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.

该科进行研究，以阐明遗传性结缔组织疾病的分子和生物化学机制，特别是骨生成障碍（OI）和Ehlers-Danlos综合征（EDS），并将这些信息应用于治疗这些疾病。我们以前已经开发了一种非致死性OI的敲入小鼠模型，我们将其命名为脆性小鼠（Brtl）。Brt 1在其胶原α 1（I）链之一中具有经典的甘氨酸取代（G349 C），并再现了OI的分子、生物化学和组织学特征。我们一直在从事合作研究的组织形态计量学和生物力学的Brtl长骨。在中段，Brt 1骨在1、2和6月龄时具有减小的横截面积，并且计算的惯性矩减小，基于其几何特性预测Brt 1骨应该更弱。在4点弯曲失效测试中，Brtl骨在1个月和2个月时以低于对照组的力断裂，但在6个月时具有相等的强度。这种弱几何性质和相等机械性质的组合预测Brtl骨本身的组成在小鼠青春期后发生了变化。这使得Brt 1小鼠成为OI的良好描述的临床特征（青春期后骨脆性降低）的极好模型。 我们还使用Brtl小鼠进行双膦酸盐的治疗试验。小鼠中的治疗试验是III型和IV型OI儿童中的治疗试验。在儿科试验中，我们正在进行一项四臂对照试验，儿童被随机分配到帕米膦酸盐，生长激素，两种药物或没有药物。正在比较治疗对椎体和长骨的影响。在药物的小鼠试验中，我们使用的是阿仑膦酸钠。对照或Brt 1小鼠已经显示响应于治疗的脊柱和长骨的骨密度增加。生物力学测试将显示这种增加的密度如何影响骨强度和脆性。 Brt 1小鼠也是一个很好的模型，其中接近OI的基因治疗，因为核酶（RZ）切割位点被工程化到Brt 1突变等位基因。通过RZ切割突变转录物可以抑制突变蛋白的表达，并且在生物化学上模拟无效等位基因，这在人类中是临床上轻微的。在过去的一年中，我们已经发表了我们的工作与RZ在培养的OI成纤维细胞，显示等位基因特异性抑制突变型胶原蛋白转录到约50%的基线水平在对照细胞。我们通过与Brt 1交配产生了用于转移治疗性分子的RZ小鼠，并且正在测试RZ对RZ转基因的正常骨的作用和比较各种RZ构建体的反义作用的过程中。 在我们的研究人类胶原突变引起OI，我们一直专注于一组突变的I型和V型胶原，影响原纤维的形成。在这些突变中的一个中，存在含有远肽结合位点的a1（I）中的外显子的缺失。即使在一小部分单体中缺少该外显子也会导致先证者纤维形成的显著延迟。的总胶原蛋白混合物。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。