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Introduction
In previous studies, scientists have successfully
modified the filamentous bacteriophages, which are capable of
delivering genes to mammalian cells using targeting ligands,
such as growth factors[1_5],
antibodies[6], and viral capsid
proteins[7]. To improve the ligand display level on phages,
we developed LMP cells (Escherichia coli bearing the
ligand-pIII-encoding helper phage) to make cell-targeted phagemid
particles[8]. We also demonstrated that it can deliver reporter
genes into target cells.
RNAi techniques are a promising way to silence
therapeutic target genes. Both non-viral vectors and viral
vectors have been used as a vehicle to deliver siRNA in vitro and in vivo. The major problem of non-viral vectors lies in its
low gene delivery efficiency and hard quality control.
Animal viruses, such as adenoviruses, have broad natural
tropism which makes them difficult to selectively transfer siRNA
genes into target cells. In contrast to these animal viruses,
filamentous bacteriophage has no natural tropism to
mammalian cells. However, it can highly efficiently infect the
targeted cells when the peptide ligand was displayed on the
surface. Like non-viral vectors, the major problem of this
vector is its low gene delivery efficiency. For-tunately,
hydroxycamptothecin (HCPT), a topoisomerase I inhibitor
and also anticancer drug, can substantially improve its gene
delivery efficiency[1,13,17,18]. In this study, we tested whether
cell-targeted phages can be used as siRNA gene delivery
tools or not.
Materials and methods
Reagents Restriction endonucleases were purchased
from TaKaRa Biotechnology (Dalian, China). Dulbecco's
modified Eagle's medium (DMEM), Dulbecco's
phosphate-buffered saline, fetal bovine serum (FBS), and
trypsin/ethylenediamine tetraacetic acid were purchased from Invitrogen
(Grand Island, NY, USA).
Cell culture H1299 (large-cell lung carcinoma; ATCC,
Manassas, VA, USA) cells were cultured at 37 ºC in medium
consisting of 90% DMEM and 10% FBS in a humidified
atmosphere of 95% air and 5% CO2.
Plasmid construction All oligonucleotides were
synthesized by Sangon (Shanghai, China) and annealed to be
double-stranded, as previously
described[10]. The selection of the coding sequences of EGFP and Akt for siRNA was
based on 2 different publications,
respectively[9,10]. Detailed information of the oligonucleotides is shown in Table 1.
To generate the pSi1.0-EGFP RNAi plasmid, a 55-nt
oligo-DNA duplex (annealed by siEGFP-f and siEGFP-r)
corresponding to nucleotides 106_127 of the pEGFP-N1 (BD
Biosciences Clontech, Palo Alto, CA, USA)-coding region was
directly inserted into the pSilencer1.0 (Ambion, Austin, TX,
USA) vector digested with ApaI (blunted) and HindIII.
The pSilencer4.0-f plasmid was generated as follows: the
pGl3-control plasmid (Promega category no.: E1741; Promega,
Madison, WI,USA) was digested with ScaI, and the 1.08 kb
fragment were used to ligate with pSilencer4.0 (Ambion,
Austin, TX, USA) which is linearized with ScaI. The inserted subclones with the reversed orientation of
cytomegalovirus (CMV) promoter are genetically selected with the
supplementary of ampicillin resistance. This modified
psilencer4.0 was prepared for the phage package and siRNA
expression.
To generate the pSi4.0-Akt plasmid, a 52 nt oligo-DNA
duplex (annealed by siAkt-f and siAkt-r) corresponding to
nucleotides 1462_1482 of the Akt1 (NM_001014432) and 1244_1264 of Akt2 (NM_001626)-coding region was
directly inserted into the pSilencer4.0 vector digested with BamHI and HindIII. The mock Akt sequence is only one
nucleotide different from siAkt, as listed in Table 1.
Preparation of phagemid particles The phagemid
particles were prepared following previously published
protocols[11] with some modifications.
M13KO7EGFCT[8] was transformed into Escherichia
coli to make LMP cells. The phagemid carrying the siRNA-encoding sequence was
transformed into LMP cells. The cells were then plated on LB
agar (containing 70 μg/mL kanamycin and 50 μg/mL ampicillin)
and incubated at 37 ºC overnight, A cell clone was picked the
next day and transferred into 1 L LB containing 70
mg/mL kanamycin and 50 mg/mL ampicillin. After shaking at 37 ºC
for 14_16 h, the supernatant of the culture was collected and
the phagemid particles were purified with polyethylene
glycol (PEG)/NaCl precipitation.
Quantification of phagemid particles The phagemid
particles were quantified by ELISA, as previously
described[12]. Briefly, serial dilutions of phagemid particles in coating buffer
(0.1 mol/L NaHCO3, pH 9.1) were coated in microtiter
plates (Corning; Corning, NY, USA) at 4 ºC overnight.
After blocking with 1% bovine serum albumin (BSA; Sigma,
St Louis, MO, USA) in phosphate-buffered saline (PBS),
the bound phages were stained with a mouse anti-M13
antibody conjugated with horseradish peroxidase (HRP;
Amersham Biosciences, Piscataway, NJ, USA) diluted at
1:200 in PBST (PBS containing 0.1% Tween-20). The
signal was developed using the
2,2´-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) substrate and
quantitated in an ELISA reader (Bio-Rad Laboratories,
Hercules, CA, USA). The M13KO7 helper phage of a known number of pfus was used for standardization.
Purification of single-strand DNA from phagemid
particles Single-strand DNA (ssDNA) was extracted from the
phagemid particles following the supplied protocol
(Ph.D.-12 phage display peptide library kit, New England Biolabs,
Beverly, MA, USA). Briefly, the phagemid particles were
precipitated by PEG/NaCl. The pellet was then suspended
with 100 μL iodide buffer. In total, 250 mL ethanol was added
and incubated for 10 min to allow for ssDNA precipitation.
The pellet was collected after centrifugation at 12
000×g for 10 min, washed in 70% ethanol, and air dried for 10 min. It
was finally dissolved in 30μL TE buffer (10 mmol/L Tris-HCl
[pH 8.0] and 1 mmol/L EDTA) and analyzed by agarose gel
electrophoresis.
In vitro phagemid particle
transfection The cells were plated into 24-well plates at densities of 10 000 cells per well,
24 h prior to the phage particle addition. The phages were
added at 1011 pfu/mL and incubated with the cells for 48 h at
37 ºC in complete media. The cells were visualized under an
epifluorescent inverted microscope (X71; OlIympus, Tokyo,
Japan). All the transfections were done in triplicate and
performed at least twice.
HCPT treatment HCPT treatments were performed 48 h
after the addition of the phages in medium containing 10%
FBS. The medium was removed at 48 h after the phage
addition, and the cells were incubated with HCPT at 2.5
μmol/L for 6 h at 37 ºC. This was followed by replacement with fresh
medium and an additional incubation of 18 h at 37 ºC.
Western blot analysis In total,
106 H1299 cells transfected transiently with a variety of phagemid particles were
lysed after HCPT treatment, as described in the Results. Prior
to the lyses, the cells were washed with PBS and collected
by scraping. Then, they were lysed in ice-cold Tris buffer
(50 mmol/L, pH 7.5) containing 5 mmol/L EDTA, 300 mmol/L
NaCl, 0.1% Igepal, 0.5 mmol/L NaF, 0.5 mmol/L
Na3VO4, 0.5 mmol/L phenylmethylsulfonyl fluoride, and antiprotease
mixture (Roche Molecular Biochemicals, Indianapolis, IN, USA),
sonicated, and centrifuged at 13 000×g for 10 min. The
supernatant was used for protein determination by the Bradford
procedure (Bio-Rad, USA) and Western blotting. The
proteins were resolved on 12% SDS-PAGE, transferred onto
nitrocellulose membranes, and incubated with the appropriate
antibodies. The following protocols are the same as
described earlier. The anti-pSer473-AKT antibody (Cell
Signaling Technology, Beverly, MA, USA) was used in 1:1000
dilutions. The goat antimouse immunoglobulin G conjugated
with HRP (ImmuClub Labs, Sunnyvale, CA, USA) was
diluted at 1:10 000 in 1% BSA in PBST. After 1 h incubation,
the filters were washed with PBST, developed using a
SuperSignal West Pico kit (Pierce Biotechnologies,
Rockford, IL, USA) for 5 min, and exposed to X-ray film.
Results
Construction of siRNA expression vectors To improve
the expression of siRNA against Akt, the pSilencer4.1-CMV
vector which carries the CMV promoter was used.
pSi4.1CMV-f1 was constructed by inserting the F1 origin sequence into
pSilencer4.1-CMV. pSilencer4.0-siAKT was obtained by
insert shRNA against Akt into pSi4.1CMV-f1.
ssDNA analysis To examine the ratio of phagemids to
helper phage genomes packaged in the phagemid particles,
we analyzed the ssDNA. The results indicate that almost all
of the DNA packaged were phagemids (Figure 1).
In vitro inhibition of EGFP expression by EGF
displaying pSil.0-siEGFP phagemid particle
infection To test whether phagemid particles carrying siRNA against EGFP
can inhibit EGFP expression, we transfected the pEGFP-N1
plasmid into NCI-H1299 cells. At 12 h after transfection, the
phagemid particles (1011 pfu/mL) were transduced into the
cells. At 24 h after transduction, HCPT was added into the
culture at a final concentration of 2.5μmol/L; the cells was
incubated for another 24 h. The data shown in Figure 2
indicate that pSil.0-siEGFP can significantly inhibit the
expression of EGFP.
Phagemid particles of pSi4.1-siAkt can inhibit Akt
expression of NCI-H1299 in the presence of
HCPT The NCI-H1299 cells was infected with pSi4.1-siAkt phagemid
particles packaged by M13KO7EGFCT. HCPT was added to
improve the siRNA expression. Western blotting was
performed to examine the Akt expression.
As shown in Figure 3A, the pSi4.1-siAKT plasmid
transfected by Lipofectamine can inhibit the Akt expression by
approximately 50%. No inhibition was found in mock vector
pSi4.1-M.
As shown in Figure 3B, in the absence of HCPT, the
phagemid particles of siAkt can not efficiently inhibit the
Akt expression. However, in the presence of HCPT (Figure
3C), the siAKT phagemid particles can inhibit the Akt
expression in a dose-dependent manner. The highest
inhibition was 50%-60% when 1×1011 pfu/mL particles were
added.
Discussion
In this study, we utilized a novel phagemid system to
deliver several siRNA into mammalian cells and estimated
their effect in vitro. The phagemid vectors have a number of
advantages over other vectors. Unlike other viral gene
delivery vector, filamentous phages have no natural
mammalian cell tropism. Phagemid particles can be easily retargeted
by displaying various ligands, such as peptides, scFv and
natural ligands (eg EGF,
FGF)[5,8,14_16]. In addition, it is very
efficient to prepare the phagemid particles rapidly (about
2_3 d). In previous studies, phages were modified so that they
could be used in gene delivery. The ability of cell-targeted
phages to deliver siRNA would be of interest. Since phage
genomes for packaging replicates by rolling circles, we
examined in this study whether the hairpin structure of the
siRNA-encoding sequence will impede its replication and
package. The results clearly demonstrate that phagemid
particles carrying siRNA can be prepared as efficiently as those
carrying other protein-encoding genes.
This study also demonstrates that cell-targeted phagemid
particles carrying siRNA can be expressed efficiently in the
presence of HCPT, although further studies need to be
performed to elucidate the mechanism.
We also tested whether the phagemid particles carrying
siRNA against Akt have some effect on cell growth. However,
no significant inhibition was obtained. This is not
uncommon, because the EGF ligand on the phagemid
particles has a cell-growth stimulatory effect that might
compensate the cell growth inhibitory effect contributed by the
siRNA. Thus, to be a cancer gene-delivery vector, phage
vectors should display ligands that do not prompt cell growth
or carry siRNA against other oncogenes, such as PI3K.
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