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1.
Gene Ther ; 27(10-11): 516-524, 2019 11.
Article in English | MEDLINE | ID: mdl-32322014

ABSTRACT

Mutations in the human desmin gene cause autosomal-dominant and recessive cardiomyopathies and myopathies with marked phenotypic variability. Here, we investigated the effects of adeno-associated virus (AAV)-mediated cardiac wild-type desmin expression in homozygous desmin knockout (DKO) and homozygous R349P desmin knockin (DKI) mice. These mice serve as disease models for two subforms of autosomal-recessive desminopathies, the former for the one with a complete lack of desmin protein and the latter for the one with solely mutant desmin protein expression in conjunction with protein aggregation pathology in striated muscle. Two-month-old mice were injected with either a single dose of 5 × 1012 AAV9-hTNT2-mDes (AAV-Des) vector genomes or NaCl as control. One week after injection, mice were subjected to a forced swimming exercise protocol for 4 weeks. Cardiac function was monitored over a period of 15 month after injection and before the mice were sacrificed for biochemical and morphological analysis. AAV-mediated cardiac expression of wild-type desmin in both the homozygous DKO and DKI backgrounds reached levels seen in wild-type mice. Notably, AAV-Des treated DKO mice showed a regular subcellular distribution of desmin as well as a normalization of functional and morphological cardiac parameters. Treated DKI mice, however, showed an aberrant subcellular localization of desmin, unchanged functional cardiac parameters, and a trend toward an increased cardiac fibrosis. In conclusion, the effect of a high-dose AAV9-based desmin gene therapy is highly beneficial for the heart in DKO animals, but not in DKI mice.


Subject(s)
Cardiomyopathies , Dependovirus , Animals , Cardiomyopathies/genetics , Cardiomyopathies/therapy , Dependovirus/genetics , Desmin/genetics , Disease Models, Animal , Genetic Therapy , Humans , Mice
2.
Gene Ther ; 25(1): 13-19, 2018 01.
Article in English | MEDLINE | ID: mdl-29350681

ABSTRACT

The downregulation of ß-adrenergic receptors (ß-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 ß-adrenergic signaling, leading to increased protein phosphatase 1 activity and dephosphorylation of key phosphoproteins, including phospholamban. Downregulated and hypophosphorylated I-1 likely contributes to ß-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.


Subject(s)
Dependovirus , Genetic Therapy/methods , Heart Failure/therapy , Intracellular Signaling Peptides and Proteins/genetics , Myocardial Contraction/genetics , Animals , Calcium-Binding Proteins/metabolism , Disease Models, Animal , Echocardiography , Gene Expression , Genetic Vectors , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Phosphorylation , Promoter Regions, Genetic , Troponin T/genetics
3.
Gene Ther ; 23(8-9): 673-9, 2016 08.
Article in English | MEDLINE | ID: mdl-27101257

ABSTRACT

Mutations of the human desmin (DES) gene cause autosomal dominant and recessive myopathies affecting skeletal and cardiac muscle tissue. Desmin knockout mice (DES-KO), which develop progressive myopathy and cardiomyopathy, mirror rare human recessive desminopathies in which mutations on both DES alleles lead to a complete ablation of desmin protein expression. Here, we investigated whether an adeno-associated virus-mediated gene transfer of wild-type desmin cDNA (AAV-DES) attenuates cardiomyopathy in these mice. Our approach leads to a partial reconstitution of desmin protein expression and the de novo formation of the extrasarcomeric desmin-syncoilin network in cardiomyocytes of treated animals. This finding was accompanied by reduced fibrosis and heart weights and improved systolic left-ventricular function when compared with control vector-treated DES-KO mice. Since the re-expression of desmin protein in cardiomyocytes of DES-KO mice restores the extrasarcomeric desmin-syncoilin cytoskeleton, attenuates the degree of cardiac hypertrophy and fibrosis, and improves contractile function, AAV-mediated desmin gene transfer may be a novel and promising therapeutic approach for patients with cardiomyopathy due to the complete lack of desmin protein expression.


Subject(s)
Cardiomyopathies/therapy , Dependovirus/genetics , Desmin/genetics , Genetic Therapy , Actin Cytoskeleton/metabolism , Animals , Cardiomyopathies/genetics , Desmin/metabolism , Genetic Vectors/genetics , Intermediate Filament Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Muscle Proteins/metabolism , Myocardial Contraction , Myocytes, Cardiac/metabolism , Ventricular Function, Left
4.
Gene Ther ; 21(2): 131-8, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24305416

ABSTRACT

Low levels of the molecular inotrope S100A1 are sufficient to rescue post-ischemic heart failure (HF). As a prerequisite to clinical application and to determine the safety of myocardial S100A1 DNA-based therapy, we investigated the effects of high myocardial S100A1 expression levels on the cardiac contractile function and occurrence of arrhythmia in a preclinical large animal HF model. At 2 weeks after myocardial infarction domestic pigs presented significant left ventricular (LV) contractile dysfunction. Retrograde application of AAV6-S100A1 (1.5 × 10(13) tvp) via the anterior cardiac vein (ACV) resulted in high-level myocardial S100A1 protein peak expression of up to 95-fold above control. At 14 weeks, pigs with high-level myocardial S100A1 protein overexpression did not show abnormalities in the electrocardiogram. Electrophysiological right ventricular stimulation ruled out an increased susceptibility to monomorphic ventricular arrhythmia. High-level S100A1 protein overexpression in the LV myocardium resulted in a significant increase in LV ejection fraction (LVEF), albeit to a lesser extent than previously reported with low S100A1 protein overexpression. Cardiac remodeling was, however, equally reversed. High myocardial S100A1 protein overexpression neither increases the occurrence of cardiac arrhythmia nor causes detrimental effects on myocardial contractile function in vivo. In contrast, this study demonstrates a broad therapeutic range of S100A1 gene therapy in post-ischemic HF using a preclinical large animal model.


Subject(s)
Arrhythmias, Cardiac/therapy , Genetic Therapy/adverse effects , Genetic Vectors/administration & dosage , Genetic Vectors/adverse effects , Heart Failure/metabolism , Heart Failure/therapy , Myocardial Infarction/complications , Myocardial Ischemia/complications , Myocardium/metabolism , S100 Proteins/therapeutic use , Animals , Dependovirus/genetics , Disease Models, Animal , Heart Failure/physiopathology , Humans , Myocardial Infarction/physiopathology , Myocardial Infarction/therapy , Myocardial Ischemia/physiopathology , Myocardial Ischemia/therapy , Myocardium/pathology , S100 Proteins/genetics , S100 Proteins/metabolism , Stroke Volume/physiology , Swine
5.
Gene Ther ; 18(2): 199-209, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21048795

ABSTRACT

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.


Subject(s)
Dependovirus/genetics , Gene Expression Regulation , Gene Transfer Techniques , Genetic Vectors , Heart , MicroRNAs/pharmacology , Animals , Injections, Intravenous , Liver , Mice , Organ Specificity , Transgenes , Untranslated Regions
6.
Transplant Proc ; 40(2): 596-7, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18374139

ABSTRACT

To overcome the shortage of human organs for transplantation, pigs are considered as xenogeneic donors. However, primarily immunological and virological barriers exist. One of the main virological obstacles, represented by the presence of functional and infectious porcine endogenous retroviruses (PERV) in the genome of the pigs, may be excluded by conventional breeding. In contrast, there are truncated proviral sequences that have the capacity to retrotranspose, causing insertional mutagenesis in the xenograft and in infected human cells. To estimate this risk we have investigated the potential of PERV to retrotranspose. Moloney Murine Leukemia Virus (MoMLV), a gamma type retrovirus and close relative to PERV, which has been described as able to retrotranspose, was implemented as a control. First results based on a neomycin indicator monitoring system indicate that PERV is able to retrotranspose at higher frequencies compared with MoMLV.


Subject(s)
Endogenous Retroviruses/pathogenicity , Retroviridae Infections/transmission , Retroviridae/genetics , Transplantation, Heterologous/adverse effects , Alternative Splicing , Animals , Endogenous Retroviruses/genetics , Humans , Introns , Retroviridae/pathogenicity , Retroviridae Infections/prevention & control , Swine , Transplantation, Heterologous/standards
7.
J Gen Virol ; 81(Pt 5): 1183-90, 2000 May.
Article in English | MEDLINE | ID: mdl-10769059

ABSTRACT

Avian polyomavirus (APV) causes an acute fatal disease in a variety of avian species. DNA laddering indicating apoptosis was demonstrated in APV-infected chicken embryo (CE) cells. DNA laddering, however, was not observed in Vero cells infected with mammalian polyomavirus simian virus 40. Expression of APV agnoprotein 1a and agnoprotein 1b induced apoptosis in insect cells and CE cells. An APV full-length plasmid transfected in CE cells induced apoptosis, and infectious virus was produced. After transfection of CE cells with a plasmid containing a mutated initiation codon for agnoprotein 1a and agnoprotein 1b, however, a considerably lower number of apoptotic cells was observed, and no infectious progeny was produced.


Subject(s)
Apoptosis , Polyomavirus/physiology , Viral Proteins/physiology , Animals , Baculoviridae/genetics , Cell Line , Cells, Cultured , Chick Embryo , Chlorocebus aethiops , DNA Fragmentation , Immunoblotting , In Situ Nick-End Labeling , Plasmids/genetics , Polyomavirus/genetics , Simian virus 40/physiology , Spodoptera , Transfection , Vero Cells , Viral Proteins/genetics , Viral Regulatory and Accessory Proteins
8.
Avian Pathol ; 28(3): 279-85, 1999 Jun.
Article in English | MEDLINE | ID: mdl-26915384

ABSTRACT

Infections of young chickens with infectious bursal disease virus (IBDV) result in depletion of lymphoid cells of the bursa of Fabricius (BF) due to necrosis and apoptotic processes. Interactions between IBDV and lymphoid cells were investigated by labelling paraffin-embedded tissue sections of infected BF with combinations of either immunohistochemistry (IHC), in situ hybridization (ISH) or in situ TUNEL reaction (IST). With regard to specificity and sensitivity, results of ISH were comparable to those of IHC. By double-labelling it was shown, for the first time, that viral antigen was present in most of the apoptotic cells. This suggests that IBDV may be directly involved in the induction of the apoptotic process. However, some cells also showed either viral antigen or DNA fragmentation, especially at the early stages of infection. It should be taken into account, therefore, that the apoptotic processes might also be induced by IBDV through indirect interaction between cells. Remarkably, in some of the infected lymphoid cells ISH signals were observed in the nucleolus.

9.
Radiologe ; 15(1): 19-20, 1975 Jan.
Article in English | MEDLINE | ID: mdl-1124285

ABSTRACT

During hystero-salpingography a sharply outlined collection of contrast medium was observed near the cervical canal of the uterus and interpreted as filling of a Gartner's duct cyst, an observation that- to the best of our knowledge-has not yet been reported as a radiological finding.


Subject(s)
Cysts/diagnostic imaging , Hysterosalpingography , Uterine Diseases/diagnostic imaging , Adult , Female , Humans , Infertility, Female/etiology
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