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饰唾液腺的分泌机制，以靶向递送治疗性蛋白质。为了实现这一目标，将开发新的脂质-佐剂复合物用于将siRNA直接呈递到唾液腺。由此产生的基因干扰旨在增强治疗性蛋白质向血流的分泌。唾液腺是基因治疗方案的有吸引力的靶点，因为腺体能够产生大量的分泌蛋白，并且可以通过非侵入性程序经由唾液管进入。将DNA构建体逆行输注到唾液腺中已被用于表达外源治疗性蛋白质，其可以靶向口腔和全身疾病。后一种应用受到唾液上皮细胞的分泌途径的限制，其90%被导向口腔（外分泌）。初步数据表明，外分泌的破坏导致内分泌增加，即分泌物从口腔转移到血流。因此，本申请基于以下发现：酸性硫酸化蛋白聚糖和碱性富含脯氨酸的蛋白质可以调节蛋白质储存，显然是通过pH依赖性机制。一个低的管腔pH值似乎也发挥了作用，在唾液腺蛋白的极化分泌。这些发现表明，调节分泌途径的pH提供了一个机会，调节治疗性蛋白质的分泌，而不改变这些蛋白质的结构。据推测，外分泌分泌可以通过提高分泌细胞器的内部pH值而减少，从而导致内分泌增加。这可以通过选择性沉默参与分泌细胞器酸化的基因来实现。为了验证这一假设，拟议的研究将解决两个主要目标：1）开发脂质和佐剂，用于用siRNA和质粒载体有效转染唾液上皮细胞; 2）使用siRNA增加体内唾液腺模型治疗蛋白hGH的内分泌。为了确保RNAi和基因治疗构建体在体内有效递送至唾液腺，将开发新型脂质制剂并与转染佐剂配对。因此，该项目将开发一种灵活的平台技术，可以适应唾液腺中多种基因的沉默。项目叙述：唾液腺是基因治疗的有吸引力的目标，旨在表达治疗性蛋白质的循环。为了正确靶向这些蛋白质，将使用小干扰RNA来修饰细胞分泌机制。siRNA将与专门用于体内唾液腺的创新脂质转染试剂一起引入。