Healing of Segmental Defects of Bone by Gene Transfer

基因转移修复节段性骨缺损

• 批准号: 8301760
• 负责人: CHRISTOPHER Howard EVANS
• 金额: $ 63.39万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8301760
• 关键词: Actins; Address; Adenovirus Vector; Adenoviruses; Animal Model; Animals; Attenuated; Autologous; Autopsy; Biodistribution; Biological; Biology; Blood Vessels; Calcified; Cartilage; Cells; Chemotaxis; Chondrocytes; Clinical; Collaborations; Complementary DNA; Contralateral; DNA; Data; Defect; Deposition; Development; Diaphyses; Dose; Effectiveness; Endothelial Cells; Euthanasia; Evaluation; Fatty acid glycerol esters; Feasibility Studies; Femur; Fluoroscopy; Fracture; Funding; Gene Expression; Gene Transfer; Genes; Genetic; Genetic Enhancement; Genetically Modified Animals; Goals; Grant; Harvest; Healed; Histology; Histopathology; Human; Image; Immune; Immune response; Immune system; Immunosuppressive Agents; Implant; In Vitro; Inbred F344 Rats; Infection; Inferior; Injection of therapeutic agent; LacZ Genes; Lesion; Luc Gene; Luciferases; Mechanics; Mediating; Methods; Modality; Modeling; Morphology; Mus; Muscle; Muscle Cells; Myosin ATPase; Nude Rats; Operative Surgical Procedures; Organ; Osteoblasts; Osteocalcin; Osteogenesis; Osteotomy; Population; Positioning Attribute; Procedures; Process; Production; Progress Reports; Property; Radiology Specialty; Rattus; Reaction; Recombinants; Reporter; Reporter Genes; Research; Research Institute; Role; Rosa; Series; Sheep; Skeletal Muscle; Staining method; Stains; Stem cells; Steroids; Switzerland; Technology; Testing; Thick; Time; TimeLine; Transcriptional Regulation; Transgenes; Translational Research; Vascular Endothelial Cell; Viral Genome; Virus; Work; X-Ray Computed Tomography; Xenograft procedure; base; bone; bone cell; bone healing; bone morphogenetic protein 2; cell type; gene therapy; head-to-head comparison; healing; implantation; improved; in vivo; insight; long bone; muscle form; new technology; novel; offspring; osteogenic; osteoprogenitor cell; programs; promoter; public health relevance; recombinant human bone morphogenetic protein-2; recombinase; research study; response; safety testing; scaffold; tissue repair; transgene expression; vector

DESCRIPTION (provided by applicant): This competing renewal seeks to continue our development of novel, gene-based methods for improving the healing of large segmental defects in bone. For reasons discussed in the body of the grant, a recombinant adenovirus vector carrying the cDNA for human bone morphogenetic protein-2 (Ad.BMP-2) is used for this project. During the first funding cycle of this grant, using a rat femoral defect model, we noted a remarkable improvement in defect healing when skeletal muscle from syngeneic rats was transduced with Ad.BMP-2 and then inserted into the defect. Under these conditions, healing of the defect was rapid, reliable and uniform. In the next cycle of this grant we propose two Specific Aims that will: i) establish the biology that underlies this phenomenon and ii) evaluate the efficacy of the technology in a large animal model, the sheep. The latter Aim will be completed in collaboration with AO Research Institute in Davos, Switzerland. Specific Aim 1 will test the hypothesis that the high effectiveness of Ad.BMP-2 modified muscle grafts in promoting bone healing reflects the ability of the graft to supply not only endogenously synthesized BMP-2, but also progenitor cells that form cartilage and then bone. Because immune responses to adenovirus is a recognized problem for certain applications of adenovirus-based gene therapy, Fischer rats will be used to determine the duration of transgene expression in the rat and the role of the immune system in curtailing expression. Based upon our preliminary data, the abbreviated ex vivo method we have developed eliminates the humoral response to the virus. To enable us to take advantage of genetically modified mice to address the other questions raised in Specific Aim 1, we have developed a new model in which muscle from genetically modified mice are inserted into femoral defects in athymic rats. Using this model we will insert Ad.BMP-2 transduced muscle grafts from the following genetically modified mice: mice containing the luciferase gene under the control of the human osteocalcin promoter; Rosa mice, all of whose cells are LacZ+; and mice derived from crosses between Tie2- Cre mice and R26R mice to enable lineage analysis of tie2+ cells derived from endothelial cells of blood vessels. The sheep studies will utilize a standard 3cm, critical size tibial defect to evaluate the reparative ability of Ad.BMP-2 transduced autologous muscle grafts. Healing will be assessed by serial radiology. After 6 months, sheep will be euthanized and healing assessed by micro-computed tomography, histology, mechanical testing and intravital fluoroscopy. The immune response of the sheep to the vector will also be assessed, and various organs will be recovered at necropsy for evaluation of histopathology and the distribution of viral genomes. PUBLIC HEALTH RELEVANCE: The proposed research investigates a novel technology for improving the healing of large bone fractures. The method involves the converting muscle into bone by transferring a gene encoding bone morphogenetic protein- 2 using an adenovirus.

描述（由申请人提供）：这种竞争性更新旨在继续我们开发基于基因的新型，基于基因的方法，以改善骨骼中较大的分段缺陷的愈合。由于赠款的主体讨论的原因，该项目使用了携带人骨形态发生蛋白-2（AD.BMP-2）cDNA的重组腺病毒载体。在该赠款的第一个融资周期中，使用大鼠股骨缺陷模型，当通过AD.BMP-2转导骨骼肌肉时，我们注意到缺陷愈合的明显改善，然后将其插入缺陷。在这些条件下，缺陷的愈合是快速，可靠和均匀的。在这笔赠款的下一个周期中，我们提出了两个具体目标：i）建立这种现象和ii的生物学，ii）评估该技术在大型动物模型绵羊中的功效。后一个目标将与瑞士达沃斯的AO研究所合作完成。具体目标1将检验以下假设：AD.BMP-2改良的肌肉移植物在促进骨骼愈合方面的高有效性反映了移植物不仅提供内源合成的BMP-2的能力，还反映了形成软骨和骨骼的祖细胞的能力。由于对腺病毒的免疫反应是基于腺病毒基因疗法的某些应用的公认问题，因此Fischer大鼠将用于确定大鼠中转基因表达的持续时间以及免疫系统在减少表达中的作用。基于我们的初步数据，我们开发的缩写离体方法消除了对病毒的体液反应。为了使我们能够利用转基因的小鼠解决特定目标1中提出的其他问题，我们开发了一种新的模型，在该模型中，从转基因小鼠的肌肉中插入了无胸腺大鼠的股骨缺陷中。使用该模型，我们将从以下遗传改性的小鼠中插入AD.BMP-2转导的肌肉移植物：在人骨钙化蛋白启动子的控制下含有荧光素酶基因的小鼠；罗莎小鼠，所有细胞都是lacz+；从扎2- CRE小鼠和R26R小鼠之间的杂交中得出的小鼠可以对源自血管内皮细胞的TIE2+细胞进行谱系分析。绵羊研究将利用标准的3cm，临界大小的胫骨缺陷来评估AD.BMP-2转导的自体肌移植物的修复能力。愈合将通过串行放射学评估。 6个月后，将对绵羊进行安乐死，并通过微型层析成像，组织学，机械测试和插入式荧光镜检查来评估绵羊。绵羊对载体的免疫反应也将进行评估，并在尸检时回收各种器官，以评估组织病理学和病毒基因组的分布。 公共卫生相关性：拟议的研究调查了一种新的技术，用于改善大骨骨折的愈合。该方法涉及通过使用腺病毒来传递编码骨形态发生蛋白2的基因，将肌肉转化为骨骼。