A cutaneous gene therapy for cocaine abuse and alcohol co-abuse

项目总结 可卡因和酒精是经常被滥用和经常共用的毒品。可用的药物不会 满足治疗持续的可卡因和酒精滥用、复发和共同滥用的需要。改造后的人类 丁酰胆碱酯酶(HBChE)对可卡因具有很强的催化活性和底物特异性 并能有效降低可卡因对啮齿动物的行为和毒性影响。胰升糖素样肽1 (GLP1)受体激动剂可以减弱可卡因和酒精对啮齿动物的增强作用。两者都有 然而，hBChE和GLP1在体内的半衰期非常短，限制了它们在治疗可卡因滥用方面的潜力 以及与酒精共同滥用。培养的自体表皮移植已在临床上用于治疗大面积皮肤溃疡。 几十年的皮肤创伤。来自工程皮肤祖细胞的皮肤移植提供了 一种新的和理想的方法，长期和有效地提供体内治疗药物。我们已经做出了 开发一种新的小鼠对小鼠皮肤移植方法的关键技术进展 稳定地将工程化的表皮祖细胞导入具有免疫活性的宿主小鼠。我们还有 使用CRISPR技术靶向hBChE基因或多西环素(DOX)诱导的修饰GLP1 将基因(DImGLP1)导入表皮祖细胞。然后我们移植了工程化的hBChE或DImGLP1 细胞分别(GhBChE和GDImGLP1)或一起(GBChEGLP1)进入小鼠。GhBChE和 GDImGLP1小鼠不形成可卡因和乙醇诱导的条件性位置偏爱(CPP) 分别进行了分析。CPP表达后立即移植hBChE细胞可减轻可卡因诱导的脑缺血再灌注损伤 CPP。GBChEGLP1小鼠不发生CPP，表现出较低的可卡因致死率和 乙醇联合给药的小鼠比对照组小鼠更强。这项提议的目标是利用这些优势 测试一种针对可卡因滥用和与酒精共滥用的创新皮肤基因疗法的新平台 老鼠。我们还将评估hBChE和mGLP1预防可卡因和/或酒精滥用的持续时间， 潜在的免疫反应和减少它们的方法。我们希望提出的皮肤基因 治疗要持久、高度特异和有效，个体差异很小。能够进行工程设计 通过表皮祖细胞稳定的体内治疗基因输送系统具有重要意义 因为它有可能减少持续的可卡因滥用以及与酒精的共同滥用和复发 在吸毒者和瘾君子身上。拟议工作将产生很大影响，因为结果将是关键 为开发高度个性化、持久和负担得起的方法奠定基础 打击可卡因滥用和与酒精共同滥用。

PROJECT SUMMARY Cocaine and alcohol are commonly abused and frequently co-abused drugs. Available medications do not meet the needs for treating ongoing cocaine and alcohol abuse, relapse and co-abuse. The modified human butyrylcholinesterase (hBChE) exhibits great catalytic potency and substrate specificity for cocaine hydrolysis and is effective in reducing the behavioral and toxic effects of cocaine in rodents. The glucagon-like peptide 1 (GLP1) receptor agonists can attenuate the reinforcing properties of cocaine and alcohol in rodents. Both hBChE and GLP1 have very short half-lives in vivo, however, limiting their potential in treating cocaine abuse and co-abuse with alcohol. Cultured epidermal autografts have been clinically used for treatment of massive skin wounds for decades. Transplantation of skin grafts derived from engineered skin progenitor cells provides a novel and ideal approach for long-term and efficient delivery of therapeutic agents in vivo. We have made key technical advancement in developing a novel mouse-to-mouse skin transplantation method that allows the stable introduction of engineered epidermal progenitor cells into immunocompetent host mice. We have also used the CRISPR technology to target either an hBChE gene or a doxycycline (dox)-inducible modified GLP1 gene (DImGLP1) into epidermal progenitor cells. We then transplanted the engineered hBChE or DImGLP1 cells into mice either separately (GhBChE and GDImGLP1) or together (GBChEGLP1). GhBChE and GDImGLP1 mice did not develop cocaine- and ethanol-induced conditioned place preference (CPP) respectively. Grafting hBChE cells right after CPP expression attenuated cocaine-induced reinstatement of CPP. GBChEGLP1 mice did not develop CPP and they exhibited a lower lethality rate induced by cocaine and ethanol co-administration than those in control mice. The goal of this proposal is to take advantage of these novel platforms to test an innovative cutaneous gene therapy for cocaine abuse and co-abuse with alcohol in mice. We will also evaluate duration of hBChE and mGLP1 protection against cocaine and/or alcohol abuse, potential immune responses and approaches to reduce them. We expect the proposed cutaneous gene therapy to be long-lasting, highly specific and efficient with little individual variation. To be able to engineer stable in vivo bio-delivery systems for therapeutic genes via epidermal progenitor cells is significant because it holds potential for reducing ongoing cocaine abuse and co-abuse with alcohol and relapse in users and addicts. The proposed work will have a high impact in that results will lay key groundwork for the development of a highly personalized, long-lasting and affordable approach for combating cocaine abuse and co-abuse with alcohol.