In vivo gene silencing in salivary glands

唾液腺体内基因沉默

• 批准号: 7532965
• 负责人: SVEN-ULRIK GORR
• 金额: $ 18.44万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-12 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7532965
• 关键词: Address; Adjuvant; Blood; Blood Circulation; Cell secretion; Communities; Complex; DNA; Data; Development; Disruption; Drug Formulations; Endocrine; Engineering; Ensure; Epithelial Cells; Gene Silencing; Genes; Gland; Goals; Inorganic Sulfates; Invasive; Lipid Chemistry; Lipids; Modeling; Oral; Oral cavity; Organelles; Pathway interactions; Plasmid Cloning Vector; Play; Procedures; Proteins; Proteoglycan; Protocols documentation; RNA Interference; Reagent; Research; Research Personnel; Role; Salivary; Salivary Glands; Salivary Proteins; Salivary duct structure; Small Interfering RNA; Somatropin; Stream; Structure; Systemic disease; Technology; Testing; Transfection; Transfusion; Unspecified or Sulfate Ion Sulfates; base; design; gene therapy; improved; in vivo; innovation; novel; proline-rich proteins; secretory protein; targeted delivery; therapeutic protein

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to use gene interference to modify the secretory machinery of salivary glands for targeted delivery of therapeutic proteins. To achieve this goal novel lipid-adjuvant complexes will be developed for direct presentation of siRNA to salivary glands. The resultant gene interference is designed to enhance the secretion of therapeutic proteins to the blood stream. Salivary glands are attractive targets for gene therapy protocols since the glands are capable of producing large amounts of secretory proteins and are accessible by non-invasive procedures via the salivary ducts. Retrograde transfusion of DNA constructs into salivary glands has been used to express foreign therapeutic proteins, which can be targeted to both oral and systemic diseases. The latter application is limited by the secretory pathways of salivary epithelial cells, which are directed 90% to the oral cavity (exocrine secretion). Preliminary data suggest that disruption of exocrine secretion leads to increased endocrine secretion, i.e. a shift of secretion from the oral cavity to the blood stream. Thus, this application is based on the findings that acidic sulfated proteoglycans and basic proline-rich proteins can regulate protein storage, apparently by a pH- dependent mechanism. A low luminal pH appears to also play a role in the polarized secretion of salivary gland proteins. These findings suggest that modulation of the pH of the secretory pathway offers an opportunity to modulate the secretion of therapeutic proteins without changing the structure of those proteins. It is hypothesized that exocrine secretion can be decreased by raising the internal pH of secretory organelles resulting in increased endocrine secretion. This can be accomplished by selectively silencing the genes involved in acidification of secretory organelles. To test this hypothesis the proposed research will address two major goals: 1) develop lipids and adjuvants for efficient transfection of salivary epithelial cells with siRNA and plasmid vectors; and 2) use siRNA to increase endocrine secretion of the model therapeutic protein hGH from salivary glands in vivo. To ensure efficient delivery of the RNAi and gene therapy constructs to salivary glands in vivo, novel lipid formulations will be developed and paired with transfection adjuvants. Thus, the project will develop a flexible platform technology that can be adapted to the silencing of a variety of genes in salivary glands. Project Narrative: The salivary glands are attractive targets for gene therapy aimed at expressing therapeutic proteins in the circulation. To correctly target these proteins, small interfering RNAs will be used to modify the cellular secretion machinery. siRNAs will be introduced with innovative lipid transfection reagents developed specifically for use in salivary glands in vivo.

描述（由申请人提供）：拟议研究的长期目标是使用基因干扰来修改唾液腺的分泌机制，以靶向递送治疗蛋白。为了实现这一目标，将开发出新型的脂质 - 辅助复合物，以直接呈现siRNA向唾液腺。所得的基因干扰旨在增强治疗蛋白向血流的分泌。唾液腺是基因治疗方案的有吸引力的靶标，因为这些腺能够产生大量的分泌蛋白，并且可以通过唾液管通过非侵入性手术来获得。将DNA构建体逆行输血到唾液腺已被用来表达异物治疗蛋白，可以针对口腔和全身性疾病。后一种应用受到唾液上皮细胞的分泌途径的限制，唾液上皮细胞被指向口腔90％（外分泌分泌）。初步数据表明，外分泌分泌的破坏会导致内分泌分泌增加，即从口腔到血液的分泌转变。因此，该应用基于以下发现：酸性硫化蛋白聚糖和碱性富含脯氨酸的蛋白可以调节蛋白质储存，这显然是通过pH-依赖性机制来调节蛋白质的。低腔pH值似乎在唾液腺蛋白的两极分化中也起作用。这些发现表明，分泌途径的pH值的调节提供了一个机会，可以调节治疗蛋白的分泌而不改变这些蛋白质的结构。假设可以通过提高分泌细胞器的内部pH来减少外分分泌，从而增加内分泌分泌。这可以通过选择性地沉默与分泌细胞器酸化有关的基因来实现。为了检验该假设，提出的研究将解决两个主要目标：1）开发脂质和辅助因子，以有效地转染唾液上皮细胞用siRNA和质粒载体； 2）使用siRNA增加体内唾液腺的模型治疗蛋白HGH的内分泌分泌。为了确保在体内有效地递送RNAi和基因治疗构建体向唾液腺，将开发新型的脂质制剂，并与转染辅助剂配对。因此，该项目将开发一种灵活的平台技术，该技术可以适应唾液腺中各种基因的沉默。项目叙述：唾液腺是旨在表达循环中治疗蛋白的基因疗法的有吸引力的靶标。为了正确靶向这些蛋白质，将使用小的干扰RNA来修饰细胞分泌机械。将引入siRNA，并使用专门用于体内唾液腺使用的创新脂质转染试剂。