rAAV9 packaging and purification.
Recombinant AAV9 vectors used in this study were generated, purified, and titered by the UMass Gene Therapy Vector Core as previously described.31
To clone in the miRNAs a 425 bp sequence containing all three miRNAs in tandem was cloned into CB intron by replacing 315 bp intron sequence between enzyme sites SgrAI and XbaI. Cloning of the miRNAs at the polyA region was accomplished by inserting the 425 bp fragment at the NotI site 5 bp upstream of the polyA region.
Cell culture and transfection. HEK-293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 mg/l of penicillin-streptomycin (Gemini Bio-products, West Sacramento, CA). 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. Cell culture supernatants were collected at 24, 48, and 72 hours, and cell lysates were collected at 72 hours.
Serum AAT ELISAs.
Human AAT ELISA: Total AAT protein was detected by ELISA. High binding extra, 96-well plate (Immulon 4; Dynatech Laboratories, Chantilly, VA) were coated with 100 µl of human specific goat anti-AAT (1:500 diluted; Biomedicals, Solon, OH) in Voller's buffer overnight at 4 °C. After blocking with 1% non-fat dry milk in phosphate-buffered saline with Tween 20, duplicate standard curves (Athens Research and Technology, Athens, GA) and serially diluted unknown samples were incubated in the plate at room temperature for 1 hour, a second antibody, goat anti-hAAT(HRP) (1:5,000 diluted; Abcam, Cambridge, MA) was incubated at room temperature for 1 hour. The plate was washed with phosphate-buffered saline-Tween 20 between reactions. After reaction with TMB peroxidase substrate (KPL, Gaithersburg, MD) reactions were stopped by adding 2 N H2SO4 (Fisher Scientific, Hudson, NH). Plates were read at 450 nm on a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA).
Z-AAT ELISA: Human Z-AAT protein levels were detected by ELISA with coating antibody specific for human Z-AAT (1:100 diluted mouse-anti-human α-1-antitrypsin-Z; Cell Sciences, Fair Lawn, NJ). Standard curves were created with PiZ mouse serum with 5% bovine serum albumin (Sigma, St Louis, MO). Serially diluted unknown samples were incubated in the plate at 37 °C for 1 hour. The secondary antibody and the next step were same as the standard human-AAT ELISA described above, except the secondary antibody was diluted in 5% bovine serum albumin and incubated in the plate at 37 °C for 1 hour.
c-Myc ELISA: c-Myc tag levels were quantified by a method similar to that described above. Plates were coated with a c-Myc antibody (1:1,000 diluted Goat anti-c-Myc; Abcam), plates were then blocked with 5% bovine serum albumin at 37 °C for 1 hour. To create the standard curve C57Bl/6 mice were dosed via tail vein with c-Myc-AAT expressing vector, at 2 weeks serum were collected and pooled. The amount c-Myc-AAT in the serums was then quantified with the human specific AAT ELISA described above. The obtained values were then used to produce a standard of c-Myc protein for the c-Myc ELISA.
RNA extraction: Flash frozen mouse liver tissue was ground in a pestle and mortar and used to extract either small or total RNA using the mirVana miRNA RNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's instructions.
microRNA qRT-PCR: mircoRNA was primed and reverse-transcribed with TagMan MicroRNA reverse transcription Kit (Applied Biosystems, Foster City, CA). Quantitative PCR was performed in duplicate with gene-specific RT-miRNA primers, and PCR assays were as designed by Applied Biosystems, using TaqMan Gene Expression Master mix (Applied Biosystems) in a StepOne Plus real-time PCR instrument (Applied Biosystems).
PiM and PiZ qRT-PCR: Total RNA was primed with oligo(dT) and reverse-transcribed with SuperScript III First-Strand Synthesis kit for RT-PCR (Invitrogen). Quantitative PCR were performed by gene-specific primer pairs. PiM and PiZ share the primers but differ in the probes. Forward primer CCAAGGCCGTGCATAAGG, reverse primer: GGCCCCAGCAGC TTCAGT, PiZ probe: 6FAM-CTGACCATCGACAAGA-MGBNFQ, and PiM probe: 6FAM-CTGACCATCGACGAGA-MGBNFQ. Reactions were performed using TaqMan Gene Expression Master mix (Applied Biosystems) in a StepOne Plus real-time PCR instrument (Applied Biosystems).
Z-AAT transgenic mice and rAAV9 delivery.
The PiZ-transgenic mice used in this study have been described.11
All animal procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School. rAAV9 vector was administered by mouse tail vein injection in a volume of 200 µl with titer of 1 × 1012
vps. The injections were performed in the most accessible vessels veins that run the length of both lateral aspects of the tail by grasping the tail at the distal end. Blood was collected through the facial vein pre-injection and every week after tail vein rAAV9 delivery until termination of the studies.
Liver histology. For determination of histological changes, liver samples were fixed in 10% neutral-buffered formalin (Fisher Scientific), and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin and PAS with or without diastase digestion.
Immunohistochemistry for hAAT, was performed as described,13
briefly tissue sections (5 µm) were deparaffinized, rehydrated, and blocked for endogenous peroxidase with 3% hydrogen peroxide in methanol for 10 minutes. To detect hAAT expression, tissue sections were incubated with primary antibody, rabbit antihuman AAT (1:800; RDI/Fitzgerald Industries, Acton, MA), for overnight at 4 °C. Staining was detected using ABC-Rb-HRP and DAB kits (Vector Laboratories, Burlingame, CA).
Histology image analysis. Slides were stained for PASD to remove glycogen. Whole digital slide images were created using an Aperio CS ScanScope (V, CA) and analyzed using the positive pixel count algorithm (version 9). PASD-positive globules were expressed as the proportion of strong positive pixels to total pixels using a hue value of 0.9, hue width of 0.15, and color saturation threshold of 0.25. The intensity threshold for strong positivity was set to an upper limit of 100.
Analysis of Z-AAT protein monomer and polymer. For soluble/insoluble protein separation, 10 mg of whole liver was added to 2 ml buffer at 4 °C (50 mmol/l Tris-HCl (pH 8.0), 150 mmol/l NaCl, 5 mmol/l KCl, 5 mmol/l MgCl2, 0.5% Triton X-100, and 80 µl of complete protease inhibitor stock). The tissue was homogenized in a prechilled Dounce homogenizer for 30 repetitions, then vortexed vigorously. A 1-ml aliquot was passed through a 28-gauge needle 10 times. The total protein concentration of the sample was determined, and a 5-µg total liver protein sample was aliquoted and centrifuged at 10,000g for 30 minutes at 4 °C. Supernatant (soluble (S) fraction) was immediately removed into fresh tubes; extreme care was taken to avoid disturbing the pellet (insoluble (I) fraction). The insoluble polymers pellet (I fraction) was denatured and solubilized via addition of 10 l chilled cell lysis buffer (1% Triton X-100, 0.05% deoxycholate, 10 mmol/l EDTA in phosphate-buffered saline), vortexed for 30 seconds, sonicated on ice for 10 minutes and vortexed. To each soluble and insoluble sample, 2.5 sample buffer (50% 5 sample buffer (5% sodium dodecyl sulfate, 50% glycerol, 0.5 mol/l Tris (pH 6.8)), 10% mercaptoethanol, 40% ddH2O) was added at a volume of 50% of the sample volume. Samples were boiled and loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); equal amounts of total liver protein were loaded per soluble–insoluble pair in quantitative experiments. Densitometry was performed using Image J Software (NIH, Bethesda, MD).
Serum chemistry. All serum samples were analyzed by UMass Mouse Phenotyping Center Analytical Core, using the NExCT Clinical Chemistry Analyzer (Alfa Wassermann Diagnostic Technologies, West Caldwell, NJ). Serum was analyzed for alanine aminotransferase and aspartate aminotransferase according the manufacturers specifications.
miRNA microarray expression analysis.
We isolated 8 µg of total RNA from flash frozen mouse livers using the mirVana miRNA isolation kit (Ambion). The experimental design included six groups with RNA samples from five mice each which were assayed on single color arrays for a total of 30 independent microarrays. In brief, the RNA was labeled with Cy5 and hybridized to dual-channel microarray µParaFlo microfluidics chips (LC Sciences, Houston, TX) containing miRNA probes to mouse mature miRNAs available in the Sanger miRBase database (Release 16.0) as described.32
Each of the spotted detection probes consisted of a nucleotide sequence complementary to a specific miRNA sequence and a long non-nucleotide spacer that extended the specific sequence away from the chip surface. Fluorescence images were collected with a laser scanner (GenePix 4000B; Molecular Devices) and digitized using Array-Pro image analysis software (Media Cybernetics, Bethesda, MD). The data were analyzed, including background subtraction, with a LOWESS (locally weighted regression) method on the background-subtracted data as described.33
The normalization is to remove system related variations, such as sample amount variations and signal gain differences of scanners. Samples were considered positive only if transcripts had a signal intensity higher than 3 times (background-subtracted data) and the spot coefficient of variation <0.5. Coefficient of variation as calculated by (subtracted data)/(signal intensity), and in which repeating probes on the array produced signals from at least 50% of the repeating probes above detection level. Data are represented as a Log2
transformation. The data were further filtered to remove miRNAs with (normalized) intensity values below a threshold value of 32 across all samples. t-
test were performed between “control ” and “test ” sample groups where T values are calculated for each miRNA, and P
values are computed from the theoretical t-distribution. If P
≤ 0.05, it is plotted as red spot in a log scatter plot.
Long-term in vivo
silencing of human AAT by rAAV9 expressed miRNAs.
Liver function in response to rAAV9 delivery.
assessment of dual-function pro-viral plasmid.