Cell culture and compound treatment
Huh7 cells and Hepa1-6 cells were purchased from JCRB and ATCC, respectively and were cultured in DMEM with 10% FBS, 2 mM glutamine. For hydroxyurea (Sigma), fludarabine (Fisher scientific), triapine (Fisher scientific) and Gallium (III) nitrate hydrate (Fisher scientific), cells were pretreated with each compound for 16 hours at indicated concentration and the cultured medium were replaced with fresh medium without compounds before rAAV transduction. For Torin-1, FK228 (Fisher scientific) (Fisher scientific), Trichostatin A, MG132 (Sigma) and Teniposide (Abcam), cells were treated with each compound for 24 hours at indicated concentration and rAAV was transduced 1 hour after compound addition.
Vectors
The AAV vectors containing ITR sequences used in this study are based on AAV type 2 backbone. CAG-Fluc, Albumin-P2A-hF9, HLP-hAAT vectors were prepared as described previously52. Albumin-P2A-GFP vector was generated by replacing hF9 coding sequence of the Albumin-P2A-hF9 vector with GFP coding sequence using In-Fusion® HD Cloning Kit (TAKARA). SaCas9-sgRNA8 vector and Albumin-P2A-GFP vectors used for Figure.5 were prepared as described previously20. For construction of the GAPDH-P2A-GFP vector, human genomic GAPDH segments were PCR-amplified using Fw: 5’-GACTGTACAGGGCTGCTCACATATTCTGG-3’ and Rv: 5’-CTGTGTACAGAGTGTATGTGGCTGTGGCCC -3’ (both containing BsRG1 sites for cloning) and inserted between AAV2 ITRs into BsrGI restriction sites in a modified pTRUF backbone53. The genomic segment spans approximately 1.7 Kb upstream and 1.7 kb downstream to the GAPDH stop codon. We then synthesized a 1,359 bp fragment spanning the region at the end of the GAPDH locus between the two SexA1 sites to be cloned in the vector. In this fragment, the GAPDH stop codon was removed and it was inserted an optimized P2A coding sequence preceded by a linker coding sequence (glycine-serine-glycine) and followed by the GFP sequence (without the start codon). For Apoe-P2A-hF9 vector construction, a genomic fragment containing sequences used for both homology arms was amplified from mouse genomic DNA. Primers (5’- TCC ACA CCT GCC TAG TCT CG -3’) and (5’- GTG CCA GAG GCA GTT GAG TT -3’) were used to amplify a 2.9kb fragment and the PCR product was directly cloned into the pCR Blunt II TOPO vector using the Zero Blunt TOPO PCR cloning kit (Invitrogen), sequence verified. The left homology arm was amplified from the cloned ApoE genomic fragment using primers (5’- ata tca tcg atc gcg atg cat taa tta agc ggc cgA AGA CTG TAG GTC CTG ACC C –3’) and (5’- ggt ggc gcc gct tcc TTG ATT CTC CTG GGC CAC -3’), the middle part containing hF9 sequence was amplified from Albumin vector using primers (5’- gcc cag gag aat caa GGA AGC GGC GCC ACC AAT -3’) and (5’- gga gaa gga tac tca TGT CAG CTT GGT CTT TTC TTT GAT CC -3’), the right homology arm was amplified from the cloned ApoE genomic fragment using primers (5’- aag acc aag ctg aca TGA GTA TCC TTC TCC TGT CCT GC -3’) and (5’- acg taa cag atc tga tat cac gcg tgt aca cta gtG CCC TGC TGA GTC CCT GAG -3’). Lower case letters indicate the overlapping sequences. Phusion Hot Start Flex (NEB) was used for all amplifications. Amplicons were assembled using the NEBuilder HiFi DNA Assembly Master Mix (NEB) according to instructions.
Animals
All animal works were performed in accordance to the guidelines for animal care at Stanford University or approved by the ICGEB review board, with full respect to the EU Directive 2010/63/EU for animal experimentation, and by the Italian Health Minister (authorization 996/2017-PR). Wild-type C57BL/6 (B6) mice were purchased from Jackson Laboratory (Bar Harbor, ME). We used 3-4-week-old juvenile mice to test the effect RNR small molecule inhibitors on gene targeting efficiency, as they are still undergoing development and growth, including in the liver.
Mouse AAV injection, drug dosing, bleeding and tissue sampling
B6 mice received tail vain injections of rAAV8 packaging each vector at the designated dose and were bled at indicated time points. Body weight of mice was measured using Scout pro portable scale (Ohaus) at indicated time points. Serum samples were obtained by centrifugation at 10,000 rpm for 10 minutes and used for ELISA assay of hF9 or hAAT, ALT enzyme measurements, and complete blood count tests. HU and fludarabine were resuspended in PBS and injected intraperitoneally with indicated dose/regimen. For our studies examining the in vivo effects of RNR inhibitors, we chose to use HU and fludarabine due to their FDA approval and well-established safety profiles. The drug dosing was based on information from several preclinical studies.
Briefly, an initial dose of 1000 mg/kg injection of HU showed severe toxicity, leading us to reduce the dose to 300 mg/kg/day. For fludarabine, an LD10 for single or 5-daily IV infusions were about 980 and 400 mg/kg per dose, respectively, in male mice according to the product monograph of fludarabine. Additionally, five-day doses of ½ the LD10 or ~200mg/kg per dose was considered safe and administration of a smaller dose (125 mg/kg) three times a day over three days (1125 mg/kg total dose) produced the greatest activity in a leukemia tumor xenograft model. Using this information, we decided to use a dose of three 125 mg/kg injections per day (375 mg/kg/day) of fludarabine for 3 days. All drug administrations were through intraperitoneal injections (i.p.). For BrdU labeling of proliferating mice hepatocytes, BrdU was resuspended in PBS and intraperitoneally injected at 200 mg/kg per day for 3 days or 7 days. Diethylnitrosamine (DEN) solution was prepared using saline. We determined an appropriate dose of DEN after finding administration at 100 mg/kg resulted in severe weight loss requiring euthanasia. Dosing at 10 or 30 mg/kg as a single injection per day for 3 sequential days was well tolerated and used to examine this drug’s genotoxic effect on gene targeting. At the end of each experiment, mice were anesthetized with isoflurane and perfused transcardially with PBS and then liver tissues were quickly harvested and cut into several pieces. The tissues for mRNA extractions were immediately submerged in RNAlater solution (Sigma) and stored at 4°C until use. For gDNA or protein extraction, tissues were snap-frozen in liquid nitrogen and stored at -80°C until use.
Hematology and liver pathology
CBC (complete blood count) and liver histopathological analysis were performed in mice injected with three 125 mg/kg injections of fludarabine per day, for 3 days. On the day of last injection and 4 weeks after that, mice were submitted to the Veterinary Service Center (VSC) at Stanford University for standard blood paneling tests and blinded analysis of liver pathology by a skilled veterinary pathologist.
AAV production
rAAV vectors were produced as previously described using a triple transfection protocol with Ca3(PO4)2 or PEI 25K, followed by purification by CsCl gradient53 or using AAVpro® Purification Kit (All Serotypes) purchased from Takara Bio. Purified rAAVs were stored at -80°C until used. rAAV genomes were extracted and purified using QIAamp MinElute Virus Spin Kit (QIAGEN) and were titered by qPCR. The sequence information of primers are shown in Table1.
Firefly luciferase assay
Luciferase assays were performed using ONE-Glo™ Luciferase Assay System (Promega) following the manufacturer's instruction. Briefly, at indicated time points after rAAV (CAG-Fluc) transduction, the equal volume of the reconstituted substrate to the cultured medium was added to the cells grown in 96 well plates and incubated for 10 minutes with gentle shaking. Luminescent activity was measured using a plate reader.
siRNA transfection
ON-TARGETplus siRNA against human RRM1 and POLR2A as well as scramble control siRNA were purchased from Dharmacon and were transfected into Huh7 cells using RNAiMAX (Life Technologies) according to the manufacturer’s instructions. Final concentration of siRNA was 20 nM. AAVDJ packaging GAPDH-P2A-GFP vectors were added into the cultured medium at a multiplicity of infection (MOI) of 20,000 vector copies/cells at 48 hours after siRNA transfection. Flow cytometry was performed for detection of GFP positive fractions at the indicated time points.
Flow cytometry
Huh7 or Hepa1-6 cells were harvested and washed with cold PBS and resuspended in cold PBS containing 3% FBS. Cells were kept on ice and protected from light until analyzed. Singlet cells were determined based on FSC/SSC plot and GFP positive fractions were gated based on negative control which was non-transduced cells. The number of GFP expressing cells was evaluated using the BD FACSCalibur™ instrument and data were analyzed using the FlowJo software package.
RNA extraction and cDNA preparation
Total RNA was extracted using RNeasy micro plus kit (QIAGEN) according to the manufacture’s protocol with DNase treatment. Liver tissue samples stabilized in RNA later solution (-100mg) were homogenized in RINO 1.5 mL Screw-Cap Tube filled with stainless steel beads and 600 mL of RLT buffer (including b-mercaptoethanol) using a bead homogenizer (Next Advance Bullet Blender Storm). Total RNA was extracted from the tissue lysates using RNeasy plus mini kit (QIAGEN) with additional on-column DNase treatment. cDNA was synthesized from 200-500 ng of total RNA using High-Capacity RNA-to-cDNA™ Kit (Life Technologies) according to the manufacturer’s instructions.
gDNA extraction
Cultured cells were collected by trypsinization and washed with PBS. Total genomic DNA was extracted using QIAamp DNA Mini Kit (QIAGEN) according to the manufacture’s protocol with RNase A treatment. Snap-frozen liver tissue (-100mg) were homogenized in RINO 1.5 mL Screw-Cap Tube filled with stainless steel beads and 600 mL of AL buffer using a bead homogenizer. Total RNA was extracted from the tissue lysates using DNeasy Blood & Tissue Kit (QIAGEN).
PCR and qRT-PCR
The polymerase chain reactions (PCRs) to amplify genomic regions where homologous integrations occurred (junction PCR) were performed using Q5® Hot Start High-Fidelity 2X Master Mix (New England Biolabs). The following cycling conditions were used: Human b-Actin (one cycle of 98°C for 30 sec, 28 cycles of 98°C for 10 sec, 60°C for 15 sec, and 72°C for 10 sec, and one cycle of 72°C for 2 min), GAPDH-P2A junction (one cycle of 98°C for 30 sec, 35 cycles of 98°C for 10 sec, 62°C for 15 sec, and 72°C for 1 min, and one cycle of 72°C for 2 min), Mouse albumin (one cycle of 98°C for 30 sec, 32 cycles of 98°C for 10 sec, 60°C for 10 sec, and 72°C for 2 min, and one cycle of 72°C for 2 min) , hF9-Albumin junction nested PCR (one cycle of 98°C for 30 sec, 20 cycles (1st PCR) and 25 cycles (2nd PCR) of 98°C for 10 sec, 62°C for 15 sec, and 72°C for 1 min, and one cycle of 72°C for 2 min). PCR products were analyzed in agarose gels containing Ethidium bromide and visualized using ChemiDoc Imaging Systems (Bio-Dad). Primer sequence information are listed in Table1.
Quantitative PCR was performed in duplicate using Apex qPCR GREEN Master Mix (Genesee Scientific) and CFX384 Touch Real-Time PCR Detection System (Bio-Rad) using the following cycling conditions: 95°C for 15 min, 45 cycles of 95°C for 10 sec, 60°C for 10 sec and 72°C for 10 sec, and one cycle of 95°C for 10 sec and 65°C for 1 min and 65-97°C (05°C/sec). Standard curves for each primer set were generated using serially diluted linearized plasmid and used for quantification. CFX Maestro Software was used for data analysis and relative mRNA expression levels were calculated by normalized against β-actin. All sequence information of primers is listed in Table1.
Protein extraction and western blotting
Total cell lysates from cultured cells or mouse liver tissues were prepared using RIPA buffer containing Halt™ Protease and Phosphatase Inhibitor Cocktail (both from Thermo Fisher). Liver tissues were homogenized in RINO 1.5 mL Screw-Cap Tube filled with stainless steel beads and 600 mL of RIPA buffer using a bead homogenizer. Protein concentration were measured using Pierce™ BCA Protein Assay Kit (Thermo Fisher) and the same amount of proteins for each sample were loaded into NuPAGE™ 4-12% Bis-Tris Protein Gels (Thermo Fisher). iBlot2 transfer system (Thermo Fisher) was used for western blotting. PVDF membranes were blocked with 5% BSA containing TBS-T buffer and the following 1st antibodies were used. Anti-RRM1 (CST, 1:1000), HRP-conjugated anti-a-tubulin (CST, 1:2000) and anti-gH2AX (Novus Biologicals, 1:2000) antibodies. HRP-conjugated secondary antibodies were used, and signals were detected using Pierce™ ECL Plus Western Blotting Substrate (Thermo Fisher) and ChemiDoc Imaging Systems (Bio-Rad)
Southern blotting
Nuclear fractions of Huh7 cells were obtained using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher) and genomic DNA was extracted using QIAamp DNA Mini Kit (QIAGEN) and digested overnight with XhoI (New England Biolabs) to cut only host genomic DNA. Digested DNA was run in a 1% TAE agarose gel at room temperature O/N. After the electrophoresis, the gel was washed with denaturing buffer (3 M NaCl, 400 mM NaOH) twice for 5 minutes and DNA was transferred to an Amersham Hybond-XL membrane using transfer buffer (3 M NaCl, 8 mM NaOH) O/N. Membrane was washed with 2xSSC buffer for 5 minutes and then blocked with UltraPure™ Salmon Sperm DNA (Thermo Fisher) in QuikHyb Hybridization Solution (Agilent Technologies) for 1 hour at 65°C. Probes for GFP (574 bp) were generated using gel-purified PCR amplicons containing GFP sequence and BcaBEST™ Labeling Kit (TAKARA) and [α-32P]-dCTP (PerkinElmer), then probe hybridization were performed O/N at 65°C with rotation. The membrane was washed with 2xSSC buffer and with 2xSSC containing 0.1% SDS buffer at 65°C. Signals were visualized using Personal Molecular Imager™ System (Bio-Rad).
Immunohistochemistry staining of liver sections
For all in situ hybridization and immunostaining experiments liver tissue was dissected into 2-3 mm pieces and fixed for 24 hours in 10% neutral buffered formalin (Sigma Aldrich, St. Louis, MO) at 4 ºC. Tissue was subsequently processed through 10%, 20%, and 30% sucrose solutions for 24 hours each, then frozen embedded into OCT media (Sakura Finetek USA, Torrance, CA) with liquid nitrogen and 2-Methylbutane (Sigma Aldrich). Frozen tissue was sectioned into 16 µm thick sections using a Microm HM550 Microtome (Thermo Scientific, Waltham MA). Tissue sections were blocked with antibody diluent comprised of 5.0% normal donkey serum (Jackson Immuno Research, West Grove, PA) and 0.1% Triton-X 100 (Sigma Aldrich). GFP was stained with an anti-GFP chicken IgY primary antibody (Invitrogen, Carlsbad, CA) and phosphorylated Ser139 γH2AX was stained with a rabbit monocolonal (20E3) (Cell Signaling Technologies, Danvers, MA). Polyclonal secondary detection antibodies consisted of anti-chicken IgY antibody conjugated to Alexa Fluor 488 (Jackson Immuno Research) and polyclonal anti-rabbit IgG antibody conjugated to Alexa Fluor 594 (Thermo Scientific).
Detection of BrdU incorporated DNA was accomplished with heat denaturing in an antigen retrieval buffer (Advanced Cell Diagnostics, Newark, CA), followed by staining with a rat monoclonal anti-BrdU antibody (BU1/75 (ICR1)) (Abcam, Cambridge, UK) and secondary Alexa Fluor 594 antibody (Thermo Scientific). All IHC slides were mounted with Prolong Diamond Antifade with Dapi (Thermo Scientific) and imaged on a Zeiss LSM 880 confocal microscope. Specificity of all staining procedures was ensured with appropriate biological controls and control slides stained with secondary antibody only.
For eGFP experiments with Cas9 specimens were frozen in optimal cutting temperature compound (BioOptica, Milano, Italy) and 4 µm slices were obtained in a cryostat. Percentage of eGFP+ cells in liver specimens were detected by natural GFP fluorescence, while nuclei were visualized by Hoechst (10 lg/ml) staining. Slides were mounted in Mowiol 4-88 (Sigma). Images were acquired on a Nikon Eclipse E-800 epi-fluorescent microscope with a charge-coupled device camera (DMX 1200F; Nikon, Amstelveen, The Netherlands). Digital images were acquired on a Nikon Ti Eclipse inverted fluorescence microscope equipped with Intensilight Epi-fluorescence Illuminator, a Perfect Focus 3 system and 20× (NA 0.45) objective. Acquisition was performed with a DS-Qi2 16 Mpixel camera (Nikon). NIS Elements microscope imaging software (Nikon, version 4.6) was used to quantify the total number of cells (Hoechst staining) and the GFP positive cells. The settings of Automated Spot Detection were set in order to detect all bright spots with a typical minimum diameter of 8 μm. The function ”Detect all objects” was activated and spot detection-output were directly exported to Excel, where the data analysis was performed. For each animal an average 10000 nuclei were counted. Measurements (GFP+ cells/total nuclei) were averaged for each animal, and the results were expressed as mean ± SD for each treatment.
RNAScope in situ hybridization of hF9
Liver tissue was processed for RNA in situ hybridization as described above. Fixed frozen tissue was sectioned into 9 µm thick sections and RNAScope hybridization was performed according to the manufacturer’s protocol (Advanced Cell Diagnostics). A custom probe was designed to detect codon-optimized human factor 9 mRNA, while control probes targeted either murine peptidylprolyl isomerase B (PPIB) (positive control for RNA quality) or bacterial 4-hydroxy-tetrahydrodipicolinate reductase (dapB) (negative control). RNA specificity was confirmed using RNase digestion of control tissue sections and slides were counterstained with 50% hematoxylin (Thermo Scientific). Imaging was performed using a Leica DM2000 brightfield microscope.
Image analysis
All image analysis was performed in a blinded manner. Analysis of BrdU incorporation was performed manually requiring nuclear colocalization of BrdU signal and greater signal intensity over background to be recorded as a positive nucleus. Signal from overtly non-hepatocyte nuclei directly associated with larger liver structures such as central veins or bile ducts were not included in tally. Analysis of BrdU incorporation and GFP colocalization was performed similarly. Scoring of phosphorylated Ser139 γH2AX was performed using ImageJ software.
Enzyme-linked immunosorbent assay (ELISA)
Mice serum samples were used to quantify hF9 or hATT protein expression levels. ELISA for hF9 was performed as previously described6 with the following antibodies: mouse anti-hF9 IgG primary antibody at 1:1,000 (Sigma Cat#F2645), and polyclonal goat anti-hF9 peroxidase-conjugated IgG secondary antibody at 1:4,000 (Enzyme Research Cat#GAFIX-APHRP). ELISA for hAAT was performed as previously described52.
Targeted Illumina Deep Sequencing
Genomic DNA was extracted from liver tissue using phenol/chloroform extraction, following incubation with RNAseA and Proteinase K. Genomic DNA was submitted to the Genome Engineering and IPSC Center (GEiC) at Washington University in St. Louis for targeted deep sequencing of the gRNA target site.
Statistics
GraphPad Prism was used for statistical analysis. Groups of two were analyzed by unpaired t test. More than two groups were compared by one-way ANOVA with Bonferroni correction. Statistical significance was assumed with P value <0.05 (*), <0.01(**) and <0.001 (***). Bars in graphs represent standard deviation for each group.
Image analysis was performed using two-tailed t-tests between groups. Normally distributed data, determined by D'Agostino Pearson test or Shapiro–Wilk test, was analyzed with parametric student’s t test if groups had equal variance. Groups with unequal variance were analyzed with an unpaired t test with Welch's correction. Significance in non-normally distributed data was determined using non-parametric Mann-Whitney U test.