Micro-dystrophin gene construction.
The human micro-dystrophin cassette contained the (R4–R23/Δ71–78) domains as previously described, and the FLAG epitope (DYKDDDDK) was added to the C-terminus as a translational fusion by PCR.3
The complementary DNA was codon optimized for human usage and synthesized by GenScript (Piscataway, NJ). It includes a consensus Kozak sequence, an SV40 intron, and synthetic polyadenylation site (53 base pairs). A muscle creatine kinase promoter/enhancer was used to drive muscle-specific gene expression. The MCK micro-dystrophin.FLAG expression cassette was cloned between AAV2 inverted terminal repeats using flanking Xba
I restriction enzyme sites in a plasmid derived from pCMVβ (Clontech, Mountain View, CA). Msc
I restriction enzyme digestions were used to confirm ITR integrity.
rAAV vector production.
rAAV vectors were produced by a modified cross-packaging approach whereby the AAV type 2 ITRs can be packaged into multiple AAV capsid serotypes.26
Production was accomplished using a standard 3 plasmid DNA CaPO4
precipitation method using HEK293 cells. Two hundred ninety-three cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin. The production plasmids were as follows: (i) pAAV.MCK.microdys.FLAG, (ii) rep2-cap8 modified AAV helper plasmids encoding cap serotype 8-like isolate rh.74, and (iii) an adenovirus type 5 helper plasmid (pAdhelper) expressing adenovirus E2A, E4 ORF6, and VA I/II RNA genes. A quantitative PCR-based titration method was used to determine an encapsidated vg titer utilizing a Prism 7500 TaqMan detector system (PE Applied Biosystems, Foster City, CA).27
The primer and fluorescent probe targeted the MCK promoter and were as follows: MCK forward primer, 5-CCCGAGATGCCTGGTTATAATT-3; MCK reverse primer, 5-GCTCAGGCA CAGGTGTTG-3; and MCK probe, 5-FAM-CCAGACATGTGGCTGCTCCCCC-TAMRA-3.
Animals and treatments
IM injection of the TA of mouse: All procedures were approved by the Research Institute at Nationwide Children's Hospital Institutional Animal Care and Use Committee. Three to four-week-old mdx mice and normal age-matched C57/BL10 were used for IM injection. Mice were anesthetized and maintained on 1–4% Isoflurane (in oxygen). Both hindlimbs were shaved, and the TA muscle was injected with 3 × 1010 vg of rAAV8.micro-dystrophin.FLAG or normal saline (30 µl volume) using a 30-gauge insulin syringe.
IM injection of the diaphragm of mouse: Briefly, mice were weighed and anesthetized with a combination of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively). A single abdominal incision was made from the base of the sternum to just above the pelvis (~1 cm incision). The diaphragm was identified, and 30 µl of the vector preparation in sterile saline was delivered using a 32-gauge needle. The abdominal wall was closed with 4.0 Vicryl Plus continuous sutures, and skin wound was closed with sterile surgical staples. Mice were treated with a postoperative dose of buprenorphine 0.01 mg/kg subcutaneously for pain. The animals are allowed to recover on a 37 °C warmer.
Diaphragm fatigue protocol in mouse: Mice were euthanized, and the diaphragm dissected with rib attachments and central tendon intact, and placed in K-H buffer (5 mmol/l KCl, 137 mmol/l NaCl, 1.2 mmol/l NaH2PO4-H2O, and 1.2 mmol/l MgSO4). A 1 mm wide section (from rib to tendon) of diaphragm was isolated and attached to a force transducer. The diaphragm strip was looped around a basket assembly attached to the transducer (the rib cartilage serves as the anchor), and the tendon was pierced by a pin. The muscle was stretched to optimal length (length at maximum twitch force) for measurement of twitch contractions and rested for 5 minutes before starting the muscle fatigue protocol. The muscle fatigue protocol measured the force exerted by the muscle when stimulated every second for 90 seconds (1 Hz amplitude, 80 ms duration, and 130 ms frequency). Following the muscle fatigue protocol, the muscle strip was removed from the apparatus, the rib cartilage removed and weighed.
NHP IM injection: Three 7-year-old Chinese rhesus macaques that did not possess AAV8-binding antibodies above background were studied (ELISA performed using 1:50 serum dilution). All NHPs were housed in pairs to promote socialization. The macaques were anesthetized using Telazol (5 mg/kg IM) and treated with buprenorphine (0.01 mg/kg) IM preoperatively. The TA of both hindlimbs was shaved and prepared with 95% EtOH and povidone solution, and the animal secured to a warming blanket (37 °C) that overlies the surgery table. The TA was visualized by blunt dissection, and 5 × 1012 vg of rAAV8.micro-dystrophin.FLAG or normal saline (1 ml volume total) was injected in three sites 0.5 cm apart. The fascial layer and skin incision were closed with 3–4 interrupted Vicryl sutures and skin bond, and the injection site was marked with tattoo ink. For the muscle biopsy at 8 weeks, an incision was made to visually expose the TA muscle, using sterile drapes and scalpels. A small muscle biopsy was obtained (block 0.75–1.0 × 0.5 cm), with bleeding controlled by direct pressure. The wound was closed with interrupted Vicryl sutures.
Isolated limb perfusion in rhesus macaques: Rhesus macaques were sedated with IM Telazol (3–6 mg/kg), intubated and secured to a heated procedure table at 37 °C. General anesthesia was administered with Isoflurane (in oxygen) 1–4% during the procedure. The left groin was shaved with extension to the mid thigh, and prepped with povidone–iodine solution followed by 95% ethanol. A groin incision was made over the femoral bundle, and the femoral artery was isolated. The femoral artery was catheterized using fluoroscopy-guided 3-0 f catheter (Cook) that was advanced to the sural branch of the popliteal artery. Prior to vector administration, a prevector flush of saline (2.5 ml/kg) was given over 1 minute. This was immediately followed by occluding blood flow to the extremity using a standard phlebotomy tourniquet placed proximal to the tip of the catheter that was typically right above the knee. A second tourniquet was placed at the base of the gastrocnemius. rAAV8.MCK.micro-dys.FLAG was infused over 60 seconds at a dose of 2 × 1012 vg/kg in 2.5 ml/kg of Tris buffered saline. The extremity remained isolated from the circulation for 10 minutes. A postvector flush (2.5 ml/kg) was infused over 1 minute and then the tourniquets were released. Direct pressure was applied for 10 minutes to control bleeding, and the wound was closed with 4-0 Vicryl suture.
Gene expression analyses. TA and gastrocnemius skeletal muscles were collected from treated and contralateral control limbs at 8 weeks and 5 months after treatment, respectively, for subjects treated by IM injection. A single 1.0 × 0.5 cm block was removed at biopsy at the 8-week time point, and the remainder of the TA was removed and blocked at necropsy at 5 months. Subjects treated by isolated limb perfusion were killed at 3 months at which time the entire gastrocnemius muscle was removed and blocked (0.75–1.0 × 0.5 cm blocks). Muscles were embedded in 7% gum tragacanth and flash-frozen in isopentane cooled in liquid nitrogen. Cryostat sections (12 µm) for FLAG immunofluorescence were incubated with anti-FLAG polyclonal primary antibody (F7425; Sigma, St Louis, MO) at a dilution of 1:175 in blocking buffer [phosphate-buffered saline (PBS), 10% goat serum, 0.1% Triton X-100] for 1 hour at 25 °C in a wet chamber. For dystrophin-specific staining, Dys2 (NCL-Dys2; Novocastra Laboratories, Newcastle, UK) primary antibody was used (1:3 in blocking buffer). Sections were then washed with PBS three times, each for 20 minutes and reblocked. Visualization was achieved by incubation for 45 minutes at 25 °C with an Alexa 568 goat anti-rabbit or Alexa 488-conjugated IgG1 isotype-specific goat anti-mouse antibody at a 1:300 dilution (Molecular Probes, Carlsbad, CA). Sections were washed with PBS three times for 20 minutes and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Fluorescence staining was visualized using a Zeiss Axioskop 2 Plus Microscope (Zeiss, Thornwood, NY), and images were captured with a Zeiss AxioCam MRC5 camera (Zeiss). The number of fibers with sarcolemmal staining were expressed as percentage of all fibers. Means for IM necropsy studies were obtained from the mean of six blocks surrounding the original injection site. Means for isolated limb perfusion studies were obtained by counting four ×10 fields from six muscle blocks in proximal, central, and distal regions for each muscle.
Western blot analysis. Tissue sections (10–20 µm thick) from micro-dys.FLAG treated and untreated muscle were collected into a microcentrifuge tube and homogenized with 100 µl homogenization buffer (125 mmol/l Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, 4 mol/l urea, 5% βME and protease inhibitor cocktail). Protein levels were quantified using RC/DC method (BioRad Laboratories, Hercules, CA). Protein samples (50 µg per lane) were electrophoresed on a 3–8% polyacrylamide Tris-Acetate gel (NuPAGE; Invitrogen, Carlsbad, CA) and then transferred to a polyvinylidene fluoride membrane (Amersham Biosciences, Pittsburgh, PA). After blocking for 1 hour in 5% nonfat dry milk in TBST (100 mmol/l Tris-HCl, pH 8.0, 167 mmol/l NaCl, 0.1% Tween), the western blot was incubated overnight with dystrophin monoclonal antibody NCL-DYS2 or DYS3 (Novocastra Laboratories) at a dilution of 1:100, followed by horseradish peroxidase–labeled goat anti-mouse IgG (GE Healthcare, Piscataway, NJ) at a dilution of 1:2,000 for 1 hour. Immunoreactive bands were visualized with the use of the ECL Plus western blotting detection system (GE Healthcare) and Hyperfilm ECL (Amersham Biosciences). Signal intensities were measured with ImageQuant software (GE Healthcare).
Quantitative PCR to detect genome copy number.
TaqMan quantitative PCR was used to quantify the number of vg copies compared to contralateral control tissue as described.27
A vector-specific primer probe set amplified a portion of the unique sequence of the MCK promoter within the micro-dystrophin.FLAG cassette. The rhesus erythropoietin gene was used as an internal control to normalize for genomic input and confirm the absence of PCR inhibitors in the sample DNA. Copy number is reported as vg per microgram of genomic DNA.
Mononuclear cell analysis. Immunohistochemistry was performed to identify immune cells. Tissue sections were incubated with monoclonal antibodies to CD4 (L200; BD Biosciences, San Jose, CA), CD8 (RPA-T8; BD Biosciences) and were diluted 1:50 with PBS. Visualization was achieved with DAB substrate using the Super Sensitive Polymer-HRP IHC Detection System for Automation (QD410-YAX; BioGenex, San Ramon, CA). The entire muscle section was analyzed, and the number of mononuclear cells were counted and expressed as total number per mm2.
IFN-γ ELISpot analysis. ELISpot assays were performed on fresh PBMCs, which were added at a concentration of 2 × 105/well in duplicate wells of a 96-well flat-bottom membrane plate (Millipore, Billerica, MA). Three peptide pools were used for the AAV8 capsid protein (Genemed Synthesis, San Antonio, TX), containing 34–36 peptides, each 18 amino-acid long and overlapping by 11 residues. Three peptide pools encompassed the micro-dystrophin.FLAG protein (Genemed Synthesis) and contained 36–42 peptides, each 20 amino-acid long and overlapping by 10 residues. The final two peptides of the micro-dystrophin.FLAG pool 3 were specific for the FLAG-tag epitope. Pool 1 is composed of aa14-240 (actin-binding domain), aa253-327 (hinge 1), and aa337-427 (first part of spectrin repeat 1). Pool 2 is composed of aa428-447 (remainder of spectrin repeat 1), aa448-556 (spectrin repeat 2), aa557-667 (spectrin repeat 3), aa668-717 (hinge 2), and aa2932-3040 (spectrin repeat 24). Pool 3 is composed of aa3041-3112 (hinge 4), aa3080-3360 (cysteine repeat region), plus the sequence of the FLAG-tag. Concanavalin A (Sigma) served as a positive control and a hepatitis B virus peptide pool as a negative control. Peptides were added directly to the wells at a final concentration of 1 µg/ml in 200 µl of AIM-HS [Aim-V lymphocyte media (Invitrogen) supplemented with 2% human AB serum (Gemini-BioScience, Basel, Switzerland)]. Monkey IFN-γ ELISpot kits were purchased from U-CyTech (Utrecht, the Netherlands). After the addition of PBMCs and peptides, the plates were incubated at 37 °C for 48 hours and then developed according to the manufacturer's protocol. IFN-γ spot formation was counted using a Cellular Technologies systems analyzer (Cellular Technologies, Cleveland, OH).
ELISA analysis. An ELISA was performed to measure the level of circulating AAV8 capsid–binding antibody in serum. Immulon-4 96-well plates (ISC BioExpress, Kaysville, UT) were coated with 100 µl of 2 × 1010 vg/ml AAV8 viral stock in carbonate buffer (pH 9.4; Pierce, Rockford, IL) per well. Plates were sealed overnight at 4 °C. Plates were blocked with 280 µl per well of a 5% nonfat dry milk and 1% normal goat serum (Invitrogen) in PBS for 3 hours at 25 °C. Rhesus plasma was diluted at a 1:50 ratio in solution identical to the blocking solution, and 100 µl added in duplicate to both wells coated with AAV8 particles in carbonate buffer and wells coated with carbonate buffer alone. Plates were incubated at 25 °C for 1 hour before being washed five times with 280 µl of PBS-T (0.05% Tween). Blocking solution was used again to dilute the secondary antibody, goat anti-monkey IgG-HRP (Sigma) at a 1:10,000 dilution. Wells received 250 µl of the secondary antibody and were incubated at 25 °C for 30 minutes before being washed five times and blotted dry. Tetramethylbenzidine (100 µl/well; Pierce) was added and incubated at 25 °C for 10 minutes in the dark, before the addition of 100 µl of 1N H2SO4 (Acros Organics, Geel, Belgium) to stop the reaction. The OD450 was measured using a Wallace 1420-050 Multilabel Counter (PerkinElmer, Waltham, MA). Samples were considered positive if the OD450 average of the antigen-coated wells was three times greater than wells coated with carbonate buffer alone.
SUPPLEMENTARY MATERIALFigure S1.
Micro-dystrophin.FLAG gene transfer does not induce histopathological changes. (a) Hematoxylin and eosin staining from isolated muscle tissue at the site of gene transfer revealed no evidence of tissue damage or cellular infiltration in all three subjects. (b,c) CD4+ and CD8+ mononuclear cell stains show sparsely, scattered positive cells (arrows) following gene transfer in perivascular and endomysial sites. No mononuclear cell invasion of muscle fibers was detected. Scale bar = 100 μm, Magnified image in panel c, scale bar = 50 μm.Figure S2.
Vector genome copy number. Histogram demonstrating micro-dystrophin.FLAG copy number determined by quantitative Taqman qPCR. Subjects treated by intramuscular injected (IM) are compared with those treated by isolated limb perfusion without (ILP AAV-) and with (ILP AAV+) pre-existing immunity to AAV8.