Transgenic Rabbits Research Articles

Therapeutic Efficacy of Mexiletine for Long QT Syndrome Type 2: Evidence From Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, Transgenic Rabbits, and Patients.

Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel-mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2. Heart rate-corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD90) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model. After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate-corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD90 by 113 ms, indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ∆QTc during the test (r= -0.8; P<0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07-0.14) to 0.04 (95% CI, 0.02-0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16-0.84), reflecting a 60% reduction in the event rate (P=0.01). Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2.

KCNQ1 suppression-replacement gene therapy in transgenic rabbits with type 1 long QT syndrome.

Type 1 long QT syndrome (LQT1) is caused by pathogenic variants in the KCNQ1-encoded Kv7.1 potassium channels, which pathologically prolong ventricular action potential duration (APD). Herein, the pathologic phenotype in transgenic LQT1 rabbits is rescued using a novel KCNQ1 suppression-replacement (SupRep) gene therapy. KCNQ1-SupRep gene therapy was developed by combining into a single construct a KCNQ1 shRNA (suppression) and an shRNA-immune KCNQ1 cDNA (replacement), packaged into adeno-associated virus serotype 9, and delivered in vivo via an intra-aortic root injection (1E10 vg/kg). To ascertain the efficacy of SupRep, 12-lead electrocardiograms were assessed in adult LQT1 and wild-type (WT) rabbits and patch-clamp experiments were performed on isolated ventricular cardiomyocytes. KCNQ1-SupRep treatment of LQT1 rabbits resulted in significant shortening of the pathologically prolonged QT index (QTi) towards WT levels. Ventricular cardiomyocytes isolated from treated LQT1 rabbits demonstrated pronounced shortening of APD compared to LQT1 controls, leading to levels similar to WT (LQT1-UT vs. LQT1-SupRep, P < .0001, LQT1-SupRep vs. WT, P = ns). Under β-adrenergic stimulation with isoproterenol, SupRep-treated rabbits demonstrated a WT-like physiological QTi and APD90 behaviour. This study provides the first animal-model, proof-of-concept gene therapy for correction of LQT1. In LQT1 rabbits, treatment with KCNQ1-SupRep gene therapy normalized the clinical QTi and cellular APD90 to near WT levels both at baseline and after isoproterenol. If similar QT/APD correction can be achieved with intravenous administration of KCNQ1-SupRep gene therapy in LQT1 rabbits, these encouraging data should compel continued development of this gene therapy for patients with LQT1.

Beneficial normalization of cardiac repolarization by carnitine in transgenic short QT syndrome type 1 rabbit models.

Short QT syndrome type 1 (SQT1) is a genetic channelopathy caused by gain-of-function variants in human-ether-a-go-go (HERG) underlying the rapid delayed-rectifier K+ current (IKr), leading to QT-shortening, ventricular arrhythmias, and sudden cardiac death. Data on efficient pharmacotherapy for SQT1 are scarce. In patients with primary carnitine-deficiency, acquired-short QT syndrome (SQTS) has been observed and rescued by carnitine supplementation. Here, we assessed whether carnitine exerts direct beneficial (prolonging) effects on cardiac repolarization in genetic SQTS. Adult wild-type (WT) and transgenic SQT1 rabbits (HERG-N588K, gain of IKr) were used. In vivo electrocardiograms (ECGs), ex vivo monophasic action potentials (APs) in Langendorff-perfused hearts, and cellular ventricular APs and ion currents were assessed at baseline and during L-Carnitine/C16-Carnitine-perfusion. Two-dimensional computer simulations were performed to assess re-entry-based ventricular tachycardia-inducibility. L-Carnitine/C16-Carnitine prolonged QT-intervals in WT and SQT1, leading to QT-normalization in SQT1. Similarly, monophasic and cellular AP duration (APD) was prolonged by L-Carnitine/C16-Carnitine in WT and SQT1. As underlying mechanisms, we identified acute effects on the main repolarizing ion currents: IKr-steady, which is pathologically increased in SQT1, was reduced by L-Carnitine/C16-Carnitine and deactivation kinetics were accelerated. Moreover, L-Carnitine/C16-Carnitine decreased IKs-steady and IK1. In silico modelling identified IKr changes as the main factor for L-Carnitine/C16-Carnitine-induced APD-prolongation. 2D simulations revealed increased sustained re-entry-based arrhythmia formation in SQT1 compared to WT, which was decreased to the WT-level when adding carnitine-induced ion current changes. L-Carnitine/C16-Carnitine prolong/normalize QT and whole-heart/cellular APD in SQT1 rabbits. These beneficial effects are mediated by acute effects on IKr. L-Carnitine may serve as a potential future QT-normalizing, anti-arrhythmic therapy in SQT1.

SGK1 inhibition normalizes action potential duration in transgenic LQT2 rabbits but not in LQT1, suggesting a novel gene-specific therapeutic approach in long QT syndrome

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): SNSF (Swiss National Science Fundation) Background Increasing evidence suggests that secondary remodeling in Na+ and Ca2+ homeostasis may further enhance the propensity to develop arrhythmias in K-channelopathies LQT1 and LQT2 syndrome. In this context, the Serum and Glucocorticoid regulated Kinase-1 (SGK-1) was recently identified as a major regulator of sodium channels in cardiomyocytes. It is mainly activated in pathological conditions, leading to increased INaLate, prolongation of action potential duration (APD) and increased susceptibility to ventricular arrhythmias, recapitulating LQTS phenotype. SGK1 inhibition might hence be a novel therapeutic target also in genetic LQTS subtypes. Methods In cardiomyocytes (CM) isolated from wild type (WT), LQT1 and LQT2 adult rabbits patch-clamp experiments were performed to record AP with SGK1-inhibitor(SGK1-inh, 3 µM) or DMSO vehicle control. Results In the perforated patch configuration at 37°C, 3 μM of SGK1-inh shortened APD90 in LQT2 CMs at 1 Hz (DMSO-control [ms]: 477.3 vs treated cells[ms]: 362.8 p &amp;lt; 0.0001) and at 2 Hz (DMSO-cells: 325.7 vs treated cells:295.4,p &amp;lt;0.05). In contrast, in WT and LQT1 CMs, no effect on APD90 was observed neither at 1 Hz (WT, DMSO-cells: 329.2 vs treated cells: 301.6 p=ns; LQT1, DMSO-cells: 429.8 vs treated cells: 431.6, p=ns) nor at 2 Hz (WT, DMSO-cells:256.8 vs treated cells:242 p=ns; LQT1,DMSO cells:310.4 vs treated cells: 315.8,p=ns). Importantly, the SGK1-inhibitor effect in LQT2 rabbit CMs was so pronounced that it normalized APD to the wildtype level (LQT2+SGK1-Inh: 362.8 vs WT:329.2, ps=ns). Furthermore, short-term-variability in APD was markedly reduced by SGK1-Inh in LQT2, indicating an antiarrhythmic effect. Conclusions SGK1-inhibition exerts a beneficial APD-shortening effect only in LQT2, indicating a gene-specific efficacy. The underlying mechanism is still unclear. We hypothesize that the SGK1 pathway/INaLate activation in LQT1 might not be as active as in LQT2. We are currently conducting more experiments on potential genotype differences in SGK1 activity and SGK1-mediated INaLate inhibition to further elucidate the differences.Effects of SGK1-inh on AP characteristic

Interplay of electrical and mechanical properties in transgenic rabbit models of long-QT and short-QT syndrome

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): SNF Introduction Long-QT syndrome (LQTS) and short-QT syndrome (SQTS) are cardiac disorders primarily affecting electrical function but secondarily also impacting on mechanical features through electro-mechanical coupling (EMC). This study delves into in vivo and cellular electro-mechanical function and explores the impact of acute mechanical changes on the electrical phenotype, termed mechano-electrical coupling (MEC), in transgenic rabbit models. Aim This study aims to investigate the interrelationship between electrical and mechanical properties in LQTS and SQTS rabbit models, examining how acute mechanical alterations influence the electrical phenotype and exploring cellular electro-mechanical characteristics. Methods We examined 12-lead ECGs in LQT2, wildtype (WT), and SQT1 adult rabbits under various preload and afterload conditions. Additionally, patch-clamp and ionoptix experiments on isolated ventricular cardiomyocytes were conducted to investigate cellular electrical and mechanical characteristics. Results Baseline heart-rate corrected QTc intervals were prolonged in LQT2 (p&amp;lt;0.0001) and shortened in SQT1 (p=0.012) compared to WT. QT-dispersion was solely increased in LQT2 (p&amp;lt;0.05). Acute increase in preload and in afterload prolonged QTc intervals in all groups, while reduced preload shortened QTc intervals. MEC (mechano-induced ΔQTc) varied among genotypes; in LQT2, QTc changes were more significant than in WT (p&amp;lt;0.0001 with increased preload, p&amp;lt;0.001 with decreased preload, and p&amp;lt;0.05 with increased afterload). SQT1 showed a trend towards less pronounced changes compared to WT only after increased preload (p=0.11). At the cellular level, action potential duration was longer in LQT2 (p&amp;lt;0.001) and shorter in SQT1 (p&amp;lt;0.0001) compared to WT. While cardiomyocytes from WT and SQT1 showed similar Ca2+ transient durations at 1Hz, 0.5Hz, and 0.25Hz, in LQT2 cardiomyocytes Ca2+ transients were significantly prolonged at 0.5 (p&amp;lt;0.001) and 0.25Hz (p&amp;lt;0.05) in line with the pronounced APD prolongation. Additionally, LQT2 exhibited significantly more pro-arrhythmic Ca2+ oscillations, which may contribute to arrhythmia formation in LQT2s. Moreover, LQT2 exhibited a slower calcium transient upstroke velocity and a reduced cellular contraction velocity and peak sarcomere shortening at all frequencies, demonstrating a mechanical impairment compared to WT and SQTS cardiomyocytes. Conclusions Acute mechanical alterations induce electrical changes through MEC in LQT2, WT, SQT1 rabbits. The magnitude of these changes correlates with baseline QTc, exhibiting the most prominent effects in LQT2. Consistent findings between cellular electro-mechanical assessments and in vivo measurements underscore the significance of cardiomyocytes in unravelling the mechanisms behind EMC and MEC. This intricate interplay between electro-mechanical properties in cardiac disorders, highlighting the necessity of considering both aspects when investigating arrhythmia mechanisms.

In vivo KCNH2-suppression-replacement gene therapy attenuates the pathogenic phenotype in transgenic rabbits with short QT syndrome type 1

Abstract Background Short QT syndrome type 1 (SQT1) is a genetic channelopathy characterized by gain-of-function variants in the KCNH2-encoded potassium channel underlying the IKr current, leading to a pathologically shortened cardiac action potential duration (APD). Patients with SQT1 manifest shortening of the QT duration on 12-lead ECG and are prone to cardiac arrhythmias that can culminate in sudden cardiac death. Purpose To investigate the effect of a KCNH2-specific suppression-replacement (KCNH2-SupRep) gene therapy on the pathogenic phenotype in transgenic SQT1 rabbits. Methods A novel dual-component KCNH2-SupRep gene therapy was developed by combining a custom-designed KCNH2 shRNA (suppression) and an shRNA-immune (shIMM) KCNH2 cDNA (replacement) into AAV9. In vivo AAV9-mediated KCNH2-SupRep / sham treatment was performed by intra-aortic root injection of the KCNH2-SupRep AAV9 (1E10 vg/kg) / NaCl 0.9% during balloon occlusion using a Swan Ganz catheter. Three weeks after administration, 12-lead ECGs were assessed in adult transgenic SQT1 (KCNH2-N588K, gain of IKr due to impaired inactivation) and wild-type (WT) rabbits to determine the effect of KCNH2-SupRep / sham treatment on the rabbit’s QTc duration and QT index. Patch-clamp recordings were performed in isolated ventricular cardiomyocytes (CMs) to evaluate the effect of KCNH2-SupRep / sham treatment on the CM’s action potential duration at 90% repolarization (APD90). Results After in vivo AAV9-mediated KCNH2-SupRep gene therapy injection (1E10 vg/kg) in 7 SQT1 rabbits, QTc duration and QT index significantly increased in all SQT1 rabbits (QTc: untreated(UT)-SQT1: 153.2±3.8ms vs treated-SQT1: 166.5±4.6ms, p=0.0002, N=7 each; QTi: UT-SQT1: 92.5±1.9% vs treated-SQT1: 99.7±2.7%, p=0.0007, N=7 each), reaching a QT index of ~100% typically observed in WT rabbits. No such changes were observed in the sham-SQT1 rabbits. Furthermore, cardiomyocytes isolated from 7 SQT1 rabbits that received in vivo SupRep gene therapy demonstrated APD90 prolongation closer to WT rabbits compared to UT-SQT1 and sham-SQT1 rabbits (APD90: UT-SQT1: 314.2±11.9ms, WT: 404.5±13.8ms, treated-SQT1: 362.9±10.6ms; UT-SQT1 vs WT: p&amp;lt;0.0001; UT-SQT1 vs treated-SQT1: p=0.019; WT vs Treated-SQT1: p=0.031). In line with these observations, first IKr measurements demonstrate smaller IKr currents in SupRep-SQT1 than in sham-SQT1 CMs. Conclusion In vivo KCNH2-SupRep gene therapy normalizes the pathologically shortened QTc duration and QT index in transgenic SQT1 rabbits. Moreover, KCNH2-SupRep treatment prolongs APD90 in SQT1 compared to UT-SQT1 and sham-SQT1 reaching levels closer to WT.

In vivo KCNQ1-suppression-replacement gene therapy in transgenic LQT1 rabbits restores a physiological QT interval at baseline and under catecholamine infusion

Abstract Background Long-QT syndrome type 1 (LQT1) is a genetic channelopathy characterized by loss-of-function variants in the repolarizing potassium channel KCNQ1/IKs which lead to QT-prolongation and predisposes patients to adverse events such as cardiac arrhythmias. Clincally, during catecholamine infusion or exercise stress test, patients with LQT1 present a paradoxical QT prolongation. Purpose To investigate whether a gene-therapy approach targeting the defective K+-channel, through a KCNQ1 suppression-and-replacement (SupRep) strategy, can restore the healthy phenotype at baseline and under catecholamine infusion in LQT1 transgenic rabbits. Methods Our proprietary dual component suppression-and-replacement (SupRep) gene therapy was created by combining a custom-designed KCNQ1 shRNA and a shRNA-immune (shIMM) KCNQ1 cDNA into AAV9. In vivo KCNQ1-SupRep gene therapy was performed by targeted intra-aortic root injection during balloon occlusion. Two or three weeks post gene therapy, 12-lead ECGs were assessed in adult transgenic LQT1 (KCNQ1-Y315S, loss-of IKs) and wild-type (WT) rabbits to determine the effect of SupRep gene therapy on QT-duration at baseline and under catecholamine infusion (10nM isoproterenol, 5mL/h). Ventricular cardiomyocytes (VCMs) were isolated to investigate the effect of SupRep gene therapy on action potential duration (APD) and calcium transients (Ca2+T) at baseline and under isoproterenol infusion. Results At the cellular level, VCMs isolated from SupRep treated LQT1 rabbits showed a significant shortening of the APD90 as compared to UT-LQT1 (p&amp;lt;0.0001). At the whole animal level, at baseline, the QTi (%) of the UT-LQT1 transgenic rabbits (122±3) was significantly longer compared to the WT rabbits (105±2, p&amp;lt;0.0001). Treatment with AAV9-KCNQ1-SupRep shortened significantly the QTi of the transgenic LQT1 rabbits as compared to the UT-LQT1 rabbits (111±2, p=0.0115). To further investigate the effect of SupRep gene therapy, 12-lead ECGs were obtained in UT-LQT1, sham-treated LQT1, SupRep-treated LQT1, and WT rabbits under ISO infusion. While reaching a 20% increase in heart rate during ISO stimulation, the UT-LQT1 and the sham treated LQT1 rabbits showed a marked QT prolongation, with ΔQTi of 31.4% and 24.5%, respectively, as compared to baseline. In contrast, the SupRep-treated LQT1 rabbit behaved nearly identical to the WT rabbit during ISO infusion, showing a ΔQTi of 16.9% as compared to 16.5% in the WT rabbit. Similarly, in contrast to the UT-LQT1, VCMs isolated from SupRep-treated LQT1 rabbits demonstrated a normalization of the APD and Ca2+T under ISO stimulation, showing a significantly shortened APD90 (p&amp;lt;0.0001) and Ca2+T90 (p=0.0025) which was similar to what was observed in WT VCMs. Conclusion In vivo KCNQ1 suppression-and-replacement gene therapy normalizes the pathologically prolonged APD/QT interval and restores a physiological response to catecholamine infusion in treated LQT1 transgenic rabbits.

Interplay of electrical and mechanical properties in transgenic rabbit models of long-QT and short-QT syndrome

Interplay of electrical and mechanical properties in transgenic rabbit models of long-QT and short-QT syndrome

In vivo KCNQ1-suppression-replacement gene therapy in transgenic rabbits with type 1 long QT syndrome

Abstract Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): Pfizer Background Type 1 long QT syndrome (LQT1) is a genetic channelopathy characterized by both haploinsufficient and dominant-negative loss-of-function pathogenic variants in the KCNQ1-encoded Kv7.1 K+ channels conducting IKs. Patients with LQT1 may manifest QT prolongation and ventricular arrhythmias that can culminate in sudden cardiac death. Purpose With this project, we aim to investigate whether our mutation independent, KCNQ1-specific suppression-replacement (SupRep) gene therapy can restore the deficient K+-channel and thereby restore the healthy phenotype. Methods Our proprietary dual component SupRep gene therapy was created by combining a custom-designed KCNQ1 shRNA and a shRNA-immune (shIMM) KCNQ1 cDNA into AAV9. In vivo AAV9-mediated KCNQ1SupRep gene therapy was performed by targeted intra-aortic root construct injection (1E12 vg/kg body weight) during balloon occlusion using a Swan Ganz catheter. 2 weeks after in vivo gene therapy, 12-lead ECGs were assessed in adult transgenic LQT1 (KCNQ1-Y315S, loss-of IKs) and wild-type (WT) rabbits to determine the effect of SupRep therapy on the rabbit’s QTc. Patch-clamp and calcium transient measurements were performed in isolated ventricular cardiomyocytes to evaluate the effect of SupRep on the cardiomyocyte’s action potential duration (APD90) and Ca2+ transient duration (Ca2+90) both, in vitro – after plasmid transfection, and in vivo – after AAV9-mediated gene therapy. AAV9-KCNQ1-construct expression was verified with immunohistochemistry targeting the GFP-tag. Results After injection of 3 LQT1 rabbits so far, no significant changes were observed in the LQT1 rabbits’ QTc before and after in vivo AAV9-mediated gene therapy. However, at the cellular level, LQT1 cardiomyocytes transfected in vitro with the SupRep-plasmid demonstrated a significant reduction of APD90 (ms, 1Hz stimulation at 37°) compared to LQT1 control cells (SupRep-in-vitro, 358±21 vs. control 447±22, p&amp;lt;0.05). No differences were observed between sham-plasmid transfected cardiomyocytes and LQT1 control cells after one day of culturing. Similarly, cardiomyocytes isolated from the 3 LQT1 rabbits that underwent in vivo SupRep gene therapy demonstrated pronounced shortening of both APD90 and Ca2+90 compared to LQT1 controls, leading to levels similar to WT controls (APD90, 1Hz, 37°: LQT1: 530±18, WT: 445±18, LQT1-SupRep: 375±25, p&amp;lt;0.0001 LQT1 vs. LQT1-SupRep, p=ns LQT1-SupRep vs. WT) (Ca2+90: LQT1: 487±23, WT: 346±16, LQT1-SupRep: 393±19, p&amp;lt;0.0001 LQT1 vs. LQT1-SupRep, p=ns LQT1-SupRep vs. WT). Conclusion In vivo KCNQ1-suppression-replacement gene therapy normalizes cellular action potential and calcium transient duration in transgenic LQT1 rabbits. Further in vivo experiments will be conducted to evaluate whether this therapeutic correction at the cardiomyocyte level will translate into normalization of the LQT1 rabbits’ QTc both at rest and during stress testing.

PO-02-210 AAV9-MEDIATED KCNQ1-SUPPRESSION-REPLACEMENT GENE THERAPY IN TRANSGENIC RABBITS WITH TYPE 1 LONG QT SYNDROME

Type 1 long QT syndrome (LQT1) is characterized by both haploinsufficient and dominant-negative loss-of-function pathogenic variants in the KCNQ1-encoded Kv7.1 K+ channels conducting IKs current, which contributes to the cardiac action potential. No current therapies target the molecular cause of LQT1. To rescue the pathologic phenotype in the transgenic KCNQ1-Y315S LQT1 rabbit model using our proprietary KCNQ1-specific suppression-replacement (SupRep) gene therapy. Our dual component SupRep gene therapy was created by combining into a single construct a custom-designed KCNQ1 shRNA (suppression) and a cardiac-specific troponin C (cTnC) promoter driven shRNA-immune KCNQ1 cDNA (replacement). The KCNQ1-SupRep construct was packaged into AAV9 (AAV9-KCNQ1-SupRep), a nominally immunogenic and cardiotropic viral vector with limited off-target delivery. AAV9-KCNQ1-SupRep was injected in vivo via a targeted intra-aortic root injection (1E12 vg/kg bodyweight) during balloon occlusion using a Swan Ganz catheter. Two weeks post in vivo gene therapy, 12-lead ECGs were assessed in adult transgenic LQT1 and wildtype (WT) rabbits to ascertain the effect of SupRep on the rabbit’s QTc. Patch-clamp and calcium transient measurements were performed in isolated ventricular cardiomyocytes (CMs) to evaluate the effect of SupRep on the CM’s action potential duration (APD90) and Ca2+ transient duration (Ca2+90) post in vivo gene therapy. AAV9-KCNQ1-SupRep expression was verified with immunohistochemistry. After injection of 3 LQT1 rabbits so far, no statistically significant changes were observed in the LQT1 rabbits’ QTc pre/post in vivo AAV9-KCNQ1-SupRep. However, CMs isolated from the 3 LQT1 rabbits that underwent in vivo SupRep gene therapy demonstrated pronounced shortening (ms) of both APD90 and Ca2+90 compared to LQT1 controls, leading to levels similar to WT controls (APD90, 1Hz, 37°: LQT1: 530±18, WT: 445±18, LQT1-SupRep: 375±25, p<0.0001 LQT1 vs. LQT1-SupRep, p=ns LQT1-SupRep vs. WT) (Ca2+90: LQT1: 487±23, WT: 346±16, LQT1-SupRep: 393±19, p<0.0001 LQT1 vs. LQT1-SupRep, p=ns LQT1-SupRep vs. WT). In vivo AAV9-KCNQ1-SupRep gene therapy normalizes cellular AP and calcium transient duration in transgenic LQT1 rabbits. Further in vivo experiments will be conducted to evaluate whether this therapeutic correction at the CM level will translate into normalization of the LQT1 rabbits’ QTc both at rest and during stress testing.

PO-01-217 FUNCTIONAL CHARACTERIZATION OF CALCIUM HANDLING KINETICS IN RE-ENGINEERED CARDIOMYOCYTES FROM A PATIENT WITH TYPE 2 LONG QT SYNDROME

Type 2 long QT syndrome (LQT2) is caused by pathogenic variants in the KCNH2-encoded Kv11.1 K+ channel resulting in prolongation of the action potential duration (APD) at the cellular level and QT prolongation in the patient, predisposing LQT2 patients to potentially lethal ventricular arrhythmias. Limited evidence, namely prolonged Ca2+ transient in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) and enhanced Ca2+ leak in transgenic LQT2 rabbit model, suggests that KCNH2 pathogenic variants may be associated with perturbations in intracellular Ca2+ handling. To characterize the APD and Ca2+ handling properties of an iPSC-CM model of LQT2. iPSC-CMs were generated from a patient with a pathogenic KCNH2 frame-shift variant (p.Q901fs/71) and an unrelated healthy control. Confocal imaging of FluoVolt voltage dye was used to measure the APD at 90% repolarization (APD90) in iPSC-CMs. Ca2+ kinetics was characterized using Ca2+ sensing dye Fluo4-AM before and after treatment with 100 nM isoproterenol (ISO). Q901fs/71-iPSC-CMs had a significantly prolonged APD90 compared to wild-type (WT) iPSC-CMs (444 ±9 ms vs 631±8 ms, p<0.001). Q901fs/71-iPSC-CMs had a higher Ca2+ amplitude (DF/F0) before (0.423±0.043 vs 0.97±0.07, p=0.001) and after ISO treatment (0.602±0.051 vs 1.056 ±0.06, p<0.001) compared to WT. The Ca2+ transient duration (CTD90) was longer in Q901fs/71-iPSC-CMs than in WT before (1.062±0.041 s vs 1.306±0.038 s, p=0.01), but not after ISO (1.071±0.062 s vs 1.092±0.044 s, p=0.96). Peak to 90% Ca2+ decay time was increased in Q901fs/71-iPSC-CMs before (0.785±0.04 s vs 0.942±0.033 s, p=0.028), but not after ISO (0.748±0.054 s vs 0.767±0.038 s, p=0.95). Upstroke time was longer in Q901fs/71-iPSC-CMs compared to WT before (0.281±0.006 s vs 0.364±0.014 s; p=0.002) but not after ISO (0.327±0.014 s vs 0.344±0.025 s; p=0.88). Upstroke velocity (DF/F0/t) was increased in Q901fs/71-iPSC-CMs before (1.092±0.122 vs 2.278±0.278, p<0.001) and after ISO (1.310±0.117 vs 2.2±0.139, p=0.005). Ca2+ leak from sarcoplasmic reticulum was higher in Q901fs/71-iPSC-CMs before (16% vs 38%) and after ISO (16% vs 55%). We show that iPSC-CMs derived from the patient with a truncating LQT2-causative variant (yielding likely Kv11.1 channel haploinsufficiency) have prolonged APD90 and display abnormal Ca2+ handling properties such as higher amplitude, increase in Ca2+ leak, prolonged transient duration, Ca2+ decay time, and upstroke velocity compared to WT iPSC-CMs.

Physiological role of SK channels in modulating cardiac repolarization: APD and dispersion at slow heart rate in long QT syndrome.

Long QT syndrome (LQTS) is a congenital disease associated with polymorphic ventricular tachycardia (pVT) and sudden cardiac death. The hallmarks of LQTS are early afterdepolarizations that initiate pVT and dispersion of repolarization allowing reentry formation. Small conductance Ca2+-activated K+ (SK) channels are directly gated by Ca2+ and provide feedback from Ca2+ to membrane voltage to accelerate cardiac repolarization, which may help normalizing LQTS. Using optical mapping, we examined the role of ISK in transgenic rabbit models of LQT1, LQT2, and LQT5 and found that enhancing ISK with NS309 consistently shortened APD, reduced APD dispersion, and suppressed pVT genesis universally in the three LQT rabbit models. Enhancement of ISK has a greater impact at longer cycle length, flattening restitution curves at slow heart rate where most pVTs occur in LQTS. We incorporated a six state ISK gating simulation combined with intrinsic rectification and divalent block into the Mahajan rabbit action potential model to investigate the role of ISK in suppressing LQT-related arrhythmias. Increasing levels of SK channel conductance (gSK) were able to shorten and reduce APD dispersion simulated by conductance gradients of gKs by ∼64% under LQT2 conditions and gtof by ∼70% in LQT1 conditions. Computer modeling indicates that ISK can reduce APD dispersion through two mechanisms; increased Ca2+ duration in the prolonged APD cells augment ISK to shorten long APD and lowering plateau Vm by ISK masks IKs heterogeneity by reducing IKs's role in repolarization. In conclusion, the Ca2+-dependent gating of ISK allows it to directly feedback on the APD especially when other K+ channels are not functional and to be a potential therapeutic target for LQTS.

Macrophage elastase derived from adventitial macrophages modulates aortic remodeling.

Abdominal aortic aneurysm (AAA) is pathologically characterized by intimal atherosclerosis, disruption and attenuation of the elastic media, and adventitial inflammatory infiltrates. Although all these pathological events are possibly involved in the pathogenesis of AAA, the functional roles contributed by adventitial inflammatory macrophages have not been fully documented. Recent studies have revealed that increased expression of matrix metalloproteinase-12 (MMP-12) derived from macrophages may be particularly important in the pathogenesis of both atherosclerosis and AAA. In the current study, we developed a carrageenan-induced abdominal aortic adventitial inflammatory model in hypercholesterolemic rabbits and evaluated the effect of adventitial macrophage accumulation on the aortic remodeling with special reference to the influence of increased expression of MMP-12. To accomplish this, we compared the carrageenan-induced aortic lesions of transgenic (Tg) rabbits that expressed high levels of MMP-12 in the macrophage lineage to those of non-Tg rabbits. We found that the aortic medial and adventitial lesions of Tg rabbits were greater in degree than those of non-Tg rabbits, with the increased infiltration of macrophages and prominent destruction of elastic lamellae accompanied by the frequent appearance of dilated lesions, while the intimal lesions were slightly increased. Enhanced aortic lesions in Tg rabbits were focally associated with increased dilation of the aortic lumens. RT-PCR and Western blotting revealed high levels of MMP-12 in the lesions of Tg rabbits that were accompanied by elevated levels of MMP-2 and -3, which was caused by increased number of macrophages. Our results suggest that adventitial inflammation constitutes a major stimulus to aortic remodeling and increased expression of MMP-12 secreted from adventitial macrophages plays an important role in the pathogenesis of vascular diseases such as AAA.

A Possible Explanation for the Low Penetrance of Pathogenic KCNE1 Variants in Long QT Syndrome Type 5.

Long QT syndrome (LQTS) is an inherited cardiac rhythm disorder associated with increased incidence of cardiac arrhythmias and sudden death. LQTS type 5 (LQT5) is caused by dominant mutant variants of KCNE1, a regulatory subunit of the voltage-gated ion channels generating the cardiac potassium current IKs. While mutant LQT5 KCNE1 variants are known to inhibit IKs amplitudes in heterologous expression systems, cardiomyocytes from a transgenic rabbit LQT5 model displayed unchanged IKs amplitudes, pointing towards the critical role of additional factors in the development of the LQT5 phenotype in vivo. In this study, we demonstrate that KCNE3, a candidate regulatory subunit of IKs channels minimizes the inhibitory effects of LQT5 KCNE1 variants on IKs amplitudes, while current deactivation is accelerated. Such changes recapitulate IKs properties observed in LQT5 transgenic rabbits. We show that KCNE3 accomplishes this by displacing the KCNE1 subunit within the IKs ion channel complex, as evidenced by a dedicated biophysical assay. These findings depict KCNE3 as an integral part of the IKs channel complex that regulates IKs function in cardiomyocytes and modifies the development of the LQT5 phenotype.

Effect of high hydrostatic pressure on the in vitro development and molecular quality of transgenic rabbit embryos derived from nano-transfected zygotes

Abstract The aim of this study was to evaluate the effect of high hydrostatic pressure (HHP) on the in vitro developmental abilities of nano-transfected rabbit zygotes, their transfection efficiency, and the molecular quality of the blastocysts generated. This quality was assessed by estimating the quantitative profiles of Oct4, Casp7, and Bcl2 mRNA transcripts. The nano-transfection efficiencies of zygotes that had been pre-treated with either 20 MPa or 40 MPa of HHP (13.5% and 13.7%, respectively) were insignificantly lower than those found in zygotes not exposed to HHP prior to their nano-transfection (20.1%; P≥0.05). Moreover, applying HHP treatment with the parameters of 20 MPa and 40 MPa followed by the nano-transfection of zygotes brought about an insignificant decrease in the rates of embryos at the blastocyst stage (30.4% and 23.0%, respectively) as compared to the control group of nano-transfected zygotes (40.4%; P≥0.05). Furthermore, analyzing the transcriptional activity of Oct4, Bcl2, and Casp7 genes revealed that HHP enhances the relative abundance (RA) of all mRNA transcripts in blastocysts derived from non-transfected rabbit zygotes. In turn, the augmented RAs found in the pro-apoptotic Casp7 and anti-apoptotic Bcl-2 transcripts confirmed the onset and progression of programmed cell death in blastocysts developed from nano-transfected zygotes that had undergone HHP pre-treatment. The conceptualization based not only on a novel nano-transfection approach used to genetically modify in vivo-fertilized rabbit zygotes but also on their HHP pre-treatment is elaborated here for the first time, with an emphasis on further investigations aimed at producing transgenic rabbit and other mammalian species embryos by somatic cell cloning.

Abstract 430: GFP-tagging Of Rabbit ApoAI Decreases Expression And Cholesterol-efflux Activity

Introduction: Gene therapy using a helper-dependent adenovirus (HDAd) to express rabbit apolipoprotein (apo) AI in carotids of fat-fed rabbits reduces atherosclerosis. We hypothesize that transgenic apoAI promotes reverse cholesterol transport from the artery wall; however, this could not be tested because the transgenic rabbit apoAI is indistinguishable from endogenous rabbit apoAI. We therefore constructed an HDAd expressing GFP-tagged rabbit apoAI. Methods: We constructed HDAdGFPapoAI by fusing the GFP sequence to the N-terminus of the rabbit APOA1 gene with a short linker. Bovine aortic endothelial cells (BAEC) were transduced with HDAdGFPapoAI, washed, and incubated in serum-free medium for 24h. Conditioned medium (CM) was collected and fluorescence measured. Cell extracts and CM were analyzed by western blotting for protein size, expression level, and presence of GFP and apoAI antigens. CM cholesterol-efflux activity was measured using cholesterol-loaded BHK cells expressing ABCA1. Results: CM of HDAdGFPapoAI-transduced BAEC had green fluorescence and contained a protein of the size predicted for the GFP-apoAI fusion (~56 kDa), which was detected with antibodies to both GFP and apoAI. A smaller protein (~30 kDa) with GFP and apoAI antigens was also detected. Compared to a vector expressing WT rabbit apoAI, GFP-tagging decreased expression of apoAI protein by ~50% and the percentage of secreted apoAI from 55% to 13%. In combination, this reduced secreted GFP-apoAI to only 12% of levels of secreted WT apoAI. While CM containing WT rabbit apoAI strongly stimulated cholesterol efflux from BHK cells (46%), GFP-apoAI-containing CM did not promote cholesterol efflux compared to CM from untransduced BAEC (7.8% vs 7.4% efflux), even when present in CM at a concentration similar to that of WT apoAI. Conclusion: GFP-rabbit apoAI protein has reduced expression and secretion (likely due to misfolding/degradation) and does not promote cholesterol efflux (n-terminal GFP may disrupt the phospholipid/cholesterol organization needed for efflux via ABCA1 to this dynamic apoprotein). It will not be useful to investigate mechanisms of atheroprotection by vessel wall-expressed apoAI. Alternatives include use of a smaller tag or expression of human apoAI.

Eptacog beta, a novel recombinant factor VIIa, for the treatment of hemophilia.

Hemophilia A and B are X-linked hereditary bleeding disorders due to factor VIII (FVIII) or factor IX (FIX) deficiency, respectively. Major advancements have been made in the care of patients with hemophilia, yet the development of inhibitors to infused FVIII or FIX continues to be a formidable challenge. The current first-line therapy for acute bleeding episodes in patients diagnosed with inhibitors are bypassing agents including activated prothrombin complex concentrates (aPCCs) and recombinant factor VIIa (rFVIIa). Eptacog beta (SevenFact; LFB Biotechnologies, Hema Biologics) is a new rFVIIa product produced via expression in the milk of transgenic rabbits. This emerging platform has demonstrated numerous cost advantages to traditional cell culture systems including a better ability to scale up production and better protein yields. Eptacog beta is currently approved by the U.S. Food and Drug Administration (FDA) for the on-demand control of bleeding episodes in patients with hemophilia aged 12 to 75 with inhibitors. A potential future expansion of its current label could occur given the recent completion of two major phase III clinical trials evaluating its efficacy in children as well as its use for perioperative management. In this paper, we describe the preclinical and clinical literature documenting the development of eptacog beta and discuss its current and future application for the management of patients with hemophilia and inhibitors.

Fat-1 expression alleviates atherosclerosis in transgenic rabbits.

Atherosclerosis is the main cause of cardiovascular diseases. The Fat‐1 gene can express the n‐3 fatty acid desaturase, which converts n‐6 polyunsaturated fatty acids (PUFA) to n‐3 PUFAs. The role of n‐3 PUFAs in atherosclerosis is widely debated. This study explored the effect of n‐3 PUFAs on atherosclerosis in rabbits. In this study, atherosclerosis was induced in Fat‐1 transgenic rabbits and their littermate (WT) rabbits by feeding a high‐cholesterol diet containing 0.3% cholesterol and 3% soybean oil for 16 weeks. Plasma lipid, fatty acid and pathological analyses of atherosclerotic lesions were conducted. Fatty acid composition in the liver and muscle showed that n‐3 PUFAs increased and n‐6 PUFAs decreased in the Fat‐1 group. Plasma high‐density lipoprotein cholesterol (HDL‐C) levels were significantly increased in the Fat‐1 group, and the atherosclerotic lesion area of the aortic arch in Fat‐1 transgenic rabbits was significantly reduced. Histological analysis showed that smooth muscle cells (SMCs) in atherosclerotic lesions decreased significantly. In conclusion, n‐3 PUFAs improve atherosclerosis in Fat‐1 transgenic rabbits, and this process may depend on the increase in plasma HDL‐C and the decrease in the amount of SMCs in atherosclerotic plaques.

Identification, purification, and pharmacological activity analysis of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAα1) expressed in transgenic rabbit mammary glands.

Desmodus rotundus plasminogen activator alpha 1(DSPAα1) is a thrombolytic protein with advantages, such as a long half-life, high accuracy and specificity for thrombolysis, wide therapeutic window, and no neurotoxicity. To date, DSPAα1 has only been expressed in the Chinese hamster ovary, insect cells, transgenic tobacco plants, and Pichia pastoris. To the best of our knowledge, we are the first to report the expression of DSPAα1 in transgenic rabbit mammary glands, extract the product, and analyze its pharmacology activity. An efficient mammary gland-specific expression vector pCL25/DSPAα1 was transferred to prokaryotic zygotes in rabbits by microinjection to generate six DSPAα1 transgenic rabbits. The recombinant DSPAα1 (rDSPAα1) expression in transgenic rabbit milk was 1.19 ± 0.26mg/mL. The rDSPAα1 purification protocol included pretreatment, ammonium sulfate precipitation, benzamidine affinity chromatography, cation exchange chromatography, and Cibacron blue affinity chromatography; approximately 98% purity was achieved using gel electrophoresis. According to sequencing results, the primary structure of rDSPAα1 was consistent with the theoretical design sequence, and its molecular weight was consistent with that of the natural protein. N-terminal sequencing results indicated rDSPAα1 to be a mature protein, as the goat signal peptide sequence of the expression vector was no longer detected. The fibrinolytic activity of rDSPAα1 was estimated to be 773,333IU/mg. Fibrin-agarose plate assay and in vitro rat blood clot degradation assay showed that rDSPAα1 had strong thrombolytic activity. In conclusion, we report recombinant DSPAα1 with high thrombolytic activity expressed in transgenic rabbit mammary glands.

Genetically modified animals for use in biopharmacology: from research to production

&#x0D; &#x0D; Introduction: In this review, the analysis of technologies for obtaining biologically active proteins from various sources is carried out, and the comparative analysis of technologies for creating producers of biologically active proteins is presented. Special attention is paid to genetically modified animals as bioreactors for the pharmaceutical industry of a new type. The necessity of improving the technology of development transgenic rabbit producers and creating a platform solution for the production of biological products is substantiated.&#x0D; The advantages of using TrB for the production of recombinant proteins: The main advantages of using TrB are the low cost of obtaining valuable complex therapeutic human proteins in readily accessible fluids, their greater safety relative to proteins isolated directly from human blood, and the greater safety of the activity of the native protein.&#x0D; The advantages of the mammary gland as a system for the expression of recombinant proteins: The mammary gland is the organ of choice for the expression of valuable recombinant proteins because milk is easy to collect in large volumes.&#x0D; Methods for obtaining transgenic animals: The modern understanding of the regulation of gene expression and the discovery of new tools for gene editing can increase the efficiency of creating bioreactors for animals and help to obtain high concentrations of the target protein.&#x0D; The advantages of using rabbits as bioreactors producing recombinant proteins in milk: The rabbit is a relatively small animal with a short duration of gestation, puberty and optimal size, capable of producing up to 5 liters of milk per year per female, receiving up to 300 grams of the target protein.&#x0D; &#x0D; &#x0D;