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The downregulation of β-adrenergic receptors (β-AR) and decreased cAMP-dependent protein kinase activity in failing hearts results in decreased phosphorylation and inactivation of phosphatase-inhibitor-1 (I-1), a distal amplifier element of β-adrenergic signaling, leading to increased protein phosphatase 1 activity and dephosphorylation of key phosphoproteins, including phospholamban. Downregulated and hypophosphorylated I-1 likely contributes to β-AR desensitization; therefore its modulation is a promising approach in heart failure treatment. Aim of our study was to assess the effects of adeno-associated virus serotype 9 (AAV9) - mediated cardiac-specific expression of constitutively active inhibitor-1 (I-1c) and to investigate whether I-1c is able to attenuate the development of heart failure in mice subjected to transverse aortic constriction (TAC). 6-8 week old C57BL/6 N wild-type mice were subjected to banding of the transverse aorta (TAC). Two days later 2.8 × 1012 AAV-9 vector particles harbouring I-1c cDNA under transcriptional control of a human troponin T-promoter (AAV9/I-1c) were intravenously injected into the tail vein of these mice (n=12). AAV9 containing a Renilla luciferase reporter (AAV9/hRluc) was used as a control vector (n=12). Echocardiographic analyses were performed weekly to evaluate cardiac morphology and function. 4 weeks after TAC pressure- volume measurements were performed and animals were sacrificed for histological and molecular analyses. Both groups exhibited progressive contractile dysfunction and myocardial remodeling. Surprisingly, echocardiographic assessment and histological analyses showed significantly increased left ventricular hypertrophy in AAV9/I-1c treated mice compared to AAV9/hRluc treated controls as well as reduced contractility. Pressure-volume loops revealed significantly impaired contractility after AAV9/I-1c treatment. At the molecular level, hearts of AAV9/I-1c treated TAC mice showed a hyperphosphorylation of the SR Ca2+-ATPase inhibitor phospholamban. In contrast, expression of AAV9/I-1c in unchallenged animals resulted in selective enhancement of phospholamban phosphorylation and augmented cardiac contractility. Our data suggest that AAV9-mediated cardiac-specific overexpression of I-1c, previously associated with enhanced calcium cycling, improves cardiac contractile function in unchallenged animals but failed to protect against cardiac remodeling induced by hemodynamic stress questioning the use of I-1c as a potential strategy to treat heart failure in conditions with increased afterload.

Adeno-associated viral (AAV) vectors yield high potential for clinical gene therapy but, like for other vectors systems, they frequently do not sufficiently transduce the target tissue and their unspecific tropism prevents their application for multifocal diseases such as disseminated cancer. Targeted AAV vectors have been obtained from random AAV display peptide libraries but so far, all vector variants selected from AAV libraries upon systemic administration in vivo retained some collateral tropism, frequently the heart. Here we explored, if this impediment can be overcome by microRNA-regulated transgene cassettes as the combination of library-derived capsid targeting and micro-RNA control has not been evaluated so far. We used a tumor-targeted AAV capsid variant (ESGLSQS) selected from random AAV-display peptide libraries in vivo with remaining off-target tropism toward the heart and regulated targeted transgene expression in vivo by complementary target elements for heart-specific microRNA (miRT-1d). Although this vector still maintained its strong transduction capacity for tumor target tissue after intravenous injection, transgene expression in the heart was almost completely abrogated. This strong and completely tumor-specific transgene expression was used for therapeutic gene transfer in an aggressive multifocal, transgenic, polyoma middle T-induced, murine breast cancer model. A therapeutic suicide gene, delivered systemically by this dual-targeted AAV vector to multifocal breast cancer, significantly inhibited tumor growth after one single vector administration while avoiding side effects compared with untargeted vectors.