BONE MARROW TRANSPLANTATION IN OIM--A GENE THERAPY MODEL

OIM 骨髓移植——基因治疗模式

• 批准号: 2006508
• 负责人: DANIEL J MCBRIDE
• 金额: $ 11.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2001-12-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2006508
• 关键词: SDS polyacrylamide gel electrophoresis; bone marrow transplantation; cell proliferation; collagen; gene expression; gene therapy; genetic transcription; genetically modified animals; laboratory mouse; messenger RNA; nucleic acid hybridization; nucleic acid sequence; osteoblasts; osteogenesis imperfecta; polymerase chain reaction; tissue /cell culture

Bone formation occurs during embryonic development, somatic growth, and during bone remodeling and repair. The ability to form bone throughout an organisms life suggests the continuous presence of a reservoir of cells capable of osteogenesis. Osteoblasts are the differentiated cell type responsible for the biosynthesis, secretion, and orderly deposition of type 1 collagen and other bone matrix elements. A number of studies have demonstrated that the population of adherent marrow stromal cells include stem cells for at least four types of connective tissue: hematopoiesis-supporting stroma, adipose, cartilage, and bone. Osteogenesis imperfecta (01), caused by mutations in type 1 procollagen genes, is one example of a disease that may benefit from somatic gene therapy. Since bone fragility in 01 is caused by structural mutations in the genes that encode type 1 procollagen, candidate cells for 01 gene therapy are osteoblast progenitor cells and/or osteoblasts. Mice with the oim mutation are a valuable tool to investigate this question since these mice provide convenient markers for wild type and oim alleles at the gene, mRNA, and protein levels. The rationale for these studies is that mice heterozygous for the oim mutation have an improved phenotype compared to homozygous mice in terms of increased body weight and bones that are less brittle. Two problems related to somatic gene therapy for 01 are addressed by this proposal: (1) the identification and characterization of the cellular target vehicle using bone marrow transplantation and (2) determining the minimal level of replacement gene expression in transgenic mice that results in phenotypic improvement. The transplantation studies will identify optimal transplantation conditions for engraftment and proliferation of donor osteoprogenitor cells into oim/oim mice. This work will form the basis of future work using genetically modified cells. Our markers for success in this project are (1) allele specific-oligonucleotides that can discriminate wild type from oim gene nucleotide sequence, (2) allele specific oligonucleotides that can discriminate wild type from oim mRNA sequence, and (3) SDS-PAGE analysis for presence of the proalpha2(I) chain in cell culture and tissue samples. Successful engraftment and expression of normal collagen in bone is only one measure of success. A more important test is the demonstration of a functional improvement in bone biomechanical properties. Variable expression of proalpha2(I) cDNA constructs in transgenic mice will establish a relationship between construct expression (assessed at the mRNA and protein levels) and functional improvement determined by ex vivo mechanical testing of femurs.

骨形成发生在胚胎发育，体细胞生长和 在骨骼重塑和修复过程中。整个骨骼的能力 生物的生活表明，不断存在 能够成骨的细胞。成骨细胞是分化的细胞 负责生物合成，分泌和有序沉积的类型 1型胶原蛋白和其他骨基质元件的元素。 许多研究 已经证明了粘附的骨髓基质细胞的种群 包括至少四种类型的结缔组织的干细胞： 造血基质的基质，脂肪，软骨和骨骼。 成骨的不完美（01），是由1型Procollagen突变引起的 基因是一种可能受益于体细胞基因的疾病的例子 治疗。由于01中的骨骼脆弱性是由结构突变引起的 编码1型Procollagen的基因，候选细胞01基因 治疗是成骨细胞祖细胞和/或成骨细胞。带有的老鼠 OIM突变是调查这个问题的宝贵工具，因为这些 小鼠为野生型和OIM等位基因提供方便的标记 基因，mRNA和蛋白质水平。这些研究的理由是 OIM突变的杂合子的小鼠具有改善的表型 与纯合小鼠相比，体重增加和骨骼 不太脆弱。与躯体基因疗法有关的两个问题 本提案解决了01：（1）身份证明和 使用骨髓来表征细胞目标车辆 移植和（2）确定最低替代基因的水平 转基因小鼠的表达导致表型改善。这 移植研究将确定最佳移植条件 用于植入和扩散供体的骨源细胞进入 OIM/OIM小鼠。这项工作将构成未来工作的基础 转基因细胞。我们在这个项目中取得成功的标记是 （1）可以区分野生类型的等位基因特异性 - 寡核苷酸 OIM基因核苷酸序列，（2）等位基因特异性寡核苷酸 可以将野生型与OIM mRNA序列区分开，（3）SDS-PAGE 分析细胞培养中存在proalpha2（i）链的存在和 组织样品。正常胶原蛋白的成功植入和表达 在骨头中只是成功的一种衡量标准。一个更重要的测试是 骨生物力学功能改善的演示 特性。 proalpha2（i）cDNA构建体的可变表达 转基因小鼠将建立构造之间的关系 表达（在mRNA和蛋白质水平上评估）和功能 通过股骨的离体机械测试确定的改进。