Extracellular Vesicle-Encapsulated Adeno-Associated Viruses for Therapeutic Gene Delivery to the Heart.

BACKGROUND: Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a significant challenge to its successful clinical use is preexisting neutralizing antibodies (NAbs), which bind to free AAVs, prevent efficient gene transduction, and reduce or negate therapeutic effects. Here we describe extracellular vesicle-encapsulated AAVs (EV-AAVs), secreted naturally by AAV-producing cells, as a superior cardiac gene delivery vector that delivers more genes and offers higher NAb resistance. METHODS: We developed a 2-step density-gradient ultracentrifugation method to isolate highly purified EV-AAVs. We compared the gene delivery and therapeutic efficacy of EV-AAVs with an equal titer of free AAVs in the presence of NAbs, both in vitro and in vivo. In addition, we investigated the mechanism of EV-AAV uptake in human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and mouse models in vivo using a combination of biochemical techniques, flow cytometry, and immunofluorescence imaging. RESULTS: Using cardiotropic AAV serotypes 6 and 9 and several reporter constructs, we demonstrated that EV-AAVs deliver significantly higher quantities of genes than AAVs in the presence of NAbs, both to human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and to mouse hearts in vivo. Intramyocardial delivery of EV-AAV9-sarcoplasmic reticulum calcium ATPase 2a to infarcted hearts in preimmunized mice significantly improved ejection fraction and fractional shortening compared with AAV9-sarcoplasmic reticulum calcium ATPase 2a delivery. These data validated NAb evasion by and therapeutic efficacy of EV-AAV9 vectors. Trafficking studies using human induced pluripotent stem cell-derived cells in vitro and mouse hearts in vivo showed significantly higher expression of EV-AAV6/9-delivered genes in cardiomyocytes compared with noncardiomyocytes, even with comparable cellular uptake. Using cellular subfraction analyses and pH-sensitive dyes, we discovered that EV-AAVs were internalized into acidic endosomal compartments of cardiomyocytes for releasing and acidifying AAVs for their nuclear uptake. CONCLUSIONS: Together, using 5 different in vitro and in vivo model systems, we demonstrate significantly higher potency and therapeutic efficacy of EV-AAV vectors compared with free AAVs in the presence of NAbs. These results establish the potential of EV-AAV vectors as a gene delivery tool to treat heart failure.

Direct reprogramming induces vascular regeneration post muscle ischemic injury.

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail, termed 7G-modRNA, of four cardiac-reprogramming genes-Gata4 (G), Mef2c (M), Tbx5 (T), and Hand2 (H)-together with three reprogramming-helper genes-dominant-negative (DN)-TGFß, DN-Wnt8a, and acid ceramidase (AC)-to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival, and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.

A novel adenylyl cyclase type 5 inhibitor that reduces myocardial infarct size even when administered after coronary artery reperfusion.

We developed a novel adenylyl cyclase type 5 (AC5) inhibitor, C90, that reduces myocardial infarct size even when administered after coronary reperfusion. This is key, since it is not practical to administer a drug to a patient with myocardial infarction before revascularization, and is one reason why so many prior drugs, which reduced infarct in experimental animals, failed in clinical trials. C90 is the most potent AC5 inhibitor, as exhibited by its IC50 value for AC5 inhibition, which was 5 times lower than the next most potent AC5 inhibitor. C90 reduced cAMP in response to forskolin in wild type mice by 42%, but no longer reduced cAMP in response to forskolin in mice with disruption of AC5, indicating that the mechanism of C90 was specific for AC5 inhibition. Compared with vehicle treatment, C90 reduced infarct size by 64% at a dose of 0.6 mg/kg. Thus, C90 is a novel, selective and potent AC5 inhibitor that reduces infarct size, when delivered after coronary artery reperfusion, rendering it potentially clinically useful. It also reduces beta-adrenergic receptor signaling, which will provide additional benefit to patients with coronary artery disease or heart failure.

Cocaine Constrictor Mechanisms of the Cerebral Vasculature.

Cocaine constriction of the cerebral vasculature is thought to contribute to the ischemia associated with cocaine use. However, the mechanisms whereby cocaine elicits relevant vasoconstriction remain elusive. Indeed, proposed intra- and intercellular mechanisms based on over 3 decades of ex vivo vascular studies are, for the most part, of questionable relevancy due to the generally low contractile efficacy of cocaine combined with the use of nonresistance-type vessels. Furthermore, the significance attached to mechanisms derived from in vivo animal studies may be limited by the inability to demonstrate cocaine-induced decreased cerebral blood flow, as observed in (awake) humans. Despite these apparent limitations, we surmise that the vasoconstriction relevant to cocaine-induced ischemia is elicited by inhibition of dilator and activation of constrictor pathways because of cocaine action on the neurovascular unit (neuron, astrocyte, and vessel) and on vessels outside the unit. Furthermore, previous cocaine exposure, that is, conditions present in human subjects, downregulates and sensitizes these dilator and constrictor pathways, respectively, thereby enhancing constriction to acute cocaine. Identification of specific intra- and intercellular mechanisms requires investigations in the isolated microvasculature and the neurovascular unit from species chronically exposed to cocaine and in which cocaine decreases cerebral blood flow.

Functional ET(A)-ET(B) Receptor Cross-talk in Basilar Artery In Situ From ET(B) Receptor Deficient Rats.

The role of endothelin (ET)(A)-ET(B) receptor cross-talk in limiting the ET(A) receptor antagonist inhibition of ET-1 constriction is revealed by the partial or complete dependency of the ET(A) receptor antagonist inhibition on functional removal of the ET(B) receptor. Although functional removal of the ET(B) receptor is generally accomplished with ET(B) receptor antagonist, a novel approach using rats containing a naturally occurring deletion mutation in the ET(B) receptor [rescued "spotting lethal" (sl) rats; ET(B)(sl/sl)] demonstrated increased ET(A) receptor antagonist inhibition of ET-1 constriction in vena cava. We investigated whether this deletion mutation was also sufficient to remove the ET(B) receptor dependency of the ET(A) receptor antagonist inhibition of ET-1 constriction in the basilar artery. Consistent with previous reports, ET-1 plasma levels were elevated in ET(B)(sl/sl) as compared with ET(B)(+/+) rats. ET(B) receptor antagonist failed to relax the ET-1 constricted basilar artery from ET(B)(+/+) and ET(B)(sl/sl) rats. Relaxation to combined ET(A) and ET(B) receptor antagonist was greater than relaxation to ET(A) receptor antagonist in the basilar artery from ET(B)(+/+) and, unexpectedly, ET(B)(sl/sl) rats. These findings confirm the presence of ET(A)-ET(B) receptor cross-talk in the basilar artery. We speculate that mutant ET(B) receptor expression produced by alternative splicing may be sufficient to allow cross-talk.

Cocaine constriction of rat basilar artery in situ: roles of nitric oxide and endothelin-1.

This study investigated whether cocaine constriction of rat basilar artery in situ is mediated by nitric oxide (NO) inhibition and/or endothelin (ET)-1 release. Cocaine (3-100 µmol/l) concentration-dependently constricted the basilar artery to a maximum of 18%. Nω-nitro-L-arginine (100 µmol/l) was without effect on constriction to 3 and 10 µmol/l cocaine. PD145065 (1 and 10 µmol/l), an ETA/B receptor antagonist, variably and at most partially inhibited the 100 µmol/l cocaine constriction. Capsaicin denervation of sensory nerves innervating the basilar, which contain ET-1 and NO synthase, also failed to influence cocaine constriction. These findings suggest that cocaine constriction of cerebral vessels (1) varies with respect to the involvement of ET-1 release and (2) unlike findings in the peripheral vasculature, the constriction is not mediated by inhibition of NO.

Metabolomic analysis of two different models of delayed preconditioning.

Recently we described an ischemic preconditioning induced by repetitive coronary stenosis, which is induced by 6 episodes of non-lethal ischemia over 3 days, and which also resembles the hibernating myocardium phenotype. When compared with traditional second window of ischemic preconditioning using cDNA microarrays, many genes which differed in the repetitive coronary stenosis appeared targeted to metabolism. Accordingly, the goal of this study was to provide a more in depth analysis of changes in metabolism in the different models of delayed preconditioning, i.e., second window and repetitive coronary stenosis. This was accomplished using a metabolomic approach based on liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques. Myocardial samples from the ischemic section of porcine hearts subjected to both models of late preconditioning were compared against sham controls. Interestingly, although both models involve delayed preconditioning, their metabolic signatures were radically different; of the total number of metabolites that changed in both models (135 metabolites) only 7 changed in both models, and significantly more, p<0.01, were altered in the repetitive coronary stenosis (40%) than in the second window (8.1%). The most significant changes observed were in energy metabolism, e.g., phosphocreatine was increased 4 fold and creatine kinase activity increased by 27.2%, a pattern opposite from heart failure, suggesting that the repetitive coronary stenosis and potentially hibernating myocardium have enhanced stress resistance capabilities. The improved energy metabolism could also be a key mechanism contributing to the cardioprotection observed in the repetitive coronary stenosis and in hibernating myocardium. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".

Modified mRNA Formulation and Stability for Cardiac and Skeletal Muscle Delivery.

Directly injecting naked or lipid nanoparticle (LNP)-encapsulated modified mRNA (modRNA) allows rapid and efficient protein expression. This non-viral technology has been used successfully in modRNA vaccines against SARS-CoV-2. The main challenges in using modRNA vaccines were the initial requirement for an ultra-cold storage to preserve their integrity and concerns regarding unwanted side effects from this new technology. Here, we showed that naked modRNA maintains its integrity when stored up to 7 days at 4 °C, and LNP-encapsulated modRNA for up to 7 days at room temperature. Naked modRNA is predominantly expressed at the site of injection when delivered into cardiac or skeletal muscle. In comparison, LNP-encapsulated modRNA granted superior protein expression but also additional protein expression beyond the cardiac or skeletal muscle injection site. To overcome this challenge, we developed a skeletal-muscle-specific modRNA translation system (skeletal muscle SMRTs) for LNP-encapsulated modRNA. This system allows controlled protein translation predominantly at the site of injection to prevent potentially detrimental leakage and expression in major organs. Our study revealed the potential of the SMRTs platform for controlled expression of mRNA payload delivered intramuscularly. To conclude, our SMRTs platform for LNP-encapsulated modRNA can provide safe, stable, efficient and targeted gene expression at the site of injection.

Multi-Center, Randomized, Double-Blind, Placebo-Controlled, Exploratory Study to Evaluate the Efficacy and Safety of HAD-B1 for Dose-Finding in EGFR Mutation Positive and Locally Advanced or Metastatic NSCLC Subjects Who Need Afatinib Therapy.

Afatinib is a target anticancer drug of the second-generation EGFR TKI type, showing an advantage in treatment effect compared to conventional chemotherapy. However, patients on EGFR-TKI drugs also usually progress after 9 to 13 months according to secondary resistance. HAD-B1 is composed of drugs that are effective against lung cancer. This study is an exploratory study to evaluate the efficacy and safety between dosage groups by conducting a clinical trial in subjects requiring afatinib drug treatment in non-small cell lung cancer with EGFR mutation positive to determine the optimal dosage for HAD-B1 administration. At the final visit compared to before administration, each change in the disease control rate was measured according to the HAD-B1 doses of the test group 1 (972 mg), the test group 2 (1944 mg), and the control group. The efficacy and safety of HAD-B1 were compared and evaluated through sub-evaluation variables. As a result of the study, there was no statistically significant difference in the disease control rate at 12 weeks after dosing, but complete and partial remission were evaluated as 1 patient each in the test group 1, and none in the other groups. There was no statistically significant difference between groups in the sub-evaluation variable. In addition, there was no problem of safety from taking the test drug. However, the initially planned number of subjects was 66, but the number of enrolled subjects was only 14, which may limit the results of this study.

Vasospasm following subarachnoid hemorrhage: evidence against functional upregulation of protein kinase C constrictor pathway.

This study tested the hypothesis that vasospasm due to subarachnoid hemorrhage involves the functional upregulation of protein kinase C. Spasm of the rabbit basilar artery was achieved using a double hemorrhage model, which we previously demonstrated was endothelin-1 dependent. In situ effects of agents were determined by direct measurement of vessel diameter following their suffusion in a cranial window. Chelerythrine, a protein kinase C inhibitor, relaxed the spasm. However, relaxations to chelerythrine were not significantly greater in endothelin-1 constricted spastic vessels initially relaxed with the endothelin converting enzyme inhibitor, phosphoramidon, as compared to endothelin-1 constricted control vessels. These results suggest that subarachnoid hemorrhage induced vasospasm does not involve functional upregulation of protein kinase C.

Vasospasm following subarachnoid hemorrhage: evidence against functional upregulation of Rho kinase constrictor pathway.

This study tested the hypothesis that vasospasm due to subarachnoid hemorrhage involves the functional upregulation of Rho kinase. Spasm of the rabbit basilar artery was achieved using a double hemorrhage model, which we previously demonstrated was endothelin-1 dependent. In situ effects of agents were determined by direct measurement of vessel diameter following their suffusion in a cranial window. Y-27632, a Rho kinase inhibitor, relaxed the spasm. However, relaxations to Y-27632 were not significantly greater in endothelin-1 constricted spastic vessels initially relaxed with the endothelin converting enzyme inhibitor, phosphoramidon, as compared to endothelin-1 constricted control vessels. These results suggest that, at least in the rabbit double subarachnoid hemorrhage model, vasospasm does not involve the functional upregulation of Rho kinase.

Repeated constriction to respiration-induced hypocapnia is not mimicked by isocapnic alkaline solution in rabbit basilar artery in situ.

The purpose of this study was to test whether constriction of the cerebral vasculature in response to respiration-induced hypocapnia was mimicked by isocapnic alkaline solution. Since the regulation of the cerebral vasculature by hypocapnia necessitates vessels to constrict repeatedly in response to hypocapnic challenge, we tested whether repeated challenge with isocapnic alkaline solution was also associated with constriction. In contrast to our previous demonstration that repeated hypocapnic challenge elicited constrictions of similar magnitudes in rabbit basilar artery in situ, repeated challenge with isocapnic alkaline solution resulted in reduced constriction. Constriction to hypocapnia was also reduced following isocapnic alkaline solution. Since we previously demonstrated that constrictions to hypocapnia and isocapnic alkaline solution were endothelin-1 dependent, we tested whether the inhibition of hypocapnia- and isocapnic alkaline solution-induced constrictions following isocapnic alkaline solution was due to reduced endothelin-1 constriction. Endothelin-1 constriction was not reduced following isocapnic alkaline solution. Thus, constriction to isocapnic alkaline solution does not mimic constriction to hypocapnia. The results further suggest that the decreased constriction to isocapnic alkaline solution is due to blockade of endothelin-1 release, and that both hypocapnia and isocapnic alkaline solution share a common step in their endothelin-1 release pathways that can be inhibited by isocapnic alkaline solution.

Sensory nerves and transient receptor potential vanilloid 1 channels in CO(2) regulation of cerebrovascular tone.

This study investigated the involvement of sensory nerves and, in particular, neuronal transient receptor potential vanilloid (TRPV) 1 channels, in the CO(2)-mediated regulation of cerebrovascular tone. Basilar artery diameter and blood flow velocity in the ventral midbrain were determined in a rat cranial window preparation by digital imaging and laser-Doppler flowmetry, respectively. Superfusion of the basilar artery with capsaicin, a selective TRPV1 receptor agonist, caused a transient relaxation, consistent with acute desensitization of neuronal TRPV1 channels. Constriction to respiratory hypocapnia remained unaffected following capsaicin superfusion. Denervation of sensory nerves by repeated capsaicin injection of neonates also did not reduce the respiratory hypocapnia constriction of the basilar artery as well as the decreased flow velocity in the ventral midbrain in adults. These findings suggest that sensory nerves and, in particular, neuronal TRPV1 channels, do not play a role in respiratory hypocapnia constriction and decreased flow, at least in rat basilar artery and ventral midbrain.

EndothelinA-endothelinB receptor cross-talk in rat basilar artery in situ.

The rationale for the therapeutic use of dual as opposed to selective endothelin (ET) receptor antagonists stems in part from cross-talk between the ET(A) and ET(B) receptors. However, whether ET(A)-ET(B) receptor cross-talk is present in the cerebral vasculature is difficult to discern since findings of cross-talk contrast even among the few reports available. Thus, this study tested whether ET(A)-ET(B) receptor cross-talk is present in the rat basilar artery. In an in situ cranial window, 0.1 µM sarafotoxin S6c, an ET(B) receptor agonist, relaxed basilar artery basal tone by 54%. ET-1 (3 nM) in the absence and presence of 10 µM BQ123, an ET(A) receptor agonist, induced 13% contraction and 15% relaxation, respectively. In contrast, the 3-nM ET-1 plateau contraction was relaxed by only ∼50% by 3-10 µM BQ123 and 10 µM BQ610, ET(A) receptor antagonists. N(ω)-nitro-L: -arginine, an NO synthase inhibitor, did not enhance contraction to 3 nM ET-1, suggesting that the partial relaxation of the ET-1 plateau contraction did not involve unmasked endothelial ET(B) receptor-mediated relaxation. The ∼50% ET-1 contraction that remained following ET(A) receptor antagonist was relaxed by 3-10 µM BQ788, consistent with an ET(B) receptor-mediated component of contraction. However, 10 µM BQ788 in the absence of prior ET(A) receptor antagonist did not cause relaxation. Subsequent BQ123 addition in the presence of BQ788 completely relaxed the ET-1 contraction. PD145065 (1 µM), an ET(A/B) receptor antagonist, completely relaxed 3-nM ET-1 contracted vessels in both the absence and presence of BQ123. These findings suggest that the inability of ET(A) receptor antagonist to completely relax the ET-1 plateau contraction in rat basilar artery is due to ET(A)-ET(B) receptor cross-talk.

pCO(2) and pH regulation of cerebral blood flow.

CO(2) serves as one of the fundamental regulators of cerebral blood flow (CBF). It is widely considered that this regulation occurs through pCO(2)-driven changes in pH of the cerebral spinal fluid (CSF), with elevated and lowered pH causing direct relaxation and contraction of the smooth muscle, respectively. However, some findings also suggest that pCO(2) acts independently of and/or in conjunction with altered pH. This action may be due to a direct effect of CSF pCO(2) on the smooth muscle as well as on the endothelium, nerves, and astrocytes. Findings may also point to an action of arterial pCO(2) on the endothelium to regulate smooth muscle contractility. Thus, the effects of pH and pCO(2) may be influenced by the absence/presence of different cell types in the various experimental preparations. Results may also be influenced by experimental parameters including myogenic tone as well as solutions containing significantly altered HCO(3) (-) concentrations, i.e., solutions routinely employed to differentiate the effects of pH from pCO(2). In sum, it appears that pCO(2), independently and in conjunction with pH, may regulate CBF.

GATA4 expression is primarily regulated via a miR-26b-dependent post-transcriptional mechanism during cardiac hypertrophy.

AIMS: GATA4 is a transcription factor that is up-regulated during cardiac hypertrophy and plays a fundamental role in myocyte growth and survival. In this study, we investigate the transcriptional vs. post-transcriptional mechanisms that are involved in regulating GATA4 in the heart during neonatal and pressure overload-induced hypertrophic growth. METHODS AND RESULTS: GATA4 protein is significantly higher during pressure overload-induced (2.9 ± 0.4-fold) and neonatal (6.8 ± 1-fold) hypertrophic growth vs. the normal adult mouse heart. Using RNA polymerase II immunoprecipitation combined with deep sequencing, we confirmed that active transcription of the Gata4 gene remained unchanged during hypertrophy, whereas it was two-fold higher in the neonatal vs. adult heart, commensurate with the mRNA levels. These results suggested a post-transcriptional mode of regulation of its expression, which prompted the identification of a conserved sequence in its 3'-untranslated region that was responsible for reduced translation via miR-26b. Overexpression of miR-26b reduced GATA4-dependent transcription, endothelin-induced hypertrophy, and sensitized the cells to apoptotic insults. Additionally, miR-26b targeted phospholipase C-ß1, which, in turn, inhibited miR-26b expression, creating a double-negative feedback loop. Accordingly, overexpression of miR-26b in the heart inhibited up-regulation of its targets and the development of hypertrophy. However, knockdown of miR-26b is not sufficient for inducing hypertrophy. CONCLUSION: Down-regulation of miR-26b in the heart is required for the up-regulation of GATA4 and the induction of pressure-induced cardiac hypertrophy. The results also underscore the functional relevance of miRNAs in regulating gene expression during cardiac hypertrophy.