Download Efficient Gene Transfer of HIV-1-Specific Short Hairpin RNA into

ARTICLE
doi:10.1016/j.ymthe.2003.11.025
Efficient Gene Transfer of HIV-1-Specific Short
Hairpin RNA into Human Lymphocytic Cells
Using Recombinant Adeno-associated
Virus Vectors
Daniel Boden,* Oliver Pusch,* Fred Lee,
Lynne Tucker, and Bharat Ramratnamy
Laboratory of Retrovirology, Division of Infectious Diseases, Department of Medicine, Brown Medical School,
Providence, RI 02903, USA
*These authors contributed equally to this article.
y
To whom correspondence and reprint requests should be addressed at the Laboratory of Retrovirology, 55 Claverick Street, 4th Floor,
Providence, RI 02903, USA. Fax: (401) 444-2939. E-mail: [email protected].
The cellular introduction of short, interfering RNA leads to sequence-specific degradation of
homologous mRNA, a process termed RNA interference (RNAi). Here, we report that
recombinant adeno-associated virus 2 (rAAV-2) can be used to transfer short hairpin (sh) RNA
expression cassettes genetically into human cells. HIV-1 replication was suppressed by >95% in
H9 cells and primary human lymphocytes that expressed shRNA targeting the first exon of the
viral transactivator protein tat compared to control cells. rAAV-2 integrated stably into the host
genome, leading to long-term expression of tat shRNA. Our findings demonstrate the utility of
rAAV-2 for the genetic transfer of shRNA expression cassettes into human cells, providing an
alternative to using retroviral vectors as RNAi delivery systems.
Key Words: adeno-associated virus, HIV-1 gene therapy, RNA interference
INTRODUCTION
RNA interference (RNAi)-mediated silencing of human
immunodeficiency virus type 1 (HIV-1) replication can
be achieved by transfection of short interfering RNA
(siRNA) or DNA vectors containing siRNA expression
cassettes. Recent work has demonstrated that cellular
expression of siRNA targeting HIV-1 proteins such as tat,
rev, and gag inhibits virus replication, raising the possibility of using RNAi as a genetic therapy for chronic
HIV-1 infection [1 – 7]. Application of antiviral RNAi in a
clinical setting will require the use of efficient vector
delivery systems capable of mediating long-term, stable
siRNA expression.
Recombinant adeno-associated virus (rAAV) is an
attractive vehicle for gene therapy that offers several
advantages over other vector systems of viral origin. A
major safety concern of retroviral vectors is the insertional activation of cellular proto-oncogenes resulting
from random integration of provirus into the host
genome. One of the most attractive features of AAV is
its ability to integrate site-specifically into the AAVS1
region of chromosome 19, thereby reducing the muta-
396
genic and carcinogenic potential of somatic gene transfer [8 – 11].
Due to the requirement of helper virus for productive
infection, adeno-associated viruses are naturally replication-deficient parvoviruses and have not been associated
with any human disease [12]. Moreover, AAV-2 exhibits a
broad host range and infects a wide variety of tissues and
cells [13 – 17]. AAV-2-derived viral vectors have been used
successfully for efficient long-term gene expression in
both dividing and nondividing cells [13 – 16]. As opposed
to retroviral vectors, recombinant AAV vectors are devoid
of endogenous promoters, thereby excluding the possibility of promoter interference between an upstream viral
promoter (e.g., LTR) and an internal promoter of an
expressed transgene [17,18].
Given previous demonstration of AAV-mediated delivery of therapeutic RNA molecules such as ribozymes
[19,20], we sought to determine the utility of AAV-2 for
the stable genetic transfer of short hairpin RNA (shRNA)
expression cassettes into a lymphocytic cell line and
primary human lymphocytes. Employing this system,
we tested the antiviral activity of shRNA that targets the
HIV-1 transactivator protein tat.
MOLECULAR THERAPY Vol. 9, No. 3, March 2004
Copyright B The American Society of Gene Therapy
1525-0016/$30.00
doi:10.1016/j.ymthe.2003.11.025
RESULTS AND DISCUSSION
Antiviral Activity of a tat shRNA Expression Construct
We introduced the shRNA expression cassette into a
recombinant AAV DNA vector. The cassette consisted of
an upstream MTD (modified-tRNA-derived) Pol III-type
promoter followed by a short hairpin DNA sense and
antisense sequence, separated by a hexaloop (Fig. 1). We
chose the MTD promoter given the recent demonstration
that it more effectively mediates HIV-1-specific RNAi
compared to other Pol III promoters such as U6, U6+1,
and H1 [21]. We determined the tat shRNA target sequence by alignments of computer-predicted secondary
structures (mfold 2.0) of HIV-1 tat exon 1. We selected
the five lowest free energy structures for the analysis and
scanned 21- to 23-nt sequence stretches that were conserved among all energy structures for potential siRNA
target sites. The resulting construct pAAV-tat contained a
22-nt target sequence with favorable predicted secondary
structure features such as free energy (DG) of the target
site, total numbers of unpaired nucleotides, and major
loop size. We constructed a control vector (pAAV-luc) by
introducing luciferase-specific sequences into the shRNA
expression construct.
To assess the antiviral potency of the tat shRNA expression vector, we cotransfected 293T cells with pAAVtat or control vector pAAV-luc and the infectious molecular clone HIV-1NL4.3. We collected cell-free supernatant
2 days after transfection and quantified HIV-1 p24 antigen levels by ELISA. HIV-1 production was reduced by 72fold in cells transfected with pAAV-tat compared to
pAAV-luc control vector.
Generation of Cell Lines Stably Expressing shRNA and
HIV-1 Challenge
We produced infectious rAAV particles with an AAV
helper-free system that consisted of cloning, packaging,
and helper vectors. The AAV cloning vector contained
the Pol III-driven shRNA expression cassettes and farther
FIG. 1. Schematic representation of a short hairpin RNA expression cassette
introduced between the inverted terminal repeats (ITRs) of a recombinant
AAV-2 DNA vector. The MTD promoter was used to drive expression of a tat/
luc hairpin transcript consisting of a 21-nt sense and antisense sequence
separated by a hexaloop. A five-thymidine transcription termination signal
was placed downstream of the hairpin sequence. To allow the generation of
shRNA-expressing cell lines, a SV40 early promoter-driven neomycin
resistance gene was introduced downstream of the hairpin expression
cassette. The neomycin cassette was replaced with a GFP reporter cassette
to determine infectious titer of rAAV-2 by flow cytometry.
MOLECULAR THERAPY Vol. 9, No. 3, March 2004
Copyright B The American Society of Gene Therapy
ARTICLE
downstream a Pol II promoter-driven neomycin resistance gene or GFP reporter gene. Infectious particles were
generated after cotransfection of 293T cells with equimolar amounts of AAV cloning, packaging, and helper
vectors. We chose H9 cells, which are highly permissive
for HIV-1 infection, for the AAV transduction. The cells
were preincubated with DNA synthesis inhibitors based
on previous reports that AAV transduction of cells is
enhanced by chemical induction using certain DNAdamaging agents [22]. We determined the transduction
efficiency of this method using rAAV-hrGFP and it
revealed consistent transduction efficiencies of about
30%, determined by flow cytometry for GFP expression.
Finally, we transduced H9 cells with rAAV-tat and rAAVluc, which contain in addition to the shRNA expression
cassette the neomycin drug resistance gene, and placed
them under drug selection for 4 weeks.
Verification of Genomic Integration of rAAV
Previous work has revealed that the most common integration site of AAV into the host genome is at AAVS1,
which is located on human chromosome 19 within
19q13.4 [8,23,24]. To verify rAAV-2 genomic integration,
we performed an AAVS1-specific AAV PCR as previously
described by Huser et al. [10] and verified the results by
Southern blot analysis. We subjected DNA extracted from
rAAV-2-transduced and neomycin-selected H9 cells to a
two-step AAVS1 AAV PCR (Fig. 2A). The first-step PCR
includes the pAAV-tat-specific forward primer PAV-s and
the reverse primer PAAVS1-as, which is complementary
to a region in the AAVS1 gene of chromosome 19. The
second-step PCR consists of two AAV-specific primers. As
shown in Fig. 2B, integration of rAAV-tat was demonstrated in the genomic DNA of stably transduced H9 cells
compared to the appropriate controls. We included,
minus-first-step PCR sample (Fig. 2B, lane 5) to rule out
contamination and/or coamplification of episomal rAAV
in the second-step PCR.
To verify these findings, we performed a Southern blot
on DNA isolated from rAAV-2-transduced and neomycinselected H9 cells. The Southern blot shows genomic
integration of recombinant AAV-2 (Fig. 2C). The two
bands observed in the EcoRV lane may indicate two
different integration events within AAVS1. It is known
that AAV can integrate in various positions in the 4-kblong AAVS1 site [10]. One EcoRV site is present within the
AAVS1 site and two additional EcoRV sites are located
approximately 5.4 kb upstream and 5.8 kb downstream of
AAVS1. It is conceivable that one integration event leaves
the internal AAVS1 EcoRV site intact, while the other
integration event removes the EcoRV site. AAV integration events can lead to disruption, deletions, and rearr a n g e m e n t s o f t h e A A V S 1 t a r g e t [ 2 5, 26 ] w it h
chromosomal deletions up to 2 kb [26]. Thus, the potential removal of the EcoRV site within AAVS1 combined
with different degrees of chromosomal deletions may
397
ARTICLE
doi:10.1016/j.ymthe.2003.11.025
explain the observed two bands indicating two separate
integration events.
Quantification of tat shRNA Expression in Stably
Transduced Cell Lines
We quantified precursor tat shRNA expression levels in
transduced H9 cells by real-time PCR over a 21-day
period. The MTD promoter belongs to the class II polymerase III promoters that are intragenic and cotranscribed with the tat shRNA. The total length of the
resulting transcript including the shRNA is 150 bp and
can therefore be readily detected by RT-PCR. We subjected total RNA to reverse transcription and real-time
PCR. Input RNA amount was normalized by GAPDH PCR.
Fig. 3 shows the expression of MTD tat shRNA transcripts
in transduced and neomycin-selected cells at different
time points after HIV-1 infection. The results demonstrate stable expression of tat-specific shRNA transcripts
up to day 21 of viral infection, corresponding to day 48
posttransduction with rAAV-tat.
HIV-1 Challenge of Transduced H9 Cells and Primary
Lymphocytes
We infected H9 cells permanently expressing tat or luc
shRNA with 100 TCID50 of HIV-1NL4-3. We assayed culture
supernatant for p24 antigen production over a 3-week
period. On day 21 HIV-1 replication was suppressed by
1200-fold in cultures of H9 cells expressing tat shRNA,
compared to cultures of cells expressing luc shRNA
(Fig. 4A). Northern blot analysis of total intracellular
HIV-1 RNA confirmed the p24 antigen results with specific degradation of full-length HIV-1 RNA in infected H9
FIG. 2. (A) Strategy of two-step AAVS1 AAV PCR for the detection of genomic
integration of recombinant AAV. The first-step PCR applies primer PAV-s,
which binds to a region within the SV40 promoter of pAAV-tat, and primer
PAAVS1-as, which is complementary to a region in the AAVS1 gene of
chromosome 19. The second-step PCR includes in addition to primer PAV-s, a
second pAAV-tat-specific primer PAV-as, which binds to a region within the
MTD promoter of pAAV-tat. (B) Gel analysis of PCR products of two-step
AAVS1 AAV PCR. Lane 1,100 bp marker; lane 2,1 pg pAAV-tat DNA; lane 3,
genomic DNA of H9 cells; lanes 4 and 5, genomic DNA of H9 cells stably
transduced with AAV-MTD-tat subjected to one-step or two-step PCR; lane 6,
H2O. (C) Southern blot analysis of genomic DNA of H9 cells transduced with
rAAV-2 and subjected to neomycin selection over a period of 4 weeks.
Restriction enzyme analysis of DNA with HindIII and EcoRV reveals integration
of rAAV-2 into the host genome. DNA isolated from parental H9 cells served as
a control.
398
FIG. 3. Expression of MTD-tat shRNA transcript in rAAV-tat-transduced and
neomycin-selected H9 cells at different time points after HIV-1 infection
determined by real-time PCR. Variation of RNA input was normalized by
GAPDH real-time PCR. The number of threshold cycles correlates inversely to
the amount of expressed shRNA. Values represent the averages of duplicate
independent experiments, with the range indicated.
MOLECULAR THERAPY Vol. 9, No. 3, March 2004
Copyright B The American Society of Gene Therapy
doi:10.1016/j.ymthe.2003.11.025
FIG. 4. (A) Inhibition of HIV-1 replication in H9 cells expressing tat shRNA.
Control cells express shRNA that targets luciferase. Cells were infected with
100 TCID50 HIV-1NL4-3, and p24 antigen was assessed from cell-free supernatant at weekly intervals by immunoassay. The y axis shows fold inhibition of
p24 production in H9 cells expressing tat shRNA compared to control cells
that express luc shRNA. (B) Northern blot analysis of full-length HIV-1 RNA.
Degradation of full-length HIV-1 RNA in HIV-1-infected H9 cells expressing tat
short hairpin RNA compared to infected control cells expressing luc shRNA is
shown. RNA isolated from parental H9 cells served as negative control. GAPDH
expression served as an internal standard.
cells expressing tat shRNA compared to control cells
expressing luc shRNA on day 21 postinfection (Fig. 4B).
Next, we assessed whether AAV could be used to
transfer HIV-1-specific RNAi to primary lymphocytes.
MOLECULAR THERAPY Vol. 9, No. 3, March 2004
Copyright B The American Society of Gene Therapy
ARTICLE
We obtained peripheral blood mononuclear cells of an
anonymous HIV-1 seronegative donor from the Rhode
Island Blood Bank and depleted them of CD8+ T lymphocytes using magnetic beads (Dynal, NY, USA). We
transduced the resulting CD4+ T lymphocyte-enriched
population with AAV-tat and AAV-luc. For these experiments, the neomycin resistance gene was replaced with
blasticidin to allow for rapid selection of transduced
cells. After 1 week of drug selection in blasticidin (1 Ag/
ml), we placed the CD4+ T lymphocytes in medium
containing IL-2 (10 U/ml) and PHA (2 Ag/ml) and challenged them with 1000 TCID50 (50% tissue culture infectious dose) of HIV-1NL4-3. Measurement of p24 antigen
level in culture supernatant 3 days after viral challenge
revealed a 21-fold reduction of HIV-1 replication in CD4+
T lymphocytes expressing tat shRNA compared to cells
expressing luc shRNA. This degree of inhibition is comparable to that of recent studies employing lentiviral
delivery of si/shRNA expression cassettes targeting both
HIV-1 gene products and accessory cellular proteins
required for the viral life cycle (e.g., the CCR5 coreceptor) [27,28].
RNA interference offers a powerful technique to
inhibit selectively the expression of disease-related
genes such as oncogenes [29]. RNAi can inhibit the
replication of viruses such as HIV-1, hepatitis C, and
human papillomavirus [30 – 32]. Thus far, permanent
expression of siRNA has been most frequently achieved
by the use of retroviral delivery systems [33]. Recent
work has demonstrated that lentiviral constructs are
able to engineer RNAi in primary lymphocytes and
stem cells [27,34]. Although these systems allow successful transduction of a wide variety of cell types, their
application to human diseases may be limited given the
possibility of insertional activation of cellular oncogenes by random integration of retrovirus into the host
genome, as recently observed in a gene therapy trial for
human severe combined immunodeficiency-X1 disease
[35,36].
Our results demonstrate the utility of rAAV-2 in attaining stable expression of HIV-1-specific shRNA in human
lymphocytic cells and primary lymphocytes. Despite the
availability of highly active antiretroviral therapy, the
dynamic replication kinetics of HIV-1 ensures the eventual selection of viral species that escape drug and immune control. Nearly 50% of individuals who initiate
highly active antiretroviral therapy will eventually fail
treatment due to the emergence of drug-resistant virus.
Currently, individuals who harbor multidrug-resistant
HIV-1 have few therapeutic options. In the absence of
new, effective antiviral agents, these patients will eventually progress to severe immunodeficiency and death.
Control of HIV-1 replication by genetic transfer of HIV-1specific shRNA to stem cells or lymphocytes ex vivo may
be an option for these individuals to delay progression to
AIDS. It is not yet known whether HIV-1-specific RNAi
399
ARTICLE
can durably suppress virus replication in a clinical setting.
It is likely that the 20-fold inhibitory effect we observed
in primary lymphocytes expressing tat shRNA in vitro will
be insufficient to contain HIV-1 replication in vivo. Thus,
an important research goal must be to devise more potent
HIV-1-specific shRNA constructs. Several aspects of the
RNAi machinery could potentially be manipulated to
increase the efficiency of the resulting gene silencing.
For example, it has been demonstrated that the choice of
Pol III promoter impacts directly the potency of HIV-1specific RNAi [21]. Alternatively, it may be possible to
design vectors capable of delivering multiple shRNAs
simultaneously and thus achieve a synergistic or additive
effect. Further investigations are warranted regarding the
optimization of HIV-1 RNAi and the clinical potential of
rAAV-mediated genetic transfer of si/shRNA expression
cassettes into human cells.
METHODS
Construction of MTD promoter-driven shRNA expression cassette. The
MTD promoter was generated by recombinational PCR using the following
primers: TS, 5V-TGCTGGGCCCATAACCCAGAGGTCGATGGATCGAAACCTGCGCCACTCCTGATGAGCCTCTAGACACTCTCGAGGGCGAT-3V; TAS, 5V-ATCGCCCTCGAGAGTGTCTAGAGGCTCATCAGGAGTGGCGCAGGTTTCGATCCATCGACCTCTGGGTTATGGGCCCAGCA3V; Not-TS, 5V-GCGGCCGCGGCAGAACAGTCGAGTGGCGCAGCGGAAGCGTGCTGGGCCCATAAC-3V, and Not-TAS, 5V-GCGGCCGCATCGATGTTAGGAGATCTAAACAGAACAGTATCGCCCTCGAGAGTGTCTAG-3V. The resulting PCR product was cloned into the NotI site of the
shuttle vector pCMV-MCS (Stratagene, Valencia, CA, USA), yielding the
construct pCMV-MTD-MCS. For the production of stable cell lines, a
neomycin resistance cassette, including the SV40 early promoter and the
SV40 polyadenylation signal, was inserted into the BglII and ClaI sites of
pCMV-MTD-MCS construct. To determine transfection efficiency of the
producer cell line and transducing viral titers, humanized Renilla reniformis green fluorescent protein was introduced into the above restriction
sites. The resulting shRNA expression cassettes were subcloned into the
NotI site of pAAV-MCS (Stratagene). To incorporate the target shRNA
sequence, two complementary DNA oligos were synthesized, annealed,
and inserted between the XbaI and the XhoI sites within the MTD
promoter of pAAV-MTD-Neo. The DNA oligo sequences consisted of 21nt sense and reverse complementary sequences separated by a 6-nt loop
and terminated by a five-thymidine termination signal. The resulting
forward sequence of shRNA of pAAV-tat was 5V-CTGCTTGTACCAATTGCTATTAGGATCAATAGCAATTGGTACAAGCAGTTTTT-3V. The 6-nt loop is
highlighted in bold. A control vector, pAAV-luc, was constructed targeting the luciferase protein of the firefly Photinus pyralis with the luc shRNA
sequence 5V-AATGTCCGTTCGGTTGGCAGAAGGATCTCTGCCAACCGAACGGACATTTTT-3V. All shRNA expression cassettes were sequence
verified.
Cell transfections. 293T cells were cotransfected with pAAV-tat or pAAVluc (1 Ag) and 1 Ag HIV-1NL4.3 using 6 Al Lipofectamine 2000 (Invitrogen,
Carlsbad, CA, USA) per reaction. Twenty-four hours prior to transfection,
cells were trypsinized and plated at 4 105 cells per well in six-well plates
in DMEM containing 10% fetal bovine serum (FBS).
Creation of recombinant AAV for the genetic delivery of shRNA
expression cassettes. To create cell lines permanently expressing tat and
luc shRNA, we used an AAV Helper-Free System (Stratagene), which
consists of a pAAV cloning vector and pAAV-RC packaging and pAAVhelper vectors. The pAAV cloning vector contains a CMV-promoter-
400
doi:10.1016/j.ymthe.2003.11.025
driven expression cassette with a multiple cloning site for the introduction of the gene of interest flanked by inverted terminal repeats (ITRs),
which are essential for chromosomal integration of AAV-2. The AAV-RC
vector encodes rep and cap DNA replication proteins necessary for the
production of infectious virus. The AAV helper plasmid contains the
adenovirus genes E2A and E4, which are critical for virus particle production. The 293T cell line stably expresses adenovirus E1A and E1B
proteins, which are essential for packaging recombinant virions. Replication-deficient, infective recombinant viral particles are produced upon
cotransfection of the three DNA vectors in 293T cells. The AAV cloning
vector was modified by removing the expression cassette flanked by the
ITR and replacing it with the tat shRNA and luc shRNA expression
cassettes including the neomycin/blasticidin drug selection marker or
the GFP reporter gene. 293T cells were plated at a concentration of 5105
in 2 ml DMEM containing 10% FBS in six-well plates 24 h prior to
transfection. Each plasmid (recombinant pAAV expression plasmid,
pAAV-RC, and pAAV-Helper; 1.5 Ag was combined, precipitated, and
eluted in 20 Al of elution buffer. The combined DNA was transfected
into 293T cells using 15 Al Lipofectamine 2000. Cells were harvested 72
h posttransfection and subjected to four rounds of freeze/thaw in dry
ice – ethanol and a 37jC water bath, respectively. Cells were spun down at
5000g for 10 min at 4jC and the supernatant was filtered and stored at
80jC. Viral titers of recombinant AAV were determined 48 h postinfection of H9 cells by flow cytometry using the pAAV-hrGFP expression
vector.
It has been previously shown that AAV vectors transduce S-phase cells
at >200 times the frequency of non-S-phase cells [22]. Treatment of non-Sphase or S-phase cells with various DNA-damaging agents increased AAV
transduction frequencies up to 750-fold [22,37]. Therefore, we chemically
induced H9 cells and CD8+-depleted primary lymphocytes with the DNA
synthesis inhibitors hydroxyurea (100 mM) and sodium butyrate (3 mM)
for 6 h at 37jC. Cells were washed and resuspended in 500 Al RPMI
containing 5 Ag/ml Polybrene and infected at a M.O.I. of 0.3 with either
rAAV-tat or rAAV-luc. Viral transduction was performed at 37jC for 2 h.
Two days later, cells were washed and resuspended in RPMI containing
600 Ag/ml neomycin and maintained for 6 weeks. The chemical induction
method combined with the addition of Polybrene gave consistent transduction efficiencies of about 30% determined by flow cytometry for GFP
expression.
Genomic integration of rAAV. To verify recombinant AAV-2 genomic
integration, we performed AAVS1-specific AAV PCR and Southern blot
analysis. DNA from rAAV-2-transduced and neomycin-selected H9 cells
was extracted from 1 107 cells using the QIAamp DNA kit (Qiagen,
Valencia, CA, USA). Genomic DNA (500 ng) was subjected to a two-step
AAVS1 AAV PCR (Fig. 2A). The first-step PCR includes the pAAV-tatspecific forward primer PAV-s, 5V-CTAACTGACACACATTCCAC-3V, and
the reverse primer PAAVS1-as, 5V-GATCCGCTCAGAGGACATCAC-3V,
which is complementary to a region in the AAVS1 gene of chromosome
19. The PCR was carried out for 30 cycles (98jC for 20 s/56jC for 30 s/
72jC for 5 min) using LA-Takara polymerase (Panvera – Invitrogen, Madison, WI, USA) according to the supplier’s instructions. One microliter of
the first-step PCR product was used as template in the seminested
second-step PCR, which contained two pAAV-tat-specific primers, PAVs (same as above) and PAV-as, 5V-CATAACCCAGAGGTCGATG-3V. The
second-step PCR was performed under Titanium Taq polymerase (Clontech) under the following thermal cycling conditions: 95jC for 1 min
and 25 (95jC for 15 s, 55jC for 30 s, 68jC for 30 s). Southern blot
analysis was performed on 6 Ag of genomic DNA, which was digested
with HindIII or EcoRV (absent restriction sites in pAAV-tat) and separated
on a 0.8% agarose gel. The gel was denatured, neutralized, and then
blotted in 20 SSC transfer buffer on a Zeta-Probe GT membrane (BioRad, Hercules, CA, USA). The membrane was probed with a 400-bp
neomycin-specific DNA probe, which was 32P-labeled using the Megaprime DNA labeling system (Amersham Biosciences, Piscataway, NJ,
USA). Hybridization was carried out at 65jC in Ultrahyb Buffer (Ambion)
and membranes were washed twice in 2 SSC, 0.1% SDS and 0.2 SSC,
0.1% SDS at 65jC.
MOLECULAR THERAPY Vol. 9, No. 3, March 2004
Copyright B The American Society of Gene Therapy
ARTICLE
doi:10.1016/j.ymthe.2003.11.025
Quantification of precursor shRNA expression level. Precursor tat shRNA
expression levels in transduced H9 cells were assayed by real-time PCR
over a 21-day period. Total cellular RNA was isolated by Trizol (Invitrogen) extraction and treated with RQ1 DNase (Promega, Madison, WI,
USA) according to the manufacturer’s protocol. One microgram of DNase
I-treated RNA was used for the RT reaction using Powerscript Reverse
Transcriptase (Clontech, Palo Alto, CA, USA) following the supplier’s
recommended usage. One microliter of cDNA was added to the PCR
mix including 1 Titanium Taq PCR buffer (Clontech); 20 pmol of sense
primer MT-S, 5V-GGCAGAACAGTCGAGTG-3V, and antisense primer MTAS, 5V-CAATTGGTACAAGCAGTTCTAGAG-3V; 1 mM dNTPs; SYBR Green I
(1:75,000); 10 nM fluorescein, and 1 Titanium Taq polymerase (Clontech). RNA normalization was performed by GAPDH PCR with the
primers G1, 5V-GATTTCTCCCCCTTCTGCTGATG-3V, and G2, 5VCCTTGGCTGGGGGTGCTAA-3V. Real-time PCR was carried out in an
iCycler (Bio-Rad) using the following thermal cycling profile: 95jC for 1
min and 35 (95jC for 15 s, 61jC for 30 s, 68jC for 30 s).
HIV-1 infection of transduced target cells. Two million 293T cells were
preplated on 10-cm dishes in DMEM supplemented with 10% FBS. After
24 h 10 Ag of the molecular infectious clone HIV-1NL4.3 was transfected
into preplated 293T cells using Lipofectamine 2000. Forty-eight hours
later viral supernatant was harvested, spun down to remove any remaining cells, and filtered through a 0.45-Am syringe filter. Ten million H9
cells were infected with 1 ml of viral supernatant for 2 h at 37jC. Cells
were washed twice in PBS, resuspended in 10 ml complete RPMI, and
maintained in culture for 14 days. Culture supernatant was collected on
day 14 postinfection, spun down, filtered through a 0.45-Am syringe filter,
and stored frozen at 70jC for later use. The resulting viral stocks were
titered on PHA-stimulated PBMCs and H9 cells and the TCID50 was
determined by the method of Reed and Muench. Five million H9 cells
transduced with MTD-luc and MTD-tat, respectively, were infected with
100 TCID50 of viral stock for 2 h at 37jC, washed twice with PBS, and
resuspended in 5 ml cRPMI (10% FBS, penicillin 100 U/ml, streptomycin
100 Ag/ml) supplemented with 600 Ag/ml neomycin. Five hundred
thousand CD8+-depleted and PHA-stimulated peripheral blood lymphocytes transduced with MTD-luc or MTD-tat were infected with 1000 TCID50
of viral stock as described above for H9 cells. Cells were resupended in
cRPMI containing IL-2 (10 U/ml), PHA (2 Ag/ml), and blasticidin (1 Ag/ml).
Northern blot for intracellular HIV-1 RNA. Total cellular RNA was
extracted from rAAV-tat/luc-transduced H9 cells 21 days after infection
with HIV-1NL4.3. Ten micrograms of total cellular RNA was incubated in
glyoxal sample loading dye (Ambion) for 1 h at 50jC, separated on a
1.2% agarose gel, and transferred to a nylon membrane using the
Turboblotter system (Schleicher & Schuell, Dassel, Germany). RNA was
blotted on a Zeta-Probe GT membrane (Bio-Rad) and immobilized by UV
crosslinking. HIV-1 RNA was detected with a 32P-random-labeled gag
DNA probe (HIV-1NL4.3 position 1631 – 2130). Hybridizations were carried
out at 65jC using Ultrahyb-Oligo hybridization buffer (Ambion). The
membranes were washed twice in 2 SSC, 0.1% SDS and 0.2 SSC, 0.1%
SDS at 60jC. As an internal standard, a 500-bp 32P-random-labeled
GAPDH DNA probe was used. Filters were stripped between hybridizations by incubation in 0.1 SCC, 0.71% SDS at 95jC.
ACKNOWLEDGMENTS
This work was supported by a Clinical Scientist Development Award from the
Doris Duke Charitable Foundation, a Daland Fellowship in Clinical
Investigation from the American Philosophical Society, and a Career Development Grant from NIAID/NIH (B.R.). We thank the Brown/Tufts/Lifespan
CFAR Retrovirology Core Laboratory and the Center for Cancer Research
Development (NIHP20RR017695) for assay support. We acknowledge the AIDS
Research and Reference Reagent Program Division of AIDS, NIAID, NIH for
supplying H9 cells (contributed by Dr. Robert Gallo) and HIVNL4-3 (contributed
by Dr. Malcolm Martin).
RECEIVED FOR PUBLICATION SEPTEMBER 10, 2003; ACCEPTED NOVEMBER
18, 2003.
MOLECULAR THERAPY Vol. 9, No. 3, March 2004
Copyright B The American Society of Gene Therapy
REFERENCES
1. Lee, N. S., et al. (2002). Expression of small interfering RNAs targeted against HIV-1 rev
transcripts in human cells. Nat. Biotechnol. 20: 500 – 505.
2. Novina, C. D., et al. (2002). siRNA-directed inhibition of HIV-1 infection. Nat. Med. 8:
681 – 686.
3. Coburn, G. A., and Cullen, B. R. (2002). Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J. Virol. 76: 9225 – 9231.
4. Yamamoto, T., et al. (2002). Double-stranded nef RNA interferes with human immunodeficiency virus type 1 replication. Microbiol. Immunol. 46: 809 – 817.
5. Surabhi, R. M., and Gaynor, R. B. (2002). RNA interference directed against viral and
cellular targets inhibits human immunodeficiency virus type 1 replication. J. Virol. 76:
12963 – 12973.
6. Capodici, J., Kariko, K., and Weissman, D. (2002). Inhibition of HIV-1 infection by small
interfering RNA-mediated RNA interference. J. Immunol. 169: 5196 – 5201.
7. Jacque, J. M., Triques, K., and Stevenson, M. (2002). Modulation of HIV-1 replication
by RNA interference. Nature 418: 435 – 438.
8. Kotin, R. M., et al. (1990). Site-specific integration by adeno-associated virus. Proc.
Natl. Acad. Sci. USA 87: 2211 – 2215.
9. Muro-Cacho, C. A., Samulski, R. J., and Kaplan, D. (1992). Gene transfer in human
lymphocytes using a vector based on adeno-associated virus. J. Immunother. 11:
231 – 237.
10. Huser, D., Weger, S., and Heilbronn, R. (2002). Kinetics and frequency of adeno-associated virus site-specific integration into human chromosome 19 monitored by quantitative real-time PCR. J. Virol. 76: 7554 – 7559.
11. Huser, D., and Heilbronn, R. (2003). Adeno-associated virus integrates site-specifically
into human chromosome 19 in either orientation and with equal kinetics and frequency. J. Gen. Virol. 84: 133 – 137.
12. Cao, L., Liu, Y., During, M. J., and Xiao, W. (2000). High-titer, wild-type free recombinant adeno-associated virus vector production using intron-containing helper plasmids.
J. Virol. 74: 11456 – 11463.
13. Chiorini, J. A., et al. (1995). High-efficiency transfer of the T cell co-stimulatory molecule B7-2 to lymphoid cells using high-titer recombinant adeno-associated virus vectors. Hum. Gene Ther. 6: 1531 – 1541.
14. Wendtner, C. M., et al. (1997). Gene transfer of the costimulatory molecules B7-1 and
B7-2 into human multiple myeloma cells by recombinant adeno-associated virus enhances the cytolytic T cell response. Gene Ther. 4: 726 – 735.
15. Maass, G., et al. (1998). Recombinant adeno-associated virus for the generation of
autologous, gene-modified tumor vaccines: evidence for a high transduction efficiency
into primary epithelial cancer cells. Hum. Gene Ther. 9: 1049 – 1059.
16. Hanazono, Y., et al. (1999). In vivo marking of rhesus monkey lymphocytes by adenoassociated viral vectors: direct comparison with retroviral vectors. Blood 94: 2263 – 2270.
17. Cullen, B. R., Lomedico, P. T., and Ju, G. (1984). Transcriptional interference in avian
retroviruses – implications for the promoter insertion model of leukaemogenesis. Nature
307: 241 – 245.
18. Proudfoot, N. J. (1986). Transcriptional interference and termination between duplicated alpha-globin gene constructs suggests a novel mechanism for gene regulation.
Nature 322: 562 – 565.
19. Horster, A., et al. (1999). Recombinant AAV-2 harboring gfp-antisense/ribozyme fusion
sequences monitor transduction, gene expression, and show anti-HIV-1 efficacy. Gene
Ther. 6: 1231 – 1238.
20. Feng, Y., et al. (2000). Inhibition of CCR5-dependent HIV-1 infection by hairpin ribozyme gene therapy against CC-chemokine receptor 5. Virology 276: 271 – 278.
21. Boden, D., et al. (2003). Promoter choice affects the potency of HIV-1 specific RNA
interference. Nucleic Acids Res. 31: 5033 – 5038.
22. Alexander, I. E., Russell, D. W., and Miller, A. D. (1994). DNA-damaging agents greatly
increase the transduction of nondividing cells by adeno-associated virus vectors. J. Virol.
68: 8282 – 8287.
23. Shelling, A. N., and Smith, M. G. (1994). Targeted integration of transfected and
infected adeno-associated virus vectors containing the neomycin resistance gene. Gene
Ther. 1: 165 – 169.
24. Dutheil, N., Shi, F., Dupressoir, T., and Linden, R. M. (2000). Adeno-associated virus
site-specifically integrates into a muscle-specific DNA region. Proc. Natl. Acad. Sci. USA
97: 4862 – 4866.
25. Dyall, J., and Berns, K. I. (1998). Site-specific integration of adeno-associated virus into
an episome with the target locus via a deletion-substitution mechanism. J. Virol. 72:
6195 – 6198.
26. Nakai, H., et al. (2003). AAV serotype 2 vectors preferentially integrate into active genes
in mice. Nat. Genet. 34: 297 – 302.
27. Li, M. J., et al. (2003). Inhibition of HIV-1 infection by lentiviral vectors expressing Pol
III-promoted anti-HIV RNAs. Mol. Ther. 8: 196 – 206.
28. Qin, X. F., An, D. S., Chen, I. S., and Baltimore, D. (2003). Inhibiting HIV-1 infection in
human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5.
Proc. Natl. Acad. Sci. USA 100: 183 – 188.
29. Brummelkamp, T. R., Bernards, R., and Agami, R. (2002). Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2: 243 – 247.
30. Jiang, M., and Milner, J. (2002). Selective silencing of viral gene expression in HPV-
401
ARTICLE
positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene 21: 6041 – 6048.
31. Kapadia, S. B., Brideau-Andersen, A., and Chisari, F. V. (2003). Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc. Natl. Acad. Sci. USA
100: 2014 – 2018.
32. Wilson, J. A., et al. (2003). RNA interference blocks gene expression and RNA synthesis
from hepatitis C replicons propagated in human liver cells. Proc. Natl. Acad. Sci. USA
100: 2783 – 2788.
33. Barton, G. M., and Medzhitov, R. (2002). Retroviral delivery of small interfering RNA
into primary cells. Proc. Natl. Acad. Sci. USA 99: 14943 – 14945.
402
doi:10.1016/j.ymthe.2003.11.025
34. An, D. S., et al. (2003). Efficient lentiviral vectors for short hairpin RNA delivery into
human cells. Hum. Gene Ther. 14: 1207 – 1212.
35. Cavazzana-Calvo, M., et al. (2000). Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288: 669 – 672.
36. Hacein-Bey-Abina, S., et al. (2003). A serious adverse event after successful gene
therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 348:
255 – 256.
37. Russell, D. W., Alexander, I. E., Miller, A. D. (1995). DNA synthesis and topoisomerase
inhibitors increase transduction by adeno-associated virus vectors. Proc. Natl. Acad. Sci.
USA 92: 5719 – 5723.
MOLECULAR THERAPY Vol. 9, No. 3, March 2004
Copyright B The American Society of Gene Therapy