Heritable Disorders Of Connective Tissue

本科开展研究，阐明结缔组织遗传性疾病的分子和生化机制，特别是成骨不全症（OI）和ehers - danlos综合征（EDS），并将这些信息应用于这些疾病的治疗。我们之前已经开发了一种非致死性成骨不全的敲入小鼠模型，我们将其命名为脆性小鼠（Brtl）。Brtl在其胶原α - 1(I)链中有一个经典的甘氨酸取代（G349C），再现了OI的分子、生化和组织学特征。我们一直在合作研究Brtl长骨的组织形态学和生物力学。在中轴处，Brtl骨在1、2和6个月时横截面积减小，计算的惯性矩减小，根据其几何特性预测Brtl骨应该更弱。在4点弯曲试验中，Brtl骨折在1个月和2个月时的强度低于对照组，但在6个月时强度相等。这种微弱的几何特性和相等的力学特性的结合预示着Brtl骨本身的组成在小鼠的青春期后发生了变化。这使得Brtl小鼠成为描述良好的成骨不全临床特征（青春期后骨质脆性降低）的绝佳模型。我们还使用Brtl小鼠进行双膦酸盐的治疗试验。小鼠的治疗试验与III型和IV型成骨不全症儿童的治疗试验平行。在儿科试验中，我们做了一个四组对照试验，孩子们被随机分配到帕米膦酸盐，生长激素，有药或无药。目前正在比较治疗对椎体和长骨的影响。在药物的小鼠试验中，我们使用的是二磷酸阿仑膦酸盐。对照或Brtl小鼠在治疗后已经显示出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。Brtl小鼠也是进行OI基因治疗的一个极好的模型，因为一个核酶（RZ）切割位点被设计到Brtl突变等位基因中。RZ切割突变体转录本可以抑制突变蛋白的表达，生化模拟一个空等位基因，在人类临床表现为轻度。在过去的一年里，我们发表了RZ在培养成骨不全成纤维细胞中的研究成果，显示在对照细胞中，突变胶原转录的等位基因特异性抑制约为基线水平的50%。我们通过与Brtl配对，培育了RZ小鼠用于转移治疗分子，并正在测试RZ转基因RZ对正常骨骼的影响，并比较不同RZ构建体的反义作用。在我们对导致成骨不全的人类胶原蛋白突变的研究中，我们一直关注影响纤维形成的I型和V型胶原蛋白的一组突变。在其中一种突变中，a1 (I)中含有端肽结合位点的外显子缺失。即使一小部分单体缺少这个外显子，也会导致先证者纤维形成的显著延迟。S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长度是对照分子的4-5倍。

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.