MATERIALS AND METHODSCell culturePC (prostate cancer) cell lines were obtained from American Type Culture Collection. LNCap cells (ATCC No. CRL-1740), PC-3 cells (ATCC No. CRL-1435) were grown in Roswell Park Memorial Institute medium 1640 (RPMI 1640, Invitrogen) supplemented with 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin and were maintained at 37°C and 5% CO2. LNCap cells were shown to be PSMA positive by Western blotting 31 (Figure (Figure22).MaterialsDOTAP, cholesterol, and DSPE-PEG2000 were purchased from Avanti Polar Lipid, Inc. unless otherwise noted, all chemicals were purchased from Sigma-Aldrich, all cell culture products were purchased from Gibco BRL/Life Technologies, a division of Invitrogen, and all restriction enzymes were obtained from New England BioLabs (NEB).Design and cloning of PLK1-specific gRNAgRNA expression plasmids were constructed according to manufacturer's protocol 32, 33 and detailed BLAST searches of the human and murine genomes were conducted to identify potential off-target binding of PLK1 gRNAs. To assess the utility of PLK1-targeting gRNAs, oligonucleotide were designed to target the complete genomic PLK1 (5′-CGGAGGCTCTGCTCGGATCG-3′). All oligonucleotides were synthesized and purified by Sangon Biotech Co (Shanghai, China). Briefly, to prepare a 100-bp double-stranded DNA insert fragment containing the target sequence (20 bp) and a protospacer-adjacent motif sequence, we used a set of oligonucleotides and generated the fragment using T4 PNK (NEB, Ipswich, MA, USA). The double-stranded DNA fragment was purified and inserted into the BbsI site of a gRNA cloning vector with T4 DNA ligase (NEB).Preparation of A10-liposome- CRISPR/Cas9 chimerasIn vitro transcription of the A10 aptamerTranscription was performed either with or without modified NTPs (2’F-dCTP, 2’F-dUTP and 3′NH2-dUTP) according to the instructions of the DuraScribe® T7 Transcription Kit (Epicentre Biotechnologies). The 20 μL transcription reactions contained 2 μL 10 × T7 reaction buffer, 1 μL 100 mM ATP, 1 μL 100 mM GTP, 2 μL 50 mM 2’F-dCTP or CTP, 2 μL 50 mM 2’F-dUTP or 3′NH2-dUTP or UTP, 2 μL 100 mM DTT, 2 μL DuraScribe T7 RNA Enzyme Mix, and 1 μg A10 double-stranded PCR template.Conjugation of DSPE-PEG2000 and modified aptamerFifty microliters of DSPE-PEG2000-COOH (10 μg/μL in DNase/RNase-free water) was incubated with 100 μL of 800 mmol/L 1-(3-dimethylaminopropyl)- 3-ethylcarbodimide hydrochloride (EDC) and 100 μL of 200 mmol/L N-hydroxysuccinimide (NHS) for 15 min at room temperature with gentle stirring. Where indicated, the resulting NHS-activated DSPE-PEG2000 was covalently linked to 50 μL of 3′-NH2–modified A10 PSMA aptamer (1 μg/μL in DNase/RNase-free water). The aptamer-DSPE-PEG2000 bioconjugates were washed, resuspended, and preserved in suspension form in DNase/RNase-free water.Preparation of liposome-CRISPR/Cas9 chimerasDOTAP and cholesterol were dissolved at a 3:1 molar ratio in a mixture of chloroform in a glass vial. The solvent was removed by reverse evaporation in a rotary evaporator at 25°C, 0.06 MPa. Five milliliters of sterile deionized water was added to the dried lipid film, and the mixture was allowed to swell overnight. The lipids were then sonicated in a bath-type sonicator for 5 min followed by extrusion through 450 and 220 nm membrane filters 5 times. This preparation was stored at 4°C before use. Novel liposome-CRISPR/Cas9 chimeras were composed of DOTAP/cholesterol liposome, protamine, and a mixture of gRNA and calf thymus DNA (0.75:1 weight ratio). To prepare the liposome-CRISPR/Cas9 chimeras, protamine (2 mg/mL), deionized water, and the gRNA/calf thymus DNA mixture (2 mg/mL) were combined in a 1.5 mL tube. The complex was allowed to stand at room temperature for 10 min before the addition of DOTAP/cholesterol liposome (300:1 molar ratio vs gRNA). The liposome- CRISPR/Cas9 chimeras were incubated at room temperature for another 10 min before further application.Conjugation of DSPE-PEG2000-A10 and liposome-CRISPR/Cas9 chimerasThe resultant mixture of DSPE-PEG2000-A10 bioconjugates (0.3:1 mole ratio vs DOTAP) and the liposome-CRISPR/Cas9 chimeras were incubated at 60°C for 1 h so that the ligand-anchors could rapidly insert into the outer liposome monolayer.Characterization of the A10-liposome-CRISPR/Cas9 chimerasThe particle size and zeta potential of the liposome-CRISPR/Cas9 chimeras were measured using the Nano Zetasizer (Malvern Instrument Ltd.). Average values were reported as the mean ± standard deviation. The liposome-gRNA chimeras were observed under transmission electron microscopy (TEM). Briefly, the liposomes were diluted ten-fold with distilled water and applied to 300 mesh, formvarcarbon-coated Cu grids. Chimeras were then negatively stained with 2% uranyl acetate (pH 4.8) for 30 s. Stained samples were characterized on a Philips CM120 TEM at a final magnification of 120,000 ×. Three grids were prepared for each sample and the grid openings were randomly selected and viewed.Cell-surface binding competition assaysLNCaP or PC-3 cells were used for cell-surface binding competitive experiments. Anti-PSMA 3C6 antibody (2 μg, R&D Systems Inc.), A10 aptamer, DSPE-PEG2000-A10, Mut-A10 aptamer or A10-liposome-CRISPR/Cas9 chimeras were used as antibody competitors. Cells were trypsinized, washed twice with 500 mL PBS, and fixed in 400 mL of FIX solution (PBS + 1% formaldehyde) for 20 min at 25°C. After washing with PBS, cells pellets were re-suspended in 1 × binding buffer (20 mM HEPES pH 7.4, 150 mM Nacl, 2 mM CaCl2, 0.01% BSA) containing 2 μg (1 μg/μL) PE modified anti-PSMA 3C6 antibody at 37°C for 20 min. Then different competitors were added to compete with PE modified anti-PSMA 3C6 antibody in PBS with 4% FBS pre-warmed at 37°C for 20 min. Cells were washed three times with PBS, fixed in 400 μL of FIX, and analyzed by flow cytometry.In the 5-α-dihydrotestosterone (DHT, Sigma) treatment experiment, LNCap or PC-3 cells were grown in RPMI 1640 medium containing 5% charcoal-stripped serum for 24 h before the addition of DHT in RPMI 1640 medium containing 5% charcoal-stripped FBS for 48 h. And then competition assays were performed with different competitors described above. Cell surface competition was assessed by flow cytometry.Total PSMA expression was analyzed by western blotting. Cells were trypsinized, and counted using a hemocytometer. Cell pellets were re-suspended in 1 × RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 % NP-40) containing 1 × protease and phosphatase inhibitor cocktails (Sigma) and incubated on ice for 20 min. Split products were boiled with 2 × protein loading buffer, and 15 μL of total protein from the supernatants were resolved on a 12.5 % SDS-PAGE gel. PSMA was detected using a 1:1000 dilution of an antibody specific to human PSMA (Abnova) and a 1:2000 dilution of secondary antibody (goat anti-mouse IgG) in blocking buffer. b-actin was detected using a 1:1000 dilution of an antibody specific to human β-actin (Cell Signaling Technology, Inc.) and a 1:2000 dilution of secondary antibody (goat anti-mouse IgG) in blocking buffer.Gene knockdown assayLNCap or PC-3 cells were seeded in 6-well plates at 60% confluency. Cells were transfected with CRISPR/Cas9 on day 3 using Lipofectamine-2000 (Invitrogen) following the manufacturer's recommendations. In parallel, cells were treated with free CRISPR/Cas9, liposome-CRISPR/Cas9 chimeras, liposome-CRISPR/Cas9 chimeras (with protamine and calf thymus), A10-liposome-CRISPR/Cas9 chimeras or A10-liposome- CRISPR/Cas9 chimeras (with protamine and calf thymus). To further verify that silencing by liposome- CRISPR/Cas9 chimeras is dependent on PSMA, LNCap or PC-3 cells were incubated with 2 nM DHT for 48 h before the addition of chimeras. Cells were collected on day 5 for analysis. Gene knowkdown was assessed by RT-PCR of mRNA (50 ng) from cells treated with the various CRISPR/Cas9 or chimeras using the One-Step RT-PCR Kit (Tiangen, Beijing). All reactions were done in a 50-mL volume in triplicate. PCR parameters were as follows: 50°C for 30 min, 5 min of Taq activation at 95°C, followed by 45 cycles of 95°C × 30 s, 57°C × 30 s, 72°C × 30 s. Standard curves were generated, and the relative amount of target gene mRNA was normalized to GAPDH mRNA. Specificity was verified by agarose gel electrophoresis.Cellular uptake studyLNCap cells (2.25 × 105 per well) were seeded in 6-well plates (Corning Inc.) and allowed to attach and grow overnight at 37°C and 5% CO2. Cells were washed with PBS 3 times and incubated with various concentrations of FAM labeled siRNAs, Lipofectamine-2000-FAM labeled CRISPR/Cas9 chimeras, liposome-CRISPR/Cas9 chimeras, liposome-CRISPR/Cas9 chimeras with protamine and calf thymus DNA, or A10-liposome-CRISPR/Cas9 chimeras with protamine and calf thymus DNA for 4 hr at 37°C. Treatments were done in triplicate. The cells were then washed with PBS 3 times and lysed with 1 mL of 0.1% Triton-X-100 in PBS. The cells were collected and measured for the fluorescence intensity at λem = 562 nm and λex = 494 nm by microscopy using a Nikon eclipse TE 300 Confocal microscope.Cell apoptosis assayThe apoptosis of LNCap cells in the presence or absence of different chimeras, or without any treatment as a blank control, was evaluated by flow cytometry. the cells were collected and stained with the Annexin V-FITC apoptosis detection kit (KeyGEN, Nanjing, China) according to the manufacturer's instructions, and were immediately analyzed via the FACScan flow cytometer with 10,000 events collected (Ex: 488 nm; Em: 530 nm).Cell viabilityThe viability of LNCap cells in the presence or absence of different chimeras, or without any treatment as a blank control, was evaluated by 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl- tetrazoliumbromide (MTT) assay over a period of 24 h. LNCap cells were seeded at a density of 5,000 cells/well (200 μL) in 96-well flat-bottomed microtiter plates over-night. Cells were treated with PBS once and incubated with 400 nM free siRNA, Lipofectamine-2000, liposomes, liposomes with protamine and calf thymus DNA, A10-liposomes, A10-liposomes with protamine and calf thymus, CRISPR/Cas9-Lipofectamine-2000 chimeras, liposome-CRISPR/Cas9 chimeras, liposome-CRISPR/Cas9 chimeras (with protamine and calf thymus DNA), or A10-liposome-CRISPR/Cas9 chimeras (with protamine and calf thymus DNA) for 24 h at 37°C, and then washed 3 times with PBS. 20 μL of MTT solution (5 mg/mL in PBS) was then added to each well and the cells were incubated further for 1 hr at 37°C. The media were removed and the cells were dissolved in DMSO. Ultraviolet absorbance at 555 nm was measured in Thermo Electron Corporation Multiskan MK3 (Thermo Fisher Scientific Inc.). The data are expressed as the percent of viable cells compared to untreated control cells.Animal experimentsMale athymic nude mice (nu/nu) of age 6–8 weeks were obtained from BioDure Technology (Beijing, China) and maintained in a sterile environment. The First Affiliated Hospital of Xi’an Jiaotong University Institutional Animal Licensing Committee approved the animal experiments undertaken, and the research protocol was in accordance with the First Affiliated Hospital of Xi’an Jiaotong University institutional guidelines for the Animal Care and Use Committee. Mice were inoculated with LNCap cells and randomly divided into five groups of nine mice per treatment group as follows: no treatment (PBS); treatment with free CRISPR/Cas9 (40 μg gRNA + 40 μg Cas9); treatment with A10-liposomes-Scrambled CRISPR/Cas9 chimeras (40 μg A10-liposomes-Scrambled gRNA+ 40 μg Cas9); treatment with liposomes-CRISPR/Cas9 chimeras (40 μg liposomes-gRNA + 40 μg Cas9); treatment with A10-liposomes- CRISPR/Cas9 chimeras (40 μg A10-liposomes-gRNA + 40 μg Cas9). Tumors were measured every other day with calipers in two dimensions. The following formula was used to calculate tumor volume: VT = W × L × L × 0.5 (W, the longest dimension; L, the shortest dimension). The growth curves are plotted as the mean tumor volume ± S.E.M. The animals were sacrificed 3 d after the last treatment, and the tumors were excised and formalin fithe for immunohistochemistry. Slides of serial sections were stained with hematoxylin and eosin (H&E).Cytokine induction assayMale athymic nude mice (nu/nu) were injected i.v. with PBS, free CRISPR/Cas9 (40 μg gRNA + 40 μg Cas9), A10-liposomes-Scrambled CRISPR/Cas9 chimeras (40 μg A10-liposomes-Scrambled gRNA + 40 μg Cas9), liposomes- CRISPR/Cas9 chimeras (40 μg liposomes-gRNA + 40 μg Cas9), A10-liposomes-CRISPR/Cas9 chimeras (40 μg A10-liposomes-gRNA + 40 μg Cas9), or poly (I:C) (25 μg). Just prior to use, the poly (I:C) stock was diluted 1:5 in PBS to provide a 500 μg/mL working solution. Two hours after the injections, blood samples were collected from the tail artery and incubated at room temperature for 0.5 h for coagulation. Serum was obtained by centrifuging the clotted blood at 16,000 rpm for 20–40 min. Cytokine levels were determined using ELISA kits for IL-12 and IFN-α (BD Biosciences, San Diego, CA).Statistical analysisStatistical analysis was conducted using one-way ANOVA, Mann Whitney. P < 0.05 was considered to indicate a significant difference. The survival rate was analyzed by the chi-square test.
Article TitleTargeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome
The potent ability of CRISPR/Cas9 system to inhibit the expression of targeted gene is being exploited as a new class of therapeutics for a variety of diseases. However, the efficient and safe delivery of CRISPR/Cas9 into specific cell populations is still the principal challenge in the clinical development of CRISPR/Cas9 therapeutics. In this study, a flexible aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery and increased flexibility. Our chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand. Cationic liposomes were linked to aptamers by the post-insertion method and were used to deliver therapeutic CRISPR/Cas9 that target the survival gene, polo-like kinase 1, in tumor cells. We demonstrate that the aptamer-liposome-CRISPR/Cas9 chimeras had a significant cell-type binding specificity and a remarkable gene silencing effectin vitro. Furthermore, silencing promoted a conspicuous regression of prostate cancerin vivo. Importantly, the approach described here provides a universal means of cell type–specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs.