No abstract

Background RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. We report on targeted RNAi for the treatment of heart failure (HF), an important disorder in humans resulting from multiple etiologies. Successful treatment of HF is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban (PLB), a key regulator of cardiac Ca2+ homeostasis. Whereas gene therapy rests on recombinant protein expression as its basic principle, RNAi therapy employs regulatory RNAs to achieve its effect. Methods and Results We describe structural requirements to obtain high RNAi activity from adenoviral (AdV) and adeno-associated virus (AAV9) vectors and show that an AdV short hairpin RNA vector (AdV-shRNA) silenced PLB in cardiomyocytes (NRCMs) and improved hemodynamics in HF rats 1 month after aortic root injection. For simplified long-term therapy we developed a dimeric cardiotropic AAV vector (rAAV9-shPLB) delivering RNAi activity to the heart via intravenous injection. Cardiac PLB protein was reduced to 25% and SERCA2a suppression in the HF groups was rescued. In contrast to traditional vectors rAAV9 shows high affinity for myocardium, but low affinity for liver and other organs. rAAV9-shPLB therapy restored diastolic (LVEDP, dp/dtmin, Tau) and systolic (fractional shortening) functional parameters to normal range. The massive cardiac dilation was normalized and the cardiac hypertrophy, cardiomyocyte diameter and cardiac fibrosis significantly reduced. Importantly, there was no evidence of microRNA deregulation or hepatotoxicity during these RNAi therapies. Conclusion Our data show, for the first time, high efficacy of an RNAi therapeutic strategy in a cardiac disease.

Adeno-associated virus (AAV) vectors with capsids of AAV serotype 9 enable an efficient transduction of the heart upon intravenous injection of adult mice but also transduce the liver. The aim of this study was to improve specificity of AAV9 vector-mediated cardiac gene transfer by microRNA (miR)-dependent control of transgene expression. We constructed plasmids and AAV vectors containing target sites (TSs) of liver-specific miR122, miR192 and miR148a in the 3¢ untranslated region (3¢UTR) of a luciferase expression cassette. Luciferase expression was efficiently suppressed in liver cell lines expressing high levels of the corresponding miRs, whereas luciferase expression was unaffected in cardiac myocytes. Intravenous injections of AAV9 vectors bearing three repeats of miR122 TS in the 3¢UTR of an enhanced green fluorescent expression (EGFP) expression cassette resulted in the absence of EGFP expression in the liver of adult mice, whereas the control vectors without miR TS displayed significant hepatic EGFP expression. EGFP expression levels in the heart, however, were comparable between miR122-regulated and control vectors. The liver-specific de-targeting in vivo using miR122 was even more efficient than transcriptional targeting with a cardiac cytomegalovirus (CMV)-enhanced myosin light chain (MLC) promoter. These data indicate that miR-regulated targeting is a powerful new tool to further improve cardiospecificity of AAV9 vectors.

Low hCAR abundance may render normal human myocardium resistant to CAR-dependent viruses, whereas re-expression of hCAR, such as that observed in DCM, may be a key determinant of cardiac susceptibility to viral infections. Asymmetric expression of hCAR in the vessel wall may be an important determinant of adenovirus tropism in humans. hCAR subcellular localization in human myocardium and hCAR targeting to cell-cell contacts in cardiomyocyte cultures suggest that hCAR may play a role in cell-cell contact formation.

Infection of cattle with the bovine leukemia virus (BLV) results in a strong permanent antibody response to the BLV antigens some weeks after infection. However, cattle may carry provirus and not have detectable antibody titers. To prove the occurrence of different BLV provirus variants in German cattle and to study the influence of special BLV variants on the immunoreaction, a 444-bp fragment of the env gene of 35 naturally BLV infected animals was analyzed. Seven different groups of BLV provirus variants were found on the basis of restriction fragment length polymorphism. Three BLV provirus variant groups and five additionally sequenced BLV isolates showed a high similarity to BLV provirus isolates from other geographical areas. The variation in nucleotide sequence of the five BLV isolates compared with nine previously sequenced BLV isolates ranged up to 5. 3%. While BLV provirus variant groups A, C, D, E, F, and G were clearly related to agar-gel immunodiffusion test (AGID)- and enzyme-linked immunosorbent assay (ELISA)-positive animals, BLV provirus variant group B was solely found in permanent AGID- and ELISA-negative or in transient ELISA-positive animals. Altogether, these results indicate that special BLV provirus variants may be responsible for atypical forms of BLV infection in cattle.

Bioprinting is a novel technology that may help to overcome limitations associated with two-dimensional (2D) cell cultures and animal experiments, as it allows the production of three-dimensional (3D) tissue models composed of human cells. The present study describes the optimization of a bioink composed of alginate, gelatin and human extracellular matrix (hECM) to print human HepaRG liver cells with a pneumatic extrusion printer. The resulting tissue model was tested for its suitability for the study of transduction by an adeno-associated virus (AAV) vector and infection with human adenovirus 5 (hAdV5). We found supplementation of the basic alginate/gelatin bioink with 0.5 and 1 mg/mL hECM provides desirable properties for the printing process, the stability of the printed constructs, and the viability and metabolic functions of the printed HepaRG cells. The tissue models were efficiently transduced by AAV vectors of serotype 6, which successfully silenced an endogenous target (cyclophilin B) by means of RNA interference. Furthermore, the printed 3D model supported efficient adenoviral replication making it suitable to study virus biology and develop new antiviral compounds. We consider the approach described here paradigmatic for the development of 3D tissue models for studies including viral vectors and infectious viruses.

No abstract

Background— The coxsackievirus-adenovirus receptor (CAR) was cloned as a receptor for both viruses, but its primary biological functions and regulatory mechanisms are unknown. CAR was low in healthy adult myocardium, whereas strong CAR reexpression was observed in human dilated cardiomyopathy. The molecular mechanisms of CAR induction in cardiomyocytes are unknown. Methods and Results— We report on CAR regulation during development, CAR induction after myocardial infarction, and cell-to-cell contact–dependent CAR regulation in the rat. The high CAR expression during development in various organs decreased up to 190-fold after birth. After infarction resulting in severe cardiac dysfunction (dP/dt max , −53%; dP/dt min , −58%; left ventricular pressure, −45%), CAR was induced locally in cardiomyocytes of the infarct zone, where it was also expressed by capillary-like CD31 + structures and CD18 + interstitial cells, whereas it remained confined to subendothelial layers of arterioles and venules. In cultured cardiomyocytes, endothelin-1, cardiotrophin-1, leukemia-inhibiting factor, and cyclic stretch had no effect on CAR, whereas at high versus low cell density, CAR was suppressed up to 10-fold ( P =0.006). Conditioned media from low- or high-density cardiomyocytes or cardiofibroblasts had no effect. Conclusions— The locally confined CAR upregulation after infarction makes induction by various humoral factors unlikely, because cardiac dysfunction results in high activities of sympathetic and renin-angiotensin systems and cytokines. The cell culture experiments identify a cell-to-cell contact–dependent mechanism of CAR regulation. Further characterization of the signals linking cell-to-cell interactions to CAR gene expression may provide insight into mechanisms and functional consequences of the generalized CAR induction in dilated cardiomyopathy, and of its local induction after myocardial infarction.