Salivary Gland Secretion Mechanisms--Normal & Pathologic

唾液腺分泌机制——正常

• 批准号: 7146098
• 负责人: BRUCE J BAUM
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7146098
• 关键词: NOD mouse; Sjogren' s syndrome; gene therapy; human tissue; ionizing radiation; physiology; radiobiology; salivary disorder; salivary glands; secretion; therapy design /development; tissue engineering

Salivary secretions maintain the health of the oral cavity. Building on our past studies of saliva formation and its alteration during pathology, we are developing novel approaches to treat salivary gland (SG) dysfunction primarily using principles of gene therapy and tissue engineering. During this reporting period we have made considerable progress in many facets of our work. Our studies are directed at fundamental questions necessary to move gene therapy into the clinic for phase-I trials in three areas: irradiation (IR)-induced salivary hypofunction, systemic single protein deficiency disorders (SSPDDs), and Sjogren?s syndrome (SS). The treatment of most head and neck cancer patients includes IR. SGs in the IR field suffer irreversible damage. Much of our effort has gone to prevent such damage, as well as to restore function to existing damaged glands. Over the last several years, we have examined the usefulness of the stable nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol) for radioprotection of the SGs and during this reporting period, we have studied its pharmacokinetic characteristics in a murine model. Tempol (137.5 or 275 mg/kg, 10 minutes prior to IR) significantly reduced IR-induced salivary hypofunction (~50 - 60%). Intravenous or subcutaneous administration was as protective as intraperitoneal administration, while intramuscular delivery was ineffective. Topical use of Tempol, either as a mouthwash or gel led to radioprotectiion. The results strongly suggest that Tempol may be useful to protect SGs in head and neck cancer patients undergoing radiotherapy. An important step in developing a clinical therapy is moving from a rodent to a large animal pre-clinical model. During this reporting period we have established the value of the miniature pig (minipig) parotid gland as a model for IR-induced salivary hypofunction. This year we began extending IR repair studies, reported last year using AdhAQP1 in this model. Those studies demonstrated that localized delivery of AdhAQP1 to IR-damaged salivary glands increases salivary secretion, without significant general adverse events, in a large animal model. We have now begun to test serotype 2 and 5 adeno-associated viral (AAV) vectors in this model to provide stable hAQP1 expression and,hopefully, stable restoration of fluid secretion. Our past studies have shown that SGs are a useful target site for treating SSPDDs, particularly when the transgene encodes a constitutive pathway secretory protein. The ability to control transgene expression is essential for clinical application. Previously, in a proof-of-concept study, we showed that the rapamycin-inducible transcriptional regulation system can regulate protein expression after adenoviral-mediated gene transfer to SGs. To evaluate the potential ability to utilize this regulatory system for long-term control of transgene expression in this tissue, we employed a ?third generation?, single AAV serotype 2 viral vector encoding human erythropoietin (hEPO) under the control of a rapamycin inducible promoter. The vector, rAAV-TF2.3-hEPO (10e10 particles/animal), was delivered to mouse SGs. No detectable increase in serum hEPO or hematocrit levels was observed in the absence of rapamycin administration. However, rapamycin induced elevation of serum hEPO levels, as well as concomitant hematocrit changes, that were dose-dependent, completely reversible and relatively stable over six months (longest time studied), with no appreciable change in rapamycin responsiveness. Unfortunately, regulated secretory pathway proteins, when delivered as transgenes to SGs, are secreted predominantly into saliva. This is not useful for those proteins whose therapeutic function is required systemically, e.g., human growth hormone (hGH). One strategy to improve the efficiency of hGH secretion into the bloodstream involves manipulation of existing sorting signals. We made several mutations in the C-terminus of the hGH cDNA and tested them. One biologically active mutant containing substitution of E174A and E186A, and with an included C-terminal extension showed, in vivo, a relative increase in the proportion of constitutive pathway secretion seen from rat SGs, with a significantly lower saliva vs. serum secretion ratio (p=0.03). Although this mutant is unlikely to be therapeutically beneficial, these results suggest that the final destination of a transgenic secretory protein may be controlled by re-engineering its? sorting determinants. We continue to use the female non-obese diabetic (NOD) mouse model of SS. Mice develop spontaneous autoimmune sialadenitis and loss of salivary flow. In the current period we used the NOD model and evaluated if transfer of the vasoactive intestinal peptide (VIP) cDNA could be useful in management of SS. We constructed a serotype 2 AAV vector encoding the human vasoactive intestinal peptide transgene (rAAV2hVIP) and, when administered prior to disease onset, observed that it led to significantly improved salivary flow and a reduction of submandibular gland cytokines IL-2, IL-10, IL-12 (p70) and TNF-alpha, and serum RANTES, compared to control vector, although no difference in focus scores was detected. In addition to our gene transfer studies, we have continued to make progress toward the development of an artificial SG, as described in previous annual reports. During the past year we have focused on establishing an autologous, polarized, non-human primate epithelial cell system to use in the device and thus facilitate in vivo functional testing. Using methods similar to those previously reported for human cells, we have achieved a reproducible means of obtaining these cells and we have begun to characterize their phenotype and function.

唾液分泌保持口腔的健康。在我们过去对唾液形成及其在病理过程中的改变的基础上，我们正在开发新的方法来治疗唾液腺（SG）功能障碍，主要使用基因治疗和组织工程原理。在此报告期间，我们在工作的许多方面取得了长足的进步。我们的研究针对将基因疗法转移到诊所中的基本问题，以在三个领域进行I期试验：辐射（IR）诱导的唾液功能不全，全身性单蛋白缺乏症（SSPDDS）和Sjogre？s综合征（SS）。 大多数头颈癌患者的治疗包括IR。 IR领域的SGS遭受了不可逆转的损害。我们的大部分努力都用于防止这种损害，并恢复现有受损腺体的功能。在过去的几年中，我们研究了稳定的硝基氧化物4-羟基-2,2,6,6-四甲基哌啶氨酸-N-氧基（tempol）对SGS的放射性降射剂的有用性，在此报告期间，我们研究了其在鼠类模型中的药代动力学特征。 Tempol（137.5或275 mg/kg，在IR前10分钟）显着降低了IR诱导的唾液功能障碍（〜50-60％）。静脉内或皮下给药与腹膜内的给药一样保护性，而肌内递送无效。 tempol的局部用途，无论是漱口水还是凝胶导致了放射线保护。结果强烈表明Tempol可能有助于保护接受放疗的头颈癌患者中的SG。开发临床疗法的一个重要步骤是从啮齿动物转变为大型动物临床前模型。在此报告期间，我们确定了微型猪（Minipig）腮腺的价值，作为IR诱导的唾液功能不全的模型。今年，我们开始扩展IR维修研究，该研究于去年使用ADHAQP1在该模型中使用。这些研究表明，在大型动物模型中，将ADHAQP1局部递送到受IR损害的唾液腺增加了唾液分泌，而没有重大不良事件。现在，我们已经开始测试该模型中与腺相关的病毒（AAV）载体（AAV）媒介的测试，以提供稳定的HAQP1表达，并希望可以稳定地恢复流体分泌。 我们过去的研究表明，SGS是治疗SSPDD的有用目标，尤其是当转基因编码本构途径分泌蛋白时。控制转基因表达的能力对于临床应用至关重要。以前，在概念证明的研究中，我们表明雷帕霉素诱导的转录调控系统可以调节腺病毒介导的基因转移到SGS后的蛋白质表达。为了评估使用该调节系统在该组织中对转基因表达的长期控制的潜在能力，我们采用了第三代？，单型AAV血清型2病毒载体编码人类红细胞生成素（HEPO），在Rapamycin诱导的启动子的控制下。将载体raav-tf2.3-hepo（10e10颗粒/动物）传递到小鼠SGS。在没有雷帕霉素给药的情况下，未观察到血清HEPO或血细胞比容水平的可检测增加。然而，雷帕霉素诱导血清HEPO水平的升高以及伴随的血细胞比容变化，这些变化依赖于剂量，完全可逆且在六个月内相对稳定（研究时间最长），雷帕霉素的响应能力没有明显变化。不幸的是，当作为转基因传递给SGS时，受调节的分泌途径蛋白主要分泌到唾液中。这对于那些有系统地需要治疗功能的蛋白质（例如人类生长激素（HGH））没有用。提高HGH分泌效率为血液的一种策略是操纵现有的分类信号。我们在HGH cDNA的C末端进行了几个突变，并测试了它们。一个含有E174A和E186A的生物活性突变体，并在随附的C末端扩展中显示，在体内，从大鼠SGS看到的本构途径分泌的比例相对增加，其唾液与血清分泌比率明显更低（P = 0.03）。尽管该突变体不太可能在治疗上有益，但这些结果表明，可以通过重新设计其转基因分泌蛋白的最终目的地？分类决定因素。 我们继续使用SS的雌性非肥胖糖尿病（NOD）小鼠模型。小鼠出现自发性自身免疫性鼻腺炎和唾液流量的丧失。在当前时期，我们使用了NOD模型，并评估了血管活性肠肽（VIP）cDNA的转移是否可以在SS的管理中有用。 We constructed a serotype 2 AAV vector encoding the human vasoactive intestinal peptide transgene (rAAV2hVIP) and, when administered prior to disease onset, observed that it led to significantly improved salivary flow and a reduction of submandibular gland cytokines IL-2, IL-10, IL-12 (p70) and TNF-alpha, and serum RANTES, compared to control矢量，尽管未检测到焦点得分的差异。 除了我们的基因转移研究外，我们还继续朝着人工SG的发展迈进，如先前的年度报告所述。在过去的一年中，我们专注于建立一种自体，两极分化的非人类灵长类动物上皮细胞系统，以便在设备中使用，从而促进体内功能测试。使用类似于先前报道的人类细胞的方法，我们已经获得了获得这些细胞的可再现方法，并且我们已经开始表征它们的表型和功能。