. Self-complementary AAV9 was produced by transient transfection procedures using a double-stranded AAV2-ITR-based CB-GFP vector, with a plasmid encoding Rep2Cap9 sequence as previously described along with an adenoviral helper plasmid pHelper (Stratagene, Santa Clara, CA) in 293 cells.50
Our serotype 9 sequence was verified by sequencing and was identical to that previously described. Virus was produced in three separate batches for the experiments and purified by two cesium chloride density gradient purification steps, dialyzed against PBS and formulated with 0.001% Pluronic-F68 to prevent virus aggregation and stored at 4 °C. All vector preparations were titered by quantitative-PCR using Taq-Man technology. Purity of vectors was assessed by 4–12% sodium dodecyl sulfate-acrylamide gel electrophoresis and silver staining (Invitrogen, Carlsbad, CA).
Animal care and use. All procedures performed were in accordance to either the Mannheimer Foundation (Homestead, FL), the Research Institute at Nationwide Children's Hospital or The Ohio State University Institutional Animal Care and Use Committees.
Nonhuman primate intravascular vector delivery. The breeding, housing and procedures performed on the young, male cynomolgus macaques (Macaca fasciculata, age P1–P90) were carried out at the Mannheimer Foundation. Briefly, veterinary staff anesthetized the subject and placed a catheter into the saphenous vein, through which either a suspension of 1–3 × 1014 vg/kg AAV9.CBA.GFP or PBS was infused over a period of 5–8 minutes. Upon recovery, subjects were returned to their mother and housed under routine conditions for the duration of the study.
At Nationwide Children's Hospital, the 3-year-old subject was infused with 2.7 × 1013 vg/kg using interventional radiological techniques to target delivery to the radicular arteries of the thoracic cord. Briefly, the subject was anesthetized and a catheter was introduced percutaneously into the brachial artery and guided to the proximal portion of the descending aorta. A second catheter delivered an occlusive balloon to the distal portion of the descending aorta at the level of the celiac trunk. Proper placement was confirmed by fluoroscopy and injection of a radiopaque dye via the proximal catheter. Before injection, the distal balloon was inflated to occlude blood flow distal to and including the celiac trunk. The viral suspension was delivered over ~1 minute, and the balloon was left inflated for another 2 minutes postinfusion. After recovery, the animal was released back to its regular environment for the duration of the study.
Seronegativity for anti-AAV9 antibodies was confirmed in all subjects by enzyme-linked immunosorbent assay. Briefly, a 2 × 1010 vg/ml solution of empty AAV9 capsids was made with a carbonate coating buffer and applied to a 96-well plate and incubated over night at 4 °C. The following day, the plate was washed and blocked with a 5% milk solution in PBS with 0.1% Tween-20. Serums were diluted from 1:50 to 1:6400 and incubated at room temperature for an hour. The wells were washed with PBS-T and then incubated with an horseradish peroxidase conjugated anti-monkey secondary (Sigma-Aldrich, St Louis, MO) for 1 hour at room temperature. The wells were washed with PBS-T then developed with TMB. The reaction was stopped with the addition of hydrochloric acid and absorbance was read at 650 nm on a plate reader.
Intrathecal Injection. Farm-bred sows (Sus scrofa domestica) were obtained from a regional farm. Five-day-old (P5) piglets received 0.5 cc/kg ketamine induction anesthesia and then were maintained by mask inhalation of 5% isoflurane in oxygen. Body temperature, electrocardiogram, and respiratory rate were monitored throughout the procedure. For lumbar puncture, piglets were placed prone and the spine was flexed in order to widen the intervertebral spaces. The anterior–superior iliac spines were palpated and a line connecting the two points was visualized. The intervertebral space rostral to this line is ~L5–L6. Intraoperative fluoroscopy confirmed rostral-caudal and mediolateral trajectories. Using sterile technique, a 25-gauge needle attached to a 1-ml syringe was inserted. Gentle negative pressure was applied to the syringe as the needle was passed until a clear flash of CSF was visualized. For cisterna puncture, the head of the piglet was flexed while maintaining the integrity of the airway. Fluoroscopy again confirmed adequate trajectory. A 25-gauge needle was passed immediately caudal to the occipital bone, and a flash of clear CSF confirmed entry into the cistern magna.
For reagent delivery, the syringe was removed while the needle was held in place. A second 1-cc syringe containing either viral solution (5.2 × 1012 vg/kg) or PBS was secured and the solution was injected into the intrathecal space at a slow and constant rate. After delivery, ~0.25 ml of sterile PBS was flushed through the spinal needle so as to ensure full delivery of reagent.
We confirmed rostral and caudal intrathecal flow by injecting a radioopaque agent (Omnipaque, GE Healthcare, Waukesha, WI) and recording intrathecal spread with real-time continuous fluoroscopy.
Perfusion and tissue-processing. All subjects (primate and porcine) were killed between 21 and 24 days postinjection. Subjects were deeply anesthetized by intramuscular injection of sodium pentobarbital solution (primates) or Telazol followed by Propofol (piglets). A midventral sternal thoracotomy was performed and a cannula was inserted in the aorta through the left ventricle. The right atrium was opened and 0.5–1 l of PBS was injected through the cannula by gravity flow, followed by perfusion with 1 l of 4% paraformaldehyde in phosphate buffer (pH 7.4). Organs were removed and post-fixed 48 hours in 4% paraformaldehyde before further processing for histological sectioning or stored long-term in 0.1% NaN3 PBS solution.
Histology and microscopy. Primate and porcine spinal cord segments were embedded in 3% agarose before cutting into 40-µm horizontal sections using a Leica VT1200 vibrating microtome (Leica Microsystems, Buffalo Grove, IL). Sections were transferred in Tris-buffered saline and stored at 4 °C until processing.
Primate and porcine brains were cryoprotected by successive incubation in 10, 20, and 30% sucrose solutions. Once sufficiently cryoprotected (having sunk in 30% sucrose solution), brains were frozen and whole-mounted on a modified Leica SM 2000R sliding microtome (Leica Microsystems) in OCT (Tissue-Tek, Torrance, CA) and cut into 40-µm coronal sections.
For immunofluorescent determination of cell types transduced, floating sections were submerged in blocking solution (10% donkey serum, 1% Triton-X100 in Tris-buffered saline) for 1 hour followed by overnight incubation in primary antibody solution at 4 °C. The following primary antibodies were used in this study: Rabbit-anti-GFP (1:500; Invitrogen), goat-anti-ChAT (1:100; Millipore, Billerica, MA), guinea-pig-anti-GFAP (1:1,000; Advanced Immunochemical, Long Beach, CA) and rabbit-anti-Iba1 (1:500; Dako, Carpentaria, CA). Primary antibodies were detected using Fitc-, Cy3-, or Cy5-conjugated secondary antibodies (1:1,000; Jackson ImmunoResearch, West Grove, PA) and mounted in PVA-DABCO medium.
For immunohistochemical staining, sections were incubated at room temperature in 0.5% H2O2/10% MeOH solution and subsequently blocked and stained as above with rabbit-anti-GFP overnight. Anti-GFP antibodies were detected using biotinylated donkey-anti-rabbit secondary antibody (1:200; Jackson ImmunoResearch) and developed using Vector NovaRed per the provided protocol (Vector Labs, Burlingame, CA). Sections were then mounted in Cytoseal 60 medium (Thermo Fisher Scientific, Kalamazoo, MI).
Non-neural tissues were cut to ~1 cm3 blocks and cryoprotected by overnight incubation in 30% sucrose solution. They were then embedded in gum tragacanth and flash-frozen in liquid nitrogen-cooled isopentane. Samples were cut by cryostat into 10–12 µm sections and slides stored at −20 °C. GFP expression was detected by a similar immunofluorescent protocol as above with the addition of DAPI in secondary antibody solution (1:1,000; Invitrogen).
Fluorescent images were captured using a Zeiss 710 Meta confocal microscope (Carl Zeiss MicroImaging, Thornwood, NY) located at TRINCH and processed with LSM software.
Whole brain sections were scanned to ×40 resolution at the Biopathology Center in the Research Informatics Core at the Research Institute at Nationwide Children's Hospital using an Aperio automated slide scanner (Aperio, Vista, CA) and resulting images were processed with ImageScope software.
In situ hybridization
. As described previously,11
we generated antisense and sense DIG-UTP-labeled GFP riboprobes. Probe yield and incorporation of DIG-UTP was confirmed by electrophoresis and dot blot. Sections of spinal cord 10-µm thick were mounted and prepared by fixation with 4% paraformaldehyde, washed in 0.5× SSC, permeabilized by incubation in proteinase K (2.5 µg/ml), washed in 0.5 × SSC and dehydrated in series of alcohol washes. Prehybridization was performed at 42 °C using RiboHybe buffer (Ventana, Tucson, AZ) for 1 hour followed by hybridization overnight at 55 °C with the respective riboprobes on AAV9-injected and PBS-control-injected cord sections. Stringency washes were performed and immunological detection using anti-Digoxigenin AP antibody (1:500; Roche, Tucson, AZ) and development with NBT/BCIP (Thermo Fisher Scientific) and Nuclear Fast Red (Vector Labs).
SUPPLEMENTARY MATERIALFigure S1.
GFP expression with the dorsal horn of spinal cord. Sections from a P1 AAV9 P1 injected monkey show GFP positive fibers within the dorsomedial white matter and dorsal horn gray matter indicating AAV9 transduction of dorsal root ganglia at the cervical (a
), thoracic (b
) and lumbar (c
) levels.Figure S2.
GFP expression in a 3-year-old monkey spinal cord. In situ
hybridization again reveals GFP expression in neurons (black arrows) and glia (white arrows) specifically in antisense (a
), but not sense (b
), probed spinal cord sections from an AAV9 injected animal. GFP (c
, black and white) expression was confirmed in motor neurons (d
, ChAT, black and white) by co-localization (Merged, GFP in green and ChAT in red e
). Scale bar = 100μmFigure S3.
GFP immunohistochemistry from a 3-year-old monkey. A scanned section at the level of the oculomotor nucleus (a
) from the AAV9 injected three year old monkey. There is extensive GFP expression throughout the section that is primarily glial. Interestingly, neurons of the third cranial nerve (b
) and glial within the pontine grey (c
) were highly transduced.Figure S4.
GFP immunofluorescence from AAV9-injected monkey brain. Representative brain section from the cortex of a AAV9 GFP P1 injected monkey indicates primarily glial transduction in the monkey brain following systemic injection of AAV9. Immunolabeling for GFP (a and d
) Iba-1 (b
,microglia) or GFAP (e
, astrocytes) indicates that both cell types are targeted in the brains of AAV9 injected monkeys (Merged images, c and f
respectively). Open arrows indicate GFP positive microglia while filled arrows indicate GFP positive astrocytes. Scale Bars =100μm.