Heritable Disorders Of Connective Tissue

该科进行研究，以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(OI)和埃勒斯-丹洛斯综合征(EDS)，并将这些信息应用于这些疾病的治疗。我们之前已经为非致命性OI开发了一种敲入小鼠模型，我们将其命名为脆性小鼠(Brtl)。Brt1在其一个胶原α1(I)链上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Brt1长骨的组织形态计量学和生物力学的合作研究。在中段，Brtl骨在1、2和6个月龄时横截面积减小，计算的转动惯量减小，根据其几何特性预测Brtl骨应该较弱。在4点弯曲试验中，Brtl骨在1个月和2个月时的断裂力量低于对照组，但在6个月时具有相同的强度。这种弱的几何特性和相等的机械特性的组合预测，小鼠的Brtl骨本身的成分在青春期后发生了变化。这使得Brtl小鼠成为OI临床特征的优秀模型，OI在青春期后降低骨骼脆性。我们还使用Brtl小鼠进行双膦酸盐的治疗试验。在小鼠身上的治疗试验与对III型和IV型OI儿童的治疗试验是平行的。在儿科试验中，我们正在进行一项四臂对照试验，将儿童随机分配到帕米磷酸钠、生长激素，两者都用药或不用药。目前正在比较这种疗法对椎体和长骨的疗效。在药物的小鼠试验中，我们使用的是双酚酸酯阿仑磷酸钠。对照组或Brtl小鼠已经显示出治疗后脊柱和长骨的骨密度增加。生物力学测试将显示这种密度增加是如何影响骨骼强度和脆性的。Brt1小鼠也是探讨OI基因治疗的一个很好的模型，因为核酶(RZ)切割位点被工程改造成Brt1突变等位基因。RZ对突变转录物的切割可以抑制突变蛋白的表达，并在生化上模仿零等位基因，这在人类的临床上是轻微的。在过去的一年里，我们在培养的OI成纤维细胞中发表了我们与RZ的工作，显示在对照细胞中，突变的胶原转录物被等位基因特异性抑制到基线水平的50%左右。我们培育了一只RZ小鼠，用于通过与Brt1配对来转移治疗分子，目前正在测试RZ对RZ转基因正常骨骼的影响，并比较各种RZ构建体的反义效果。在我们对引起OI的人类胶原突变的研究中，我们一直专注于一系列影响纤维形成的I型和V型胶原突变。在其中一个突变中，A1(I)中包含长肽结合位点的外显子缺失。即使在一小部分单体中缺少这个外显子，也会导致先证者S总胶原蛋白混合物的纤维形成显著延迟。由于这种延迟，主要是纤维组装动力学的纵向部分，在体外形成的胶原纤维是对照分子长度的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.