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

描述(申请人提供)：对于许多已知遗传原因的疾病，基因疗法似乎是最有希望的治疗方法。最常见的情况是，转基因是通过病毒载体传递的，而病毒载体往往不能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。为了解决这些关键的限制，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可以弥补培养细胞中的基因缺陷，对转基因大小没有限制，不会造成插入突变的风险，具有高度的稳定性，并提供长期的基因表达。在这个项目中，我们的目标是建立HAC作为非整合的、高容量的载体，用于在人成纤维细胞和角质形成细胞中进行基因治疗，并将其作为最常见的XPC的模式疾病。XP是一种遗传性疾病，其特征是皮肤癌的发展和进行性神经功能障碍，由核苷酸切除DNA修复途径的突变引起。XPC蛋白是一种损伤敏感和DNA结合因子。我们希望证明XP患者的细胞可以通过基因转移完整的XPC基因及其调控序列来保护细胞免受紫外线损伤。为了弥补这一缺陷(目标1)，我们将在患者来源的成纤维细胞和角质形成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形成细胞生成XP的功能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摩纳哥的实验室是第一个展示基于HAC的人类细胞遗传缺陷互补的实验室。Daniela Moralli开发了一种基于HSV-1的技术，允许在各种类型的细胞中直接产生HAC，并在将该技术应用于人类胚胎干细胞方面发挥了重要作用。乔纳森·加里克是3D人类皮肤样组织生物工程领域的领导者，他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，将整个基因组位点输送到人类细胞中，以补充遗传缺陷。

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.