SUPPLEMENTARY MATERIAL
Supplementary
Figure
1.
PBGD
expression
in
the
liver
of
the
M#06
immunosuppressed macaque and the M#04 control non-immunosuppressed macaque 14
weeks after HDA-hPBGD injection. (A) Representative microphotographs of formalinfixed and paraffin-embedded liver sections showing hepatocytes over-expressing
hPBGD of the indicated animals upon immunostaining with an anti-PBGD polyclonal
antibody (H-300 Santacruz, Heidelberg, Germany). B) Micrographs of the
corresponding liver sections stained with H&E showing normal histology. C)
Immunoblot assay showing PBGD over-expression in both injected and non-injected
liver lobes in the liver of the immunosuppressed M#06 non-human primate that was not
observed in the samples from the non-immunosuppressed M#04 animal. HDA, helperdependent adenovirus; PBGD, porphobilinogen deaminase; GAPDH, glyceraldehyde-3phosphate dehydrogenase; IS, immunosuppression protocol.
Supplementary Figure 2. Follow-up of transgene expression in non-directly
injected liver regions after the first administration of HDA-hPBGD vector. Followup of PBGD activity (A) and HDA vector DNA quantification (B) in serial liver
biopsies of the non-injected liver lobe taken at the indicated time points. The normal
range of PBGD activity (defined as mean ± 2 x standard deviations) was estimated in
liver samples taken at necropsy in 8 non-injected age-matched NHP. The proviral DNA
content was measured by real time QPCR in these liver biopsies. The amount of human
PBGD transcript was expressed according to the formula 2Ct(GAPDH)-Ct(gene), where Ct is
1
the cycle at which the fluorescence rises appreciably above background fluorescence.
Background threshold value was estimated in liver samples from 8 non-injected NHP.
Samples were run in duplicate or triplicate. Closed symbols represent NHP receiving
the IS regime and open symbol animals without immunosuppression. An unsupervised
clustering analysis (as described in Materials and Methods) identified two clusters: one
cluster group including samples of NHP with 3-month IS (M#05 and M#06) and the
other groups (control without IS and NHP#03 under 1-month IS). HDA, helperdependent adenovirus; PBGD, porphobilinogen deaminase. IS, immunosuppression
protocol.
Supplementary Figure 3. Sequential follow-up of vector DNA detection from serum
samples of the injected non-human primates and a naïve control animal by quantitative
PCR. Data are showed as mean ± standard deviation. Statistical analysis was performed
using the non-parametric Kruskal-Wallis test with Dunn's Multiple Comparison test as
post hoc analysis (ns, non-significant; * p<0.05; **, p<0.01 vs non-injected non-human
primates group at the indicated time points). Upper background limit was defined as
mean ± 2 x standard deviations in three unrelated naïve non-injected macaques. HDA,
helper-dependent adenovirus; hPBGD, human porphobilinogen deaminase; IS,
immunosuppression protocol.
Supplementary Figure 4. Animal well-being. Follow-up of body weight in the six
non-human primates used in the study. No external signs of disease or discomfort were
observed in any of the animals; all had normal appetite and gained weight as expected
2
for gender and age. The black arrows point to the date of HDA-hPBGD injection. The
increase shows weight gain during the period of time elapsed between the two HDAhPBGD administrations. The shaded areas represent immunosuppression treatment
periods. Closed symbols represent NHP receiving the immunosuppression protocol and
open
symbols
animals
without
immunosuppression.
HDA,
helper-dependent
adenovirus; hPBGD, human porphobilinogen deaminase. IS, immunosuppression
protocol.
Supplementary Figure 5. Absence of in vitro mitogenic responses of peripheral
blood T cells to human PBGD protein. Mitogenic responses were measured by 3HThymidine incorporation in each peripheral blood T cell sample drawn at the indicated
time-points in response to the addition of recombinant human PBGD protein or a mix of
the phorbol ester 12-myristate- 13-acetate (0.01 µg/ml) and Ionomycin (1 µg/ml) as a
positive control. The dotted line indicates the stimulation detection threshold. PBGD,
porphobilinogen
deaminase;
PMA/Iono,
phorbol
12-myristate
13-acetate
and
ionomycin; PBMC, peripheral blood mononuclear cells.
Supplementary Figure 6. Absence of transgene expression in the non-directly
injected liver region after re-administration of HDA-hPBGD vector. While upon
first exposure HDA-hPBGD vector recirculated to infect non-injected liver tissue (Fig.
2) these data are complementary to those in Fig. 4 and indicate that transduction of the
non-injected
liver
region
failed
to
occur
upon
re-administration
despite
immunosuppression. (A) PBGD enzymatic activity in homogenates from biopsies (open
3
bars) and at sacrifice (closed bars) in three control NHP (M#01-M#03) and three
animals receiving immunosuppression upon secondary exposure to the vector (M#04M#06). Importantly, these biopsies were taken from liver lobes unrelated to the
ultrasound-guided percutaneous
intrahepatic injections
in
the second vector
administration. (B) DNA vector detection in homogenates from the same samples as in
A. The results obtained at sacrifice (11 days after re-administration) are statistically
compared to biopsy material obtained before HDA re-administration. The nonparametric Mann-Whitney U-test was used for comparison of two groups (two-tailed pvalues). PBGD, porphobilinogen deaminase; HDA, helper-dependent adenovirus; IS,
immunosuppression protocol; ns, non-significant.
4