Abstract 2881: Cardiac Gene Transfer of shRNA Directed Against Phospholamban Effectively Knocks Down Gene Expression But Causes Myocarditis and Death in Canines
Background: Derangements in calcium cycling have been described in failing hearts of several etiologies, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in the failing heart. The goal of this study was to evaluate the safety and efficacy of using AAV-mediated cardiac gene transfer of shRNA to knock down expression of PLB.
Methods: Six dogs (5–10kg) were treated with 5x1011 gc/kg scAAV6 expressing shRNA (20-mer) directed against PLB under control of the U6 promoter. Three dogs were treated with empty AAV6 capsid as controls. Vector was delivered via a percutaneously inserted cardiac injection catheter as previously described by our group. Three of the six shRNA dogs were sacrificed to analyze PLB mRNA and protein expression between 2 and 4 weeks. The other three shRNA and three control dogs were followed with serial serum cardiac TnI and 3-D echocardiography to assess cardiac safety. ELISpot was used to screen for T cells reactive to AAV6 capsid in dogs with elevated TnI.
Results: PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold in treated dogs at 2 weeks. The remaining shRNA treated dogs experienced severe myocarditis at 4 weeks with TnI elevations 100-fold over baseline. Echo demonstrated decreased ejection fraction, and one dog transitioned into CHF and was euthanized. ELISpot failed to detect any T cells reactive to AAV6 capsid in PBMC’s, heart, or spleen. None of the control animals treated with AAV6 empty capsid have experienced myocarditis or decreased ejection fraction throughout a six month follow up.
Conclusion: AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe and sometimes fatal myocarditis. Since controls treated with AAV6 empty capsid did not experience toxicity and since no AAV6 capsid specific T cells were detected, the toxicity does not appear to have resulted from a capsid-specific T-cell response. Toxicity may result from saturation of endogenous micro RNA pathways. shRNA overexpression in the heart may not be a viable therapeutic option.