709. Adeno-Associated Virus Delivery of siRNAs Leads to a Reduction in Phospholamban Levels

Abstract

Regulation of Ca2+ flux in the heart via the blockade of the |[beta]|-adrenergic receptor affects the phosphorylation state of phospholamban (PLN), a regulator of the sarcoplasmic reticulum calcium ATPase (SERCA2a). Reducing the activity of PLN enhances cardiac function in some animal models of congenital and ischemic cardiac disease. Objective: As an alternative to pharmacological regulation of PLN, the aim of this study is to use viral vectors to deliver short hairpin RNAs (shRNAs) targeting the PLN mRNA through the RNA interference pathway. We employed Adeno-associated virus serotype 1 (AAV1) and also a self-complementary version of AAV1 (scAAV1). ScAAV allows a more rapid expression of the gene of interest. The regulated expression of these PLN small interfering RNAs (siRNAs) could lead to a gene therapy for myocardial dysfunction. Methods: Two PLN-specific siRNAs, si248 and si750, and a control siRNA were designed. Co-transfection of the siRNAs in combination with a plasmid expressing PLN was performed in HEK 293 cells. Subsequently, DNA versions of small hairpins containing the three siRNAs were each cloned into AAV vectors. All vectors contained GFP in addition to one of the three siRNAs driven by the H1 promoter. Co-transfection experiments were performed using a 1:4 molar ratio of PLN:siRNA plasmids. RT-PCR was utilized for quantitation of PLN mRNA levels. Immunoblots were used to assay PLN protein levels. The siRNA plasmids were then packaged into AAV-1 capsids. These viruses were used for infection of primary ventricular neonatal cardiomyocytes. Infected myocytes were GFP positive and assessed for PLN mRNA and protein reduction. Additionally, temporal vein injections were performed into neonatal mice. Animals were assessed 4 weeks post-injection. Results: In co-transfected HEK 293 cells, si248 and si750 yielded a 55% and 33% reduction in PLN mRNA levels respectively, compared to the control siRNA. Protein levels were reduced by much higher levels (>85% for both). Similarly, a |[sim]|40|[ndash]|47% reduction was seen in PLN mRNA levels 48 hours post-transfection using the PLN-targeting shRNAs expressed from the scAAV plasmids. Again, the reduction in protein levels was much higher, >80% for both. In the primary cells, levels of RNA and protein were reduced by 46% and 51% respectively after infection with scAAV virus. In addition, indirect immunofluorescence staining showed a marked reduction in PLN protein levels in cultured myocytes infected with the AAV-shRNA targeting PLN and not in cells infected with the control expressing shRNA virus. Furthermore, in vivo mRNA reduction of PLN was observed to be proportional to transduction levels in the mouse myocardium. Conclusions: AAV mediated delivery of shRNAs is a powerful tool for disrupting PLN expression. RNA interference mediated by both AAV and scAAV proved to be successful in producing targeted reduction of PLN mRNA and protein. Additional experiments are currently underway to assess the effects of in vivo RNA interference on PLN following cardiac transduction of neonatal mice.