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

患者特异性诱导多能干细胞（iPSCs）在个性化细胞替代治疗中具有巨大的前景。异色性脑白质营养不良（MLD）是一种神经退行性疾病，像许多其他单基因疾病一样，是使用校正的iPSCs进行基因治疗的候选者。实现iPSCs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导致插入突变的风险。基于簇状规则间隔短回语重复(CRISPR)/CRISPR相关（Cas9）和转录激活因子样效应（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可以促进在特定序列上引入转基因的同源重组。然而，未知位点的脱靶DNA切割可能导致难以检测的突变。另外，我们最近使用了一种可靶向的piggyBac （pB）转座酶来指导整合到单个基因组地址。目前，还没有对这两种策略之间的遗传毒性进行直接比较。重要的是，目前所有的方法都能在脱靶位点显著整合，需要一个可靠的系统来识别安全修饰的细胞。我们的目标是利用定向基因添加和靶事件检测的结合来提高纠正人类iPSC基因缺陷的安全性和效率。我们设计了一种可调的富集策略，称为事件检测，这将允许识别和分离罕见的正确修饰的细胞后靶向基因添加。我们将使用CRISPR， TALEN和我们最近开发的靶向转座酶系统将我们的事件检测盒靶向到ROSA26基因组安全港。在人类细胞系中验证多种策略后，我们将应用事件检测用于鉴定源自MLD患者的基因校正iPSCs。此外，我们将验证来自安全修饰的iPSCs的神经前体的MLD转基因表达。最后，外显子组测序将在iPSC操作前后进行，以确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.