In vivo validation of HS-CRMs. (a) Semi-high
throughput HS-CRM screening in vivo after intravenous
hydrodynamic liver-directed injection with pAAV-HS-CRM-TTR-FIX and
pAAV-TTR-FIX plasmids at a dose of 2 µg DNA.
Significant differences compared to the control without HS-CRM were
indicated (t-test, *P ≤ 0.05, mean ± SD).
(b) FIX expression levels after intravenous administration of
AAV9-HS-CRM8-Palm-FIX and AAV9-Palm-FIX
(1 × 1011 vg/mouse) (n = 5 per
cohort, C57Bl/6). The difference in FIX expression levels was significant
(t-test, *P ≤ 0.0001). (c) FIX expression
levels after intravenous administration of AAV9-HS-CRM8-TTR-FIX and
AAV9-TTR-FIX control vectors (5 × 109
vg/mouse) (n = 5 per cohort, C57Bl/6). The difference in FIX
expression levels was significant (t-test, *P ≤
0.00005). FIX levels were determined using a hFIX-specific ELISA. (d)
Hepatocyte-specificity of AAV9 containing HS-CRM8. RT-PCR analysis
on 20 ng total RNA from different organs of C57/Bl6 mice (n
= 3) injected intravenously with AAV9-HS-CRM8-TTR-FIX vectors
(3 × 1012 vg/mouse); RNA liver
samples were amplified with and without RT to exclude genomic DNA
amplification. Amplification of hFIX mRNA was not detectable in
these control samples without RT (data not shown). (e) Corresponding
qRT-PCR analysis of hFIX mRNA levels in the different organs
expressed relative to hFIX mRNA levels in the liver. Expression
levels (mean ± SD) relative to liver are shown. H2O, water
control; MW, molecular weight marker; PBS liver, liver sample of
PBS-injected control mice; RT, reverse transcription.