well. Cells were co-transfected with the
indicated DNA construct (1, 5 or 25 ng), 100 ng of the firefly luciferase
expression plasmid and 1 ng of Renilla luciferase expression plasmid (pRL)
using Lipofectamine 2000 reagent (Invitrogen). We added pBS plasmid to
create an equal DNA concentration for each transfection. Cells were lysed
48 h post transfection, to measure firefly and Renilla luciferase activities
using the Dual-Luciferase Reporter Assay System (Promega). The ratio
between firefly and Renilla luciferase activity was used for normalization of
experimental variations such as differences in transfection efficiency. The
pBS plasmid and an unrelated shRNA (shNef) served as negative controls.
The luciferase activity scored with shNef activity was set at 100%. We
performed three independent transfections, each in duplicate. Values were
corrected for between-session variation. The resulting six values were used
to calculate the s.d. shown as error bar.
Lentiviral vector production and transduction
The LV was produced and titrated as described previously.27 LV plasmids
encoding the AgoshRNAs are derived from the construct JS1
(pRRLcpptpgkgfppreSsin).57 The vector was produced by co-transfection
of packaging plasmids pSYNGP,58 pRSV-rev and pVSV-g59 and LV plasmid
with Lipofectamine 2000 (Invitrogen). After transfection, the medium was
replaced with OptiMEM (Invitrogen). The LV containing supernatant was
collected, filtered (0.45 μm) and aliquots were stored at − 80 °C. The
transduction titer was measured via GFP expression. SupT1 and THP-1 cells
were transduced at a multiplicities of infection of 0.15. Three days after
transduction, live cells were selected by fluorescence-activated cell sorting
for GFP expression.
Transduced SupT1 T cells (1 × 106 cells in 5 ml medium) were challenged
with HIV-1 LAI (0.01 ng CA-p24). Transduced THP1 cells (0.2 × 106 cells in
96-well plate in 0.2 ml medium) were challenged with HIV-1 LAI (5 ng CA-
p24). Transduced THP-1 infected cells were centrifuged at 1000 g for 2 h at
18 °C. Each cell pellet was resolved in 2 ml of media and transferred to its
own well in a six-well plate tissue culture plate. Virus spread was monitored
by measuring CA-p24 production and we scored HIV-induced syncytia
formation twice a week.
Sequencing proviral target regions
When virus replication was observed after infection with HIV-1 LAI, cellular
DNA of the infected cells with the integrated provirus was isolated as
previously described.60 Integrated proviral DNA sequences were PCR
amplified with the following primer pairs (5′–3′, the position within pLAI is
indicated): Gag sense (5′-CAGACCATCAATGAGGAAGCTGCAGAATGGGAT;
position 1445-3′) and antisense (5′-CCCTGGCCTTCCCTTGTAGGAAAA
CCAGATCTTCCC-3′; position 2141); Protease, sense (5′-GTCAGAGCAGACCA
GAGCCAACAG-3′; position 2183) and antisense (5′-GATATTTCTCATGTTCAT
CTTGGGCCTTATCTATTCC-3′; position 2659); Integrase, sense (5′-GGCAA
CTAGATTGTACACATTTAGAAGG-3′; position 4499) and antisense (5′-CTCT
TTTTCCTCCATTCTATGGAGA-3′; position 5377) and Tat-Rev sense (5′-ATATC
AAGCAGGACATAACAAGG-3′; position 5525) and antisense (5′-TGCTTTAG
CATCTGATGCACAAAATA-3′; position 6458) with 30 cycles (1 min denatura-
tion at 96 °C, 1 min annealing at 62 °C and 2 min extension at 72 °C). The
PCR products were sequenced with the Big Dye Terminator Cycle
Sequencing kit (ABI) using the same primers.
Northern blot analyses
Northern blot experiments were performed as previously described.15,56
For siRNA analyses, 1.5 × 106 HEK293T cells were seeded in T25 flasks. The
cells were transfected with 5 μg AgoshRNA and shRNA constructs using
Lipofectamine 2000 reagent. Total cellular RNA was extracted two days
post transfection using the mirVana miRNA isolation kit (Life Technologies,
Invitrogen). RNA concentrations were determined on the Nanodrop 1000
machine (Thermo Fisher Scientific, Wilmington, DE, USA). Five micrograms
of total RNA was electrophoresed in a 15% denaturing polyacrylamide gel
(Precast Novex TBU gel, Life Technologies, Invitrogen). [γ − 32P]-labeled
decade RNA marker (Life Technologies, Invitrogen) was run along-side for
size estimation. To check for equal sample loading, the RNA in the gel was
stained with 2 μg ml − 1 ethidium bromide and visualized under UV light.
The RNA was electro-transferred to a positively charged nylon membrane
(Boehringer Mannheim, GmbH, Mannheim, Germany) and cross-linked to
Gene Therapy (2017) 453 – 461
the membrane using UV light (1200 μJ × 100). The membranes were
hybridized overnight with locked nucleic acid oligonucleotides. Locked
nucleic acid oligonucleotide probes were 5′-end labeled with the
kinaseMax kit (Life Technologies, Invitrogen) in the presence of 1 μl
[γ − 32 P] ATP (0.37 MBq μl − 1, Perkin Elmer, Waltham, MA, USA). The
probes were purified on Sephadex G-25 columns (Amersham Biosciences
Inc., Sunnyvale, CA, USA) to remove unincorporated nt. We used the
following oligonucleotides probes (locked nucleic acid positions under-
lined) to detect the 5′ and 3′ strand of the siRNA, respectively: guide:
5′-GAAGAAATGATGACAGCAT-3′ and passenger: 5′-ATGCTGTCATCATTTC
TTC-3′. The signals were captured by the Typhoon FLA 9500 (GE Healthcare
Bio-Sciences AB, Uppsala, Zweden) and quantifying using the Molecular
Dynamics ImageQuant (5.2) software package (GE Healthcare, Waukesha,
WI, USA).
Competitive cell growth assay
SupT1 T cells were transduced at a multiplicities of infection of 0.15 and
1.5, and transduced cells were screened for a negative impact of lentiviral
integration and/or AgoshRNA expression on cell growth using the
competitive cell growth assay.61 In brief, the mixture of transduced (GFP
+/AgoshRNA+) and untransduced (GFP − ) cells was analyzed for the GFP
+/ − ratio up to 50 days by fluorescence-activated cell sorting. The impact
on cell growth was converted as percentage reduction in cell growth.
Statistical analysis
Results are presented as mean ± s.d. P-values o0.05 were considered
statistically significant. Comparisons between three or more groups were
analysed by one-way analysis of variance followed by a Tukey’s post-hoc
test. Statistical analysis was performed using GraphPad Prism 7.02
(GraphPad Prism, La Jolla, CA, USA).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This work was supported by the Nederlandse Organisatie voor Wetenschappelijk
Onderzoek—Chemische Wetenschappen (NWO-CW, Top Grant) and Zorg Onderzoek
Nederland—Medische Wetenschappen (ZonMw, Translational Gene Therapy Grant).
We thank Berend Hooibrink for expertise in cell sorting and maintenance of the flow
cytometry facility. We thank Cosimo Cristella for valuable help with the statistical
analyses.
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