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表现出具有明显代谢变化的临床表型的遗传疾病建立合适的动物模型，然后开发有效的治疗方法来逆转这些变化。为了开展这样的课程，我们选择研究法布里紊乱症，因为它的独特性和它在开发急需的治疗方法方面所面临的挑战。法布里病是一种x连锁的糖脂代谢遗传性疾病，由溶酶体酶a-半乳糖苷酶（AGA）活性不足引起。具有末端a-连接半乳糖基的中性鞘糖脂，球三烷基神经酰胺（神经酰胺三己外苷，CTH: Gala1-4Gal/1-4Glc/1-1Cer）和半烷基神经酰胺（Gala1-4Gal/1-1Cer），在该疾病患者的肝脏、心脏、脾脏、肾脏、血管内皮细胞和血浆中积累。主要的疾病表现包括四肢感觉异常、角膜营养不良、血管角化瘤以及心、肾和脑的闭塞性血管疾病，导致过早死亡。已分离出人类AGA cDNA和基因组克隆，并绘制了图谱。法布里患者的AGA基因分析显示异质分子病变，如点突变和部分基因重排。法布里病没有特殊的治疗方法。在法布里患者中进行肾移植有不同的结果。酶替代和体细胞基因治疗有潜力成为溶酶体贮积病的有效治疗方法。据报道，在NIH-3T3细胞中，利用双电子多药耐药基因（MDR）逆转录病毒高效表达了人A - gal A。利用重组逆转录病毒在法布里患者的成纤维细胞中证明了酶缺陷的体外纠正。然而，需要一种合适的法布里病动物模型来评估这些疗法的体外和体内潜力。一个合适的法布里病模型对于研究这种人类遗传疾病的分子病理生理学以及开发有效的治疗策略是非常宝贵的。为了通过同源重组破坏AGA基因建立法布里病小鼠模型，我们分离并鉴定了小鼠AGA基因。我们发现小鼠和人类的AGA基因在大小、基因组织和编码区的核苷酸序列上高度相似。利用基因组克隆构建靶向载体。该载体携带1 kb的缺失，横跨部分外显子III和内含子III。三个目标克隆被鉴定为基因破坏阳性，并用于产生AGA null小鼠。虽然AGA阴性小鼠在10周龄时没有临床表型，但EM分析显示，在法布里病患者的肾小管细胞溶酶体中，存在具有电子致密同心板层结构的脂质包涵体。其他细胞成分形态正常。利用荧光标记的Griffonia (Bandeiraea) simplicifolia凝集素选择性结合a- d -半乳糖基残基，我们用共聚焦显微镜分析了10周龄小鼠的肾脏和胚胎成纤维细胞。该分析显示，突变小鼠的肾脏中有强烈的荧光，表明含有a- d -半乳糖基残基的化合物显著积累。从AGA阴性小鼠胚胎中培养的成纤维细胞也显示出AGA底物的显著积累，这可以通过细胞质室中强烈荧光的颗粒染色来反映。对AGA小鼠肝脏和肾脏中性鞘脂的分析显示CTH显著积累。通过与NCI细胞生物学实验室的Michel Gottesman博士合作，我们成功地使用了两种携带人类AGA cDNA和MDR1 cDNA的双胞逆转录病毒来纠正成纤维细胞中的酶缺陷。这些研究被进一步扩展以评估其体内基因治疗的潜力。在最近的研究中，我们描述了疾病随年龄增长的进展，并探讨了骨髓移植（BMT）对表型的影响。我们的研究结果表明，BMT可能在法布里病患者的治疗中具有潜在的作用。此外，底物剥夺和基因治疗方法逆转这些小鼠的代谢缺陷已被证明是非常令人鼓舞的。我们与牙科诊所、NIDCR和NINDS合作，开展了一组法布里病患者口腔和颅面特征的研究，以促进对这种疾病的早期识别和及时治疗其表现。通过标准化问卷调查、临床检查、全景和头侧x线片以及磁共振成像收集数据。无正畸治疗史的有齿受试者（n=7）均有错牙合。7例患者中有5例出现裂口，尤其是前牙。其余患者中，2例有多次拔牙史，14岁时完全全牙，37岁时部分全牙，4例有正畸治疗史。部分患者出现牙发育和出牙异常。这些包括保留的下颌第一门牙，继发恒牙发育不全，第二磨牙嵌塞和延迟出牙史。在我们的观察中，所有患者均无广泛性肉眼牙釉质缺损。1例患者发现上颌前牙牙釉质表面异常，肉眼可见“水平波”状，颈牙釉质缺失。此外，一名患者的牙列有全身性黄棕色变色。拔出的磨牙的组织学检查显示牙本质小管数量、结构和取向正常，牙骨质和牙髓细胞正常（即没有细胞质空泡化）。本病例系列再次证实了以前病例报告的口腔和颅面发现，最重要的是，它确定了以前未描述的法布里病的其他特征。识别特定的疾病表现有助于Fabry病患者的早期诊断和干预。这些发现也将被证明对监测新出现的实验性疗法治疗这种痛苦和致命疾病的有效性有价值。许多法布里病患者的临床症状之一是唾液减少，导致口干。为了确定AGA缺乏对唾液腺的分子效应，我们分析了Fabry小鼠的腮腺和颌下唾液腺。采用组织病理学和电镜对腮腺和颌下唾液腺进行分析。用薄层色谱法测定Gb3含量。与年龄匹配的一岁野生型相比，腮腺和颌下唾液腺的AGA活性显著降低（~ 88%和91%）。脂质分析显示腮腺和颌下腺的Gb3水平显著升高。野生型小鼠唾液腺中未见或很少见Gb-3积累。电镜分析证实唾液腺溶酶体内存在典型的Gb3片层包涵体。在各种唾液腺细胞类型中大量积累的Gb-3可能损害法布里病患者的唾液腺功能。