Heritable Disorders Of Connective Tissue

结缔组织遗传性疾病

• 批准号: 6551108
• 负责人: Joan C Marini
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/6551108
• 关键词: Ehlers Danlos syndrome; clinical research; collagen; connective tissue metabolism; disease /disorder model; gene mutation; genetic disorder; human genetic material tag; human subject; laboratory mouse; molecular pathology; osteogenesis imperfecta; ribozymes

The Section conducts studies to elucidate the molecular and biochemical mechanisms of heritable disorders of connective tissue, specifically osteogenesis imperfecta (OI) and Ehlers-Danlos Syndrome (EDS), and to apply this information to the treatment of those disorders. We have previously developed a knock-in murine model for the non-lethal form of OI, which we have named the Brittle mouse (Brtl). Brtl has a classic glycine substitution (G349C) in one of its collagen alpha 1(I) chains and reproduces the molecular, biochemical and histological features of OI. We have been engaged in collaborative studies of the histomorphometry and biomechanics of Brtl long bones. At the midshaft, Brtl bone has a decreased cross-sectional area at 1,2, and 6 months of age and the calculated moment of inertia is reduced, predicting that Brtl bone should be weaker based on its geometric properties. In 4-point bending tests to failure, Brtl bone breaks with a lower force than controls at 1 and 2 months but has equal strength at 6 months. This combination of weak geometric properties and equal mechanical properties predicts that the composition of Brtl bone itself has changed after puberty in the mouse. This makes the Brtl mouse an excellent model for the well-described clinical feature of OI, decreased bone fragility after puberty. We are also using the Brtl mouse to conduct treatment trials of bisphosphonate. The treatment trials in the mouse are paralleled by treatment trials in children with types III and IV OI. In the pediatric trial, we are doing a four-arm controlled trial in which children are randomly assigned to pamidronate, growth hormone, both drugs or no drug. The effects of the treatment on vertebral bodies and on long bones are being compared. In the murine trial of the drug, we are using the bisposphonate alendronate. Controls or Brtl mice have already shown increased bone density of spine and long bone in response to treatment. Biomechanical testing will show how this increased density affects bone strength and brittleness. The Brtl mouse is also an excellent model in which to approach the gene therapy of OI, because a ribozyme (RZ) cleavage site was engineered into the Brtl mutant allele. Cleavage of mutant transcript by RZ can suppress expression the mutant protein and biochemically mimic a null allele, which is clinically mild in humans. In the past year, we have published our work with RZ in cultured OI fibroblasts, showing allele-specific suppression of mutant collagen transcript to about 50% of baseline levels in control cells. We generated a RZ mouse for transferring the therapeutic molecule by matings with Brtl and are in the process of testing the effect of the RZ on normal bones of the RZ transgenic and of comparing the anti-sense effects of various RZ constructs. In our studies of human collagen mutations causing OI, we have been focusing on a set of mutations in types I and V collagen that affect fibril formation. In one these mutations, there is a deletion of the exon in a1 (I) that contains the telepeptide binding site. Absence of this exon in even a small fraction of monomers results in a dramatic delay in fibril formation for the proband?s total collagen mix. Since this delay, primarily the longitudinal portion of fibril assembly kinetics, the collagen fibrils formed in vitro are 4-5 times the length of control molecules.

该科开展研究以阐明结缔组织遗传性疾病，特别是成骨不全症 (OI) 和埃勒斯-当洛斯综合征 (EDS) 的分子和生化机制，并将这些信息应用于这些疾病的治疗。我们之前开发了非致死性成骨不全症的敲入小鼠模型，我们将其命名为脆性小鼠 (Brtl)。 Brtl 在其胶原蛋白 α 1(I) 链之一中具有经典的甘氨酸取代 (G349C)，并再现了 OI 的分子、生化和组织学特征。我们一直致力于 Brtl 长骨的组织形态计量学和生物力学的合作研究。在中轴，Brtl 骨的横截面积在 1,2 和 6 个月龄时减小，计算出的惯性矩也减小，根据其几何特性预测 Brtl 骨应该更弱。在 4 点弯曲失败测试中，Brtl 骨折在 1 个月和 2 个月时的断裂力低于对照组，但在 6 个月时具有相同的强度。这种弱几何特性和同等机械特性的结合预示着小鼠青春期后 Brtl 骨本身的成分发生了变化。这使得 Brtl 小鼠成为描述成骨不全临床特征（青春期后骨质脆性降低）的优秀模型。 我们还使用 Brtl 小鼠进行双膦酸盐治疗试验。小鼠治疗试验与 III 型和 IV 型 OI 儿童的治疗试验同时进行。在儿科试验中，我们正在进行一项四组对照试验，其中儿童被随机分配服用帕米膦酸钠、生长激素、两种药物或不服用药物。正在比较治疗对椎体和长骨的影响。在该药物的小鼠试验中，我们使用双膦酸盐阿仑膦酸盐。对照小鼠或 Brtl 小鼠已经表现出对治疗的反应，脊柱和长骨的骨密度增加。生物力学测试将显示密度的增加如何影响骨骼强度和脆性。 Brtl 小鼠也是进行 OI 基因治疗的绝佳模型，因为 Brtl 突变等位基因中被设计了核酶 (RZ) 切割位点。 RZ 对突变转录本的切割可以抑制突变蛋白的表达，并在生化上模拟无效等位基因，这在人类中临床上是轻微的。去年，我们发表了 RZ 在培养的 OI 成纤维细胞中的研究成果，显示突变胶原转录物的等位基因特异性抑制达到对照细胞基线水平的约 50%。我们培育了一只 RZ 小鼠，用于通过与 Brtl 交配来转移治疗分子，并且正在测试 RZ 对 RZ 转基因正常骨骼的影响，并比较各种 RZ 构建体的反义效应。 在我们对导致成骨不全的人类胶原蛋白突变的研究中，我们一直关注影响原纤维形成的 I 型和 V 型胶原蛋白的一系列突变。在其中一个突变中，a1 (I) 中含有远肽结合位点的外显子被删除。即使一小部分单体中缺少该外显子，也会导致先证者总胶原混合物的原纤维形成显着延迟。由于这种延迟，主要是原纤维组装动力学的纵向部分，体外形成的胶原原纤维的长度是对照分子的4-5倍。