Augmentation of AAV-mediated cardiac gene transfer after systemic administration in adult rats

Müller, Oliver; Schinkel, Stefanie; Kleinschmidt, Jürgen A.; Katus, Hugo A.; Bekeredjian, Raffi

doi:10.1038/gt.2008.111

Cited by 58 publications

(47 citation statements)

References 47 publications

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“…In AAV9 vectors, the HMGB1 expression cassette is under the control of a cytomegalovirus (CMV)-enhanced 260-bp myosin light chain (MLC)-2v promoter, which ensures cardiomyocyte-specific expression (43,44). Here we observed myocardial inflammation and fibrosis independent of TnI immunization in both HMGB1-overexpressing wt and in RAGE-ko mice, suggesting that HMGB1 may induce cardiac inflammation by mechanisms independent of RAGE.…”

Section: Discussionmentioning

confidence: 70%

Critical role of RAGE and HMGB1 in inflammatory heart disease

Bangert

Andrassy

Müller

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

130

111

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Autoimmune response to cardiac troponin I (TnI) induces inflammation and fibrosis in the myocardium. High-mobility group box 1 (HMGB1) is a multifunctional protein that exerts proinflammatory activity by mainly binding to receptor for advanced glycation end products (RAGE). The involvement of the HMGB1-RAGE axis in the pathogenesis of inflammatory cardiomyopathy is yet not fully understood. Using the well-established model of TnI-induced experimental autoimmune myocarditis (EAM), we demonstrated that both local and systemic HMGB1 protein expression was elevated in wild-type (wt) mice after TnI immunization. Additionally, pharmacological inhibition of HMGB1 using glycyrrhizin or anti-HMGB1 antibody reduced inflammation in hearts of TnI-immunized wt mice. Furthermore, RAGE knockout (RAGE-ko) mice immunized with TnI showed no structural or physiological signs of cardiac impairment. Moreover, cardiac overexpression of HMGB1 using adeno-associated virus (AAV) vectors induced inflammation in the hearts of both wt and RAGE-ko mice. Finally, patients with myocarditis displayed increased local and systemic HMGB1 and soluble RAGE (sRAGE) expression. Together, our study highlights that HMGB1 and its main receptor, RAGE, appear to be crucial factors in the pathogenesis of TnI-induced EAM, because inhibition of HMGB1 and ablation of RAGE suppressed inflammation in the heart. Moreover, the proinflammatory effect of HMGB1 is not necessarily dependent on RAGE only. Other receptors of HMGB1 such as Toll-like receptors (TLRs) may also be involved in disease pathogenesis. These findings could be confirmed by the clinical relevance of HMGB1 and sRAGE. Therefore, blockage of one of these molecules might represent a novel therapeutic strategy in the treatment of autoimmune myocarditis and inflammatory cardiomyopathy. myocarditis | cytokines | AAV

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Section: Discussionmentioning

confidence: 70%

Critical role of RAGE and HMGB1 in inflammatory heart disease

Bangert

Andrassy

Müller

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

130

111

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“…47 AAV9 vectors were produced and purified as described previously. 19,20 Purification of virus was carried out by a filtration cascade followed by an iodixanol step gradient centrifugation. 19,20 Vector titers were determined by quantitative real-time PCR using the primers 5¢-tgc cca gta cat gac ctt atg g-3¢ and 5¢-gaa atc ccc gtg agt caa acc-3¢ and the 6-fam-agt cat cgc tat tac cat gg-MGB-labeled probe directed against a target sequence located in the CMV promoter enhancer present in all AAV vectors genomes by use of the StepOnePlus Realtime PCR System (Applied Biosystems Inc.).…”

Section: Generation Of Aav Vectorsmentioning

confidence: 99%

“…18 A heterologous promoter containing the strong cytomegalovirus (CMV) enhancer and a cardiac myosin light chain (MLC)-2v promoter allowed an efficient and predominant gene transfer into the rodent heart, but could not completely prevent a low level hepatic gene expression. 19,20 MicroRNAs (miRs) are a group of endogenous, short and non-coding RNA molecules that have a central role as key posttranscriptional regulators of gene activity including development, differentiation, apoptosis and proliferation. 21,22 The molecules are transcribed by polymerase II as long hairpin precursor transcripts.…”

Section: Introductionmentioning

confidence: 99%

microRNA122-regulated transgene expression increases specificity of cardiac gene transfer upon intravenous delivery of AAV9 vectors

et al. 2010

Self Cite

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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.

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“…Before the establishment of the animal models, some rats were injected with recombinant AAV9 at 2×10 12 viral genomes per animal via the right internal jugular vein for 1 to 2 weeks 18. Fluorescence and quantitative real‐time RT‐PCR analyses were performed to determine whether the microinjection of AAV9 resulted in increased or decreased myocardial expression levels of Kcna2 and Kcna2 AS in vivo.…”

Section: Methodsmentioning

confidence: 99%

Long Noncoding RNA Kcna2 Antisense RNA Contributes to Ventricular Arrhythmias via Silencing Kcna2 in Rats With Congestive Heart Failure

Long

Wang

Gao

et al. 2017

JAHA

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BackgroundCongestive heart failure (CHF) is a common cardiovascular disease that is often accompanied by ventricular arrhythmias. The decrease of the slow component of the delayed rectifier potassium current (IK s) in CHF leads to action potential (AP) prolongation, and the IK s is an important contributor to the development of ventricular arrhythmias. However, the molecular mechanisms underlying ventricular arrhythmias are still unknown.Methods and ResultsKcna2 and Kcna2 antisense RNA (Kcna2 AS) transcript expression was measured in rat cardiac tissues using quantitative real‐time reverse transcription–polymerase chain reaction and Western blotting. There was a 43% reduction in Kcna2 mRNA in the left ventricular myocardium of rats with CHF. Kcna2 knockdown in the heart decreased the IKs and prolonged APs in cardiomyocytes, consistent with the changes observed in heart failure. Conversely, Kcna2 overexpression in the heart significantly attenuated the CHF‐induced decreases in the IKs, AP prolongation, and ventricular arrhythmias. Kcna2 AS was upregulated ≈1.7‐fold in rats with CHF and with phenylephrine‐induced cardiomyocyte hypertrophy. Kcna2 AS inhibition increased the CHF‐induced downregulation of Kcna2. Consequently, Kcna2 AS mitigated the decrease in the IKs and the prolongation of APs in vivo and in vitro and reduced ventricular arrhythmias, as detected using electrocardiography.ConclusionsVentricular Kcna2 AS expression increases in rats with CHF and contributes to reduced IK s, prolonged APs, and the occurrence of ventricular arrhythmias by silencing Kcna2. Thus, Kcna2 AS may be a new target for the prevention and treatment of ventricular arrhythmias in patients with CHF.

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Augmentation of AAV-mediated cardiac gene transfer after systemic administration in adult rats

Cited by 58 publications

References 47 publications

Critical role of RAGE and HMGB1 in inflammatory heart disease

Critical role of RAGE and HMGB1 in inflammatory heart disease

microRNA122-regulated transgene expression increases specificity of cardiac gene transfer upon intravenous delivery of AAV9 vectors

Long Noncoding RNA Kcna2 Antisense RNA Contributes to Ventricular Arrhythmias via Silencing Kcna2 in Rats With Congestive Heart Failure

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