Materials and Methods

Blood-brain barrier–penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy

MATERIALS AND METHODSAnalysis of RNase protection assayTo evaluate the stability of ANCSS(Cas9/sgPLK1) in the presence of RNase A, the free Cas9/sgPLK1 and ANCSS(Cas9/sgPLK1) (Cas9: 200 nM) was incubated in the RNase A (1 mg/ml) solution at 37°C for 30 min, followed by the addition of targeted DNA, and incubated at 37°C for 60 min to assess its ability to induce DNA double-stranded breaks in target DNA. The RNase protection effect was analyzed using gel electrophoresis.Western blotting to determine the LRP-1 and PLK1 proteinU87MG human glioblastoma cells; GL261 mouse glioblastoma cells; CSC2 GSCs; 83NS GSCs; normal glial cell HA 1800; astrocytes cell BV2 and endothelial cell hCMEC/D3; and liver, lung, and kidney organs were lysed using lysis buffer (Beyotime, China). The protein concentrations were quantified by BCA Protein Assay. Lysates were separated by SDS–polyacrylamide gel electrophoresis and were transferred onto polyvinylidene difluoride (PVDF) membranes. PVDF membranes were incubated with primary antibody against PLK1 mouse monoclonal antibody (mAb) 35-206; 1:1000; Abcam, LRP-1 rabbit mAb (EPR3724); 1:50,000; Abcam, and secondary antibody (LI-COR IRDye 800CW). Protein bands were displayed by an ECL detection system and were analyzed using ImageJ software.Gene editing and sequencingU87MG cells were seeded in 24-well plates at 5 × 104 cells per well and were cultured for 24 hours. The cells were then incubated with ANCSS(Cas9/sgPLK1), ANC(Cas9/sgPLK1), NCSS(Cas9/sgPLK1), ANCSS(Cas9/sgScr), Lipo(Cas9/sgPLK1), or free Cas9/sgPLK1 (Cas9, 20 nM) overnight, and the medium was replaced with fresh medium containing 10% fetal bovine serum (FBS). The cells were incubated at 37°C for another 48 hours and then were collected and processed to harvest genomic DNA using the Universal Genomic DNA Kit (CWBIO, China). The sgRNA-targeted genomic locus was amplified with High-Fidelity KOD-Plus-Neo (TOYOBO, Japan). After purification by gel extraction (CWBIO, China), T7E1 cleavage assays were conducted. Briefly, 200 ng of the purified polymerase chain reaction (PCR) product was denatured and reannealed in 2 μl of NEBuffer 2 (10×) using the following protocol: 95°C, 5 min; 95° to 85°C, −2°C/s; 85° to 25°C, −0.1°C/s; and then held at 4°C. Then, 1 μl of T7E1 (M0302S) was added to the annealed PCR products and incubated at 37°C for 1 hour. Products were analyzed on 2% agarose gels and imaged with a GelDoc imaging system (Bio-Rad). PCR products with mutations indicated by the T7E1 assay were subjected to DNA sequencing and subcloned into T-clone vectors (Vazyme Biotech, China). Colonies were picked randomly and further analyzed by Sanger sequencing using an M13F primer (Sangon Biotech). The in vivo gene editing and sequencing was similar to the previous procedure, except that the tumor genomic DNA samples was harvested using the Universal Genomic DNA Kit after treatment with ANCSS(Cas9/sgPLK1), ANCSS(Cas9/sgScr), or PBS.Western blotting to measure PLK1 protein expression in vitro and in vivoIn in vitro evaluation, U87MG cells were seeded in six-well plates at 1 × 106 cells per well, cultured for 24 hours, and incubated with ANCSS(Cas9/sgPLK1), ANC(Cas9/sgPLK1), NCSS(Cas9/sgPLK1), ANCSS(Cas9/sgScr), Lipo(Cas9/sgPLK1), or free Cas9/sgPLK1 (Cas9, 20 nM) overnight; then, medium was replaced with fresh medium containing 10% FBS. The cells were incubated at 37°C for another 72 hours, and cells were treated with radioimmunoprecipitation assay lysis buffer (Beyotime, China). The concentrations of resulting proteins were quantified by BCA Protein Assay (Beyotime, China). The lysates were separated by SDS–polyacrylamide gel electrophoresis and were transferred onto PVDF membranes (Beyotime, China). PVDF membranes were incubated with primary antibody against PLK1 (mouse mAb 35-206; Abcam) at a dilution of 1:1000 and secondary antibody (LI-COR IRDye 800CW). Protein bands were visualized using an ECL detection system. Indel formation efficiencies were calculated using the value of β-actin as the denominator using ImageJ v.1.8.0. For in vivo evaluation, tumors from mice treated with ANCSS(Cas9/sgPLK1), ANCSS(Cas9/sgScr), and PBS were lysed using lysis buffer (Beyotime, China). The following procedure was the same as the previous described.Cell viability and in vitro luciferase assayU87MG-Luc cells were seeded in 96-well plates at 2 × 103 cells per well and were incubated in 100 μl of Dulbecco’s modified Eagle’s medium containing 10% FBS for 24 hours. The culture medium was removed; the cells were incubated with ANCSS(Cas9/sgLuc), ANC(Cas9/sgLuc), NCSS(Cas9/sgLuc), or free Cas9/sgLuc (Cas9, 20 nM) overnight, and the medium was replaced with fresh medium containing 10% FBS. Then, after incubation for another 72 hours, CCK-8 solution (10/100 μl of medium) was added to each well, and after incubation for another 30 min, optical density at 450 nm (OD450) value (absorbance at 450 nm) was detected by a microplate reader (Devices/13×, Molecular Device, USA). Cell viability (%) was obtained by calculating the ratio of OD450 of the cells under targeted conditions to those of untreated control cells (n = 5). For the luciferase knockout assays, luminescence intensities of cell lysates were measured with the Firefly Luciferase Reporter Gene Assay Kit (Beyotime, China). Twenty microliters was used to quantify protein concentration with a BCA protein assay kit (Beyotime, China). The luciferase signal was divided by the amount of total protein for normalization (n = 5).Apoptosis assayTo assess apoptosis, U87MG and U251 cells were seeded in 24-well plates at 5 × 104 cells per well and were cultured for 24 hours. The cells were then incubated with ANCSS(Cas9/sgPLK1), ANC(Cas9/sgPLK1), NCSS(Cas9/sgPLK1), ANCSS(Cas9/sgScr), or free Cas9/sgPLK1 (Cas9, 20 nM) overnight, and the medium was replaced with fresh medium containing 10% FBS. The cells were incubated at 37°C for another 72 hours, after which the cells were incubated with Alexa Fluor 488–annexin V and propidium iodide (PI) (Beyotime, China). The stained cells were collected and washed with PBS, and fluorescence fluorescein isothiocyanate: excitation (Ex.), 488 nm and emission (Em.), 520 nm; PI: Ex., 488 nm and Em., 630 nm was measured following the manufacturer’s protocol using a flow cytometer (Becton Dickinson, USA) and FlowJo v10 software. The efficacy of ANCSS(Cas9/sgPLK1) in low PLK1-expressed U87MG cells was similar as above, except that the U87MG cells were pretreated with PLK1 siRNA (100 nM) using polyethyleneimine as a transfection agent for 48 hours.In vitro BBB penetration evaluationThe in vitro BBB model was constructed with endothelial hCMEC/D3 cells using a transwell cell culture system. The transendothelial electrical resistance (TEER) instrument (World Precision Instruments Inc. Sarasota, FL, USA) was used to monitor the intactness of the cell monolayer. The following experiments were carried out only when the TEER value of the endothelial hCMEC/D3 cell monolayer was above 200 ohm·cm2. ANCSS(Cas9/sgRNA), ANC(Cas9/sgRNA), NCSS(Cas9/sgRNA), or free Cas9/sgRNA (AF647-Cas9: 20 nM) was added to the upper chamber. Then, FBS-free medium was added to the lower chamber. After 2, 6, and 12 hours of incubation, the supernatant in the upper chamber and the medium in the lower chamber were extracted with dimethyl sulfoxide and detected. The amounts of AF647-Cas9 in the supernatant and filtered compartments were determined using a standard microplate assay. The AF647-Cas9 ratio in each compartment was calculated compared with the feeding amount.In vivo luminescence reduction in orthotopic U87MG-Luc glioblastoma–bearing nude miceANCSS(Cas9/sgLuc), ANCSS(Cas9/sgScr) (1.5 mg of Cas9 equiv./kg), or PBS was intravenously injected into the tail vein of U87MG-Luc orthotopic tumor-bearing nude mice (n = 3). Luminescence intensity in the brain was determined before injection and at 24, 48, and 72 hours after injection using an IVIS III instrument. Mice were anesthetized with isoflurane, and luciferin was injected intraperitoneally at a dosage of 150 mg/kg (100 μl). Measurements were performed at 10 min after luciferin injection. Photons emitted from the brain region were quantified using live imaging software.Ex vivo imaging, penetration, and biodistributionANCSS(Cas9/sgRNA), ANC(Cas9/sgRNA), NCSS(Cas9/sgRNA), or free Cas9/sgRNA (1.5 mg of Cas9 equiv./kg) in PBS was administrated intravenously via the tail vein into orthotopic U87MG-Luc, 83NS GSC tumor–bearing nude mice, GL261 with or without luciferase expression–bearing C57BL/6 mice. Luminescence intensity in the brain was determined at different time points using an IVIS III instrument. At 4 hours after injection, the tumor-bearing mice were euthanized. The heart, liver, spleen, lung, kidney, brain, and tumors were collected, washed, and weighed. Fluorescence images were acquired with a Lumina IVIS III near-infrared fluorescence imaging system. To evaluate tumor penetration, cancerous brains were harvested, fixed in 4% formalin overnight, embedded in paraffin, and sliced for immunofluorescence staining analysis. Blood vessels were counterstained with an Alexa Fluor 488–donkey anti-rat secondary antibody (1:1000 dilution in PBS) in a humidified chamber at 37°C for 1 hour, followed by washing three times with PBS and staining with DAPI (5 μg/ml) for 10 min, and were observed with a CLSM imaging system (Zeiss 880). To quantify the amount of AF647-Cas9 delivered to the tumor and different organs, each tumor and organ was individually homogenized in 0.6 ml of 1% Triton X-100 with a homogenizer (70,000 Hz) for 6 min. The samples were then centrifuged at 15,000 rpm for 30 min. The content of AF647-Cas9 in the supernatant was determined by fluorometry (Ex., 649 nm; Em., 670 nm) based on a calibration curve. The penetration and biodistribution evaluation of ANCSS(Cas9/sgRNA) in GL261 mice was similar to that in U87MG mice except for the euthanasia of mice at 12 hours after injection of the nanocapsules.Penetration evaluation in 3D spheroid tumor modelThe 3D tumor spheroids of U87MG were established according to following steps. Briefly, U87MG cells (2 × 103 per well) were plated in PrimeSurface 96-well plates (Sumitomo Bakelite, Japan). After 3 days, the tumor spheroids were treated with the ANCSS(Cas9/sgRNA), NCSS(Cas9/sgRNA), ANCSS(Cas9/sgRNA), and free Cas9/sgRNA (the AF647-Cas9 concentration was 20 nM) for another 4 hours of incubation. Then, tumor spheroids were washed and fixed with 4% paraformaldehyde. The permeability of different nanoparticles into tumor spheroids was investigated by CLSM (Zeiss 880; ×110 magnification).Effect of nanocapsules on the growth of GBM tumors in vivoTumor-bearing mice aged 6 to 8 weeks (n = 11; 7 for monitoring survival rate, 1 for histological analysis, and 3 for protein and gene editing evaluation) received an intravenous injection of ANCSS(Cas9/sgPLK1), ANCSS(Cas9/sgScr), or PBS every other day. At 48 hours after injection, the mice were anesthetized, and the Lumina IVIS III system was used to evaluate the tumor luminescence intensity. The relative photon flux was normalized to the initial intensity. On day 20, the treatment was terminated, and four mice from each group were euthanized. The brain tumors and organs, including heart, liver, spleen, lung, and kidney, were harvested, weighed, and fixed with 4% paraformaldehyde for immunohistochemical analysis, including staining for PLK1, cleaved caspase-3, and Ki-67, and hematoxylin and eosin (H&E) staining. Kaplan-Meier survival curves were determined for each treatment group, and the body weights of mice were measured individually.In vivo DNA deep sequencingTo assess the off-target effects of nanocapsules, we predicted potential off-target sites based on the online database (https://cm.jefferson.edu/Off-Spotter/) according to the following acknowledged criteria (42, 43): (i) Cas9 tolerates single-base mismatches in the protospacer adjacent motif (PAM)-distal region to a greater extent than in the PAM-proximal region. (ii) Three or more mismatched base pairs eliminated detectable Cas9 cleavage in the vast majority of loci.Briefly, after being treated with ANCSS(Cas9/sgPLK1), the genomic DNAs of tumor, normal brain tissue, liver, and kidney were harvested from mice bearing U87MG or CSC2 GSCs, using the Universal Genomic DNA Kit (CWBIO, China) according to the manufacturer’s instructions. One hundred nanograms of genomic DNA was used as a template to perform PCR using primers designed against on-target and off-target sites. Purified DNA was amplified again by PCR with primers containing sequencing adapters and then sequenced and analyzed by Sangon Biotechnology Company (Shanghai, China) to detect indels around target sites.Safety evaluationBALB/c mice were weighed and divided randomly into two groups (n = 5). ANCSS(Cas9/sgPLK1) (1.5 mg of Cas9 equiv./kg), or PBS was intravenously injected via the tail vein. Blood serum was collected at 24, 48, 72, and 96 hours and centrifuged at 800g for 5 min. The blood levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and serum albumin were determined by Wuhan Servicebio Technology Co. We also performed serial daily blood monitoring of white blood cell, platelet, and red blood cell levels. Daily body weights were recorded throughout the course of the nanocapsule and control treatments.

Article TitleBlood-brain barrier–penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy

Abstract

We designed a unique nanocapsule for efficient single CRISPR-Cas9 capsuling, noninvasive brain delivery and tumor cell targeting, demonstrating an effective and safe strategy for glioblastoma gene therapy. Our CRISPR-Cas9 nanocapsules can be simply fabricated by encapsulating the single Cas9/sgRNA complex within a glutathione-sensitive polymer shell incorporating a dual-action ligand that facilitates BBB penetration, tumor cell targeting, and Cas9/sgRNA selective release. Our encapsulating nanocapsules evidenced promising glioblastoma tissue targeting that led to high PLK1 gene editing efficiency in a brain tumor (up to 38.1%) with negligible (less than 0.5%) off-target gene editing in high-risk tissues. Treatment with nanocapsules extended median survival time (68 days versus 24 days in nonfunctional sgRNA-treated mice). Our new CRISPR-Cas9 delivery system thus addresses various delivery challenges to demonstrate safe and tumor-specific delivery of gene editing Cas9 ribonucleoprotein for improved glioblastoma treatment that may potentially be therapeutically useful in other brain diseases.


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