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

• 批准号: 8771055
• 负责人: Zoia Monaco
• 金额: $ 17.3万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-06 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8771055
• 关键词: Address; Affect; Alternative Splicing; Biological Assay; Biology; Biomedical Engineering; Cell Line; Cell division; Cells; Centromere; Chromosome Structures; Chromosomes; Chromosomes, Artificial, Human; Complement; Cultured Cells; DNA; DNA Binding; DNA Repair; DNA Repair Disorder; DNA Repair Pathway; Defect; Dental Schools; Development; Disease; Disease model; Engineering; Ensure; Environment; Euchromatin; Excision; Fibroblasts; Gene Delivery; Gene Expression; Gene Mutation; Gene Transduction Agent; Gene Transfer; Generations; Genes; Genetic; Genetic Materials; Genome; Genomics; Goals; Health; Hereditary Disease; Herpesvirus 1; Heterochromatin; Human; Hypoxanthine Phosphoribosyltransferase; In Vitro; Insertional Mutagenesis; Malignant Neoplasms; Mediating; Medicine; Methods; Modeling; Monaco; Mutate; Mutation; Neurologic; Nucleic Acid Regulatory Sequences; Nucleotides; Oncogenic; Organ; Other Genetics; Pathway interactions; Patients; Phenotype; Proteins; Reporter; Research; Research Personnel; Resistance; Risk; Site; Skin; Skin Cancer; Skin Tissue; Stem cells; 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; drug discovery; gene therapy; human disease; human embryonic stem cell; keratinocyte; novel; prevent; public health relevance; skin disorder; stem cell differentiation; therapeutic gene; ultraviolet damage; vector

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，转基因与病毒载体一起递送，其通常不能容纳具有其所有调控序列和可变剪接位点的整个基因组基因座。这些载体随机整合外源遗传物质，可能导致插入诱变。为了解决这些关键的限制，我们建议使用人类人工染色体（HAC）作为载体的基因治疗研究。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，没有插入诱变的风险，高度稳定，并提供延长的基因表达。在这个项目中，我们的目标是建立HAC作为非整合，高容量载体的基因治疗在人类成纤维细胞和角质形成细胞，重点作为一个模型疾病的着色性干皮病（XP）在其最常见的形式，XPC。XP是一种遗传性疾病，其特征是发生皮肤癌和进行性神经障碍，是由核苷酸切除DNA修复途径中的突变引起的。XPC蛋白是一种损伤敏感和DNA结合因子。我们希望证明，从XP患者的细胞可以保护免受紫外线损伤的基因转移的一个完整的XPC基因沿着其调控序列。为了弥补缺陷（目的1），我们将引入患者来源的成纤维细胞和角质肿细胞的HAC载体含有整个XPC基因。其次，我们将使用校正的成纤维细胞和角质形成细胞生成XP的功能性3D皮肤等效模型，并评估补充XP缺陷是否恢复正常的DNA修复（目的2）。我们的研究团队包括了许多我们将使用的方法的先驱专家。Zoia Monaco的实验室是第一个证明人类细胞中遗传缺陷的基于HAC的互补作用的实验室。Daniela Moralli开发了一种基于HSV-1的技术，该技术允许在各种细胞类型中直接产生HAC，并有助于使该技术适用于人类胚胎干细胞。Jonathan Garlick是3D人类皮肤样组织生物工程的领导者，用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是一位国际知名的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，将整个基因组位点递送到人类细胞中以补充遗传缺陷。