Abstract 56: Shifting AAV Mediated Expression Profile to Heart by an Integrated Transcriptional Targeting Strategy
Adeno-associated viral (AAV) vectors are promising platforms for cardiac gene transport, but their broad tropism is a drawback after systematic delivery. To divert gene expression of AAV6 or 9 to heart, an integrated transcriptional targeting strategy was explored. By comparing cardiac GFP expression of scAAV6 and 9-CMV-GFP in C57/B6 or Balb/C mice, a heart-bias in C57/B6 and liver bias in Balb/C was seen. This makes the native liver biased tropism of AAV6 or 9 in Balb/C mice more suitable for the transcriptional targeting study. Luciferase (Luc) gene expression driven by cardiac specific promoters; brain natriuretic peptide (BNP), cardiac ankyrin repeat kinase (CARK), myoglobin (Myo), alpha cardiac actin (a-CARD), Troponin I (Tni), and Troponin T (Tnt), were compared to CMV promoter post retro orbital injection of 2.5x1010 viral particles of AAV6 or 9. In contrast to the previously reported heart biased signal seen in C57/B6 mice, signal was observed mainly in the liver of Balb/C mice. In vitro Luc activity, mRNA abundance and viral genomes (VG) assays were performed in liver and heart samples. Surprisingly, ∼10 folds higher Luc activity and RNA abundance in heart were observed for Tnt, Tni and a-CARD promoters than CMV promoter post AAV9 delivery, while a reduction of 100 fold was observed in liver. Copy number assay revealed six fold higher VG in liver than in heart for all promoters used including CMV. This indicates Tnt, Tni and a-CARD promoters have strong heart biased activity, without a tropism change as determined by VG copy numbers. To further develop the transcriptional targeting strategy three copies of mir122 binding site were inserted into the 3'UTR of AAV9-CMV-Luc and Balb/C mice were injected with 5x1010 viral particles. This resulted in ∼30 fold less Luc activity when compared to AAV9 without mir122 binding sites, with no change in VG copy number. In conclusion, in a mouse strain biased for liver expression after systemic delivery, transcriptional targeting to heart can be accomplished by using strong cardiac biased promoter integrated with mir122 binding sites in the 3’UTR.
- © 2012 by American Heart Association, Inc.