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Human artificial chromosome as vectors for gene therapy of a model skin disease

人类人工染色体作为模型皮肤病基因治疗的载体

基本信息

批准号：
8906759
负责人：
Zoia Monaco
金额：
$ 20.85万
依托单位：
TUFTS UNIVERSITY MEDFORD
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-06 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8906759
关键词：
Address Affect Alternative Splicing Biological Assay Biology Biomedical Engineering Cell Line Cell division Cells Centromere Chromosome Structures Chromosomes Chromosomes, Artificial, Human Clinical Complement Cultured Cells DNA DNA Binding DNA Repair DNA Repair Disorder DNA Repair Pathway DNA Sequence Alteration Defect Dental Schools Development Disease Disease model Engineering Ensure Environment Euchromatin Excision Fibroblasts Gene Delivery Gene Expression Gene Transduction Agent Gene Transfer Generations Genes Genetic Genetic Materials Genome Genomic Segment Genomics Goals Health Hereditary Disease Herpesvirus 1 Heterochromatin Human Hypoxanthine Phosphoribosyltransferase In Vitro Insertional Mutagenesis International Malignant Neoplasms Mediating Medicine Methods Modeling Monaco Mutate Mutation Neurologic Nucleic Acid Regulatory Sequences Nucleotides Oncogenic Organ Other Genetics Patients Phenotype Physiological Proteins Reporter Research Research Personnel Resistance Risk Site Skin Skin Cancer Skin Tissue System Techniques Technology Testing Tissue Model Tissues Transfection Transgenes UV Radiation Exposure Undifferentiated United States National Institutes of Health Universities Viral Viral Vector Xeroderma Pigmentosum base cell type complement deficiency complement system drug discovery gene complementation gene therapy human disease human embryonic stem cell keratinocyte novel prevent public health relevance skin disorder skin organogenesis stem cell differentiation stem cell therapy therapeutic gene ultraviolet damage vector

项目摘要

DESCRIPTION (provided by applicant): For many diseases with known genetic causes, gene therapy appears to be the most promising approach to treatment. Most frequently, transgenes are delivered with viral vectors, which often cannot accommodate the entire genomic locus with all of its regulatory sequences and alternative splicing sites. These vectors randomly integrate the exogenous genetic material, possibly leading to insertional mutagenesis. To address these critical limitations, we propose to use human artificial chromosomes (HAC) as vectors for gene therapy studies. HAC can complement gene deficiencies in cultured cells, place no limitation on transgene size, pose no risk of insertional mutagenesis, are highly stable, and deliver prolonged gene expression. In this project, we aim to establish HAC as non-integrating, high capacity vectors for gene therapy in human fibroblast and keratinocyte cell, focusing as a model disease on Xeroderma pigmentosum (XP) in its most common form, XPC. XP, a genetic disorder characterized by development of skin cancers and progressive neurological disturbances, is caused by mutations in the nucleotide excision DNA repair pathway. The XPC protein is a damage-sensing and DNA-binding factor. We expect to demonstrate that cells from XP patients can be protected from UV damage by gene transfer of an intact XPC gene along with its regulatory sequences. To complement the deficiency (Aim 1), we will introduce into patient-derived fibroblasts and keratynocytes an HAC vector containing the entire XPC gene. Secondly, we will generate a functional 3D skin equivalent model for XP using the corrected fibroblasts and keratinocytes and assess whether complementing the XP deficiency restores normal DNA repair (Aim 2). Our research team includes experts who pioneered many of the methods we will use. Zoia Monaco's lab was the first to demonstrate HAC-based complementation of a genetic deficiency in human cells. Daniela Moralli developed an HSV-1 based technique that allows direct generation of HAC in a variety of cell types and was instrumental in adapting the technique to human embryonic stem cells. Jonathan Garlick is a leader in the bioengineering of 3D human skin-like tissues to study stem cell differentiation and cancer, and to provide tissue platforms for drug discovery. Ken Kraemer is an internationally renowned XP expert. Successful completion of these aims will demonstrate the feasibility of HAC-based gene therapy vectors, delivering entire genomic loci into human cells for complementation of genetic deficiencies.

描述（由申请人提供）：对于许多已知遗传原因的疾病，基因治疗似乎是最有希望的治疗方法。最常见的是，转基因是用病毒载体传递的，病毒载体通常无法容纳整个基因组位点及其所有调控序列和选择性剪接位点。这些载体随机整合外源遗传物质，可能导致插入突变。为了解决这些关键限制，我们建议使用人类人工染色体（HAC）作为基因治疗研究的载体。 HAC可以补充培养细胞中的基因缺陷，对转基因大小没有限制，不存在插入突变的风险，高度稳定，并能延长基因表达。在这个项目中，我们的目标是建立 HAC 作为人类成纤维细胞和角质形成细胞基因治疗的非整合、高容量载体，重点关注着色性干皮病 (XP) 最常见形式 XPC 的模型疾病。 XP 是一种遗传性疾病，其特征是发展为皮肤癌和进行性神经系统紊乱，是由核苷酸切除 DNA 修复途径的突变引起的。 XPC 蛋白是一种损伤感应和 DNA 结合因子。我们希望证明，通过完整 XPC 基因及其调控序列的基因转移，可以保护来自 XP 患者的细胞免受紫外线损伤。为了弥补这一缺陷（目标 1），我们将向患者来源的成纤维细胞和角质细胞中引入包含整个 XPC 基因的 HAC 载体。其次，我们将使用校正后的成纤维细胞和角质细胞生成 XP 的功能性 3D 皮肤等效模型，并评估补充 XP 缺陷是否可以恢复正常的 DNA 修复（目标 2）。我们的研究团队包括开创了我们将使用的许多方法的专家。 Zoia Monaco 的实验室是第一个证明基于 HAC 的人类细胞遗传缺陷补充的实验室。 Daniela Moralli 开发了一种基于 HSV-1 的技术，可以在多种细胞类型中直接生成 HAC，并有助于将该技术应用于人类胚胎干细胞。 Jonathan Garlick 是 3D 人体皮肤样组织生物工程领域的领导者，致力于研究干细胞分化和癌症，并为药物发现提供组织平台。 Ken Kraemer 是国际知名的 XP 专家。这些目标的成功完成将证明基于 HAC 的基因治疗载体的可行性，将整个基因组位点传递到人类细胞中以补充遗传缺陷。