Computational protocol: Somatic Therapy of a Mouse SMA Model with a U7 snRNA Gene Correcting SMN2 Splicing

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[…] Cell lines. HeLa S2 are HeLa cells that have been stably transformed with a human SMN2 minigene as described. AAV-293 cells containing adenovirus type 5 DNA and expressing the adenovirus E1 gene were obtained from Agilent. All cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (BioConcept), 100 U/ml penicillin and 100 U/ml streptomycin (both Lubio Science). This medium is called DMEM+/+; medium without fetal calf serum and antibiotics will be referred to as DMEM−/−. The cells were kept at 37°C in a humid atmosphere with 5% CO2.AAV production and purification Expression plasmids. To accommodate multiple copies of the U7-ESE-B cassette in AAV vectors, we shortened the original cassette at the 5′ and 3′ ends while retaining all necessary promoter and 3′ elements. The resulting short U7-ESE-B cassettes (sU7) contain 253 and 104 bp of 5′ and 3′ flanking sequences, respectively. As recipient plasmids to generate ss and scAAV, we used pAAV-CMV-eGFP and pAAVsc_U7Dtex23, respectively. We generated constructs containing one to five copies of sU7. In some vectors, we inserted an EGFP expression cassette in the plasmid backbone to be able to monitor the transfection efficiency during virus production. Maps are shown in Supplementary Figure S1. Sequence files and further information on the cloning are available upon request.Helper plasmids. For AAV6 production, we used the helper plasmid pDF6. For AAV9, the separate helper plasmids Rep2-Cap9 and pHelper were used.Detailed procedures for the production and purification of AAV vectors are described in the Supplementary Materials and Methods and Supplementary Table S1. An overview of the method is presented in Supplementary Figure S5.qPCR based virus titration. Virus suspensions were treated with DNase (10 U DNase I, Roche Diagnostics, Rotkreuz, Switzerland) for 20 minutes at 37°C. Then, the viral capsids were destroyed by an incubation for 20 minutes at 95°C that simultaneously inactivated the DNase. Serial dilutions of the lysed virus preparation and of a reference plasmid were compared by qPCR. Reactions consisted of 10 µl MesaGreen qPCR Mastermix Plus for SYBR Assay (Eurogentec, Liège, Belgium), 0.2 µl of each primer (12 µmol/l; Microsynth AG, Switzerland), 5.6 µl H2O and 4 µl of sample. The qPCR was performed in a Rotor-Gene Q (Qiagen AG, Hombrechtikon, Switzerland) with initial denaturation for 10 minutes at 95°C, followed by 40 cycles of 95°C for 15 seconds, 56–60°C (according to the primers used) for 15 seconds, and 10 seconds at 70°C. A melting curve from 65–95°C was then used to assess the purity of the amplicon. The run was evaluated with Rotor Gene Q Series Software (Version 2.2.3, Build 11), Qiagen AG, Hombrechtikon, Switzerland. Primers are listed in Supplementary Table S2.Plasmid transfections and viral transduction of cells. HeLa S2 cells were seeded at a density of 105 cells per well of a six-well plate. On the next day, the medium was replaced by DMEM −/−. For each well, 1.5 µg of plasmid and 4 µl Fugene HD (Roche) were mixed in 100 µl DMEM -/- and allowed to form complexes at room temperature for 15 minutes. The mixture was then distributed dropwise over the cells. After incubation for 4 hours at 37°C, the medium was replaced by DMEM +/+.Viral transductions were performed in the mnimal sufficient volume of medium to completely cover the cells. After 6 hours at 37°C, the medium was completed to the standard volume for the corresponding culture flask.Mouse -breeding and genotyping. SMNΔ7 mice were purchased from Jackson Laboratories (FVB.Cg-Tg(SMN2)89Ahmb Smn1tm1MsdTg(SMN2*delta7) 4299AhmB/J; stock number: 005025; Bar Harbor, ME, USA) and kept at the central animal facility, Inselspital Bern under standard IVC conditions according to legal requirements (Swiss federal permit BE14/13). For genotyping, DNA from tail biopsies taken on the day of birth was extracted and analyzed with KAPA Mouse Genotyping Kits (Axonlab, Baden Switzerland) according to the manufacturers' instructions. The primers used (see Supplementary Table S2) allowed us to determine the Smn1 genotype and sex of the animals.Injections for somatic gene therapy in mice. AAV preparations or PBS (for controls) were injected into the left cerebral ventricle of newborn mice. Prior to injection, the virus was mixed with 0.025% Fast Green (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) to be able to evaluate the success of the injection. For better visibility, the head of the mouse was held onto a small LED torch lamp. Usually, 5 µl of virus were injected at P0 (day of birth) and again at P1 through a glass capillary (microhematocrit tubes non heparinized, ø ext. 1.45 mm, ø int. 0.95 mm, long 75.0 mm, Sovirel France, pulled in a micropipette puller model P97, Sutter Instrument S, Novato, CA) connected via a tube to a 1 ml syringe that was used to control the liquid flow.Monitoring of mouse development and performance. The overall condition and weight of the mice were initially recorded on a daily basis. As the mice reached a higher age, the intervals were increased to 2–3 days. The motility and strength were determined by the righting reflex (time to right from an inverted position). A second test consisted of measuring how long the mice could hold themselves with their hind legs grabbing the rim of a 50 ml Falcon tube, head downward, before falling into the tube that was padded with paper tissue to soften the fall. Additionally, a score from 0 to 4 was given depending on how far the hind legs were splayed, with 0 indicating that the legs touched each other and 4 representing the maximal possible distance.RNA extraction and alternative splicing analysis of SMN2, Uspl1, and SMN2 exon 7 specific qPCR. Total RNA from transfected or virus-infected HeLa S2 cells was extracted with homemade Tri-Reagent as described. Mouse tissues were homogenized in Tri-Reagent before extraction. Between 0.5–2 µg of RNA was reverse-transcribed with 20 pmoles of random hexamer or oligo d(T) with Affinity-Script Multi-Temp Reverse Transcriptase (Agilent Technologies, Basel, Switzerland) or with the PrimeScript RT Master Mix kit (Takara bIO eUROPE sas, Saint-Germain-en-Laye, France) according to the manufacturers' instructions. PCR for splicing analysis on agarose gels (SMN minigene and Uspl1) was performed with the PCR FastStart 2× PCR Master Mix (Roche) and appropriate primers (see Supplementary Table S2). For the detection of exon 7 containing SMN2 transcripts, the cDNA was analyzed by a SYBR-green based qPCR assay (mix as described for qPCR-based virus titration) using SMN-Ex7-Re(Fw) and SMN-Ex8-Re primers.Quantitation of U7-ESE-B expression. To analyze U7-ESE-B expression, cDNA was synthesized in a coupled polyadenylation reverse transcription reaction designed for short nonpolyadenylated RNAs. This cDNA was then used for a SYBR-green based qPCR analysis (mix as described for qPCR-based virus titration) with qPCR ESE Fw and universal reverse primers. As normalizer, U6 snRNA was analyzed with qPCR WT_U6 Fw and the universal reverse primers. The primer sequences are listed in Supplementary Table S2.Immunofluorescence analysis. Spinal cords were fixed in 4% paraformaldehyde (PFA) followed by incubation in 30% sucrose (w/v) overnight at 4°C each. The following day, the spinal cords were embedded in cryomolds using Tissue-Tek O.C.T. compound (Sakura Finetec, VWR International, Dietikon, Switzerland) and stored at −20°C. Sections of 50 µm thickness were cut with a microtome and transferred into 0.8% sodium azide in PBS. For all further treatments, the sections were incubated freely floating in wells of a 12-well plate, and transfers were done with a thin paint brush. First they were blocked in 3% bovine serum albumin (BSA) in PBS for 3 hours at RT followed by primary antibody incubation in 3% BSA and 0.3% TritonX-100 in PBS overnight at 4°C on a rocker (for antibodies and dilutions see Supplementary Table S3). Following three wash steps in 0.1% TritonX-100 in PBS for 20 minutes each, the sections were incubated with secondary antibody in 3% BSA and 0.3% TritonX-100 in PBS for 5 hours at RT or overnight at 4°C on a rocker in the dark. Subsequently, the sections were washed three times 20 minutes in the dark with 0.1% TritonX-100 in PBS. Finally they were transferred onto glass slides, air-dried, mounted with homemade Moviol and stored at 4°C. The slides were analyzed with an Olympus Fluoview 1000-BX61 microscope and Imaris 7.6 (Bitplane AG, Zürich, Switzerland) software (Olympus Schweiz AG, Volketswil, Switzerland).Diaphragms were dissected and stored at −20°C in RNAlater (Sigma-Aldrich) for further analysis. During all staining steps, they were incubated freely floating in wells of a 24-well plate. After washing in PBS for 30 minutes, NMJs were detected by incubation with α-Bungarotoxin in 3% BSA and 0.5% TritonX-100 in PBS at RT. Next, the samples were blocked for 3 hours at RT followed by primary antibody incubation in 3% BSA and 0.5% TritonX-100 in PBS for 72 hours at 4°C on a rocker (for antibodies and dilutions see Supplementary Table S3). The incubation with secondary antibodies, all wash steps and the mounting were performed as described above for the spinal cord samples. The samples were analyzed with a Leica DMi8 microscope (Leica Microsystems, Heerbrugg, Switzerland) and Leica application suite X software and further evaluated with FiJi (Image J). Figure S1. AAV vectors used in this work. Figure S2. Muscle performance of SMA Δ7 mice injected with scAAV9 4xsU7 compared to uninjected or PBS-injected mice and wt or carrier littermates. Figure S3. Photographs of SMNΔ7 Smn1−/− mice injected with scAAV9 4xsU7. Figure S4. Conservation of active zones in diaphragm neuromuscular junctions (NMJs) of SMNΔ7 Smn1−/− mice injected with scAAV9 4xsU7. Figure S5. Scheme of AAV production and purification and EM pictures of purified viruses. Supplementary Methods (virus production) and Tables Table S1. Amounts of cells/reagents used for virus production. Table S2. Oligonucleotides used in this work. Table S3. Antibodies and probes used in this work. Description of supplementary videos Supplementary File S2. Male SMA mouse D54-2-1, injected with 2.73 × 1014 vg/kg scAAV9 4xsU7. Video taken on P78. Supplementary File S3. Female SMA mouse D54-2–9, injected with 2.27 × 1014 vg/kg scAAV9 4xsU7, together with slightly larger female carrier littermate D54-2–3 and mother. Video taken on P78. Supplementary File S4. Female SMA mouse D57-1-1, injected with 6.25 × 1013 vg/kg scAAV9 4xsU7, together with adult female. Video taken on P49. Supplementary References […]

Pipeline specifications

Software tools Imaris, LAS X
Application Microscopic phenotype analysis
Organisms Mus musculus, unidentified adenovirus, Human poliovirus 1 Mahoney, Homo sapiens
Diseases Hereditary Central Nervous System Demyelinating Diseases