Salivary Gland-Based Gene Therapy for Lysosomal Storage Diseases

最近的研究表明，唾液腺中存在一种内在的内分泌途径，与 从经典的外分泌途径产生消化酶并分泌到唾液中 在正常生理条件下由唾液腺引起。因此，这种独特的内分泌途径 最近被证明能够强大地合成和分泌非受调控的(即构成的)基因 作为基因治疗载体逆行给药的结果，产物进入血管空间。 法布里病是溶酶体贮积症家族的一员，是一组单基因疾病 缺乏一类酶的(如法布里病中的α-半乳糖苷酶A) 参与大分子的溶酶体内分解代谢。这些疾病的一个有趣的特征是 观察到缺乏与疾病有关的特定溶酶体酶的组织能够 从血管间隙摄取特定的溶酶体酶。这种生理学导致了这项提议 以及随后产生缺失的酶的“酶替代疗法”的有效性 人工合成并静脉输注给Fabry患者，从而缓解疾病症状。 我们寻求将这些新兴技术结合到一种基于唾液腺的基因治疗策略中 在高度相关的小鼠基因敲除模型中，即B6；129-GLA(tm1/Kul/J)，一种α-GLA 半乳糖苷酶A(-/O)模型。我们试图检验这样一种假设，即一个包含AAV序列的载体 人α-半乳糖A注射到α-半乳糖A缺陷“Fabry”小鼠的唾液腺将导致 全身性替换酶和逆转潜在的特定全身性生化缺陷 法布里病。我们的具体目标包括：1)将含有α-GalA的载体输送到唾液中 法布里小鼠的腺体和全身性递送酶的生化测试，2)仔细检查 酶的器官生物分布，以及3)优化给药和免疫生理，以实现 该酶的长期表达。这些研究将产生对技术的重要见解 该系统的参数，包括：转基因表达的寿命、宿主免疫反应和 减轻与Fabry相关的病理，并形成其他系统性疾病可能的基础 使用唾液腺作为系统治疗的生物合成部位。

Recent studies have demonstrated that an intrinsic endocrine pathway is present in salivary glands, distinct from the classical exocrine pathway whereby digestive enzymes are produced and secreted into the saliva by the salivary glands under normal physiological conditions. As such, this distinct endocrine pathway has recently been shown to be capable of robust synthesis and secretion of non-regulated (i.e. constitutive) gene products into the vascular space as a result of retroductal administration of a gene therapy vector. Fabry disease is a member of the family of lysosomal storage diseases, a group of monogenic disorders characterized by deficiency or lack of a class of enzymes (e.g. alpha-Galactosidase A in Fabry disease) involved in intra-lysosomal catabolism of macromolecules. An interesting feature of these diseases is the observation that tissues lacking the specific lysosomal enzyme implicated in the disease are capable of uptake of the specific lysosomal enzyme from the vascular space. This physiology has led to the proposal and subsequent validation of "Enzyme Replacement Therapy" wherein the missing enzyme is produced syntheically and infused intravenously to Fabry patients, resulting in a mitigation of disease symptoms. We seek to combine these emergent technologies into a salivary gland-based gene therapy strategy for Fabry disease in highly relevant mouse knockout model, namely B6;129-Gla(tm1/Kul/J), an alpha- Galactosidase A (-/O) model. We seek to test the hypothesis that an AAV vector containing the sequence for human alpha-Gal A delivered to the salivary glands in alpha-Gal A deficient "Fabry" mice will result in systemic replacement of the enzyme and reversal of the specific systemic biochemical defect underlying Fabry disease. Our specific aims involve: 1) delivery of an alpha-Gal A containing vector to the salivary glands of Fabry mice and biochemical testing of systemic delivery of the enzyme, 2) careful examination of the organ biodistribution of the enzyme, and 3) optimization of dosing and immune physiology to achieve long-term expression of the enzyme. These studies will produce important insights into the technical paramaters of this system, including: longevity of transgene expression, host immune response, and mitigation of Fabry-associated pathology, and form the basis whereby other systemic disorders could be addressed using salivary glands as the biosynthetic site for systemic therapeutics.