Targeted Correction of Dominant Mutations of Type I Collagen Causing Severe OI

针对性纠正导致严重成骨不全的 I 型胶原蛋白显性突变

• 批准号: 9038760
• 负责人: David W. Rowe
• 金额: $ 32.73万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9038760
• 关键词: Adult; Affect; Back; Blood; Bone Matrix; Bone Regeneration; COL1A1 gene; Calvaria; Cell Line; Cell Therapy; Cells; Child; Chromatin; Cleaved cell; Clinical; Clone Cells; Code; Collagen; Collagen Gene; Collagen Type I; Connective Tissue Diseases; DNA; DNA Sequence; DNA Sequence Alteration; Defect; Derivation procedure; Disease; Embryo; Engraftment; Exons; Extracellular Protein; Fibroblasts; Fingers; Fluorescence; Gene Expression; Gene Mutation; Gene Silencing; Gene Targeting; Gene-Modified; Genes; Genetic; Genetic Recombination; Genome; Genomics; Goals; Grant; Healed; Health; Human; Immune; Immunocompromised Host; Individual; Induced Mutation; Inherited; Institutes; Introns; Laboratories; Mechanics; Methods; Modeling; Molecular; Mus; Mutation; Nonhomologous DNA End Joining; Normal Cell; Osteoblasts; Osteogenesis Imperfecta; Outcome; Pathway interactions; Patients; Pharmaceutical Preparations; Point Mutation; Population; Positioning Attribute; Process; Production; Proteins; Protocols documentation; Publishing; Reporter; Reporter Genes; Reporting; Research Personnel; Severity of illness; Simplexvirus; Site; Skin; Source; Stem cells; Techniques; Technology; Testing; Therapeutic; Tissues; Transfection; Validation; Viral; Viral Genes; Work; adeno-associated viral vector; base; bone; bone cell; bone turnover; cost; gel electrophoresis; gene correction; healing; homologous recombination; human embryonic stem cell; improved; in vivo; induced pluripotent stem cell; long bone; mutant; nanoindentation; neonate; novel strategies; nuclease; osteoblast differentiation; osteogenic; osteoprogenitor cell; progenitor; repaired; skeletal; stem cell biology; stem cell technology; stem cell therapy; three dimensional structure; tool; transcription activator-like effector nucleases; transcription factor; whole genome

DESCRIPTION (provided by applicant): Historically, osteogenesis imperfecta (OI) has been the paradigm for the study of a dominantly inherited disease of connective tissue resulting from a mutation that disrupts the three dimensional structure of a major extracellular protein. The study of OI mutations has shown that the disease results not only from an improperly organized bone matrix that is continually undergoing remodeling (high turnover bone) but also from impaired matrix production due to the high intracellular content of misfolded collagen chains (low collagen secretion) and retarded osteoblast differentiation. While therapies directed at any of these mechanisms can improve disease severity, everyone agrees that correction of the underlying genetic mutation is the ultimate therapeutic goal. With advances in stem cell biology and in methods for homologous recombination at a defined genomic site, a potential roadmap for a gene/stem cell therapy for OI has become feasible. This application will test the practicalit of the roadmap as we envision it. Fibroblasts obtained from normal and OI subjects with genetically defined mutations resulting in the severest forms of the disease will be converted to iPS cell using methods that do not integrate the transcription factors into the host genome. To follow the osteogenic fate of these cells, a bone restricted reporter gene (Col2.3GFP) will be inserted into the AAVR1 locus using a proven Zn finger technology. Subsequently the genome of OI-iPS line will be cleaved near the site of the mutation in the Col1A1 gene using TALEN targeting proteins to direct correcting DNA into the host chromatin repair mechanism. To enhance the possibility the cleaved DNA will be repaired by the desired homology-driven mechanism rather than the non-homologous end joining (NHEJ) mechanism, transient co-transfection of genes encoding herpes derived recombination enhancement proteins (UL12 and ICP8) will be added to the targeting protocol to determine if a higher proportion of correctly targeted clones is obtained. Once corrected OI-iPS clonal cell lines are obtained, their ability to correct a skeletal repair defect as well as control iPS clones, and certainly better than the uncorrected OI-iPS clones, will be evaluated by fluorescence-based cryohistological, molecular expression and mechanical criteria. This proposal is a proof of principle that correction of OI mutations will restore normal osteoblast differentiation and fully functional bone matrix production, which will yield the most desired cellular tool to determine the best manner to deliver the cells back to the affected host and achieve the greatest clinical impact on skeletal health and function. In addition, the technical pathway will be applicable to many other dominantly inherited diseases by providing a rapid and relatively low cost method for gene correction in IPS cells that are still capable of multi-lineage differentiation.

描述（由申请人提供）：从历史上看，成骨不全（OI）一直是研究由破坏主要细胞外蛋白三维结构的突变引起的结缔组织显性遗传性疾病的范例。OI突变的研究表明，这种疾病不仅是由于骨基质组织不正常，不断进行重塑（高转换骨），而且由于错误折叠的胶原蛋白链的细胞内含量高（低胶原蛋白分泌）和成骨细胞分化迟缓而导致基质产生受损。虽然针对这些机制的治疗可以改善疾病的严重程度，但每个人都同意纠正潜在的基因突变是最终的治疗目标。随着干细胞生物学和在确定的基因组位点进行同源重组的方法的进步，用于OI的基因/干细胞治疗的潜在路线图已经变得可行。本申请将测试我们设想的路线图的实用性。从正常和OI受试者中获得的成纤维细胞具有导致疾病最严重形式的遗传定义突变，将使用不将转录因子整合到宿主基因组中的方法转化为iPS细胞。为了跟踪这些细胞的成骨命运，将使用经证实的锌指技术将骨限制性报告基因（Col2.3GFP）插入AAVR 1基因座中。随后，将使用TALEN靶向蛋白在Col 1A 1基因中的突变位点附近切割OI-iPS系的基因组，以将校正DNA引导到宿主染色质修复机制中。为了提高切割的DNA通过所需的同源驱动机制而不是非同源末端连接（NHEJ）机制修复的可能性，编码疱疹衍生重组增强蛋白（UL 12和ICP 8）的基因的瞬时共转染将被添加到靶向方案中，以确定是否获得更高比例的正确靶向克隆。一旦获得校正的OI-iPS克隆细胞系，则它们的表达能力将降低。 通过基于荧光的冷冻组织学、分子表达和机械标准来评价校正骨骼修复缺陷以及对照iPS克隆，并且肯定优于未校正的OI-iPS克隆。该提议是一个原则证明，即矫正OI突变将恢复正常的成骨细胞分化和全功能的骨基质生产，这将产生最需要的细胞工具，以确定最佳的递送方式。 这些细胞返回受影响的宿主，并对骨骼健康和功能产生最大的临床影响。此外，该技术途径将通过提供用于仍能够进行多谱系分化的IPS细胞中的基因校正的快速且相对低成本的方法而适用于许多其它显性遗传疾病。