Figure 1.
Schematic of an AAV genome carrying a lacZ shRNA expressed from the murine U6 promoter, together with an RSV-alkaline phosphatase cassette used to monitor vector transduction.
No viral genes are present, and the inverted terminal repeats are the only portion derived from wildtype AAV. Single-stranded vector genomes are encapsidated following co-transfection of the vector genome plasmid and a helper plasmid into human 293D cells, and after purification are injected into the tail vein of mice.
Figure 2.
Systemic delivery of rAAV6 vectors expressing shRNAs can achieve body wide gene transfer to striated muscles.
Heat-resistant human placental alkaline phosphatase staining of heart, gastrocnemius, quadriceps and tibialis anterior muscles of a mouse 6 weeks after tail vein injection of 2×1012 vector genomes of rAAV6/lacZ-shRNA21 (left) or rAAV6/lacZ-shRNA19 (right).
Figure 3.
β-galactosidase and alkaline phosphatase expression in ROSA26 muscles.
Shown are serial cryosections prepared from quadriceps muscles of untreated mice (top) or mice sacrificed 6 weeks after injection with 2×1012 vector genomes of rAAV6/lacZ-shRNA21 (middle) or rAAV6/lacZ-shRNA19 (bottom). Untreated muscles express high levels of β-gal (left), but do not express the heat-resistant human placental alkaline phosphatase enzyme (right). Both shRNA vectors effectively knock-down β-gal expression in myofibers expressing a variety of levels of AP, used as a marker for tissue transduction. Visible in the middle panel is a β-gal expressing artery and a number of capillaries, which are not transduced by AAV6.
Figure 4.
Histochemical analysis of cryosections from heart, quadriceps or liver of ROSA26 mice.
Either untreated mice or mice injected with 2.8×1012 vector genomes (A) or 1×1012 vector genomes (B) of rAAV6/lacZ-shRNA21 were analyzed. On the left of each panel are shown hematoxylin and eosin stained sections, while on the right are shown sectioned stained for human placental alkaline phosphatase following heat-inactivation of the endogenous AP activity.
Figure 5.
Analysis of cardiac muscle following systemic rAAV6/lacZ-shRNA delivery.
A) Hematoxylin and eosin stained hearts from injection of the 21 and 19 nucleotide shRNA vectors showing a dilated cardiomyopathy that was mitigated by the shortened shRNA. Hearts were isolated 6 weeks after tail vein injection with 2×1012 vector genomes of rAAV6/lacZ-shRNA21 (left) or rAAV6/lacZ-shRNA19 (right). B) Cardiac weight in control and injected ROSA26 mice, obtained 2, 6 and 12 weeks post-injection. Note that several of the mice injected with the 21 nucleotide shRNA vector died, as indicated. All of the other mice persisted during the duration of the study.
Figure 6.
Systemic RNAi for β-gal knockdown.
Enzymatic assays for quantitation of RNAi knockdown of β-gal and to detect viral transduction (AP). A) β-gal and AP activity in ROSA26 mouse heart and quadriceps muscles following systemic injection of 1×1012 vg of AAV6 β-gal shRNA (21 nucleotide target sequence). Tail vein injection was performed into ROSA26 mice at 5 weeks of age (n = 3 per time point), and analyzed 2 and 4 weeks post injection and compared with age-matched ROSA26 (positive control) and C57BL/6 mice (negative control). Note the log scale. B) Relative β-gal activity in ROSA26 mouse gastrocnemius and quadriceps muscles following systemic injection of 2×1012 vg of AAV6 β-gal shRNA (19 nucleotide target sequence). Analysis was at 6 weeks post-injection. Statistical analyses were performed using the Student's t test or 1-way ANOVA with Dunnett's multiple comparison test.
Figure 7.
Comparison of siRNA levels produced from shRNA expression cassettes from rAAVshRNA plasmid transfected HEK293 cells.
Small Northern analysis of 25 ug of total RNA size separated on a 15% denaturing acrylamide gel identified small precursor hairpins and siRNAs using radiolabeled DNA sense oligonucleotides. The first 2 lanes contain antisense DNA sequence identical to the 21-mer expression cassette corresponding to the shRNA guide strand included for siRNA size estimation.