Salivary Gland-Based Gene Therapy for Pulmonary Arterial Hypertension

描述（由申请人提供）：本提案的总体目标是设计和测试肺动脉高压（PAH）的基因治疗策略。多环芳烃是一种罕见的、致命的、无法治愈的疾病，如果不及时治疗，从出现症状起的平均生存期为2.8年。在已批准的内皮素受体拮抗剂、磷酸二酯酶抑制剂和前列环素这三类治疗药物中，前列环素是最有效的治疗药物。然而，复杂的给药系统和与目前前列腺素（如前列环素）制剂相关的潜在副作用使一些患者和护理人员不敢使用这类高效药物。该建议需要解决的挑战是需要一种治疗方案，允许患者自身体内内源性生产前列环素治疗，贯穿患者的整个生命周期。由于前列环素可以通过表达前列环素合成酶（PGIS）内源性产生，基因治疗已经在动物模型中通过使内源性产生前列环素和逆转实验性多环芳烃显示了原理性疗效。该提案将建立在这一概念的基础上，但将使用新开发的基因转移技术来执行这一策略，该技术消除了先前研究中使用的病毒基因传递载体。病毒载体在早期研究中非常有用，但表达时间有限，由于宿主免疫反应，不能影响终身治疗，也不能重新给药。我们的基因传递系统使用超声波诱导的微泡空化，允许非病毒DNA载体进入细胞，被认为可以逃避宿主免疫反应，理论上允许在患者整个生命周期内重新给药prostocyclin Synthase治疗性转基因作为周期性助推器。为了实现内源性前列环素生产的广泛目标，该提议的另一个创新是选择唾液腺作为治疗性生物合成位点。唾液腺可以通过不流血的门诊手术进入，并且包含一个强大的内分泌分泌途径，能够将转基因产品分泌到血管内空间。唾液腺导管内迷路的包裹、固定体积也允许精确控制传递系统，使超声辅助基因转移（UAGT）比在其他器官（如心脏或胰腺）中观察到的更加实用和一致。该提案的最后一个创新是首次在体内应用Cox-1/PGIS融合蛋白，该蛋白产生的前列环素水平明显高于单独使用PGIS。总之，该项目旨在创新基于PGIS的PAH基因治疗的诱人想法，整合三种使能技术：1)实用的，可重新给药的基因传递技术，2)可通过门诊程序访问的新型生物合成位点（唾液腺），以及3)Cox-1/PGIS融合蛋白转基因，产生高水平的前列腺素治疗。这种基因治疗策略的有效性将在高度相关的严重PAH大鼠模型中进行测试。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to design and test a gene therapy strategy for Pulmonary Arterial Hypertension (PAH). PAH is a rare, deadly and incurable disease with a mean survival of 2.8 years from onset of symptoms if left untreated. Of the three classes of approved therapeutics, endothelin receptor antagonists, phospodiesterase inhibitors, and prostacyclins, prostacyclin is the most effective therapy. However, complicated delivery systems and potential side effects associated with the present formulation of prostanoids (e.g. prostacyclin) have deterred some patients and caregivers from instituting this highly effective class of agents. The challenge to be addressed by this proposal is the need for a therapeutic regimen that allows for endogenous production of prostacyclin therapy within the patient's own body, throughout the entire lifetime of the patient. Because prostacyclin can be produced endogenously through expression of the enzyme Prostacyclin Synthase (PGIS), gene therapy has previously shown proof-of-principle efficacy in animal models by enabling endogenous production of prostacyclin and reversal of experimental PAH. This proposal will build upon this concept, but will execute this strategy using newly developed gene transfer technology that obviates the viral gene delivery vectors that have been used in prior studies. Viral vectors have been extremely useful in earlier studies, but have limited duration of expression, and due to host immune response, cannot affect life- long therapy nor can they be re-dosed. Our gene delivery system, which used ultrasound-induced microbubble cavitation to allow entry of non-viral DNA vectors into cells, is thought to evade host immune responses, theoretically allowing re-dosing of the Prostacyclin Synthase therapeutic transgene as a periodic boosters throughout the entire lifespan of the patient. An additional innovation of this proposal toward achieving the field-wide goal of endogenous prostacyclin production is the choice of the salivary glands as the therapeutic biosynthesis site. The salivary glands can be accessed through a bloodless, outpatient procedure, and contain a robust endocrine secretory pathway capable of secreting transgene products into the intravascular space. The encapsulated, fixed volume of the intraductal labyrinth of the salivary glands also allow precise control of the delivery system, making ultrasound-assisted gene transfer (UAGT) far more practical and consistent than has been observed in other organs (e.g. heart or pancreas). A final innovation of this proposal is the first in vivo application of a Cox-1/PGIS fusion protein that produces dramatically higher levels of prostacyclin than PGIS alone. In summary, this project seeks to innovate the enticing idea of PGIS- based gene therapy for PAH aggregating three enabling technologies: 1) a practical, re-dosable gene delivery technique, 2) a novel biosynthesis site (salivary glands) that can be accessed through an outpatient procedure, and 3) a Cox-1/PGIS fusion protein transgene that produces superior levels of prostacyclin therapeutic. The efficacy of this gene therapy strategy will be tested in a highly relevant rat model of severe PAH.