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

患者特异性诱导多能干细胞（iPSC）为个性化细胞替代疗法带来了巨大的希望。异染性脑白质营养不良（MLD）是一种神经退行性疾病，与许多其他单基因疾病一样，是使用纠正的iPSC进行基因治疗的候选者。实现iPSC的全部潜力需要可靠的基因靶向方法。一个主要的挑战是减少由于随机插入而引起的插入诱变的风险。基于成簇的规律间隔的短回文重复序列（CRISPR）/CRISPR相关的（Cas9）和转录激活因子样效应物（TALE）核酸酶（TALEN）系统的靶向核酸酶能够诱导双链断裂（DSB）。这些DSB可以增强同源重组以在特定序列处引入转基因。然而，在未知位点的脱靶DNA切割可能导致难以检测的突变。或者，我们最近使用可靶向的piggyBac（pB）转座酶来直接整合到单个基因组地址中。目前，尚未对任何一对这些策略之间的遗传毒性进行直接比较。重要的是，所有目前的方法都在脱靶位点显著整合，需要可靠的系统来鉴定安全修饰的细胞。我们的目标是使用定向基因添加和靶事件检测的组合来提高纠正人iPSC基因缺陷的安全性和效率。我们已经设计了一种可调的富集策略，称为事件检测，这将允许在靶向基因添加后鉴定和分离罕见的正确修饰的细胞。我们将使用CRISPR，TALEN和我们最近开发的可靶向转座酶系统将我们的事件检测盒靶向ROSA 26基因组安全港。在人细胞系中验证多种策略后，我们将应用事件检测用于鉴定最初来源于MLD患者的基因校正的iPSC。此外，我们将验证来自安全修饰的iPSC的神经前体的MLD转基因表达。最后，将在iPSC操作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。

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.