Systemic RNAi Delivery to the Muscles of ROSA26 Mice Reduces lacZ Expression

Abstract

RNAi has potential for therapeutically downregulating the expression of dominantly inherited genes in a variety of human genetic disorders. Here we used the ROSA26 mouse, which constitutively expresses the bacterial lacZ gene in tissues body wide, as a model to test the ability to downregulate gene expression in striated muscles. Recombinant adeno-associated viral vectors (rAAVs) were generated that express short hairpin RNAs (shRNAs) able to target the lacZ mRNA. Systemic delivery of these rAAV6 vectors led to a decrease of β-galactosidase expression of 30–50-fold in the striated muscles of ROSA26 mice. However, high doses of vectors expressing 21 nucleotide shRNA sequences were associated with significant toxicity in both liver and cardiac muscle. This toxicity was reduced in cardiac muscle using lower vector doses. Furthermore, improved knockdown in the absence of toxicity was obtained by using a shorter (19 nucleotide) shRNA guide sequence. These results support the possibility of using rAAV vectors to deliver RNAi sequences systemically to treat dominantly inherited disorders of striated muscle.

Therapeutic impact of systemic AAV-mediated RNA interference in a mouse model of myotonic dystrophy

“…S3C). To control for non-specific or vector related effects on the disease phenotype, we injected 4 × 10 12 vgs of a rAAV6 containing either the miRNA sequence for targeting bacterial lacZ mRNA (βgal) or only the hPLAP gene without an RNAi hairpin sequence (47). There was not a significant difference in the percent exclusion of exon 22 of the Atp2α1 mRNA in the HSA LR TA muscles using either control vector compared with untreated mice (Fig.…”

Endogenous MicroRNA Competition as a Mechanism of shRNA-Induced Cardiotoxicity

“…shRNA continued to accumulate in all muscle tissues over the 6-week period assessed (Figure 1B; described below), and two mice injected with the 21-nt shRNA died by 4 weeks post-injection and one by 8 weeks. 12 The 21-nt injection also led to transient toxicity in the liver, indicated by significantly increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at 2 weeks (p = 0.0201 and p = 0.0122, respectively, Welch's t test), but this toxicity was resolved by 12 weeks after the 21-nt shRNA was eliminated (Figure 1C). At 6 weeks post-injection, lacZ expression was successfully reduced in quadriceps and heart tissues of animals treated with the 19-nt and 21-nt shRNA to 5%-11% of untreated levels, whereas hPLAP levels were not significantly different across these same tissues, confirming that transduction efficiency was similar (Figure 1D).…”

“…shRNA Design and Generation shRNAs vectors were designed, generated, and injected previously, 12 following the protocol detailed in Harper and Davidson. 34 In brief, the lacZ shRNA expression cassette was generated with DNA nucleotide extension of overlapping antisense oligonucleotides, followed by ligation into a plasmid containing the mouse U6 constitutive promoter.…”

“…Vectors were designed and generated previously. 12 Vectors were produced by shRNA plasmid and pDG6 (AAV6 capsid) plasmid cotransfection of subcultured HEK293 cells, as described in Blankinship et al 13 In brief, rAAV6 shRNA vectors were harvested from cell pellets with homogenization followed by passing the homogenate through a 0.22-mm filter and further purification with HiTrap heparin column chromatography on an AKTA10 high-performance liquid chromatography (HPLC) machine (Amersham, Piscataway, NJ, USA). Vectors were titered by heating aliquots at 95 C for 10 min and subjected to agarose gel electrophoresis and Southern blotting analysis using the hPLAP SV40 polyadenylation sequence 32 P-labeled antisense DNA probe.…”

“…Vectors were injected previously. 12 Purified shRNA vectors were administered systemically to ROSA26 and HSA LR mice (Jackson Laboratories, Bar Harbor, ME, USA) via tail vein injection at 2 Â 10 12 vector genomes. HSA LR mice express a human skeletal actin gene carrying a microsatellite expansion in the 3 0 UTR as a model of myotonic dystrophy in skeletal muscle, but do not express the transgene in cardiac muscle.…”