RESUMEN
Nonviral mRNA delivery is an attractive therapeutic gene delivery strategy, as it achieves efficient protein overexpression in vivo and has a desirable safety profile. However, mRNA's short cytoplasmic half-life limits its utility to therapeutic applications amenable to repeated dosing or short-term overexpression. Here, we describe a biomaterial that enables a durable in vivo response to a single mRNA dose via an "overexpress and sequester" mechanism, whereby mRNA-transfected cells locally overexpress a growth factor that is then sequestered within the biomaterial to sustain the biologic response over time. In a murine diabetic wound model, this strategy demonstrated improved wound healing compared to delivery of a single mRNA dose alone or recombinant protein. In addition, codelivery of anti-inflammatory proteins using this biomaterial eliminated the need for mRNA chemical modification for in vivo therapeutic efficacy. The results support an approach that may be broadly applicable for single-dose delivery of mRNA without chemical modification.
Asunto(s)
Materiales Biocompatibles , Cicatrización de Heridas , Animales , Técnicas de Transferencia de Gen , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Managing myocardial infarction (MI) to reduce cardiac cell death relies primarily on timely reperfusion of the affected coronary site, but reperfusion itself induces cell death through a toxic, ROS-mediated process. In this study, we determined whether the PrC-210 aminothiol ROS-scavenger could prevent ROS-induced damage in post-MI hearts. In a series of both in vitro and in vivo experiments, we show that: (a) in vitro, PrC-210 was the most potent and effective ROS-scavenger when functionally compared to eight of the most commonly studied antioxidants in the MI literature, (b) in vitro PrC-210 ROS-scavenging efficacy was both immediate (seconds) and long-lasting (hours), which would make it effective in both (1) real-time (seconds), as post-MI or cardiac surgery hearts are reperfused with PrC-210-containing blood, and (2) long-term (hours), as hearts are bathed with systemic PrC-210 after MI or surgery, (c) systemic PrC-210 caused a significant 36% reduction of mouse cardiac muscle death following a 45-minute cardiac IR insult; in a striking coincidence, the PrC-210 36% reduction in cardiac muscle death equals the 36% of the MI-induced cardiac cell death estimated 6 years ago by Ovize and colleagues to result from "reperfusion injury," (d) hearts in PrC-210-treated mice performed better than controls after heart attacks when functionally analyzed using echocardiography, and (e) the PrC-210 ROS-scavenging mechanism of action was corroborated by its ability to prevent >85% of the direct, H2O2-induced killing of neonate cardiomyocytes in cell culture. PrC-210 does not cause the nausea, emesis, nor hypotension that preclude clinical use of the WR-1065/amifostine aminothiol. PrC-210 is a highly effective ROS-scavenger that significantly reduces IR injury-associated cardiac cell death.
Asunto(s)
Diaminas/administración & dosificación , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Sulfhidrilo/administración & dosificación , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Diaminas/farmacología , Modelos Animales de Enfermedad , Peróxido de Hidrógeno/efectos adversos , Masculino , Ratones , Infarto del Miocardio/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitter, the channels desensitize (close). The flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, and 9) to the transmembrane channel domain (M2-M3 loop) are essential for coupling ligand binding to channel gating. Comparing the crystal structures of two bacterial pLGIC homologues, ELIC and the proton-activated GLIC, suggests channel gating is associated with rearrangements in these loops, but whether these motions accurately predict the motions in functional lipid-embedded pLGICs is unknown. Here, using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and functional GLIC channels reconstituted into liposomes, we examined if, and how far, the loops at the ECD/TMD gating interface move during proton-dependent gating transitions from the resting to desensitized state. Loop 9 moves â¼9 Å inward toward the channel lumen in response to proton-induced desensitization. Loop 9 motions were not observed when GLIC was in detergent micelles, suggesting detergent solubilization traps the protein in a nonactivatable state and lipids are required for functional gating transitions. Proton-induced desensitization immobilizes loop 2 with little change in position. Proton-induced motion of the M2-M3 loop was not observed, suggesting its conformation is nearly identical in closed and desensitized states. Our experimentally derived distance measurements of spin-labeled GLIC suggest ELIC is not a good model for the functional resting state of GLIC, and that the crystal structure of GLIC does not correspond to a desensitized state. These findings advance our understanding of the molecular mechanisms underlying pLGIC gating.