Ansätze zur kausalen Gentherapie einer hereditären Kardiomyopathie im Tiermodell durch gezielten Gentransfer mit Adeno-assoziierten viralen Vektoren

Die Duchenne Muskeldystrophie ist eine ist eine X – Chromosomal rezessive Erkrankung, welche durch den Verlust des Dystrophins zu einer Skelettmuskeldystrophie mit einer Beteiligung des Herzens (Kardiomyopathie) führen kann. Da konventionelle Therapien jedoch keine Heilung ermöglichen, könnte ein Gentransfer mittels Adeno-assoziierter Viren (AAV), einen Therapieansatz darstellen. Daher wurde das Potential transkriptionell und transduktionell zielgerichteter AAV hinsichtlich der Effizienz und Spezifität bei systemischer Applikation in Mäusen untersucht. Die verwendete Kombination aus AAV9 und dem CMV verstärkten Myosin – Leichtketten Promotor (CMV-MLC1.5kb) zeigte gegenüber AAV2(R484E;R585E) und CMV-MLC1.5kb eine höhere Transduktionseffizienz im Herzen. Im anschließenden Vergleich des CMV-MLC1.5kb mit dem unspezifischen CMV – Promotor konnte eine höhere transkriptionelle Effizienz und Spezifität zu Gunsten des kardial spezifischen Promotors erzielt werden. Als Voraussetzung zur Verpackung größerer Transgene in AAV wurde ein auf 260 Basenpaare (bp) verkürzter CMV-MLC hinsichtlich der Effizienz und Spezifität einer kardialen Transkription untersucht. Da die kardiale Transkriptionseffizienz im Vergleich zum CMV-MLC1.5kb unverändert war, wurde die Kombination aus CMV-MLC0.26kb und AAV9 für einen kardialen therapeutischen Gentransferansatz gewählt. Als Tiermodell diente die Duchenne Muskeldystrophiemaus (Mdx), welche aufgrund einer X – chromosomalen Punktmutation eine Kardiomyopathie entwickelt. Aufgrund phänotypischer Unterschiede des in dieser Arbeit verwendeten Mausstammes zu dem in der Literatur beschriebenen Originalstamm, wurde eine Geno und Phänotypisierung vorgenommen, um die Vergleichbarkeit beider Stämme zu untermauern. Auch wenn genomweit nur eine 75%ige Übereinstimmung beider Stämme erreicht werden konnte, so zeigte das X Chromosom eine 100%ige Übereinstimmung mit dem Originalstamm. Auch bei weiteren Parametern, wie Histopathologie, Blutwerten und kardialen Funktionsmessungen, konnten keine gravierenden Abweichungen zum Originalstamm gezeigt werden, weshalb beide Linien als gleichwertig gelten. Daraufhin wurden die verfügbaren Mdx – Mäuse mit einer verkürzten Dystrophin cDNA (µDys), verpackt in AAV9 und unter der Kontrolle des CMV-MLC0.26kb bzw. CMV Promotors, systemisch behandelt. Im direkten Vergleich zeigte sich, dass der CMV-MLC0.26kb durch eine hohe Transduktionseffizienz des Herzens gegenüber dem CMV Promotor überlegen ist. Selbst bei Tieren, welche in hohem Alter (42 Wochen) injiziert wurden, war eine erfolgreiche Expression des µDys unter Kontrolle des CMV-MLC0.26kb Promotors zu beobachten. Um eine systemische Applikation in größeren Tieren und Menschen zu ermöglichen, wurde ein Gentransfer mit dem kardial spezifischen CMV-MLC1.5kb Promotor in Ratten getestet. Dazu wurden mit AAV6 bzw. AAV9 beladene Mikrosphären durch Applikation eines transthorakalen Ultraschalls im Herzlumen zerstört. Dies führte im Vergleich zur systemischen Applikation ohne Mikrosphären zu einer höheren Spezifität und Effektivität der kardialen Transduktion. Die Sicherheit dieser Applikationsmethode konnte durch das Ausbleiben gewebespezifischer Komplikationen (Hämorrhagien, Arrhythmien) bestätigt werden. Somit könnte die Anwendung dieser Herzmuskel spezifischen Applikation in Kombination mit transkriptionell und transduktionell an das Zielgewebe angepassten AAV neue Wege in der Therapie von angeborenen Kardiomyopathien beim Menschen eröffnen

Hemodynamic Study of a Patient-Specific Intracranial Aneurysm: Comparative Assessment of Tomographic PIV, Stereoscopic PIV, In Vivo MRI and Computational Fluid Dynamics

Introduction: Wall shear stress (WSS) is associated with the growth and rupture of an intracranial aneurysm. To reveal their underlying connections, many image-based computational fluid dynamics (CFD) studies have been conducted. However, the methodological validations using both in vivo medical imaging and in vitro optical flow measurements were rarely accompanied in such studies. Methods: In the present study, we performed a comparative assessment on the hemodynamics of a patient-specific intracranial saccular aneurysm using in vivo 4D Flow MRI, in silico CFD, in vitro stereoscopic and tomographic particle imaging velocimetry (Stereo-PIV and Tomo-PIV) techniques. PIV experiments and CFD were conducted under steady state corresponding to the peak systole of 4D Flow MRI. Results: The results showed that all modalities provided similar flow features and overall surface distribution of WSS. However, a large variation in the absolute WSS values was found. 4D Flow MRI estimated a 2- to 4-fold lower peak WSS (3.99 Pa) and a 1.6- to 2-fold lower mean WSS (0.94 Pa) than Tomo-PIV, Stereo-PIV, and CFD. Bland-Altman plots of WSS showed that the differences between PIV-/CFD-based WSS and 4D Flow MRI-based WSS increase with higher WSS magnitude. Such proportional trend was absent in the Bland-Altman comparison of velocity where the resolutions of PIV and CFD datasets were matched to 4D Flow MRI. We also found that because of superior resolution in the out-of-plane direction, WSS estimation by Tomo-PIV was higher than Stereo-PIV. Conclusions: Our results indicated that the differences in spatial resolution could be the main contributor to the discrepancies between each modality. The findings of this study suggest that with current techniques, care should be taken when using absolute WSS values to perform a quantitative risk analysis of aneurysm rupture

Clinical Pharmacokinetics and Pharmacodynamics of the Cyclin-Dependent Kinase 4 and 6 Inhibitors Palbociclib, Ribociclib, and Abemaciclib

Palbociclib, ribociclib, and abemaciclib are inhibitors of the cyclin-dependent kinases 4 and 6 approved for the treatment of locally advanced or metastatic breast cancer. In this review, we provide an overview of the available clinical pharmacokinetic and pharmacodynamic characteristics of these novel drugs, summarize the results of food–effect and drug–drug interaction studies, and highlight exposure–response and exposure–toxicity relationships. All three drugs exhibit a large inter-individual variability in exposure (coefficient of variation range 40–95% for minimum plasma concentration), are extensively metabolized by cytochrome P450 3A4, and have their brain penetration limited by efflux transporters. Abemaciclib has three active metabolites with similar potency that are clinically relevant (i.e., M2, M20, M18), whereas the metabolites of palbociclib and ribociclib are not of clinical significance. Pharmacokinetic exposure increases in a dose-proportional manner for palbociclib, whereas exposure increases under- and over-proportionally with an increasing dose for abemaciclib and ribociclib, respectively. High exposure is associated with an increased risk of neutropenia, and for ribociclib also to corrected QT prolongation. For abemaciclib, a clear exposure–efficacy relationship has been described, while for palbociclib and ribociclib exposure–response analyses remain inconclusive. Future studies are needed to address exposure–efficacy relationships to further improve dosing

AAV6.βARKct cardiac gene therapy ameliorates cardiac function and normalizes the catecholaminergic axis in a clinically relevant large animal heart failure model

AIMS: G protein-coupled receptor kinase 2 (GRK2), which is markedly upregulated in failing human myocardium, has been implicated as a contributing factor or consequence of heart failure (HF). Importantly, cardiac-specific GRK2 knockout mice have recently proved the pathological nature of GRK2 in HF. Targeted inhibition of GRK2 is possible using a peptide inhibitor known as the βARKct, which has rescued several disparate small animal HF models. This study was designed to evaluate long-term βARKct expression in a clinically relevant large animal HF model, using stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). METHODS AND RESULTS: A porcine model of HF subsequent to left ventricular (LV) myocardial infarction (MI) was used to study the effects of retrograde injection into the anterior interventricular vein of either AAV6.βARKct or AAV6.luciferase as a control 2 weeks after MI. Echocardiography and LV hemodynamics were performed before and 6 weeks after gene transfer. Robust and long-term βARKct expression was found after AAV6-mediated delivery, leading to significant amelioration of LV haemodynamics and contractile function in HF pigs compared with AAV6.luciferase-treated control animals that showed a continued decline in cardiac function. Interestingly, the neurohormonal axis was virtually normalized in AVV6.βARKct-treated HF animals, represented by reductions in plasma norepinephrine levels, whereas AAV6.luciferase-treated pigs showed further increases in plasma catecholamine levels. As a result, LV remodelling and foetal gene expression was reversed by AVV6.βARKct gene therapy. CONCLUSION: These data—showing sustained amelioration of cardiac function in a post-MI pig HF model—demonstrate the therapeutic potential of βARKct gene therapy for HF

Cardiac AAV9-S100A1 gene therapy rescues post-ischemic heart failure in a preclinical large animal model

As a prerequisite to clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated viral (AAV) S100A1 gene therapy in a preclinical, large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and various animal models, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium Ca(2+) handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon-occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered through retrograde coronary venous delivery, AAV9-S100A1 to the left ventricular non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed this phenotype by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca(2+) cycling, sarcoplasmic reticulum calcium handling and energy homeostasis. Transgene expression was restricted to cardiac tissue and extra-cardiac organ function was uncompromised indicating a favorable safety profile. This translational study shows the pre-clinical feasibility, long-term therapeutic effectiveness and a favorable safety profile of cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our study presents a strong rational for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure