Models Of Inherited Metabolic Disorders

遗传性代谢紊乱模型

• 批准号: 6507208
• 负责人: Ashok B. KULKARNI
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6507208
• 关键词: Fabry's disease; alpha galactosidase; congenital oral /facial /cranial defect; dental disorder; disease /disorder model; enzyme therapy; gene targeting; gene therapy; laboratory mouse; metabolism disorder; model design /development; orphan disease /drug; salivary glands

The ultimate success of the "Functional Genomics" approach will depend on generating suitable animal models for genetic disorders that manifest clinical phenotypes with obvious metabolic changes and then developing effective therapeutic approaches to reverse these changes. In order to pursue such a course, we chose to work on Fabry disorder because of its unique nature and the challenges it presents in developing much needed therapeutic approaches. Fabry disease is an X-linked inherited disorder of glycolipid metabolism resulting from deficient activity of the lysosomal enzyme, a-galactosidase (AGA). Neutral glycosphingolipids with terminal a-linked galactosyl moieties, globotriaosylceramide (ceramidetrihexoside, CTH: Gala1-4Gal/1-4Glc/1-1Cer) and galabiosylceramide (Gala1-4Gal/1-1Cer), accumulate in the liver, heart, spleen, kidney, vascular endothelial cells and in plasma of the patients with this disorder. Major disease manifestations include paresthesia in the extremities, corneal dystrophy, angiokeratoma, and occlusive vascular disease of the heart, kidney, and brain, leading to premature mortality. Human AGA cDNA and the genomic clone have been isolated, and mapped. Analysis of the AGA gene in Fabry patients revealed heterogeneous molecular lesions such as point mutations and partial gene rearrangements. There is no specific therapy for Fabry disease. Renal transplantation has been performed in Fabry patients with varying outcomes. Enzyme replacement and somatic gene therapy have potential as effective therapies for lysosomal storage diseases. Efficient expression of the human a-Gal A has been reported in NIH-3T3 cells using bicistronic multidrug-resistant gene (MDR) retroviruses. In vitro correction of enzyme deficits has been demonstrated in fibroblasts derived from Fabry patients using recombinant retrovirus. However, a suitable animal model for Fabry disease is required to evaluate the ex vivo and in vivo potential of these therapies. An appropriate model of Fabry disease would be invaluable for the study of the molecular pathophysiology of this human genetic disease as well as for the development of effective therapeutic strategies. In order to develop a mouse model for Fabry disease by disrupting AGA gene by homologous recombination, we isolated and characterized the mouse AGA gene. We found that the mouse and human AGA genes are highly similar in size, gene organization and nucleotide sequence of the coding regions. A targeting vector was constructed using the genomic clones. This vector carries a 1 kb deletion spanning part of exon III and intron III. Three targeted clones were identified as positive for gene disruption and used for generation of AGA null mice. Although the AGA null mice had no clinical phenotype at 10 weeks of age, EM analysis revealed lipid inclusions with electron dense concentric lamellar structures in the lysosomes of renal tubular cells typical of those seen in patients with Fabry disease. Other cellular components appeared morphologically normal. Using fluorescent-labeled Griffonia (Bandeiraea) simplicifolia lectin which selectively binds to a-D-galactosyl residues, we analyzed kidneys of 10 week-old mice and embryonic fibroblasts by confocal microscopy. This analysis revealed intense fluorescence in the kidneys of the mutant mice indicating significant accumulations of compounds containing a-D-galactosyl residues. Cultured fibroblasts from AGA null mouse embryos also displayed significant accumulation of AGA substrates as reflected by the granular staining with intense fluorescence in the cytoplasmic compartment. Analysis of neutral sphingolipids in liver and kidneys of AGA null mice showed a striking accumulation of CTH. In collaboration with Dr. Michel Gottesman, Laboratory of Cell Biology in NCI, we have demonstrated successful use of two bicistronic retroviruses carrying both human AGA cDNA and MDR1 cDNA to correct the enzyme deficits in fibroblasts. These studies were further extended to assess their potential for in vivo gene therapy. In the recent study, we characterized the progression of the disease with aging, and explored the effects of bone marrow transplantation (BMT) on the phenotype. Our findings suggest that BMT may have a potential role in the management of patients with Fabry disease. Additionally, substrate deprivation and gene therapy approaches to reverse the metabolic defects in these mice have proven to be very encouraging. In collaboration with the Dental Clinic, NIDCR, and NINDS we carried out studies to assess oral and craniofacial characteristics in a cohort of patients with Fabry disease in order to facilitate early recognition of this condition and prompt treatment of its manifestations. Data were collected by means of a standardized questionnaire, clinical examination, panoramic and cephalometric radiographs, and magnetic resonance imaging. All dentate subjects without a history of orthodontic treatment (n=7) had malocclusion. Diastemas, particularly among the anterior teeth, were present in 5 of these 7 patients. Of the remaining patients, two had a history of multiple dental extractions resulting in complete edentulism at age 14 and partial edentulism at age 37, and four patients had a history of orthodontic treatment. Anomalies of dental development and eruption were found in some patients. These included a retained mandibular primary incisor with agenesis of the succedaneous permanent tooth, a second molar impaction and a history of delayed eruption. None of the patients had generalized macroscopic enamel defects at the time of our observation. An unusual enamel surface with macroscopic "horizontal waves" of the maxillary anterior teeth and loss of cervical enamel was noticed in one patient. Additionally, one patient had generalized yellow-brown discoloration of his dentition. Histologic evaluation of an extracted molar showed normal dentin tubular number, structure and orientation, normal cementum and pulpal cells (i.e., without cytoplasmic vacuolization;). This case series has reconfirmed oral and craniofacial findings from previous case reports, and most importantly, it has identified additional features of Fabry disease not previously described. Identification of specific disease manifestations will facilitate early diagnosis and intervention of the patients with Fabry disease. These findings will also prove to be valuable in monitoring effectiveness of emerging experimental therapies to treat this painful and fatal disease. One of the clinical symptoms noted in many patients of Fabry disease is reduced saliva resulting in a dry mouth-like condition. In order to identify molecular effects of AGA deficiency on salivary glands, we have analyzed parotid and submaxillary salivary glands of Fabry mice. Parotid and submaxillary salivary glands were analyzed by histopathology and electron microscopy. Gb3 level was determined by thin layer chromatography. AGA activities in parotid and submaxillary salivary glands were significantly lower (~88 and 91%) when compared to age matched one-year-old wild types. Lipid analysis revealed dramatic increases in Gb3 levels in both parotid and submaxillary glands. None or very little Gb-3 accumulation was seen in the salivary glands of wild-type mice. Electron microscopic analysis confirmed the presence of typical lammellar inclusion bodies of Gb3 within the lysosomes of the salivary glands. Large accumulations of Gb-3 in various salivary gland cell types potentially impair salivary gland function in patients with Fabry disease.

“功能基因组学”方法的最终成功将取决于为遗传疾病产生合适的动物模型，这些遗传疾病表现出具有明显代谢变化的临床表型，然后开发有效的治疗方法来扭转这些变化。为了追求这样的课程，我们之所以选择从事Fabry障碍，是因为其独特的性质及其在开发急需的治疗方法方面面临的挑战。 Fabry病是糖脂代谢的一种X连锁遗传疾病，是由于溶酶体酶（AGA）活性不足而导致的。与末端A连接半乳糖基部分，球环蛋白酶酰胺（ceramidetrihexoside，cth，CTH：Gala1-4Gal/1-4GLC/1-1cer）和Galabiosylceramide（Gala1-4gal/1-1cer）的中性糖磷脂醇（Ceramidetrihexoside，CTH：GALA1-4GAL/1-4GLC/1-1CER） ，血管内皮细胞和该疾病患者的血浆中。主要的疾病表现包括四肢的感觉异常，角膜营养不良，血管疾病以及心脏，肾脏和大脑的闭塞性血管疾病，导致早死亡。人AGA cDNA和基因组克隆已被分离并映射。 Fabry患者中AGA基因的分析揭示了异质的分子病变，例如点突变和部分基因重排。没有针对法布里病的特定疗法。肾移植已在较大的结果的Fabry患者中进行。酶替代和体细胞基因疗法具有溶酶体储存疾病的有效疗法。已经在NIH-3T3细胞中使用双科性多药基因（MDR）逆转录病毒在NIH-3T3细胞中有效地表达了人类A-GAL A。使用重组逆转录病毒衍生自Fabry患者的成纤维细胞证明了酶缺陷的体外校正。但是，需要适合法布里病的动物模型来评估这些疗法的离体和体内潜力。 Fabry病的合适模型对于研究这种人类遗传疾病的分子病理生理以及有效治疗策略的发展将是无价的。为了通过通过同源重组破坏AGA基因来开发Fabry疾病的小鼠模型，我们分离并表征了小鼠AGA基因。我们发现，小鼠和人AGA基因在编码区域的大小，基因组织和核苷酸序列上高度相似。使用基因组克隆构建靶向载体。该矢量带有1 kb的缺失，跨越了外显子III和内含子III。三个靶向克隆被确定为基因破坏阳性，并用于产生AGA无效小鼠。尽管AGA NULL小鼠在10周龄时没有临床表型，但EM分析显示，在肾小管疾病患者中典型的肾小管细胞溶酶体中，电子密集的同心层状结构脂质夹杂物。其他细胞成分在形态上似乎正常。使用荧光标记的格里富尼（Bandeiraea）简单凝集素，其与A-D-半乳糖基残基有选择地结合，我们通过共聚焦显微镜分析了10周大的小鼠和胚胎成纤维细胞的肾脏。该分析表明，突变小鼠的肾脏强烈荧光，表明含有A-D-半乳糖基残基的化合物的显着积累。来自Aga Null小鼠胚胎的培养的成纤维细胞也显示出AGA底物的显着积累，这是由细胞质区室中强烈的荧光反射的。分析Aga Null小鼠的肝脏和肾脏中的中性鞘脂，表明CTH的积累显着。与NCI细胞生物学实验室的米歇尔·戈特斯曼（Michel Gottesman）博士合作，我们证明了两种携带人AGA cDNA和MDR1 cDNA的双科逆转录病毒成功地使用了成纤维细胞中的酶缺陷。进一步扩展了这些研究，以评估其体内基因治疗的潜力。在最近的研究中，我们表征了通过衰老的疾病进展，并探讨了骨髓移植（BMT）对表型的影响。我们的发现表明，BMT在Fabry病患者的管理中可能具有潜在的作用。此外，证明这些小鼠的代谢缺陷的底物剥夺和基因治疗方法已被证明是非常令人鼓舞的。通过与牙科诊所，NIDCR和NINDS合作，我们进行了研究，以评估一批Fabry病患者的口腔和颅面特征，以促进早期认识到这种情况并迅速治疗其表现。通过标准化问卷，临床检查，全景和头部射线照相以及磁共振成像收集数据。所有没有正畸治疗史的牙齿受试者（n = 7）均有错误的牙合。在这7例患者中，有5例存在疾病，特别是在前牙中，有5例。在其余患者中，两名患者有多种牙齿提取的病史，导致14岁时完全剥离，而37岁的部分e部分ed术，四名患者有正畸治疗史。在某些患者中发现牙齿发育和喷发异常。其中包括保留的下颌主要门牙，并具有琥珀层永久牙齿的发育作用，第二摩尔撞击和延迟喷发病史。在我们观察时，没有一个患者概括了宏观搪瓷缺陷。在一名患者中发现了一个不寻常的搪瓷表面，上颌前牙的宏观“水平波”和宫颈搪瓷的丧失。此外，一名患者对他的牙列进行了概括的黄褐色变色。提取的摩尔的组织学评估显示出正常的牙本质管状数量，结构和方向，正常的牙骨质和牙髓细胞（即没有细胞质液泡化；）。该病例系列已从以前的病例报告中重新确认了口头和颅面的发现，最重要的是，它已经确定了先前未描述的Fabry疾病的其他特征。特定疾病表现的鉴定将有助于早期诊断和干预法布里病患者。这些发现还将被证明在监测新兴实验疗法的有效性方面非常有价值，以治疗这种痛苦而致命的疾病。许多法布里病患者注意到的临床症状之一是唾液减少，导致口腔样病。为了鉴定Aga缺乏对唾液腺的分子作用，我们分析了Fabry小鼠的腮腺和颌下唾液腺。通过组织病理学和电子显微镜分析腮腺和颌下唾液腺。 GB3水平由薄层色谱法确定。与年龄相匹配的一年一年的野生型相比，腮腺和间质唾液腺中的AGA活性显着降低（〜88和91％）。脂质分析表明，腮腺和间质腺的GB3水平急剧增加。在野生型小鼠的唾液腺中没有看到或很少的GB-3积累。电子显微镜分析证实了唾液腺溶酶体内GB3的典型lammellar包含体的存在。在各种唾液腺细胞类型中，GB-3的大量积累可能会损害Fabry病患者的唾液腺功能。