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生成合适的遗传性疾病动物模型，这些模型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逆转这些变化。为了追求这样的课程，我们选择研究法布里障碍，因为它的独特性质以及它在开发急需的治疗方法方面面临的挑战。法布里病是一种 X 连锁遗传性糖脂代谢疾病，由溶酶体酶、α-半乳糖苷酶 (AGA) 活性缺陷引起。具有末端a-连接半乳糖基部分的中性糖鞘脂、三糖基神经酰胺（ceramidetrihexoside，CTH：Gala1-4Gal/1-4Glc/1-1Cer）和半乳糖神经酰胺（Gala1-4Gal/1-1Cer）在肝脏、心脏、脾脏、肾脏、血管内皮细胞和血浆中积累 患有这种疾病的患者。主要疾病表现包括四肢感觉异常、角膜营养不良、血管角化瘤以及心脏、肾脏和大脑的闭塞性血管疾病，导致过早死亡。人类 AGA cDNA 和基因组克隆已被分离并作图。对 Fabry 患者 AGA 基因的分析揭示了异质性分子病变，例如点突变和部分基因重排。法布里病没有具体的治疗方法。法布里患者已接受肾移植，结果各异。酶替代和体细胞基因疗法有潜力成为溶酶体贮积病的有效疗法。据报道，使用双顺反子多重耐药基因 (MDR) 逆转录病毒可在 NIH-3T3 细胞中有效表达人 a-Gal A。使用重组逆转录病毒在来自法布里患者的成纤维细胞中已证实可以体外纠正酶缺陷。然而，需要合适的法布里病动物模型来评估这些疗法的离体和体内潜力。法布里病的适当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具有不可估量的价值。为了通过同源重组破坏 AGA 基因来开发法布里病小鼠模型，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。使用基因组克隆构建靶向载体。该载体携带跨越外显子 III 和内含子 III 部分的 1 kb 缺失。三个目标克隆被鉴定为基因破坏阳性，并用于产生 AGA 缺失小鼠。尽管 AGA 缺失小鼠在 10 周龄时没有临床表型，但电镜分析显示，肾小管细胞溶酶体中存在具有电子致密同心层状结构的脂质包涵体，这是法布里病患者中常见的典型现象。其他细胞成分在形态上看起来正常。使用荧光标记的单叶加纳凝集素（选择性结合 a-D-半乳糖基残基），我们通过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。该分析揭示了突变小鼠肾脏中的强烈荧光，表明含有α-D-半乳糖基残基的化合物显着积累。从 AGA 无效小鼠胚胎培养的成纤维细胞也显示出 AGA 底物的显着积累，如细胞质区室中具有强烈荧光的颗粒染色所反映的那样。对 AGA 缺失小鼠肝脏和肾脏中中性鞘脂的分析显示 CTH 显着积累。我们与 NCI 细胞生物学实验室 Michel Gottesman 博士合作，成功使用两种携带人类 AGA cDNA 和 MDR1 cDNA 的双顺反子逆转录病毒来纠正成纤维细胞中的酶缺陷。这些研究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，我们描述了疾病随衰老的进展，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发挥潜在作用。此外，事实证明，逆转这些小鼠代谢缺陷的底物剥夺和基因治疗方法非常令人鼓舞。我们与牙科诊所、NIDCR 和 NINDS 合作开展研究，评估一组法布里病患者的口腔和颅面特征，以促进早期识别这种疾病并及时治疗其表现。通过标准化问卷、临床检查、全景和头影测量X光片以及磁共振成像来收集数据。所有没有正畸治疗史的有齿受试者（n = 7）都有咬合不正。这 7 名患者中有 5 名存在纵裂，尤其是前牙间纵裂。其余患者中，两名患者有多次拔牙史，导致 14 岁时完全无牙颌，37 岁时部分无牙颌，四名患者有正畸治疗史。一些患者发现牙齿发育和萌出异常。其中包括保留下颌乳切牙、继发恒牙发育不全、第二磨牙阻生以及延迟萌出史。在我们观察时，没有患者出现全身性的宏观牙釉质缺陷。在一名患者中发现了不寻常的牙釉质表面，上颌前牙有肉眼可见的“水平波”，并且颈牙釉质缺失。此外，一名患者的牙列普遍出现黄棕色变色。拔除磨牙的组织学评估显示牙本质管数量、结构和方向正常，牙骨质和牙髓细胞正常（即无细胞质空泡化；）。该病例系列再次证实了之前病例报告中的口腔和颅面部发现，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将有助于法布里病患者的早期诊断和干预。这些发现对于监测新兴实验疗法治疗这种痛苦且致命的疾病的有效性也将被证明是有价值的。许多法布里病患者的临床症状之一是唾液减少，导致口干样症状。为了确定 AGA 缺陷对唾液腺的分子影响，我们分析了法布里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水平通过薄层色谱法测定。与年龄匹配的一岁野生型相比，腮腺和颌下唾液腺的 AGA 活性显着降低（~88% 和 91%）。脂质分析显示腮腺和颌下腺的 Gb3 水平显着增加。在野生型小鼠的唾液腺中未观察到或很少观察到 Gb-3 积累。电子显微镜分析证实唾液腺溶酶体内存在典型的 Gb3 层状包涵体。各种唾液腺细胞类型中 Gb-3 的大量积累可能会损害法布里病患者的唾液腺功能。