Supplementary Material and Methods
AAV1/SERCA2a Injection into Göttingen Minipigs.
Göttingen minipigs (~ 10kg) were injected with atropine (40µg/kg) and Telazol
(tiletamine/zolazepam; 6 mg/kg). The animals were intubated and ventilated with
100% oxygen. The animals were kept anesthetized with Propofol (5-8 mg/kg/hr)
for the duration of vector application.
Immediately prior to vector administration, 2.0 ml of diluted AAV1/SERCA2a (5 x
1011 vg/ml) was brought to ambient temperature and mixed with 1.0 ml of whole
native blood from the animal being injected, resulting in a final vector
concentration of 3.3 x 1011 vg/ml. After placement of a 5Fr femoral artery sheath,
a coronary artery infusion catheter was engaged into the left main coronary
artery ostium and the placement was confirmed by coronary angiography.
AAV1/SERCA2a was infused using a programmable syringe pump. After priming
the circuit with native blood, the diluted AAV1/SERCA2a/blood mixture was
delivered at a constant rate of 0.2 ml/min over a 15 min period. The vector
injection was followed by flushing the catheter dead volume with a native blood
from a second programmable syringe pump.
Two days later the pigs were euthanized with cardioplegic solution (Plegisol,
Hospira Inc., Lake Forest IL) and heart samples were collected from 12 different
areas of the heart (anterior wall, posterior wall, septum and free wall in the basal
and middle layers of the left ventricle; anterior wall and posterior wall in the apical
layer of the left ventricle; and the free wall from the basal layer and apical layers
of the right ventricle).
qPCR for AAV1/SERCA2a Vector Genomes
A quantitative polymerase chain reaction (qPCR) Taqman assay was used to
quantify levels of AAV1/SERCA2a DNA in the tissues collected. It detects a 107
base pair sequence unique to the AAV1/SERCA2a vector sequence using
primers spanning the CMV promoter and the SERCA2a cDNA. The probe used
lies within the SERCA2a 5’ region. The primer and probe sequences are shown
below: SERCA2a Forward Primer: 5’- AAC CCT CCC ACA AGT CTA AAA TC -3’
SERCA2a Reverse Primer: 5’- CTC GGC TTT CTT CAG AGC AG -3’ SERCA2a
Probe: 5’-6FAM AGC ATC GTT CAC GCC A MGBNFQ -3’ The ABI Prism 7700
Sequence Detection System was used for DNA amplication and signal detection.
The number of copies of AAV1/SERCA2a vector DNA detected in one μg of
genomic DNA extracted from each specimen was determined by interpolation
from a standard curve generated using serial dilutions of a plasmid containing the
target sequences (pcDNA3.1_huSERCA2) as standards.
Supplementary Figure 1: FBS Inhibits Transduction by AAV Serotypes 1
and 6 but Not by Serotypes 2 and 9. Two-fold dilutions of FBS were incubated
with constant amounts of AAV serotypes 1, 2, 6 and 9 and tested for
neutralization of transduction using the in vitro assay described in Material and
Methods. Transduction is expressed as mean percent transduction of no serum
control +/- SD.
Supplementary Figure 2: Immunoglobulin Depletion of 1/32 Diluted Dog
Serum Alleviates Inhibitory Activity on AAV6 Transduction.
(a) Two-fold dilutions of pooled dog serum (from 1/64 to 1/4096) were
incubated for 30 min. with constant amounts of AAV serotypes 1, 2, 6 and 9
encoding for luciferase and then added to 293T cells. 24 hrs post-infection,
luciferase expression was measured. Transduction is expressed as percent
transduction of no serum control samples. (b) Pooled dog serum diluted 1/32 in
DMEM was depleted of immunoglobulins by incubation with protein A (two
incubations). The effect of two-fold dilutions (1/64 to 1/4096) of the pre-cleared
serum on AAV transduction was assayed as described in Supp. Fig. 2a.
Supplementary Figure 3: Even Pigs with Low Neutralizing Antibody Titers
Show Reduced Virus Uptake into Cardiomyocytes
Göttingen mini pigs (~10 kg body weight) received 10 12 vg of AAV1/SERCA2a
(AAV1 vector with the sarcoplasmatic calcium ATPase 2a (SERCA2a) cDNA
under the control of a CMV promoter). The vector was delivered via antegrade
epicardial coronary artery infusion. Neutralizing antibody titers were determined
before vector injection. The animals were sacrificed two days post-injection and
AAV1 vector genomes were quantified by qPCR. Samples from 12 different
areas of the heart were taken (anterior wall, posterior wall, septum and free wall
in the basal and middle layers of the left ventricle; anterior wall and posterior wall
in the apical layer of the left ventricle; and the free wall from the basal layer and
apical layers of the right ventricle). No vgs were detected in a non-injected
control animal. The values shown are the mean +/- SD of the vgs in each
sample.