An Identification System for Targeted Gene Addition to MLD Patient Derived iPSCs

用于向 MLD 患者衍生的 iPSC 添加靶向基因的鉴定系统

• 批准号: 8822754
• 负责人: STEFAN MOISYADI
• 金额: $ 17.64万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8822754
• 关键词: Address; Antibiotics; Arylsulfatases; Automobile Driving; Binding; Biological Assay; Brain Diseases; CCR5 gene; CMV promoter; Candidate Disease Gene; Cells; Cessation of life; Chimeric Proteins; Clonal Expansion; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Binding Domain; Detection; Disease; Event; Flare; Fluorescence-Activated Cell Sorting; Gene Expression; Gene Targeting; Genes; Genome; Genomics; Goals; Human; Human Cell Line; Insertional Mutagenesis; Lead; Messenger RNA; Metachromatic Leukodystrophy; Methods; Modification; Mutation; Neomycin; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Outcome; Parents; Paste substance; Patients; Population; Population Heterogeneity; Production; Replacement Therapy; Reporter; Risk; Safety; Site; System; Therapeutic; Transcription Coactivator; Transgenes; Transposase; base; exome; exome sequencing; gene correction; gene therapy; genotoxicity; homologous recombination; improved; induced pluripotent stem cell; infancy; nano; nerve stem cell; nuclease; public health relevance; transgene expression

DESCRIPTION: Patient-specific induced pluripotent stem cells (iPSCs) hold enormous promise for personalized cell replacement therapy. Metachromatic leukodystrophy (MLD) is a neurodegenerative disease that, like many other monogenetic disorders, is a candidate for gene therapy using corrected iPSCs. Realizing the full potential of iPSCs requires reliable methods for performing gene targeting. A major challenge is reducing the risk of insertional mutagenesis due to random insertion. Targetable nucleases based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas9) and transcription activator like effector (TALE) nuclease (TALEN) systems are capable of inducing double stranded breaks (DSBs). These DSBs can enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, a targetable piggyBac (pB) transposase has recently been used by us to direct integration into a single genomic address. Currently, no direct comparison of genotoxicity between any pair of these strategies has been conducted. Importantly, all current methods significantly integrate at off-target sites necessitating a reliable system for the identification of safely modified cells. Our goal is to improve both the safety and efficiency in correcting a human iPSC gene deficiency using a combination of directed gene addition and target event detection. We have devised a tunable enrichment strategy, termed event detection, that will allow for the identification and isolation o rare correctly modified cells following targeted gene addition. We will use CRISPR, TALEN, and our recently developed targetable transposase system to target our event detection cassette to the ROSA26 genomic safe harbor. After validating multiple strategies in a human cell line we will apply event detection for use in identifying gene corrected iPSCs originally derived from an MLD patient. Furthermore, we will verify MLD transgene expression from neural precursors derived from safely modified iPSCs. Finally, exome sequencing will be performed before and after iPSC manipulation to identify mutations arising from different gene addition strategies.

描述：患者特异性诱导的多能干细胞（IPSC）对个性化细胞置换疗法具有巨大的希望。正常白细胞营养不良（MLD）是一种神经退行性疾病，与许多其他单基因疾病一样，它是使用校正的IPSC进行基因治疗的候选者。实现IPSC的全部潜力需要可靠的方法来执行基因靶向。一个主要的挑战是降低由于随机插入而导致插入诱变的风险。基于群集的定期间隔短的短质体重复序列（CRISPR）/CRISPR相关（CAS9）和转录激活因子（Tale）核酸酶（Talen）核酸酶（TALEN）系统的靶向核酸酶，能够诱导双链断裂（DSB）。这些DSB可以增强以特定序列引入转基因的同源重组。但是，未知位点上的靶向DNA裂解可能会导致难以检测的突变。另外，我们最近使用了可靶向的piggybac（PB）转座酶将整合到单个基因组地址中。当前，尚未对这些策略中的任何一对遗传毒性进行直接比较。重要的是，所有当前方法都在脱离靶向位点上显着整合，因此需要一个可靠的系统来识别安全修饰的单元。我们的目标是通过使用定向基因添加和目标事件检测的组合来提高纠正人IPSC基因缺乏症的安全性和效率。我们已经设计了一种可调节的富集策略，称为事件检测，该策略将允许识别和隔离o在靶向基因添加后罕见地正确修饰细胞。我们将使用CRISPR，TALEN和我们最近开发的目标转座酶系统将我们的事件检测盒瞄准Rosa26基因组安全港。在验证人类细胞系中的多种策略后，我们将应用事件检测，以识别最初从MLD患者衍生的基因校正的IPSC。此外，我们将验证来自来自安全修改IPSC的神经前体的MLD转基因表达。最后，将在IPSC操纵之前和之后进行外显子组测序，以鉴定由不同基因添加策略引起的突变。