Abstract
SummaryHuman neural stem cells (NSCs) offer therapeutic potential for neurodegenerative diseases, such as inherited monogenic nervous system disorders, and neural injuries. Gene editing in NSCs (GE-NSCs) could enhance their therapeutic potential. We show that NSCs are amenable to gene targeting at multiple loci using Cas9 mRNA with synthetic chemically modified guide RNAs along with DNA donor templates. Transplantation of GE-NSC into oligodendrocyte mutant shiverer-immunodeficient mice showed that GE-NSCs migrate and differentiate into astrocytes, neurons, and myelin-producing oligodendrocytes, highlighting the fact that GE-NSCs retain their NSC characteristics of self-renewal and site-specific global migration and differentiation. To show the therapeutic potential of GE-NSCs, we generated GALC lysosomal enzyme overexpressing GE-NSCs that are able to cross-correct GALC enzyme activity through the mannose-6-phosphate receptor pathway. These GE-NSCs have the potential to be an investigational cell and gene therapy for a range of neurodegenerative disorders and injuries of the central nervous system, including lysosomal storage disorders.
Highlights
Human neural stem cells (NSCs) are self-renewing, multilineage-producing cells that hold great promise for treating a large number of central nervous system (CNS) disorders
The CRISPR/Cas9 system (CRISPRs) system generated an average of 15% insertions or deletions (INDELs) compared with 5% using the transcription activator-like effector nucleases (TALENs) (Figure S1A, Related to Figure 1)
We report an under-representation of on-target alleles compared with GFP+ cells for targeting CCR5 and HBB, which may be due to non-target integrations of the donor-plasmid DNA, incomplete homologous recombination (HR) that interrupted digital droplet PCR (ddPCR) primer or probe sequences in the bovine growth hormone polyadenylation (BGH) poly(A) region, or the fact that there is still some episomal DNA expressing GFP after 30 days in culture in CRISPR-treated human NSCs
Summary
Human neural stem cells (NSCs) are self-renewing, multilineage-producing cells that hold great promise for treating a large number of central nervous system (CNS) disorders. No genetic modification or immortalization was required, and these banked cells continue to express NSC markers such as CD133 and Sox (Carpenter et al, 1999; Cummings et al, 2005; Tamaki et al, 2002, 2009; Tsukamoto et al, 2013; Uchida et al, 2000, 2012). They have biological NSC activities with multiple mechanisms of actions, providing neuroprotection, myelination, and retinal preservation via site-appropriate global migration
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