In vivo gene silencing in salivary glands

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目标，因为唾液腺能够产生大量的分泌蛋白，并且可以通过非侵入性的方法通过唾液腺导管进入。向唾液腺逆行输入DNA构建体已被用于表达外源治疗蛋白，该蛋白可靶向治疗口腔和全身疾病。后一种应用受到唾液上皮细胞分泌途径的限制，其90%被定向到口腔（外分泌）。初步数据表明，外分泌紊乱会导致内分泌分泌增加，即分泌从口腔转移到血液。因此，这一应用是基于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的发现，显然是通过pH依赖的机制。低腔内pH值似乎也在唾液腺蛋白的极化分泌中起作用。这些发现表明，调节分泌途径的pH值提供了在不改变这些蛋白质结构的情况下调节治疗性蛋白质分泌的机会。据推测，可以通过提高分泌细胞器的内部pH值导致内分泌分泌增加来减少外分泌。这可以通过选择性地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)在体内利用siRNA增加模型治疗蛋白hGH从唾液腺的内分泌分泌。为了确保RNAi和基因治疗结构在体内有效地递送到唾液腺，新的脂质制剂将被开发并与转染佐剂配对。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。项目描述：唾液腺是基因治疗的目标，旨在表达循环中的治疗蛋白。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将与创新的脂质转染试剂一起引入，专门用于体内唾液腺。

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.