Vector design, construction, and production.
Perfectly complementary miRNA-binding sites were designed based on the annotated miR-1 and miR-122 sequences in miRBase32
and inserted into the BstB
I restriction site in the 3′ UTR of the nLacZ expression cassette of the ubiquitously expressed pAAVCB nuclear-targeted β-galactosidase (nLacZ) plasmid using synthetic oligonucleotides (
and Supplementary Table S1
). This vector uses a hybrid cytomegalovirus enhancer/CB promoter cassette that is active in most cells and tissues. To express miR-122 and miR-1, pri-miR-122 and pri-miR-1 fragments were amplified by PCR from C57/B6 mouse genomic DNA (Supplementary Table S1
) and inserted into the XbaI restriction site 3′ to a firefly luciferase cDNA in the pAAVCBFLuc plasmid. The identity of each pri-miRNA was verified by sequencing. AAV9 vectors used in this study were generated, purified, and titered as described.33
Cell culture and transfection. HEK-293 and HuH7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 mg/l of penicillin-streptomycin (Hyclone, South Logan, UT). Cells were maintained in a humidified incubator at 37 °C and 5% CO2. Plasmids were transiently transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
Mouse studies. Male C57BL/6 mice (Charles River Laboratories, Wilmington, MA) were obtained and maintained and all animal procedures performed according to the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School. To monitor lipid profiles of the study animals, serum samples were collected 4 weeks after rAAV9 injection and analyzed for total cholesterol, high-density lipoprotein and low-density lipoprotein on a COBAS C 111 analyzer (Roche Diagnostics, Lewes, UK). To evaluate endogenous miRNA-mediated, CNS-restricted EGFP gene transfer, 10-week-old male C57BL/6 mice were injected intravenously (tail vein) with AAV9CBnLacZ-[miR-122-binding site (BS)1], AAV9CBnLacZ-(miR-122BS)3, AAV9CBnLacZ-(miR-1BS)1, AAV9CBnLacZ-(miR-1BS)3, AAV9CBnLacZ-(miR-1BS)1-(miR-122BS)1, and AAV9CBnLacZ-(miR-1BS)3-(miR-122BS)3122BS)3, respectively, at 5 × 1013 GC/kg body weight) or scAAV9CBEGFP at 2 × 1014 GC/kg body weight). Animals receiving nLacZ vectors were necropsied 4 weeks later; 8 µm cryosections of liver, heart, and pancreas tissues were prepared for X-gal-histochemical staining. Animals that received EGFP vectors were necropsied 3 weeks later and fixed by transcardial perfusion with 4% (wt/vol) paraformaldehyde. Brain, spinal cord, liver, heart, and muscle were harvested for cryosectioning. Brain and cervical spinal cord tissue were stained as floating sections in a 12-well plate using rabbit anti-EGFP antibody (Invitrogen) diluted 1:500, followed by goat anti-rabbit secondary antibody (Invitrogen) diluted 1:400. Outside the CNS, EGFP expression was detected directly by fluorescence. EGFP and antibody fluorescence was recorded using a Nikon TE-2000S inverted microscope at ×10 magnification and an exposure time of 3 seconds for liver, heart, and muscle, and 5 seconds for thalamus (brain) and cervical spinal cord.
Vector genome quantification by qPCR. Genome DNA was extracted from the selected tissues using QIAamp DNA Mini Kit (Qiagen, West Sussex, UK), according to the manufacturer's instructions. Quantitative PCR were carried out in triplicate using 50 ng DNA and 0.3 µmol/l EGFP-specific primers (EGFP-F and EGFP-R) using GoTaq qPCR master mix (Promega, Madison, WI) in a StepOne Plus real-time PCR instrument (Applied Biosystems, Foster City, CA).
qRT-PCR analysis. RNA was extracted using Trizol (Invitrogen), according to the manufacturer's instructions. Total RNA (0.5–1.0 µg) was primed with random hexamers or oligo(dT) and reverse-transcribed with MultiScribe Reverse Transcriptase (Applied Biosystems). Quantitative PCR were performed in triplicate with 0.3 µmol/l gene-specific primer pairs (nLacZ5′F/5′R, nLacZ 3′F/3′R, cyclinG1F/R and EGFP-F/EGFP-R) using the GoTaq qPCR master mix in a StepOne Plus Real-time PCR device. The specificity of qRT-PCR products derived from the 5′ and 3′ ends of nLacZ mRNA was confirmed by gel electrophoresis.
Northern blot analysis.
Total RNA was extracted from mouse liver and analyzed by Northern hybridization.34
To detect nLacZ
mRNA, a 618 bp fragment of nLacZ
cDNA was isolated by NcoI and PciI digestion of pAAVCBnLacZ
and labeled with α-32
P dCTP by random priming (Takara, Shiga, Japan). To detect 3′ fragments of the cleaved nLacZ mRNA, an 111 bp fragment of the poly(A) sequence in the vector genome was cloned into pCR4-TOPO (Invitrogen) for preparation of antisense RNA probe labeled with α-32
P CTP during in vitro
transcription using the Riboprobe System T7 kit (Promega). To detect miR-122, miR-26a, miR-22, and let-7 or U6 in total liver RNA, small RNAs were resolved by denaturing 15% polyacrylamide gels, transferred to Hybond N+ membrane (Amersham BioSciences, Pittsburgh, PA), and crosslinked with 254 nm light (Stratagene, La Jolla, CA). Synthetic oligonucleotides, 5′ end-labeled with γ-32
P ATP using T4 polynucleotide kinase (New England Biolabs, Beverly, MA), were used as DNA probes (Supplementary Table S1
) and hybridized in Church buffer (0.5 mol/l NaHPO4
, pH 7.2, 1 mmol/l EDTA, 7% (w/v) sodium dodecyl sulphate) at 37 °C. Membranes were washed using 1× SSC (150 mM sodium chloride, 15 mM sodium citrate), 0.1% sodium dodecyl sulphate buffer, and then visualized using an FLA-5100 Imager (Fujifilm, Tokyo, Japan).
Western blot analysis. Proteins were extracted with radioimmunoprecipitation assay buffer [25 mmol/l Tris–HCl, pH 7.6, 150 mmol/l NaCl, 1% (vol/vol) NP-40, 1% (wt/vol) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulphate] containing a protease inhibitor mixture (Boston BP, Boston, MA). Protein concentration was determined using the Bradford method (Bio-Rad, Melville, NY). Protein samples, 50 µg each, were loaded onto 12% polyacrylamide gels, electrophoresed, and transferred to nitrocellulose membrane (Amersham BioSciences). Briefly, membranes were blocked with blocking buffer (LI-COR Biosciences, Lincoln, NE) at room temperature for 2 hours, followed by incubation with either anti-GAPDH (Millipore, Billerica, MA), anti-cyclin G1 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-calmodulin (Millipore) for 2 hours at room temperature. After three washes with PBS containing 0.1% (vol/vol) Tween-20, membranes were incubated with secondary antibodies conjugated to LI-COR IRDye for 1 hour at room temperature, and then antibodies detected using the Odyssey Imager (LI-COR).
β-Galactosidase assay. Proteins were extracted with radioimmunoprecipitation assay buffer and quantified as described above. Fifty micrograms of protein was used for each β-galactosidase assay using the Galacto-Star System (Applied Biosystems), according to the manufacturer's instructions.
5′ RACE was performed as described.35
The 5′ RACE Outer Primer and the nLacZ
gene-specific primer bGHpolyAR (Supplementary Table S1
) were used for the first round of nested PCR. The 5′ RACE Inner Primer and the nLacZ gene-specific primer nLacZpolyR, which is located near the stop codon of nLacZ cDNA, were used for the second round of nested PCR (Supplementary Table S1
). PCR products were TOPO-cloned into pCR-4.0 (Invitrogen) and sequenced.
Statistical analysis. All results are reported as mean ± SD and compared between groups using the two-tailed Student's t-test.
Figure S1. Quantification of β-galactosidase activities in liver tissue from animals that received rAAVnLacZ vectors with and without miRNA-binding sites. Adult male C58BL/6 mice were injected intravenously with 5 × 1013 GC/kg each of rAAV9CBnLacZ (no miRNA binding site), rAAVCB9nLacZ-miR-122BS (one miR-122-binding site) or rAAV9CBnLacZ-(miR-122BS)3 (three miR-122-binding site), rAAV9CBnLacZ-miR-1BS-miR-122BS (one miR-1 and one miR-122-binding site), rAAV9CBnLacZ-(miR-1BS)3-(miR-122BS)3 (three miR-1 and three miR-122-binding sites) and rAAV9CBnLacZ-(miR-1BS)3 (three miR-1-binding sites). The animals were necropsied 4 weeks after vector administration, and liver tissues were harvested for total proteins and β-Galactosidase assays. Percentages report the β-Galactosidase activity of each vector group compared to the control group, rAAV9CBnLacZ.
Table S1. Oligonucleotide primers and probes used in the study.