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

基本信息

批准号：
8638894
负责人：
David W. Rowe
金额：
$ 39.34万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
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/8638894
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 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 alpha Collagen 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 public health relevance repaired skeletal stem cell biology stem cell technology stem cell therapy three dimensional structure tool transcription factor

项目摘要

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，成骨不完美（OI）一直是研究由突变导致的主要遗传结缔组织疾病的范式，该突变破坏了主要细胞外蛋白的三维结构。 OI突变的研究表明，该疾病不仅来自不当组织的骨基质，该骨基质不断进行重塑（高离职骨），而且还来自由于错误折叠胶原链的高细胞内含量（低胶原蛋白链）和持耐受性的骨质分化而导致的基质产生受损。尽管针对这些机制的任何一种疗法都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。随着干细胞生物学的进步和在定义的基因组部位的同源重组的方法，OI基因/干细胞疗法的潜在路线图变得可行。该应用程序将在我们设想的情况下测试路线图的实践。从具有遗传定义突变的正常和OI受试者获得的成纤维细胞，导致疾病的最严重形式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。为了遵循这些细胞的成骨命运，使用验证的Zn手指技术将骨限制的记者基因（COL2.3GFP）插入AAVR1基因座。随后，使用TALEN靶向蛋白将OI-IPS系的基因组裂解在COL1A1基因的突变部位附近，以将DNA引导到宿主染色质修复机制中。 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.一旦获得了校正的OI-IPS克隆细胞系，它们的能力纠正骨骼修复缺陷以及对照IPS克隆，肯定比未矫正的OI-IPS克隆更好，将通过基于荧光的冷冻组织学，分子表达和机械标准来评估。该建议是原理证明，校正OI突变将恢复正常的成骨细胞分化和功能齐全的骨基质的产生，这将产生最需要的蜂窝工具，以确定最佳的交付方式细胞回到受影响的宿主，并对骨骼健康和功能产生最大的临床影响。此外，通过在仍然能够具有多条件分化的IPS细胞中提供快速且相对较低的成本方法，该技术途径将适用于许多其他主要遗传性疾病。