Salivary gland-based gene therapy for lysosomal storage diseases

描述(申请人提供)：最近的研究表明，唾液腺中存在一种内在内分泌途径，不同于经典的外分泌途径，即消化酶在正常生理条件下由唾液腺产生并分泌到唾液中。因此，这种独特的内分泌途径最近被证明能够强劲地合成和分泌非调控(即结构性)基因产物到血管空间，这是基因治疗载体逆行给药的结果。Fabry病是溶酶体储存病家族的一员，是一组单基因疾病，其特征是参与大分子溶酶体内分解代谢的一类酶(如Fabry病中的α-半乳糖苷酶A)的缺乏或缺乏。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“酶替代疗法”的提出和随后的验证，在这种疗法中，缺失的酶被合成出来，并通过静脉注射给Fabry患者，从而缓解了疾病症状。我们试图将这些新兴技术结合到高度相关的小鼠基因敲除模型B6；129-GLA(tm1/Kul/J)中以唾液腺为基础的Fabry病基因治疗策略中，这是一种α-半乳糖苷酶A(-/O)模型。我们试图测试这样一种假设，即含有人α-GalA序列的AAV载体被输送到α-GalA缺陷的“Fabry”小鼠的唾液腺，将导致酶的系统替代和Fabry病潜在的特定系统生化缺陷的逆转。我们的具体目标包括：1)将含有α-GalA的载体输送到Fabry小鼠的唾液腺中，并对该酶的系统输送进行生化测试；2)仔细检查该酶在器官中的生物分布；以及3)优化剂量和免疫生理，以实现该酶的长期表达。这些研究将对该系统的技术参数产生重要的见解，包括：转基因表达的寿命、宿主免疫反应和减轻Fabry相关的病理，并形成使用唾液腺作为系统治疗的生物合成部位来解决其他系统性疾病的基础。

DESCRIPTION (provided by applicant): 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.