Gene Therapy (2014) 21, 131–138 & 2014 Macmillan Publishers Limited All rights reserved 0969-7128/14 www.nature.com/gt

ORIGINAL ARTICLE Therapeutic safety of high myocardial expression levels of the molecular inotrope S100A1 in a preclinical heart failure model

C Weber1,2, I Neacsu1,2, B Krautz1,2, P Schlegel2, S Sauer3, P Raake2, J Ritterhoff1,2, A Jungmann2, AB Remppis2, M Stangassinger4, WJ Koch5, HA Katus2,6,OJMu¨ ller2, P Most1,2,6,7 and ST Pleger1,2

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 Â 1013 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.

Gene Therapy (2014) 21, 131–138; doi:10.1038/gt.2013.63; published online 5 December 2013 Keywords: heart failure; DNA-based therapy; S100A1; AAV; therapeutic window; translational science

INTRODUCTION SERCA2a in HF patients was recently initiated, whereas a second Despite advances in clinical therapy, heart failure (HF) is not phase I/II HF gene therapy trial using adenylyl cyclase VI currently curable as available treatments only alleviate symptoms and seeks the Food and Drug Administration investigational drug 22,23 cannot reverse underlying causes of the disease.1–4 Molecular status. However, there is still legitimate concern about abnormalities in cardiomyocyte calcium (Ca2 þ ) handling and potential cardiac adverse effects of myocardial gene therapy. beta-adrenergic receptor signal transduction have been identified First, adverse cardiac effects due to modulation of intracellular 2 as the main key factors of HF pathogenesis and transition to Ca þ cycling and beta-adrenergic signal transduction have been failure and death.5–7 Cardiomyocyte-targeted correction of distinct already demonstrated in transgene animal models in terms of intracellular molecular defects by viral-based therapeutic DNA ventricular arrhythmia, deterioration of cardiac function and delivery offers the opportunity to address these pathways in the increased mortality.24–28 Second, myocardial gene delivery diseased myocardium and reverse the HF phenotype in clinically appeared largely inhomogenous in various rodent and relevant large animal models.8–11 preclinical animal models, potentially increasing susceptibility to The Ca2 þ sensor protein S100A1 has emerged as an attractive malignant ventricular arrhythmia and limiting therapeutic target for genetically targeted HF therapy in various in vivo HF effects.6,29,30 Third, high-level overexpression of a gene product models because of its molecular profile.12 The S100A1 protein might even impair cardiac contractile function as target protein regulates a network in cardiomyocytes that controls sarcoplasmic expression levels might be beyond the therapeutic window and reticulum Ca2 þ cycling and mitochondrial function through beneficial effects might be dose dependent. As therapeutic effects interaction with the ryanodine receptor, sarcoplasmic reticulum of S100A1 in HF are partly mediated by increased SERCA2a 2 þ Ca -ATPase (SERCA2) and mitochondrial F1-ATPase activity, activity, it is important to mention that Mercadier’s group showed causing antihypertrophic, positive inotrope and antiarrhythmic that SERCA2a-mediated delay of HF after myocardial infarction is effects and reducing energy depletion in HF.13–19 Importantly, the at a cost of increased acute arrhythmia.31 Thus, as a prerequisite to S100A1 protein is significantly downregulated in human end- clinical application, a careful analysis of cardiac adverse effects stage HF, rendering S100A1 an appropriate target for cardiac gene especially at high vector doses is necessary prior to clinical therapy.20,21 translation of myocardial S100A1 gene therapy trials. In the The first-ever clinical HF gene therapy phase I/II (CUPID) trial present study the AAV6-S100A1 construct was used to achieve addressing abnormal intracellular Ca2 þ handling by overexpressing high-level myocardial S100A1 protein overexpression in order to

1Center for Molecular and Translational Cardiology, Heidelberg University Hospital, Heidelberg, Germany; 2Department of Internal Medicine III, Division of Cardiology, University of Heidelberg, Heidelberg, Germany; 3Department of Pediatrics, University of Heidelberg, Heidelberg, Germany; 4Institute for Animal Physiology, Ludwig-Maximilians-University Munich, Munich, Germany; 5Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA, USA; 6Deutsches Zentrum fu¨r Herz-/Kreislaufforschung, University Hospital Heidelberg, Heidelberg, Germany and 7Laboratory for Cardiac Stem Cell and Gene Therapy, Temple University School of Medicine, Philadelphia, PA, USA. Correspondence: Dr ST Pleger, Center for Molecular and Translational Cardiology, Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany. E-mail: [email protected] Received 7 May 2013; revised 29 August 2013; accepted 30 September 2013; published online 5 December 2013 S100A1 gene therapy biosafety study C Weber et al 132 investigate the therapeutic window and safety profile of cardiac coronary artery has been demonstrated by myocardial perfusion S100A1 gene therapy in HF. echocardiography.10 At 2 weeks after myocardial infarction, post- Investigation of adverse cardiac effects as well as the myocardial infarction (MI) pigs (n ¼ 30) demonstrated systolic LV therapeutic window of myocardial S100A1 gene therapy needs dysfunction with significantly reduced LV ejection fraction (LVEF) to be accomplished in a preclinical large animal model closely and enlargement of the LV (Figures 1c–e) as compared with sham approximating human physiology, function and anatomy.10,32 pigs (n ¼ 11). Post-MI pigs (n ¼ 30) were randomized to saline As HF is mainly caused by ischemic cardiomyopathy, we used a (n ¼ 7), control virus (AAV6-luc, n ¼ 11) and AAV6-S100A1 treat- preclinical post-myocardial infarction pig model enabling ment (n ¼ 12) 2 weeks post MI. A time course of the study protocol investigation of cardiac arrhythmia and contractile function, as is given in Figure 1f. sarcomeric proteins, heart rate and, most importantly, ratio of SERCA2a/NCX activity are closer to humans as compared with rodent models.33,34 Overall, this study provides a profound and High-level myocardial S100A1 overexpression after AAV6-S100A1 gene transfer essential preclinical safety analysis of high-level myocardial High-level S100A1 myocardial overexpression after AAV6-S100A1 S100A1 protein overexpression on cardiac contractile function 13 and susceptibility to malignant arrhythmia using a preclinical gene delivery (1.5 Â 10 tvp) was compared with low-level model of post-ischemic HF. S100A1 control samples obtained from a previous study using AAV9-S100A1 (1.5 Â 1013 tvp)-treated pigs and the same pig in the post-ischemic HF model.10 Because of retrograde AAV-mediated S100A1 gene delivery via the anterior cardiac vein (ACV) into RESULTS failing pig hearts, myocardial S100A1 overexpression was localized Model of porcine post-ischemic HF in the anterior and septal remote myocardium (Figures 2a–d). By taking advantage of a model of percutaneous catheter-based S100A1 overexpression was pronounced close to the ACV running intermittent balloon occlusion of the proximal left circumflex in parallel to the LAD irrespective of whether AAV6 or AAV9 was coronary artery, lateral left ventricular (LV) transmural myocardial used as the vector (Figures 2c and d). No significant S100A1 infarction was achieved, as shown by triphenyltetrazolium overexpression occurred in the posterior segments of the heart chloride staining (Figures 1a and b). Reproducibility of the (Figures 2c and d). Distinct differences were observed in the myocardial area at risk during occlusion of the left circumflex magnitude of S100A1 expression between AAV6-S100A1- and

Figure 1. Preclinical model of pig post-ischemic heart failure. (a) Radioscopic image showing contrast dye in the left anterior descending (LAD) coronary artery as well as (arrow) catheter-based left circumflex coronary artery occlusion. (b) Representative triphenyltetrazolium chloride staining of a mid-ventricular section and a section below demonstrating scar formation 14 weeks after MI. (c–e) Echocardiography revealed significantly decreased left LVEF as well as significant LV dilation (EDD, end-diastolic diameter) 2 weeks after MI (n ¼ 30) as compared with sham-operated pigs (n ¼ 11), whereas heart rate (HR) remained unaltered. (f) Study protocol and time flow. RVS; right ventricular stimulation. w ECG; electrocardiogram. Po0.05 vs post-MI. Data are presented as mean±s.e.m.

Gene Therapy (2014) 131 – 138 & 2014 Macmillan Publishers Limited S100A1 gene therapy biosafety study C Weber et al 133

Figure 2. Cardiac AAV/S100A1 gene therapy. (a) Radioscopic image showing the retroperfusion catheter (lower arrow) inserted into the coronary sinus as well as contrast dye in the ACV (upper arrow) for retrograde delivery of the AAV vectors. (b) Retroperfusion catheter inserted into the ACV (lower arrow) and contrast dye within the LAD (upper arrow), which runs in parallel to the ACV. (c and d) Myocardial S100A1 protein expression pattern of three representative pigs 12 weeks after retrograde delivery of (c) 1.5 Â 1013 tvp of AAV9-S100A1 and (d) 1.5 Â 1013 tvp of AAV6-S100A1 via the ACV using western blot analysis in 36 segments of the heart. S100A1 overexpression was high and locally excessive in AAV6-S100A1-treated myocardium as compared with AAV9-S100A1. (e) Representative western blot analysis showing S100A1 protein expression in the myocardium 12 weeks after AAV6-S100A1 gene delivery. S100A1 protein expression is significantly increased in anterior (high-level; 48±7.1-fold) and anteroseptal (medium level; 12±2.4-fold) myocardium as compared with AAV6-luciferase (average S100A1 overexpression: 22.9±3.2-fold; Po0.001; n ¼ 36 regions of 12 animals each), whereas S100A1 protein overexpression could not be observed in the posterior (none) myocardium. Low-level S100A1 overexpression was found in AAV9-S100A1-treated anterior myocardium (3.2±0.3-fold vs AAV9-luciferase; n ¼ 36 regions of 6 animals each). Myocardial S100A1 overexpression does not alter the expression and phosporylation state of representative proteins involved in intracellular Ca2 þ cycling (n ¼ 5).

AAV9-S100A1-treated failing pig hearts. Although the same as Ser16-PLN, Thr17-PLN and Ser2808-RyR phosporylation sites dosage of AAV-S100A1 vectors was applied (1.5 Â 1013 tvp per (Figure 2e). animal; B5 Â 1011 tvp per kg) and the same cardiomyocyte- specific promoter (CMV-MLC0.26) was used to express the human High-level myocardial S100A1 overexpression does not abet the S100A1 cDNA, average LV S100A1 overexpression was signifi- occurrence of monomorphic ventricular tachyarrhythmia cantly increased following AAV6-S100A1 gene delivery as Using a clinically relevant protocol of right ventricular stimulation compared with AAV9-S100A1 (22.9±3.2-fold vs 3.2±0.3-fold of in pigs 14 weeks after myocardial infaction (12 weeks after cardiac appropriate AAV-luciferase control; Po0.01) (Figures 2c and d). gene delivery), we found that the percentage of pigs with Moreover, AAV6-S100A1-mediated myocardial S100A1 protein inducible monomorphic ventricular tachyarrhythmia (MVT) was overexpression showed various regions with 50-fold and higher not statistically different between AAV6-luc, AAV6-S100A1 and (up to 95-fold) S100A1 overexpression (Figures 2c and d). saline groups (Figure 3a). Despite locally extreme myocardial Of note, these ‘hot spots’ were frequently located close to an S100A1 expression, there was a nonsignificant trend toward an area with missing S100A1 or modest S100A1 overexpression in increased inducibility of MVT in saline pigs 14 weeks post MI AAV6-S100A1-treated hearts (Figures 2c and d). In contrast, (Figure 3a; Supplementary Table 3). Moreover, high-level myo- AAV9-S100A1 resulted in an up to 5.4-fold myocardial S100A1 cardial S100A1 expression did not abet the occurrence of right overexpression, and thus the gradient of S100A1 expression ventricular stimulation-induced MVT (Figure 3b). As expected, MVT between neighboring myocardial areas was mitigated (Figures 2c could not be induced in non-infarcted saline control pigs and d). Western blot analysis revealed that the level of chronic (Figure 3a). Importantly, extreme myocardial S100A1 expression myocardial S100A1 overexpression does not affect the expression did not cause significant alterations in heart rate, PQ interval, QRS of distinct proteins involved in intracellular Ca2 þ cycling, such as interval and corrected QT interval (QTc) (Table 1) as compared SERCA2a, ryanodine receptor and phospholamban (PLN), as well with failing and non-failing control groups.

& 2014 Macmillan Publishers Limited Gene Therapy (2014) 131 – 138 S100A1 gene therapy biosafety study C Weber et al 134

Figure 3. High-Level AAV6/S100A1 gene therapy does not abet the inducibility of monomorphic ventricular tachyarrhythmia. (a) Absolute numbers of pigs with inducible MVT 12 weeks after gene therapy (14 weeks after myocardial infarction). Neither AAV6/S100A1 nor AAV6/ luciferase increased the occurrence of MVTs, whereas MVTs were not inducible in non-infarcted sham-operated pigs. (b) Of note, high-level cardiac AAV6/S100A1 gene therapy does not abet right ventricular stimulation-induced occurrence of MVTs.

High-level myocardial S100A1 overexpression increases cardiac function and attenuates LV remodeling in failing pig myocardium Table 1. Excessive and inhomogenous myocardial S100A1 expression in vivo does not impact characteristics of the electrocardiogram at rest 12 Despite the patchy nature and local extreme myocardial S100A1 weeks after cardiac AAV6-S100A1 gene delivery (n ¼ 12) as compared expression after AAV6-S100A1 gene delivery, LVEF in failing pig with control (n ¼ 11 for sham; n ¼ 11 for HF/luc and n ¼ 6 for HF/saline) hearts was significantly improved as compared with HF control Sham HF-Saline HF-Luc. HF-S100A1 groups (LVEF: Sham: 67±2%, HF-Saline: 45±4%, HF-AAV6-Luc: 44±2%, HF-AAV6-S100A1: 55±3%), whereas HR remained Electrocardiogram at rest unchanged (Figures 4a and b). Plus dP/dt, as a further marker of HR min À 1 64±370±467±366±3 LV contractile function, showed a strong trend toward increased PQ interval 121±3 119±5 110±3 125±5 values as compared with HF control groups (Figure 4c). In line QRS interval 91±292±389±294±2 with the improvement in global cardiac function, enlargement QTc interval 507±10 515±8 494±8 488±8 of the end-diastolic LV diameter (LVEDD: Sham: 4.9±0.1 cm, Data are presented as mean±s.e.m. HF-Saline: 5.3±0.2 cm, HF-AAV6-Luc: 5.4±0.3 cm, HF-AAV6-S100A1: 5.0±0.2 cm), and thus cardiac remodeling, was significantly attenuated because of high-level AAV6-S100A1 gene therapy as compared with HF control groups (Figure 4d). Markers of chronic genomic DNA. Human S100A1 cDNA in isolated genomic DNA was cardiac failure, such as BNP and NCX expression, were significantly detectable by PCR in liver, skeletal muscle and cardiac tissue, reduced in AAV6-S100A1-treated failing pig hearts compared with demonstrating AAV6 infection in these organs (Figure 5). HF control groups (BNP expression (fold of Sham): Sham: 1.0±0.3- However, the majority of AAVs were still delivered to the fold, HF-Saline: 12.3±2-fold, HF-AAV6-Luc: 8.4±1.8-fold, HF-AAV6- myocardium as human S100A1 cDNA was 17±8-fold higher as ± S100A1: 2.6 1.5-fold) (Figure 4e and Supplementary Figure 6, compared with the liver (206±66-fold for skeletal muscle; n ¼ 5 online only supplement). Of note, high-level AAV6-S100A1 over- pigs). Human S100A1 cDNA in isolated genomic DNA was not expression showed a nonsignificant trend toward increased detectable in lung and brain tissue (Figure 5). mitochondrial F1-ATP synthase activity, whereas low-level AAV9- Three months after AAV6-S100A1 gene delivery, leukocyte, S100A1 overexpression significantly increased F1-ATP synthase erythrocyte and platelet counts as well as hemoglobin concentra- activity as compared with the control (Figure 4f). tions were unchanged between sham and HF animals treated with either saline, AAV6-luc or AAV6-S100A1 (Table 2). Accordingly, in Level of S100A1 expression determines intracellular Ca2 þ cycling all groups, sodium, potassium and glucose levels did not show any in neonatal cardiomyocytes differences, and pancreas enzymes, kidney retention parameters and serum liver enzymes were also found within the physiological Low (4-fold) and moderate (10-fold) S100A1 overexpression normal range for pigs (Table 2). significantly increases intracellular Ca2 þ transients in isolated neonatal rat cardiomyocytes (NRCM), whereas this effect was abrogated under extreme (45-fold) S100A1 protein overexpression (Supplementary Figure 7, online-only supplement). Of note, DISCUSSION increase in intracellular Ca2 þ cycling was pronounced at low Cardiac S100A1 gene therapy enters the stage of clinical S100A1 overexpression. translation as it was proven to have additive beneficial effects beyond that in current pharmacological HF treatment of b-adrenergic receptor blockade and to provide long-term AAV6-S100A1 biodistribution and safety profile after high-level therapeutic effects using a preclinical large animal HF myocardial S100A1 overexpression model.10,12,35 Moreover, S100A1 gene therapy was shown to As myocardial S100A1 expression was locally extreme after AAV6- rescue HF in small and large animal models as well as in isolated S100A1 gene delivery, we were interested in investigating a failing human cardiomyocytes.21,30,35,36 However, potential cardiac potential ‘spill-over’ of the AAV6-S100A1 vector to non-cardiac adverse effects such as contractile dysfunction and ventricular organs. As S100A1 was expressed in a cardio-selective manner arrhythmia due to high-level myocardial S100A1 overexpression employing the CMV-MLC2 promoter, we analyzed S100A1 cDNA in are of potential concern.6,24,26–28,31 Therefore, we decided to

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Figure 4. AAV6/S100A1 gene therapy increases global cardiac function and attenuates myocardial remodeling in the failing myocardium. (a–e) Twelve weeks after myocardial infarction, contractile function of post-ischemic failing hearts was significantly reduced as shown by a significant reduction in þ dP/dt and LVEF, whereas HR remained unchanged as compared with sham-operated controls (n ¼ 11). AAV6/S100A1 treatment (n ¼ 12) significantly increased LVEF as compared with HF/AAV6-luciferase (n ¼ 11) and HF/saline (n ¼ 6) controls. Plus dP/dt showed a nonsignificant trend toward increased values in AAV6/S100A1-treated failing hearts. (d and e) Myocardial remodeling of failing pig hearts was significantly attenuated 10 weeks after AAV6/S100A1 treatment as LV end-diastolic diameter (LVEDD) and expression of the brain natriuretic peptide (BNP) were significantly reduced as compared with HF/saline and HF/AAV6-luciferase controls. (f) F1-ATPase activity was significantly increased in low-level AAV9/S100A1 myocardium and showed a nonsignificant trend in high-level AAV6/S100A1 samples as y compared with appropriate controls (n ¼ 5). *Po0.05 vs AAV6-luciferase and HF/saline. Po0.05 vs AAV6-S100A1. #Po0.05 vs w. Data are presented as mean±s.e.m. investigate the therapeutic window and safety of high-level efficacy, the ability to overcome the endothelial barrier and the S100A1 overexpression prior to clinical use. interstitial space efficiently, and escape neutralizing antibodies, In order to systematically assess regional myocardial S100A1 which might in part explain different myocardial S100A1 protein expression 12 weeks after AAV-S100A1 gene delivery, the overexpression, although the same AAV particle number was non-infarcted remote myocardium was divided into 36 segments used.7 to perform western blot analysis. Retrograde delivery of 1.5 Â 1013 Despite the high-level and locally extreme myocardial S100A1 tvp of AAV6-S100A1 (1.5 Â 1013 tvp of AAV9-S100A1) via the ACV overexpression, AAV6-S100A1 significantly increased LVEF, resulted in robust myocardial S100A1 overexpression in the whereas þ dP/dt showed a nonsignificant trend toward improved anterior and anteroseptal target area (22.9±3.2-fold compared LV contractile function. Thus, S100A1 gene therapy showed a with AAV6-Luc control), whereas AAV9-S100A1 gene delivery was broad therapeutic window without detrimental effects on the shown to result in modest S100A1 overexpression (3.2±0.3-fold of contractile function. This might be explained in part by indirect AAV9-Luc control).10 Detailed analysis of S100A1 expression effects such as reduction in myocardial wall stress and regression pattern in the myocardium showed inhomogenous S100A1 of maladaptive hypertrophy mediated by the myocardium with expression, which is in line with previous cardiac in vivo gene moderate S100A1 overexpression and affecting the myocardium transfer studies.29,30,35,36 Moreover, use of AAV6-S100A1 caused with extreme or absence of S100A1 overexpression. Data from ‘hot spots’ of cardiac S100A1 overexpression (up to 95-fold) next isolated cardiomyocytes might support this hypothesis as non- to segments without S100A1 overexpression, whereas the S100A1-treated cardiomyocytes isolated from AAV-S100A1-treated maximal gradient of S100A1 expression was 5.4-fold in AAV9- failing rat hearts (B40% infection rate) displayed increased functional S100A1-treated pigs. Of note, AAV vector production and properties.35 Consistently, high-level patchy S100A1 overexpression quantification was carried out in the same vector production significantly attenuated LV enlargement, normalized NCX core lab and the same cardio-selective promoter was used. AAV6 expression and reduced BNP expression, which is in line with and AAV9 serotypes exhibit an inherent cardiotropism, which is studies on less-efficient S100A1 overexpression.10,15,17,21,30,35,36 due to distinct capsid characteristics causing transductional However, low-level S100A1 expression (3.2±0.3-fold) without

& 2014 Macmillan Publishers Limited Gene Therapy (2014) 131 – 138 S100A1 gene therapy biosafety study C Weber et al 136 local ‘hot spots’ of excessive myocardial S100A1 overexpression extreme ratios are beyond the optimum. However, dose-dependent mediated by AAV9-S100A1 rescued þ dP/dt and showed a effects of S100A1 are not fully explained but are in line with pronounced increase in LVEF as compared with AAV6-S100A1.10 previous results from isolated skeletal muscle fibers demonstrating a In line, mitochondrial F1-ATPase activity was significantly smaller increase in peak Ca2 þ released from the sarcoplasmic increased in low-level S100A1-overexpressing myocardial reticulum at high S100A1 concentration.37 samples, whereas high-level S100A1-overexpressing specimens Occurrence of ventricular tachyarrhythmia is a potential major showed a nonsignificant trend. Low and moderate S100A1 cardiac adverse effect of myocardial gene delivery that might be protein overexpression significantly increased intracellular Ca2 þ triggered by high-level and inhomogenous myocardial S100A1 transients in NRCM, whereas extreme S100A1 overexpression protein overexpression. The ‘multicenter unsustained tachycardia abrogated this effect. However, even extreme S100A1 over- trial’ demonstrated the possibility of electrophysiologically guided expression had no deleterious effects on intracellular Ca2 þ cycling risk stratification to identify patients at risk for sudden cardiac as compared with sham. Notably, expression and phosphorylation death.38,39 Although the multicenter unsustained tachycardia trial status of proteins involved in intracellular Ca2 þ cycling, such as included patients with ischemic cardiomyopathy and moderately PLN, ryanodine receptor and SERCA2a, remained unaltered after reduced LV function, we investigated the inducibility of MVT in S100A1 overexpression. Therefore, S100A1/SERCA2a, S100A1/RyR post-ischemic pigs with moderately reduced LV function as a and S100A1/PLN ratios are altered and we might speculate that marker of potential occurrence of malignant ventricular arrhythmia. Inducibility of MVTs was not increased following high-level myocardial S100A1 overexpression in HF as comparedwith HF control groups, whereas, as expected, MVTs could not be induced in sham-operated non-infarcted pigs. Moreover, repolarization and depolarization intervals of the ecg, including the QTc interval, which might be used as a biomarker of the occurrence of ventricular tachyarrhythmia, were not statistically altered in AAV6- S100A1-treated pigs. Of note, Vo¨lkers et al.10,19,36 demonstrated that S100A1 functions as an inhibitory modulator of ryanodine receptor function, reducing Ca2 þ -spark frequency at diastolic cytosolic Ca2 þ concentration and thus susceptibility to malignant ventricular arrhythmia. We further analyzed the biodistribution of S100A1 cDNA in isolated genomic DNA of non-cardiac organs. Retrograde high- dosage AAV6-S100A1 gene therapy via the ACV resulting in an average of more than 20-fold myocardial S100A1 protein expression mainly targets the heart, which is line with previous studies using AAV9-S100A1.10 However, AAV biodistribution ratios between the myocardium and liver (AAV6: 17:1 vs AAV9: 63:1) and between the myocardium and skeletal muscle (AAV6: 206:1 vs AAV9: 2564:1) were reduced in AAV6-S100A1-treated pigs as compared with AAV9-S100A1-treated pigs, suggesting an Figure 5. AAV biodistribution. Human S100A1 DNA delivered by increased spill-over and reduced myocardial absorption of AAV6- AAVs was detectable using PCR in isolated genomic DNA from liver, S100A1 as compared with AAV9-S100A1.10 Despite wider AAV skeletal muscle and cardiac tissue, demonstrating AAV infection of vector biodistribution to non-cardiac organs in AAV6-S100A1- these organs. However, the majority of AAVs were delivered to the treated pigs, blood count, sodium, potassium and glucose blood myocardium as human S100A1 DNA isolated from genomic DNA concentrations were similar and pancreas enzymes, kidney was 206±66-fold higher in cardiac tissue (anterior wall) as compared with skeletal muscle and 17±8-fold higher compared retention parameters and serum liver enzymes were within the with the liver (n ¼ 5 pigs). Human S100A1 DNA was not detectable in physiological normal range for pigs, suggesting no major lung and brain tissue. Representative gel analysis of human S100A1 detrimental impact of the AAV6 vector on the functional PCR products (PCR run at holded at cycle 27). properties of these organs.

Table 2. Preserved blood biomarkers 12 weeks after AAV6-S100A1 gene therapy (n ¼ 9) as compared with HF/AAV6-luciferase (n ¼ 6), HF/saline (n ¼ 7) and sham operation (n ¼ 8) controls

Sham HF-Saline HF-Luc. HF-S100A1

Blood parameters: Sodium (139–152 mmol l À 1) 141±0.6 140±1.2 138±2.2 140±1.4 Potassium (3.5–4.5 mmol l À 1) 3.9±0.07 4.1±0.25 4.9±0.45 3.7±0.08 Leukocytes (11–22 nl) 15.4±0.96 15.1±0.5 15.9±1.8 17.0±1.1 Erythrocytes (5.0–7.0 pl) 5.9±0.2 6.0±0.1 5.8±0.1 6.2±0.12 Platelets (200–500 nl) 376±38 430±33 302±42 455±37 Hemoglobin (9.0–13.0 g dl À 1) 9.8±0.2 10.1±0.2 9.8±0.2 10.0±0.1 Creatinine (0.8–2.3 mg dl À 1) 1.7±0.13 1.8±0.11 1.4±0.05 1.9±0.09 Urea (20–53 mg dl À 1)26±2.6 24±1.9 30±2.1 31±2.6 Glucose (mg dl À 1)82±683±773±476±7 Lipase (15–60 u l À 1)14±0.4 15±1.3 15±1.4 17±2.1 Glutamate-Pyruvate-Transaminase (GPT) (22–47 U l À 1)49±4.8 42±2.3 46±2.1 46±3.3 Glutamate–Oxalacetate-Transferase (GOT) (20–66 U l À 1)38±4.8 33±3.3 33±4.0 35±2.2

Gene Therapy (2014) 131 – 138 & 2014 Macmillan Publishers Limited S100A1 gene therapy biosafety study C Weber et al 137 Overall, high-level myocardial S100A1 protein expression CONFLICT OF INTEREST mediated by AAV6-S100A1 does not cause cardiac adverse effects, P Most and HA Katus report potential competing interests as they have filed US and such as an increased susceptibility to ventricular tachyarrhythmia EU patent applications on the therapeutic use of the S100A1 protein to treat heart or impairment of contractile function in vivo. The current study failure. CW, IN, BK, PS, PR, JR, AJ, WJK, OJM and STP declare no conflict of interest. demonstrates a broad therapeutic window of S100A1 gene therapy in post-ischemic HF, which might mitigate potential concern of cardiac adverse effects prior to clinical use of S100A1 ACKNOWLEDGEMENTS gene therapy to treat end-stage HF. We thank Barbara Leuchs and the DKFZ vector core production unit for generating high-titer AAV vector stocks. This work was supported by the following NIH grants: R01HL92130 and R01HL92130-02S1 (PM); P01HL075443, R01HL56205 and R01HL061690 (WJK); Deutsche Forschungsgemeinschaft: 1654/3-2 (OJM), 562/1-1 MATERIALS AND METHODS (PM and STP); the Bundesministerium fu¨r Bildung und Forschung: 01GU0527 (PM, All animal procedures and experiments were performed in accordance OJM, HAK); Deutsche Gesellschaft fu¨r Kardiologie Otto Hess Promotionsstipendium with the ‘Guide for the Care and Use of Laboratory Animals’ (NIH) and were (CW); and the German Cardiovascular Research Center (DZHK to PM, HAK). approved by the local ‘Animal Care and Use Committee’ of Baden- Wu¨rttemberg. REFERENCES Model of post-ischemic HF and catheter-based cardiac gene 1 AHA. Heart disease and stroke statistics: 2010 update. Circulation 2010; 21: delivery e1–e170. 2 Flather MD, Yusuf S, Køber L, Pfeffer M, Hall A, Murray G et al. 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