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.

Public Health Relevance

Metachromatic leukodystrophy (MLD) is a devastating brain disease that leads to death in infancy and is caused by mutations in a single gene. The goals of this project are to improve the safety and efficiency in correcting this mutation in induced pluripotent stem cells (iPSCs) to demonstrate a potential treatment for MLD.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Exploratory/Developmental Grants (R21)
Project #
Application #
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Morris, Jill A
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Hawaii
Anatomy/Cell Biology
Schools of Medicine
United States
Zip Code