Dopaminergic transcription factor delivery to fibroblast derived neurons

多巴胺能转录因子递送至成纤维细胞衍生的神经元

• 批准号: 8334940
• 负责人: PAUL S FISHMAN
• 金额: --
• 依托单位: BALTIMORE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-10-01 至 2014-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8334940
• 关键词: Achievement; Address; Adult; Affect; Aging; Alzheimer' s Disease; Brain; Cell Line; Cell Transplantation; Cells; Characteristics; Clinical; Clinical Trials; Culture Media; Cytosol; Development; Dopaminergic Cell; Elements; Endocytic Vesicle; Event; Fibroblasts; Gene Delivery; Gene Targeting; Generations; Genes; Genetic; Genome; Germ Lines; Goals; Growth Factor; Human; Laboratories; Lentivirus Vector; Light; Link; Long-Term Effects; Longitudinal Studies; Male Circumcision; Membrane; Methods; Motor; Neurodegenerative Disorders; Neuronal Differentiation; Neurons; Nuclear; Oncogenic; Parkinson Disease; Patients; Peptides; Population; Process; Production; Proteins; Replacement Therapy; Research; Risk; Safety; Services; Site-Directed Mutagenesis; Skin; Solutions; Somatic Cell; Source; Stem Cell Research; Stem cells; Substantia nigra structure; Sushi Domain; Symptoms; Teratoma; Therapeutic; Tissues; Transcription factor genes; Translations; Transplant Recipients; Transplantation; Vesicle; Veterans; Vietnam; Viral Vector; Work; age related; agent orange; base; cell type; cellular transduction; design; dopaminergic differentiation; dopaminergic neuron; embryonic stem cell; experience; human tissue; improved; induced pluripotent stem cell; link protein; nerve stem cell; pars compacta; penis foreskin; protein expression; public health relevance; relating to nervous system; transcription factor; tumor; vector-induced

DESCRIPTION (provided by applicant): A new cell type has been very recently created with clear relevance for therapeutic transplantation into patients with Parkinson's disease (PD). Fibroblasts derived from human skin have been induced to become neurons, and some of these cells can be differentiated into functional dopaminergic neurons, the cell type whose loss leads to most of the motor symptoms of PD. This direct trans-differentiation process does not require the creation of an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC), cell types with a risk of tumor formation (teratoma) after long term transplantation into the brain. The strategy behind this achievement is the identification of transcription factors associated with key developmental events, and the transduction of human fibroblasts with several lentiviral vectors containing the genes for the selected factors. However this strategy also raises potential safety issues which must be resolved prior to clinical trials. These viral vectors integrate into the genome of the hos cells with the potential for tumor induction. The long term effects of sustained and unregulated expression of these multi-functional and potent genes are also unknown. Alternatives to multiple lentiviral vectors for inducing trans-differentiation of somatic cells are under active investigatin. Our proposed project will assess the delivery of transcription factor protein as an alternative to viral vector based transcription factor gene delivery in the induction of dopaminergic differentiation of human fibroblasts. Our group is one of few with experience in the production of neural transcription factor protein modified to include a small peptide (cell penetrating peptide o CPP) to allow the protein to enter target cells, through a process, known as protein transduction. Such a process is particularly suited for this setting where multiple factors need to be delivered in sequence or in combination, with each purified factor added to the culture medium in a manner analogous to current use of natural growth factors. We propose to produce CPP linked forms of the transcription factors Lmx1a and FoxA2, two factors that have been shown to be involved in the dopaminergic differentiation of neurons induced form human fibroblasts. We will determine the extent to which delivery of these modified transcription factors via protein transduction can promote the dopaminergic differentiation of fibroblast derived induced neurons. We will directly compare dopaminergic differentiation driven by CPP linked factors to that seen with lentiviral vector transduction. Cells will be assessed using immuno-histochemical methods to detect and quantitate expression proteins associated with neuronal and dopaminergic differentiation, virtually never seen in normal fibroblasts. The major limitation of CCP based delivery is the very low efficiency of passage of active protein into the free cytosol of the cells where it can then access nuclear gene targets for transcription factors. Most of the presented protein is engulfed and trapped in endocytic vesicles. Our laboratory is one of few actively investigating methods to overcome this limitation. A promising method is photochemical internalization (PCI) where a photosensitizing molecule and appropriate wavelength of light results in release of CPP-linked proteins from membrane enclosed vesicles. We will determine the extent to which the efficiency of protein transduction based delivery of transcription factors and resulting dopaminergic differentiation of fibroblast derived induced neurons can be enhanced through the application of PCI. In this manner we will address two major issues that currently limit the potential clinical translation of this promising new cell type for transplantaton based therapy for Parkinson's disease.

描述（由申请人提供）： 最近已经创建了一种新的细胞类型，与治疗性移植到帕金森氏病（PD）患者中具有明显相关性。源自人皮肤的成纤维细胞已被诱导成为神经元，其中一些细胞可以分化为功能性多巴胺能神经元，该细胞类型会导致大多数PD运动症状。这种直接差异过程不需要产生胚胎干细胞（ESC）或诱导的多能干细胞（IPSC），即长期移植到大脑后具有肿瘤形成风险（Teratome）风险的细胞类型。这项成就的策略是鉴定与关键发育事件相关的转录因子，以及与几个含有基因的慢病毒载体的人类成纤维细胞转载。但是，该策略还提出了潜在的安全问题，必须在临床试验之前解决这些问题。这些病毒载体整合到HOS细胞的基因组中，具有肿瘤诱导的潜力。这些多功能和有效基因的持续和不受监管表达的长期影响也未知。多个慢病毒载体诱导体细胞的跨分化的替代方案正在活跃研究。我们提出的项目将评估转录因子蛋白的递送，作为基于病毒载体的转录因子基因递送的替代品，在诱导人成纤维细胞的多巴胺能分化。我们的小组是少数在生产神经转录因子蛋白质的经验中的少数之一，其中包括小肽（细胞穿透肽O CPP），以使蛋白质通过一种称为蛋白质转导的过程进入靶细胞。这样的过程特别适合这种环境，在这种情况下，需要以序列或组合进行多个因素，每个纯化因子都以类似于当前使用自然生长因子的方式添加到培养基中。我们建议生产CPP链接的转录因子LMX1A和FOXA2的形式，这两个因子已证明与神经元的多巴胺能分化有关，引起了人类成纤维细胞。我们将确定通过蛋白质转导的这些修饰转录因子的递送的程度可以促进成纤维细胞衍生的诱导神经元的多巴胺能分化。我们将直接比较由CPP链接的因子驱动的多巴胺能分化与慢病毒载体转导的因子。将使用免疫 - 归化方法评估细胞，以检测和定量与神经元和多巴胺能分化相关的表达蛋白，实际上在正常成纤维细胞中从未见过。基于CCP的递送的主要局限性是，活性蛋白进入细胞的游离胞质液的效率非常低，然后可以在其中访问转录因子的核基因靶标。大多数提出的蛋白质被吞噬并捕获在内吞囊泡中。我们的实验室是少数积极研究这一限制的方法之一。一种有希望的方法是光化学内在化（PCI），其中光敏分子和适当的光长度导致从膜封闭囊泡中释放CPP连接的蛋白质。我们将确定基于转录因子的基于蛋白质转导的效率以及导致衍生诱导神经元的多巴胺能分化的效率可以通过PCI的应用来提高。通过这种方式，我们将解决两个主要问题，这些问题目前限制了这种有希望的新细胞类型的临床翻译，用于基于移植的帕金森氏病治疗。