RNAi hairpin design and cloning.
The original RNAi FRG1
targets were select using a program previously available on the Promega website based on parameters specified in the scientific literature (siRNA Designer, contact Promega, Madison, WI for details). Three FRG1
siRNAs were tested in vitro
and two were selected for their knockdown efficiency of exogenously expressed FRG1 as analyzed by western blot (data not shown). From the first target sequence from FRG1
exon 8 shown in
, the full-length (FRG1
sh1) and a shortened sequence by 2 nts from the 5′ end (FRG1
sh2) to increase safety in vivo32
were cloned as hairpins using the overlapping oligonucleotides: sh1 = full-length 5′ ACGCGTCGACAAAGGCTCGGAAAGATGGATTCTTCCTGTCAAA 3′ and 5′ TGCTCTAGAAAAGGCTCGGAAAGATGGATTTGACAGGAAGAA 3′ sh2 = 5′ ACGCGTCGACAGGCTCGGAAAGATGGATTCTTCCTGTCAAA 3′ and 5′ TGCTCTAGAAGGCTCGGAAAGATGGATTTGACAGGAAGAA 3′. Oligonucleotides designed from the second selected siRNA from exon 3 to make FRG1
sh3 are as follows: 5′ ACGCGTCGACCCATAGCCATTGAAATGGACTTCCTGTCATC 3′ and 5′ TGCTCTAGACCATAGCCATTGAAATGGATGACAGGAAGTC 3′. These oligonucleotide pairs were annealed, extended, then restricted with Sal
I and Xba
I and cloned into the Sal
I and Xba
I sites of the U6+27 expression cassette cloned into pARAP4. The full cassette, U6+27-Sal I/Xba I-TTTTT, was derived from lentiviral plasmid pLGINm and inserted into pARAP4 (existing Sal
I and Xba
I sites destroyed)47
by blunt-end cloning of Cla
I and EcoR
I restricted fragment into the SnaB
I site 5′ of the RSV-hPLAP
gene (kind gift from Chao-Zhong Song, University of Washington, Seattle, WA) described in ref. 48
. AAV6 production was essentially as described in ref. 28
with modification described in ref. 49
. Viral genomes were quantitated by Southern analysis using a probe to the hPLAP
SV40 polyadenylation signal as described in ref. 50
Manipulation of experimental animals
. C57BL/6J transgenic mice overexpressing human FRG1
under the control of the human skeletal α-actin gene promoter5
were maintained by crossing them with wild-type C57BL/6J mice. FRG1
-high mice and control C57BL/6J littermates were maintained at Charles River (Calco, Italy). All procedures were approved by the Institutional Animal Care and Use Committee of the Fondazione San Raffaele del Monte Tabor and were communicated to the Ministry of Health and local authorities according to Italian law.
Mice progeny were genotyped by PCR using the primers 5′-GATCTAGCGGCCGCCATGGCCGAGTACTCCTATGTGAAGTCT-3′ and 5′-GCGCGCTTAATTAATCACTTGCAGTATCTGTCGGCTTTCA. PCR conditions were as follows: initial denaturation at 95 °C for 2 minutes, 42 cycles with denaturation at 95 °C for 30 seconds, annealing at 68 °C for 30 seconds and amplification at 72 °C for 1 minute. Virus was administered by intravenous injection via tail vein to 6-weeks-old transgenic female mice. In detail, 200 µl containing 2 × 1012 or 5 × 1012 vg of vector in physiological solution were injected into the tail vein using a 25-gauge Terumo syringe (Terumo, Leuven, Belgium). Mice injected with an equal volume of physiological solution (vehicle) and wild-type sex and age-matched C57BL/6J were used as controls. All mice were evaluated during 12 weeks subsequent to treatment.
Cell culture and viral infection
overexpressing C2C12 myoblast cells were previously described.5
Cells were grown in DMEM-HIGH (Dulbecco's modified Eagle's medium, high glucose with sodium pyruvate and -glutamine; EuroClone, Milano, Italy) supplemented with 10% fetal bovine serum (EuroClone), 1% penicillin/streptomycin (100 U/ml final concentration; EuroClone) and 1 µg/µl Neomycin (G-418; InvivoGen, San Diego, CA). All cells were maintained at 37 °C with 5% CO2
in a humidifier incubator.
Cells were seeded in 6-well plates with 1 × 104 cells/well and were infected 24 hours later with 1010 vg/well of rAAV6 expressing sh1, sh2, and sh3. After 3 days, cells were collected for RNA and protein analysis.
Histological analysis. Mice were euthanized by CO2 inhalation or by cervical dislocation. Skeletal muscle tissue specimens were rapidly mounted in OCT (Optimal Cutting Temperature compound; Sakura, Zoeterwoude, The Netherlands) and then frozen in isopentane cooled in liquid nitrogen. Transverse sections (8 µm) were performed on a cryostat and used for the analysis described below.
The percentage of centrally nucleated fibers and the CSA of single muscle fibers were determinate by using ImageJ software (NIH, Bethesda, MD) on calibrated images of cryosections stained with Gömöri Trichrome.
Characterization of fibrotic and fat tissue in the muscle was performed after Sirius Red and Oil Red O staining, respectively. The tissues were quantified measuring the percentage of red area in the sections (ImageJ, NIH).
For CD45 immunohistochemistry, cryostat sections were air-dried and fixed in prechilled acetone for 10 minutes. Endogenous peroxidase was inhibited by incubation for 10 minutes at room temperature with methanol plus 0.03% hydrogen peroxide. Tissue sections were incubated overnight at 4 °C with rat, anti-mouse CD45 antibody (BD, Franklin Lakes, NJ) diluted 1:500. Then samples were incubated with 1:300 dilution of an anti-rat antibody conjugated with biotin (#13-4813-85; eBioscience, San Diego, CA). Avidin-biotin-peroxidase complex was added (Vector lab, Burlingame, CA) and sections were incubated for 5–10 minutes with a DAB substrate (Dako, Glostrup, Denmark) and counterstained with hematoxylin and mounted.
For dystrophin immunofluorescence, cryostat sections were permeabilized with 0.2% TritonX100 and 1% bovine serum albumin for 15 minutes. After a blocking of 30 minutes in 0.2% TritonX100, 1% bovine serum albumin and 10% goat serum, the sections were incubated O/N with a mix of two anti-mouse Dystrophin antibodies (NCL-Dys1 and NCL-Dys2; Novacastra, Leica Microsystem, Wetzlar, Germany) diluted 1:50. Then samples were incubated for 1 hour with 1:500 dilution of a goat anti-mouse Alexa Fluor 555 (Invitrogen, Cergy-Pontoise, France). Finally, nuclei were stained by Hoechst3342 and section were mounted in phosphate-buffered saline 90% glycerol.
For the analysis of human alkaline phosphatase (hPLAP) activity, muscles cryosections were fixed in 4% paraformaldehyde for 10 minutes, incubated at 65 °C for 90 minutes to inactivate endogenous APs and stained at room temperature protected from the light with BCIP/NBT substrate (Roche, Basel, Switzerland) in a buffer containing 100 mmol/l Tris-HCl pH 9, 150 mmol/l NaCl, 1 mmol/l MgCl2, 0.001% Tween20. Next, sections were dehydrated in an alcohol scale and mounted.
Following euthanization, mouse livers and heart specimens were collected and paraffin embedded. Sections of 8 µm were cut and stained with hematoxylin and eosin.
RNA isolation and real-time RT-PCR. Total RNA was extracted from cells with PureLink RNA MiniKit (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Dissected muscles were disrupted by TissueLyser (Qiagen, Hilden, Germany) and RNA was isolated using RNeasy fibrous tissue kit (Qiagen).
Equal amounts of RNA for each sample were retrotranscribed with SuperScript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad, CA) following the manufacturer's conditions. In brief, RNA was retrotranscribed with a 1:1 mix of oligo(dT) and random hexamer primers and with SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA). After treatment, the complementary DNAs were digested with RNaseH for 20 minutes at 37 °C.
For gene expression analysis, real-time PCR with Sybr GreenER qPCR kit (Invitrogen, Carlsbad, CA) was used. Each sample was run in triplicate with specific primers for FRG1 (forward 5′-AGTCCTCCAGAGCAGTTTAC-3′, reverse 5′-AATAAAGCAGCTATTTGAGGC-3′) and hPLAP (forward 5′-GGTGAACCGCAACTGGTACT-3′, reverse 5′-CCCACCTTGGCTGTAGTCAT-3′). IL-10 (forward 5′-ATTTG-AATTCCCTGGGTGAGAAG-3′, reverse 5′-CACAGGGGAGAAATCGATGACA-3′) and TNF-α (forward 5′-TCCCAGGTTCTCTTCAAGGGA-3′, reverse 5′-GGTGAGGAGCACGTAGTCGG-3′).
For sample normalization, GAPDH (forward 5′-TCAAGAAGGTGGTGAAGCAGG-3′, reverse 5′-ACCAGGAAATGAGCTTGACAAA-3′) or ACTN3 (forward 5′-GGGGCGGCGAGTACATGGAAC-3′, reverse 5′-CAGTGAAGGTTTTCCGCTGCTGT-3′) were used in cell or muscle samples, respectively. The PCR conditions were the following: initial denaturation at 95 °C for 10 minutes, 40 cycles with denaturation at 95 °C for 30 seconds, annealing at 58 °C for 30 seconds and amplification at 72 °C for 30 seconds.
Gene expression of IL-10 (forward 5′-ATTTG-AATTCCCTGGGTGAGAAG-3′, reverse 5′-CACAGGGGAGAAATCGATGACA-3′) and TNF-α (forward 5′-TCCCAGGTTCTCTTCAAGGGA-3′, reverse 5′-GGTGAGGAGCACGTAGTCGG-3′) was normalized versus GAPDH and the conditions of the PCR were the following: 50 °C for 2 minutes, 95 °C for 10 minutes, 40 cycles with 95 °C for 15 seconds and 60 °C for 1 minute. Melting curves were performed at the end of each amplification reaction to monitor product specificity.
Viral genome quantification. Vector genomes per nuclei from injected mice were determined by real-time qPCR on an ABI Prism 7500 (Perkin Elmer, Waltham, MA) with primers to the SV40 polyadenylation signal [forward- 5′ TTTTCACTGCATTCTAGTTGTGGTT 3′ and reverse- 5′ CATGCTCTAGTCGAGGTCGAGAT 3′ with probe 5′ 6FAM-ACTCATCAATGTATCTTATCATG-MGBNFQ 3′ (Applied Biosystems, Austin, TX)]. Nuclear mouse Ldlr was quantified [forward primer- 5′ CGTGCTCCCAGGATGACTTC 3′ and reverse primer 5′ CTCCATCACACACAAACTGCG 3′ and detected with probe- 5′ 6FAM-ATGCCAGGATGGCAAGTGCATCTCC-TAMRA (Operon, Huntsville, AL)] and used to determine the vector genomes per nuclei (vg/n) based on the number of genomes/(number of copies of Ldlr/2).
Western blotting. Cells or dissected muscles were homogenized in RIPA buffer (50 mmol/l Tris pH = 7.4, 150 mmol/l NaCl, 1% Triton X100, 1% deoxycholic acid, 0.1% sodium dodecyl sulfate) supplemented with a cocktail of protease inhibitors (Complete Tablets; Roche, Basel, Switzerland) using a TissueLyser (Qiagen). Samples were incubated for 30 minutes on ice and centrifuged at 16,500g for 10 minutes at 4 °C. The supernatant was removed and centrifuged again. Protein concentration of the final supernatant was measured using the Bradford assay (Pierce, Rockford, IL). Ten micrograms of protein extracts were separated using a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred onto a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK). Immunoblots were incubated with primary antibodies against either human FRG1 (mouse monoclonal, 1:500, sc-101050; Santa Cruz, San Diego, CA) or HA (mouse monoclonal, 1:500 #MMS-101; Covance, Princeton, NJ). Subsequently, 1:10,000 dilution peroxidase-conjugated donkey anti-mouse IgG (#715-035-015; Jackson ImmunoResearch, West Grove, PA) was added. Detection was performed by super signal west pico chemioluminescence substrate (Thermo Scientific, Waltham, MA). Homogeneous sample loading was verified with an anti-α tubulin antibody (T9026; Sigma, St Louis, MO) at 1:10,000 dilution.
Blood analysis. The extent of hepatocellular injury was monitored by measuring serum alanine aminotransferase activity at multiple time points after AAV injection. Serum alanine aminotransferase activity was measured with a IFCC (International Federation of Clinical Chemistry and Laboratory Medicine) optimized kinetic UV method in a SABA chemical analyzer (SEAC-RADIM, Firenze, Italy) and expressed as U/L (Units/Liter).
Functional analysis. Three groups of mice (wild type n = 4 age- and sex-matched, transgenic FRG1 vector-injected n = 5, transgenic FRG1 vehicle-injected n = 5) were subjected to an exhaustion treadmill test from 1 to 12 weeks after treatment. Each mouse was placed on the belt of a six-lane motorized treadmill (Exer 3/6 Treadmill; Columbus Instruments, Columbus, OH) supplied with shocker plates. The treadmill was run at an inclination of 0° initially at 4 m/minute for 2 minutes and the speed was then increased 2 m/minute every 2 minutes at an inclination of 0°. The test was stopped when the mouse remained on the shocker plate for more than 20 seconds without attempting to re-engage the treadmill, and the time to exhaustion was determined.
miRNA extraction and quantitative RT-PCR analyses. Small RNAs were isolated from the heart, liver and skeletal muscles of FRG1 overexpressing mice infected with AVV6-sh1FRG1 (n = 3) or vehicle alone (n = 3) using miRNeasy FFPE kit (liver and heart) (Qiagen) or miRNeasy mini kit (skeletal muscles) (Qiagen). For miRNA detection, RNA was retrotranscribed and quantified by qPCR using the TaqMan MicroRNAs assay (Applied Biosystems). SnoRNA55 was used to normalize the expression and quantitative PCR was performed on a ViiA7 (Applied Biosystems).
Statistical analysis. All data are expressed as means ± SD. Difference between two groups was tested by means of two-tail t-test for independent samples. A Kolmogorov–Smirnov test was performed to determine whether the fiber distribution was similar in different samples.
SUPPLEMENTARY MATERIALFigure S1.
Absence of toxicity in liver and heart of rAAV6-sh1FRG1
treated mice.Figure S2.
Absence of toxicity in muscle and normal miRNA expression following rAAV6-sh1FRG1
reduces fibrosis in FRG1
overexpressing mice muscles.Figure S4.
reduces fat deposition in FRG1
overexpressing mice muscles.Figure S5.
reduces inflammation in FRG1
overexpressing mice muscles.Video S1.
Treated FRG1 mice show improved running endurance.