Reactive oxygen species enhance rAAV transduction by promoting its escape from late endosomes

Cell culture and chemicals

Human embryonic kidney cell line HEK 293-T (Chinese Academy of Sciences Cell Bank, GNHu17) and human cervical cancer cell line Hela (ATCC, CRM-CCL-2, Manassas, VA, USA) were used in this study. We also used human primary hepatocytes LO-2 (Procell Life Science & Technology, HL-7702) in this study. All cells were maintained in monolayer cultures in DMEM or RPMI-1640 supplemented with 1% penicillin–streptomycin (PS, Procell Life Science&Technology Co.,Ltd., Wuhan, China) and 10% fetal bovine serum (FBS). Cells were subject to incubation in a humidified chamber at 37 °C with 5% CO2. Bafilomycin A1 (Shanghai YuanYe Bio-Technology, Shanghai, China) solution was prepared in dimethylsulfoxide (DMSO) at the concentration of 10 mM. D-Luciferin (Beyotime, Shanghai, China) was prepared in PBS at 150 mg/mL concentration. In addition, cathepsin B inhibitor CA-074 (Sigma-Aldrich, C5732) and cathepsin L Inhibitor I (Sigma-Aldrich, 219421) were prepared at a concentration of 5 mM in DMSO.

Recombinant AAV vector generation

CMV promoter was employed in all recombinant AAV vectors as previously described [15]. In brief, three plasmids were transfected into 293 T cells using polyethyleneimine, Linear, molecular weight 25,000 (PEI 25 K, Polysciences). Following 72 h of transfection, cells were collected, freeze-thawed for 3 cycles, and treated with Benzonase (50 U/mL crude lysates) at 37 °C for 60 min. After that, cesium chloride gradient centrifugation was performed to purify viral vectors. Viral vectors were concentrated by ultracentrifugation followed by three PBS washes and finally resuspended in PBS. qPCR was later performed to measure the titers of rAAV vector stocks. Sequences of real time PCR primers were shown below; forward (F), 5′-tgaccctgaagttcatctgc-3′; reverse (R), 5′-gaagtcgtgc tg cttcatgt-3′.

Plasmids construction

To generate the pcDNA3.1-CTSB and pcDNA3.1-CTSL plasmids, pAAV plasmid was linearized using EcoRI and HindIII enzymes. CTSB (NM_001908.5) and CTSL (NM_001382757) coding sequences were synthesized. The following primers were utilized for the amplification of CTSB coding sequence: forward, 5′-ttgaattcatgtggcagctctgggcctccctctg-3′; and reverse, 5′-ggaagcttttaga tcttttcccagtactgatcggt-3′. The following primers were utilized for the amplification of CSTL coding sequence; forward, 5′-ccgaattcatgaat cctacactcatccttgctgc-3′; and reverse, 5′-ggaagctttta cgtagccacccat gccccattc tt-3′. PCR amplified CTSB, and CTSL DNA fragments were cloned into pcDNA3.1. pcDNA 3.1 vector containing CTSB and CTSL coding sequences was transformed into Escherichia coli TOP10 strains.

Recombinant AAV2 vector transduction assay

HeLa, 293 T, and LO-2 cells (1 × 104/well) were seeded in a 96-well plate and incubated for 12 h at 37 °C. Following pretreatment with shikonin or H2O2 for 4 h, the culture medium was replaced with DMEM containing FBS. Cells were transduced with different multiplicities of infection (MOI) with different rAAV2 vectors for 72 h at 37 °C. GFP expression levels were monitored with IncuCyte cell imaging system (Essen BioScience, MI, USA) and were analyzed by extracting the average fluorescence intensity at three different fields of each well.

siRNA transfection

Target sequences for siRNAs of CTSB and CTSL were designed and synthesized by GenePharma (Shanghai, China) (Table 1). HeLa cells were transfected with siRNAs specific for CTSB and CTSL. Scrambled siRNA was used as the control. Total cellular or tissue proteins were collected after 24 h of transfection, and gene silencing efficiency was measured by Western-blot (WB) assay. In some assays, Hela cells, were transfected with siRNA for 24 h, were further transduced with rAAV2. The endosomes were isolated, and qPCR analysis was carried out to identify the genome of rAAV2.

Table 1 Sequences of siRNA adopted for ablating CTSL and CTSB protein expressionCapsid labeling and confocal microscopy

Confocal imaging of tetramethylrhodamine (TAMRA)-labeled rAAV2 was performed as previously described [16]. Pure rAAV2 virions were incubated with mono-NHS-TAMRA (100 molecules/genome) (Sangon Biotech, Shanghai, China) at room temperature for 45 min. Then SpinOUT™ GT-600 column (G-Biosciences, MO, USA) was used to remove the unconjugated dye. qPCR was performed to determine the titer of the labeled vectors. Regarding confocal imaging experiments, Hela cells were treated with H2O2 for 4 h, and then TAMRA-labeled rAAV2 was later used to transduce HeLa cells at 2000 vg/cell. The rAAV2 distribution was analyzed at 1 h, 5 h, and 10 h after incubation. Cells were harvested, washed thrice with PBS, and subsequently fixed for 15 min with 4% paraformaldehyde (PFA) at ambient temperature. Cells were rinsed thrice with PBS, followed by a double-distilled H2O wash and were labeled using 4,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich, MO, USA). To analyze the location of rAAV2 virions, we employed the Zeiss LSM 710 spectral confocal laser-scanning microscope (Zeiss, Oberkochen, Germany).

Fractionation of cytoplasmic and nuclear protein extracts

Cytoplasmic and nuclear fractions were separated from HeLa cells following the previously described protocol [17]. Briefly, 0.01% trypsin was added to rAAV2-GFP infected cells, followed by rinsing with PBS five times to remove unabsorbed virus particles. Later, 200 µL hypotonic buffer (10 mmol/L HEPES, pH 7.9 10 mmol/L KCl, 1.5 mmol/L MgCl2, 0.5 mmol/L phenylmethanesulfonylfluoride fluoride, 0.5 mmol/L dithiothreitol) was supplemented to each tube and the cells were resuspended and incubated on ice for 5 min. After that, 10% NP-40 (10 µL) was added to each tube, and incubated for 3 min. The cells were observed under a light microscope (Nikon, Tokyo, Japan). After gentle mixing, samples were centrifuged for 5 min at 500 rpm at 4 °C, and the supernatants (cytoplasmic fraction) were collected and preserved on ice. The pellets (nuclear fraction) were rinsed twice with hypotonic buffer (1 mL) twice and preserved on ice. Cytoplasmic fraction and nuclear fraction) were analyzed to determine the purity of each fraction as described previously [18, 19]. The purity of nuclear and cytoplasmic fractions was > 95%.

In-vivo transduction assays

Animal experiments were carried out following the guidelines from the University of Huaqiao at the School of Medicine Animal Care and Use Committee. To conduct in vivo transduction assays, the shikonin solution was injected intraperitoneally into each mouse (n = 6) at 2 mg/kg body weight, and DMSO was injected intraperitoneally into six mice as the control. Next, ss-rAAV-Fluc (1 × 1011 vg/mouse) vectors carrying the fluc gene were injected intravenously into mice, and luciferase expressions were detected on day1, day 3, day 5, day 7, and day 9. Live imaging of rAAV8 vector luciferase activity was depicted in a previous study [16]. In brief, D-luciferin (Beyotime, Shanghai, China) was intraperitoneally injected (150 mg/kg) into each mouse, and an IVIS-Lumina imaging system (FluoView 100, Guangzhou Biolight Biotechnology Co., Ltd) was utilized to determine bioluminescence activity. Living Image software was adopted for quantifying the signal intensity of bioluminescence, which was shown in units of photons/s/cm2/steradian.

rAAV2 binding and internalization analysis

For assessing rAAV2 binding to the cell, cells were inoculated with rAAV2 for 30 min at 4 °C. After incubation (5000 vg/cell rAAV), cells were rinsed thrice with pre-chilled PBS. Afterwards, the TIANamp Virus DNA/RNA Kit (Tiangen, Beijing, China) was employed for extracting total cellular DNA as described in the manufacturer's guidance. qPCR was conducted with the AceQ qPCR SYBR Green Master Mix kit (SYBR Green I, Vazyme Biotechnology, Nanjing, China) using transgene-specific primers to measure the infection efficiency of rAAV as described previously [20]. For internalization assays, cells were infected for 1 h at 4 °C and cultured for 1 h at 37 °C with 5% CO2, followed by trypsin treatment to remove those non-internalized surface-bound virions. After washing thrice with pre-chilled PBS, total cellular DNA was extracted, and qPCR was conducted in order to measure total viral DNA using transgene-specific primers.

Early and late endosome separation

Early and Late endosomes were isolated according to precious description [21, 22]. At first, the cell dish was placed on ice and washed thrice with pre-chilled PBS. It was scraped and transferred into the 15 ml tubes to centrifuge at 200 g–400 g at 4 °C for 5 min. The PBS was removed and 5 mL of Homogenization Buffer being added (HB, Sucrose, 250 mM, EDTA, 1 mM; Imidazole,3 mM; Cycloheximide, 0.03 mM; Phosphatase inhibitors cocktail; Protease inhibitors cocktail) to loosen the cell pellet. Later, the suspension was transferred into the syringe for homogenization. The homogenization was monitored through phase-contrast microscopy. The homogenate was then diluted in HB, followed by 10-min centrifugation at 1600–2000 g at 4 °C. Supernatants were collected, and soluble fractions were transfected into the 13.5-ml ultracentrifuge tube on ice. Later, pre-chilled 18% percoll/sucrose buffer (9 ml) was delivered to generate one layer under the input. Ultracentrifuge tubes were placed into a rotor followed by an additional 1-h ultracentrifugation at 30,000 × g at 4 °C. Subsequently, gradient fractions were harvested from the bottom of the ultracentrifuge tube. WB assay was performed on samples obtained in percoll fractions.

Western-blot (WB) assay

We carried out WB assay according to the previous description [23]. To analyze cell protein, ~ 5 µg WCL sample was isolated using 10% SDS-PAGE, followed by transfer onto PVDF membranes (Millipore, MA, USA). After 12-h blocking using 5% BSA within TBST (150 m NaCl, 20 mmol/L Tris–HCl, pH 7.5) at 4 °C, followed by overnight incubation using specific primary antibodies at 4 °C, which included rabbit anti-CTSB (A19005, ABclonal); rabbit anti-CTSL (A4986, ABclonal); rabbit-EEA1 (#3288, CST), rabbit-RAB7 (#9367, CST); rabbit anti-GAPDH (#5174, CST). Membranes were then rinsed, followed by 1-h incubation using HRP-conjugated anti-rabbit (1:1000; R & D Systems) at an ambient temperature. Later, ECL–plus chemiluminescence substrate (Amersham Pharmacia Biotech, NJ, USA) was employed for detecting protein bands. Image J was utilized to quantify signal intensity.

Statistical analysis

Results were represented by mean ± SD. Student’s t-test or one-way ANOVA was conducted for data analysis, with p < 0.05 indicating the statistical significance. Graphpad v5.0 (GraphPad, San Diego, CA, USA) was adopted for statistical computations.

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