Cardiac Gene Therapy Research Articles

Abstract 4135301: Cardiac specific gene therapy to treat diabetic heart failure with preserved ejection fraction

Background: We have previously demonstrated that diabetes mellitus (DM) causes cardiac inflammation leading to heart failure with preserved ejection fraction (HFpEF). Arachidonate 5-lipoxygenase (5-LOX), encoded by the ALOX5 gene, is an enzyme that is highly expressed in leukocytes. 5-LOX synthesizes specialized pro-resolving mediators (SPMs) including Resolvin D1 (RvD1), which reduce inflammation. Hypothesis: We hypothesize that cardiac-specific ALOX5 upregulation could prevent diabetes-associated HFpEF without inducing systemic immunosuppression. Methods: DM was induced by feeding mice with a high fat diet (HFD, 60% fat by kcal) starting from 6 weeks of age for 22-24 weeks. At 24 weeks of age, DM mice were treated with RvD1, by intraperitoneal injection at 100 ng/mouse for 2 weeks. To specifically upregulate 5-LOX in the heart, another cohort of DM mice were injected with either AAV9-plain or AAV9- ALOX5 viral vectors under the control of the α-myosin heavy chain (MHC) at 22 weeks old. At 28 weeks, we performed echocardiographic measurement of diastolic function and heart extraction. Results: HFD-induced DM mice showed lower cardiac 5-LOX expression (0.72 ± 0.05 a.u. vs. 1.09 ± 0.12, p < 0.05) and RvD1 levels (1 ± 0.08 vs. 1.9 ± 0.14 fold change over DM mice, p<0.0001) than the mice with normal diet. RvD1 treatment significantly improved DM-associated diastolic function (19.7 ± 1.4 a.u. vs. 22.9 ± 0.4, p<0.05). With cardiac specific upregulation of 5-LOX, cardiac 5-LOX expression (0.89 ± 0.06 vs. 0.70 ± 0.04 a.u., p<0.05) and RvD1 levels were increased (2.1 ± 0.3 a.u. vs. 1.2 ± 0.2, normalized to AAV9-plain, p<0.05). AAV9- ALOX5 also reduced NLRP3 inflammasome activity (0.80 ± 0.02 a.u. vs. 0.90 ± 0.02, p<0.01) and improved DM-associated diastolic dysfunction (17.0 ± 1.2 a.u. vs. 20.5 ± 1.0, p<0.05) without affecting glucose metabolism and obesity in DM mice. Moreover, no alterations of cardiac macrophage infiltration, inflammatory IL1β signaling, or macrophage phenotype were observed in response to cardiac-specific ALOX5 upregulation. Conclusion: In conclusion, both direct RvD1 treatment and cardiac-specific gene therapy to enhance resolution of inflammation in cardiomyocytes reversed DM-associated HFpEF. Gene therapy avoided off-target immunosuppression. Therefore, AAV-directed cardiac ALOX5 upregulation represents a novel cardiac-specific gene therapy for HFpEF that avoids systemic immunosuppression.

Gene therapy for cardiac diseases: methods, challenges, and future directions.

Gene therapy is advancing at an unprecedented pace, and the recent success of clinical trials reinforces optimism and trust among the scientific community. Recently, the cardiac gene therapy pipeline, which had progressed more slowly than in other fields, has begun to advance, overcoming biological and technical challenges, particularly in treating genetic heart pathologies. The primary rationale behind the focus on monogenic cardiac diseases is the well-defined molecular mechanisms driving their phenotypes, directly linked to the pathogenicity of single genetic mutations. This aspect makes these conditions a remarkable example of 'genetically druggable' diseases. Unfortunately, current treatments for these life-threatening disorders are few and often poorly effective, underscoring the need to develop therapies to modulate or correct their molecular substrates. In this review we examine the latest advancements in cardiac gene therapy, discussing the pros and cons of different molecular approaches and delivery vectors, with a focus on their therapeutic application in cardiac inherited diseases. Additionally, we highlight the key factors that may enhance clinical translation, drawing insights from previous trials and the current prospects of cardiac gene therapy.

Poor in vivo transduction of myocardium by lentiviral vectors is likely caused by a post-entry block of transduction

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Stichting LSH-TKI Introduction In cardiac gene therapy, two prototypic viral vectors are typically considered for long-term transgene expression, i.e. lentivirus (LV) and adeno-associated virus (AAV) vectors. Compared to AAV vectors, LV vectors have the advantages of a larger insert capacity and the ability to confer permanent transgene expression due to integration of the vector genome in the host cell’s chromosomal DNA . However, standard VSV G protein-pseudotyped LV vectors (VSV G-LV vectors) has shown suboptimal transduction of cardiac myocytes in vivo. Purpose In this study, we aimed to improve LV-mediated transduction of cardiomyocytes in vivo by evaluating LV vectors carrying various different envelopes and compared the most potent variants with AAV1 capsid-pseudotyped AAV2 vectors (AAV2/1 vectors). Methods We made a panel of LV vectors pseudotyped with envelope proteins from 76 different viruses carrying an expression cassette for enhanced green fluorescent protein (eGFP). Transduction efficiencies in neonatal rat ventricular myocytes (NRVMs), human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cardiomyocyte progenitor cells (CMPCs) were determined by fluorescence-activated cell sorting (FACS) as percentage eGFP-positive cells. Male NOD -SCID mice aged 3-6 months were injected directly into the left ventricular myocardium with eGFP-encoding VSV G-LV and AAV2/1 vectors at doses of 1.4 x 10^7 infectious units and 2.0 x 10^10 genome copies, respectively, or PBS as a control. Mice were sacrificed one and four weeks after surgery. Per heart, left ventricular and eGFP-positive area was determined from 40 – 50 slides and the ratio was used as a measure of transduction efficiency. Results Of the panel of pseudotyped LVs none was able to consistently outperform VSV G-LV vectors, which reached transduction efficiencies in vitro of 58% in NRVMs to 96% in hiPSC-CMs and 100% in CMPCs . In AAV2/1 vector-injected hearts, the eGFP expressing area in the left ventricle increased from 1.07% in week 1 to 5.42% in week 4. In contrast, eGFP expression in LV-injected hearts was 0.31% and 0.16% after 1 week and 4 weeks, respectively (figure 1). Conclusion In vitro screening of a panel of pseudotyped LV vectors showed standard VSV G-LV vectors as top performer. Despite encouraging in vitro results and previously reported findings, VSV G-LV vectors was unable to provide for robust in vivo transduction. The observation that none of the 76 different envelopes yielded a meaningful improvement of lentiviral transduction rates together with the discrepancy between in vitro and in vivo transduction efficiencies, suggest that a post-entry block of transduction likely represents an important component of the poor in vivo performance.Figure 1in vivo transduction

Transcriptomic insights into cardiac healing - unveiling promising targets for effective gene therapies in diseased hearts

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation Identifying therapeutical target genes is the first step towards developing new gene therapies to promote cardiac repair after injury. In the last decades, despite the many steps forward, not a single therapy has shown to be effective and suitable for human treatment of the injured heart. Considering that cardiac diseases represent one of the most prevalent causes of morbidity, disability, and mortality in the world, it is, therefore, essential to develop new and improved therapies to induce cardiac regeneration. In this study, we aimed to identify novel therapeutical targets that could be used for future purposes of cardiac gene therapy. Unlike previous studies that investigated fetal hearts that are physiologically and functionally different from mature hearts, we focused on investigating adult human hearts that we collected from human donors with post-mortem time below 5 hours. Samples of left ventricles were molecularly and morphologically characterized for the presence of stress, cardiac damage, and cardiac fibrosis. Results of the molecular analyses were cross-referenced with the donors' medical history to classify each specimen as "control" or "diseased." Next, we performed single nuclei RNA sequencing of samples of the left ventricle from 2 "control" and 3 "diseased" hearts. Due to the adult tissue's complexity and the specimen's high-fiber content, we optimized the nuclei isolation protocol. Eventually, we successfully sequenced an average of 8.000 nuclei per sample with a gene coverage of approximately 800 genes per nucleus, identifying, via unsupervised cluster analysis, 17 different cell populations. In cardiomyocytes, we identified over 500 genes differentially expressed in diseased hearts when compared to healthy controls. Among these genes, we discovered several novel target genes whose expression is increased only in diseased cardiomyocytes. Gene ontology analysis of the known interactors of the newly identified target genes shows enrichment in terms such as sarcomere organization, muscle contraction, mitochondrial energetics, calcium handling, and fatty acid metabolism. Currently, we are investigating the molecular effects of the gain and loss of function of these genes in healthy iPS-derived cardiomyocytes and human-derived myocardial tissue slices to better understand their role in the pathophysiology of the adult human heart. From a therapeutic perspective, our ongoing research holds promises for developing new and improved intervention therapies to promote cardiac regeneration.

Gene Therapy for Cardiovascular Disease: Clinical Perspectives.

Cardiovascular disease (CVD) stands as one of the leading causes of death in the United States, with its prevalence steadily on the rise. Traditional therapeutic approaches, such as pharmacological treatment, cardiovascular intervention, and surgery, have inherent limitations. In response to these challenges, cardiac gene therapy has emerged as a promising alternative for treating CVD patients. However, several obstacles persist, including the low efficiency of gene transduction, immune reactions to vectors or transduced cells, and the occurrence of off-target effects. While preclinical research has demonstrated significant success in various CVD model in both small and large animals, the translation of these findings to clinical applications has, for the most part, yielded disappointing results, except for some early, albeit small, trials. This review aims to provide a comprehensive summary of recent preclinical and clinical studies on gene therapy for various CVDs. Additionally, we discuss the existing limitations and challenges that hinder the widespread clinical application of cardiac gene therapy.

Abstract 18864: A Novel Technique to Improve Vector Uptake for Cardiac Gene Therapy

Introduction: Cardiac gene therapy with adeno-associated virus (AAV) has promise. However, clinical trials with intracoronary (IC) delivery have been challenged by low efficacy. Hypothesis: We predicted that AAV uptake could be enhanced using a catheter-based mechanical cardiac support device (MCS) to facilitate IC delivery with brief coronary balloon occlusions in a pig model of myocardial infarction (MI). Aims: We aimed to: 1) confirm that MCS could support hemodynamics during coronary balloon occlusions, and 2) determine whether this technique could improve vector uptake and cardiac gene expression after HF onset. Methods: A week after MI, 12 Yorkshire pigs (36.6±1.45 kg, male n=6) received AAV6 encoding for luciferase by one of four delivery methods (n=3/group) (Figure 1). Tissues were harvested 1 mo later for analysis. A similar experiment was conducted with gold nanoparticles (GNPs) in place of AAV by either continuous or CAO+CSO delivery (n=4) with same-day cardiac harvest. GNP presence was seen by electron microscopy. Results: MCS maintained blood pressure and prevented arrhythmias during coronary balloon occlusions. Application of MCS during continuous infusion did not significantly alter AAV genome uptake (vg) or luciferase expression (luc). CAO+CSO delivery increased both vg and luc across heart regions, with notable significance at the infarct-border region vs continuous delivery (vg: p=0.027; luc: p=0.032). Vg was also positively associated with luc (Figure 2). Improved uptake was further supported by greater GNP presence with CAO+CSO delivery. Conclusion: MCS can be utilized to optimize IC gene delivery in a safe and clinically-applicable way.

Cardiac gene therapy treats diabetic cardiomyopathy and lowers blood glucose.

Diabetic cardiomyopathy, an increasingly global epidemic and a major cause of heart failure with preserved ejection fraction (HFpEF), is associated with hyperglycemia, insulin resistance, and intracardiomyocyte calcium mishandling. Here we identify that, in db/db mice with type 2 diabetes-induced HFpEF, abnormal remodeling of cardiomyocyte transverse-tubule microdomains occurs with downregulation of the membrane scaffolding protein cardiac bridging integrator 1 (cBIN1). Transduction of cBIN1 by AAV9 gene therapy can restore transverse-tubule microdomains to normalize intracellular distribution of calcium-handling proteins and, surprisingly, glucose transporter 4 (GLUT4). Cardiac proteomics revealed that AAV9-cBIN1 normalized components of calcium handling and GLUT4 translocation machineries. Functional studies further identified that AAV9-cBIN1 normalized insulin-dependent glucose uptake in diabetic cardiomyocytes. Phenotypically, AAV9-cBIN1 rescued cardiac lusitropy, improved exercise intolerance, and ameliorated hyperglycemia in diabetic mice. Restoration of transverse-tubule microdomains can improve cardiac function in the setting of diabetic cardiomyopathy and can also improve systemic glycemic control.

Improved Cardiac Function in Postischemic Rats Using an Optimized Cardiac Reprogramming Cocktail Delivered in a Single Novel Adeno-Associated Virus.

Cardiac reprogramming is a technique to directly convert nonmyocytes into myocardial cells using genes or small molecules. This intervention provides functional benefit to the rodent heart when delivered at the time of myocardial infarction or activated transgenically up to 4 weeks after myocardial infarction. Yet, several hurdles have prevented the advancement of cardiac reprogramming for clinical use. Through a combination of screening and rational design, we identified a cardiac reprogramming cocktail that can be encoded in a single adeno-associated virus. We also created a novel adeno-associated virus capsid that can transduce cardiac fibroblasts more efficiently than available parental serotypes by mutating posttranslationally modified capsid residues. Because a constitutive promoter was needed to drive high expression of these cell fate-altering reprogramming factors, we included binding sites to a cardiomyocyte-restricted microRNA within the 3' untranslated region of the expression cassette that limits expression to nonmyocytes. After optimizing this expression cassette to reprogram human cardiac fibroblasts into induced cardiomyocyte-like cells in vitro, we also tested the ability of this capsid/cassette combination to confer functional benefit in acute mouse myocardial infarction and chronic rat myocardial infarction models. We demonstrated sustained, dose-dependent improvement in cardiac function when treating a rat model 2 weeks after myocardial infarction, showing that cardiac reprogramming, when delivered in a single, clinically relevant adeno-associated virus vector, can support functional improvement in the postremodeled heart. This benefit was not observed with GFP (green fluorescent protein) or a hepatocyte reprogramming cocktail and was achieved even in the presence of immunosuppression, supporting myocyte formation as the underlying mechanism. Collectively, these results advance the application of cardiac reprogramming gene therapy as a viable therapeutic approach to treat chronic heart failure resulting from ischemic injury.

Site-specific prolongation of repolarization prevents postmyocardial infarction tachycardia.

Site-specific prolongation of repolarization prevents postmyocardial infarction tachycardia.

PO-02-244 AAV6-MEDIATED GFP GENE TRANSFER INDUCES TACHYARRHYTHMIAS IN A PORCINE MODEL OF AV BLOCK

Green fluorescent protein (GFP) is a reporter widely utilized to evaluate gene transfer efficiency and used as control in therapeutic overexpression strategies. Although it is frequently considered to be an innocuous protein, there is also evidence indicating interference with contractile function and electrophysiological properties, potentially leading to cardiac dilation and functional deterioration. Cognizant of these possible limitations, we considered GFP gene transfer to provide a valuable starting point to evaluate local gene transfer and its potential deleterious effects. To evaluate gene transfer efficiency and potential pro-arrhythmic effects in a porcine model of complete atrioventricular block (CAVB). A total of eight pigs were implanted with a pacemaker, after which a radiofrequency ablation CAVB was generated. Four weeks after induction of the model the animals received catheter-based intramyocardial injections of saline, AAV6-CMV-GFP or were not injected at all. All animals were then observed for four more weeks. Monitoring included weekly pacemaker interrogations. Ventricular arrhythmias were assessed by means of increase in maximum heart rates and pacemaker dependency. Four weeks post-ablation all pigs displayed maximal heart rates of 75 ± 16,9 bpm which notably increased in AAV6-CMV-GFP-injected animals two weeks post-treatment (240 ± 10 bpm; Figure A), but not in the control animals (n = 5, 78 ± 29,5 bpm; Figure A). Electronically paced beats showed a remarkable reduction in transduced animals two weeks post-injection (40 ± 37,36 %; Figure B). Although GFP is commonly used in gene transfer studies, our experiments reveal the occurrence of ventricular arrhythmias after treatment. Mechanistically, these arrhythmias could be triggered by local tissue damage stemming from a possible inflammatory response exacerbated by a reaction to the capsid and transgene. As a result, locally injected AAV6-CMV-GFP should not be used as a control transgene in large animal studies to validate cardiac gene therapies.

Progress in Clinical Gene Therapy for Cardiac Disorders.

Despite significant advances in novel treatments and approaches, cardiovascular disease remains the leading cause of death globally. Gene therapy is a promising option for many diseases, including cardiovascular diseases. In the last 30 years, gene therapy has slowly proceeded towards clinical translation and recently reached US Food and Drug Administration approval for several diseases such as Leber congenital amaurosis and spinal muscular atrophy, among others. Previous attempts at developing gene therapies for cardiovascular diseases have yielded promising results in preclinical studies and early-phase clinical trials. However, larger trials failed to demonstrate consistent benefits in patients with ischemic heart disease and heart failure. In this review, we summarize the history and current status of clinical cardiac gene therapy. Starting with angiogenic gene therapy, we also cover more recent gene therapy trials for heart failure and cardiomyopathies. New programs are actively vying to be the first to get Food and Drug Administration approval for a cardiac gene therapy product by taking advantage of novel techniques.

Beneficial effects of a cardiac gene therapy with phosphodiesterase pde2a in a mouse model of heart failure

Beneficial effects of a cardiac gene therapy with phosphodiesterase pde2a in a mouse model of heart failure

Abstract P2100: Scalable Ultra-purification Of Adeno-associated Viral Vectors - A Novel Standard To Boost Transduction Efficacy And Potency For Cardiac Gene Therapy Development

Introduction: A high transduction efficacy and potency of AAV-based cardiac gene therapies is key for the clinical translation in which hereditary and acquired cardiac disorders will be targeted. As such, ultrapure vectors with superior biological and therapeutic capabilities are a must for these therapies. Hypothesis: We hypothesized that our developed affinity chromatography (AC) based purification system will increase AAV recovery and - potency. Methods: AAV vector production, AC based - or iodixanol density gradient (DG) based purification, Q-PCR, WB, EM, LC-MS/MS, P-loop Results: Using the same vector input quantity, the AC-based purification enabled an approximately 13-fold greater vector genome copy (vgc) recovery than the DG-based purification. Mass spectrometry analysis demonstrated ultrapure AAV9 vectors as a result of an AC purification (AAV9 93.24% vs. 6,76% contaminants) whereas corresponding DG preparations resulted in highly contaminated vectors (AAV9 5.49% vs. 94,51% contaminants). Biological potency of AC- and DG-purified AAV9 vectors towards cardiac transduction were determined by systemic injections of 1·10 10 , 1·10 11 or 1·10 12 vgc of AAV9-EGFP in C57BL/6 mice (n=8 each group). AC-purified AAVs achieved a significantly higher cardiac transduction efficacy for every dosage assessed by comparative bulk myocardial DNA, RNA and protein level analysis after 2 weeks. Therapeutic potency was examined for a recently published novel target for chronic heart failure namely the RFXP1-RLN system. To this end, a dosage of 5·10 11 vgcs of either AC- or DG purified AAV9-RXFP1 vectors were systemically injected and the cardiac contractile performance increase was captured after 2 weeks in mice of both groups. Of note, 10 minutes after RLN administration, the rise in LV +dp/dt max. was already significantly greater in the AC- than the DG-vector treated group (AC: 13594+/-1972 vs. DC: 9822+/-801 mmHg/s; n=8 per group, p<0.01). Conclusion: The data clearly promote AC-based AAV purification as a novel standard for cardiovascular basic and translational research. Higher consistency in results, higher therapeutic effects and superior biological potency can be expected from higher production yields of ultrapure AAVs even at lower vector dosages.

Abstract P3041: DWORF Overexpression In The Heart Results In Enhanced Mitochondrial Function

Background: Calcium (Ca 2+ ) plays a pivotal role in regulating physiological processes in the heart including excitation-contraction coupling, Ca 2+ signaling, and mitochondrial function. It is well established that both Ca 2+ dysregulation and altered cardiac energy metabolism are universal features of heart failure (HF). Dwarf open reading frame (DWORF), a newly discovered microprotein, increases SERCA2a activity in part by relieving SERCA2a inhibition by phospholamban (PLN). DWORF overexpression via cardiac specific transgene or gene therapy is cardioprotective in genetic and experimental HF mouse models. In addition to activation of SERCA2a by PLN displacement, preliminary data indicates that enhanced mitochondrial function contributes to the cardioprotective effects of DWORF. Here we tested the effects of DWORF overexpression on cardiac mitochondrial function and Ca 2+ dynamics. Methods and Results: We isolated mitochondria from wildtype (WT) and DWORF transgenic (Tg) mouse hearts and measured O 2 consumption rates using a Seahorse mito stress test. We found that DWORF Tg mice have increased respiratory capacity compared to WT (DWORF Tg 1150.0 pmol/min, WT 799.4 pmol/min +/-146.2) and enhanced spare respiratory capacity (DWORF Tg 489.1 pmol/min, WT 229.4 pmol/min +/- 102.1). To measure mitochondrial Ca 2+ dynamics, we performed Ca 2+ retention capacity assays and found that DWORF Tg mice have increased Ca 2+ uptake rates compared to WT (DWORF Tg 32.32 s, WT 46.46 s +/- 4.39). Western blot analysis of the mitochondrial Ca 2+ uniporter (MCU) in mito extracts from WT and DWORF Tg hearts indicated elevated levels of MCU. However, electron transport chain (ETC) protein expression indicated no significant differences, indicating the enhanced mitochondrial activity observed in DWORF Tg mice was not due to increased ETC levels. Conclusions: DWORF Tg mouse heart mitochondria exhibit increased maximal respiration rates and enhanced spare respiratory capacity. However, ETC protein levels are unchanged, indicating altered enzyme activity rather than expression. Additionally, mitochondria from DWORF Tg hearts uptake Ca 2+ faster into the mitochondria through the increased levels of MCU, suggesting enhanced mitochondrial Ca 2+ dynamics.

Novel Designed Proteolytically Resistant VEGF-B186R127S Promotes Angiogenesis in Mouse Heart by Recruiting Endothelial Progenitor Cells.

Background: Previous studies have indicated that vascular endothelial growth factor B186 (VEGF-B186) supports coronary vascular growth in normal and ischemic myocardium. However, previous studies also indicated that induction of ventricular arrhythmias is a severe side effect preventing the use of VEGF-B186 in cardiac gene therapy, possibly mediated by binding to neuropilin 1 (NRP1). We have designed a novel VEGF-B186 variant, VEGF-B186R127S, which is resistant to proteolytic processing and unable to bind to NRP1. Here, we studied its effects on mouse heart to explore the mechanism of VEGF-B186-induced vascular growth along with its effects on cardiac performance. Methods: Following the characterization of VEGF-B186R127S, we performed ultrasound-guided adenoviral VEGF-B186R127S gene transfers into the murine heart. Vascular growth and heart functions were analyzed using immunohistochemistry, RT-PCR, electrocardiogram and ultrasound examinations. Endothelial progenitor cells (EPCs) were isolated from the circulating blood and characterized. Also, in vitro experiments were carried out in cardiac endothelial cells with adenoviral vectors. Results: The proteolytically resistant VEGF-B186R127S significantly induced vascular growth in mouse heart. Interestingly, VEGF-B186R127S gene transfer increased the number of circulating EPCs that secreted VEGF-A. Other proangiogenic factors were also present in plasma and heart tissue after the VEGF-B186R127S gene transfer. Importantly, VEGF-B186R127S gene transfer did not cause any side effects, such as arrhythmias. Conclusion: VEGF-B186R127S induces vascular growth in mouse heart by recruiting EPCs. VEGF-B186R127S is a novel therapeutic agent for cardiac therapeutic angiogenesis to rescue myocardial tissue after an ischemic insult.

Gab1 Overexpression Alleviates Doxorubicin-Induced Cardiac Oxidative Stress, Inflammation, and Apoptosis Through PI3K/Akt Signaling Pathway.

Grb2-associated binding protein 1 (Gab1), an intracellular scaffolding adaptor, was involved in several cardiovascular diseases. However, the role of Gab1 in doxorubicin (DOX)-induced cardiotoxicity remains largely unknown. The present study investigated whether Gab1 protected against DOX-induced cardiotoxicity and the underlying mechanism. We overexpressed Gab1 in the hearts using an adeno-associated virus 9 system through tail vein injection. C57BL/6 mice were subjected to DOX (15 mg/kg/d, i.p.) to generate DOX-induced cardiotoxicity. Echocardiography, histological analysis, immunofluorescence and enzyme-linked immunosorbent assay (ELISA) kits, Western blotting, and quantitative real-time polymerase chain reaction (PCR) evaluated DOX-induced cardiotoxicity and the underlying mechanisms. Myocardial Gab1 protein and messenger RNA (mRNA) levels were markedly decreased in DOX-administered mice. Overexpression of Gab1 in myocardium significantly improved cardiac function and attenuated cardiac oxidative stress, inflammatory response, and apoptosis induced by DOX. Mechanistically, we found that PI3K/Akt signaling pathway was downregulated after DOX treatment, and Gab1 overexpression activated PI3K/Akt signaling pathway, whereas PI3K/Akt signaling pathway inhibition abolished the beneficial effect of Gab1 overexpression in the heart. Collectively, our results indicated that Gab1 is essential for cardioprotection against DOX-induced oxidative stress, inflammatory response, and apoptosis by mediating PI3K/Akt signaling pathway. And cardiac gene therapy with Gab1 provides a novel therapeutic strategy against DOX-induced cardiotoxicity.

Cardiac gene therapy with PDE2A limits ventricular remodeling, dysfunction and arrhythmias promoted in mice by chronic infusion with catecholamines

Chronic ß-AR activation is detrimental because it promotes cardiac remodeling and leads to heart failure (HF). Multiple cyclic nucleotide phosphodiesterases (PDEs) finely tune ß-AR responses by degrading and compartmentalizing cAMP. PDE2A is upregulated in HF, and PDE2A-transgenic mice are protected against catecholamine-induced arrhythmias. Since chronic treatment with PDE inhibitors increases mortality in HF and PDE2 overexpression seems cardioprotective, we postulated that decreasing cAMP levels by overexpressing PDE2A in the heart using gene therapy may have therapeutic effects. C57BL/6N male mice were injected with serotype 9 adeno-associated viruses (AAV9, 10e12 viral particles) encoding for PDE2A, or luciferase (LUC). Two weeks later, mice were implanted with osmotic pumps infusing NaCl or isoprenaline (Iso) and phenylephrine (IP, 30 mg/kg/day each) for 2 weeks. Cardiac function was assessed by echocardiography, susceptibility to arrhythmias by catheter-mediated ventricular pacing after Iso (1.5 mg/kg) and atropine (1 mg/kg) injection. Ventricular cardiomyocytes were isolated, loaded with 1 μM Fura-2AM and stimulated at 1 Hz to record calcium transients (CaT), sarcomere shortening (SS) and pro-arrhythmogenic spontaneous calcium waves (SCW). In LUC mice, IP treatment increased left ventricular weight over body weight ratio by 35 ± 5.7% ( P = 0.0001) and decreased ejection fraction by 29 ± 2.4% ( P < 0.0001). Both parameters were improved in animals subjected to AAV9-PDE2A injection ( P = 0.039 and P = 0.0003, respectively) increasing by ∼10 fold PDE2A levels. Ventricular tachycardias were triggered in IP-LUC mice ( P = 0.016) but not in animals treated with AAV9-PDE2A. In isolated myocytes from control mice, ß-AR stimulation (3 nM Iso) increased SS by 355% and CaT by 86% ( P < 0.0001). These inotropic effects were blunted in cells from IP treated animals with SS increasing by 157% ( P = 0.0002, vs. LUC-NaCl) and CaT by only 55% ( P = 0.06 vs. IP-LUC at baseline). However, the Iso effects were preserved in cardiomyocytes from animals injected with AAV9-PDE2A (SS: P < 0.0001, CaT: P < 0.0001 vs. IP-PDE2A at baseline), which exhibited less SCW upon maximal ß-AR stimulation (Iso, 100 nM, P = 0.02 vs. IP-LUC). Our results suggest that gene therapy with PDE2A limits cardiac hypertrophy and dysfunction induced by catecholamines as well as ventricular arrhythmias.

Cas9/AAV9-Mediated Somatic Mutagenesis Uncovered the Cell-Autonomous Role of Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2 in Murine Cardiomyocyte Maturation.

Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) is a key player in cardiomyocyte calcium handling and also a classic target in the gene therapy for heart failure. SERCA2 expression dramatically increases during cardiomyocyte maturation in the postnatal phase of heart development, which is essential for the heart to acquire its full function in adults. However, whether and how SERCA2 regulates cardiomyocyte maturation remains unclear. Here, we performed Cas9/AAV9-mediated somatic mutagenesis (CASAAV) in mice and achieved cardiomyocyte-specific knockout of Atp2a2, the gene coding SERCA2. Through a cardiac genetic mosaic analysis, we demonstrated the cell-autonomous role of SERCA2 in building key ultrastructures of mature ventricular cardiomyocytes, including transverse-tubules and sarcomeres. SERCA2 also exerts a profound impact on oxidative respiration gene expression and sarcomere isoform switching from Myh7/Tnni1 to Myh6/Tnni3, which are transcriptional hallmarks of cardiomyocyte maturation. Together, this study uncovered a pivotal role of SERCA2 in heart development and provided new insights about SERCA2-based cardiac gene therapy.

Engineered bacterial voltage-gated sodium channel platform for cardiac gene therapy

Therapies for cardiac arrhythmias could greatly benefit from approaches to enhance electrical excitability and action potential conduction in the heart by stably overexpressing mammalian voltage-gated sodium channels. However, the large size of these channels precludes their incorporation into therapeutic viral vectors. Here, we report a platform utilizing small-size, codon-optimized engineered prokaryotic sodium channels (BacNav) driven by muscle-specific promoters that significantly enhance excitability and conduction in rat and human cardiomyocytes in vitro and adult cardiac tissues from multiple species in silico. We also show that the expression of BacNav significantly reduces occurrence of conduction block and reentrant arrhythmias in fibrotic cardiac cultures. Moreover, functional BacNav channels are stably expressed in healthy mouse hearts six weeks following intravenous injection of self-complementary adeno-associated virus (scAAV) without causing any adverse effects on cardiac electrophysiology. The large diversity of prokaryotic sodium channels and experimental-computational platform reported in this study should facilitate the development and evaluation of BacNav-based gene therapies for cardiac conduction disorders.

Cardiac gene therapy with type 2 phosphodiesterase (PDE2) in experimental heart failure: Complementary or alternative to β–blockers?

Chronic activation of the β-adrenergic (β-AR) pathway induces cardiac remodeling and worsens outcome in heart failure (HF). β-blockers counteract β-AR pathway membranous activation and reduce mortality and morbidity in HF. Various cyclic nucleotide phosphodiesterases (PDEs) finely tune β-AR responses by degrading and compartmentalizing cAMP. Recent results suggest that cardiac PDE overexpression protects against maladaptive remodeling induced by chronic catecholamines infusion in mice. To determine if a cardiac PDE gene therapy would treat HF induced by pressure overload in mice, alone or in combination with β-blockers. HF was induced by transverse aortic constriction (TAC) and mice were injected with adeno-associated virus (AAV9 to allow preferential cardiac expression, 10^12 viral particles per mice) encoding PDE2A or the Luciferase (Luc) as a control for cardiac overexpression 1 week after surgery. Metoprolol (Meto) or NaCl were administered via osmotic minipumps at 2 weeks post-surgery during 4 weeks. Four groups were compared: TAC-Luc-NaCl, TAC-PDE2A-NaCl, TAC-Luc-Meto, TAC-PDE2A-Meto. Cardiac function was monitored by serial echocardiography and physical capacity tested by treadmill exhaustion test. Following euthanasia, hearts were harvested for histological and molecular analysis. In a pilot study, healthy mice were treated with Meto (30 mg/kg/day, n = 4) or NaCl ( n = 2). Meto decreases phospholamban phosphorylation, as well as heart rate (−18%) and ejection fraction (−17%). Following this validation, TAC mice with aortic gradient > 60 mmHg were randomized. Both PDE2A-AAV9 injection and Meto were well tolerated. On-going experiments should be completed by July. Preliminary results indicate that cardiac PDE2A gene therapy is well tolerated in mice with HF induced by pressure overload.