CRISPR/Cas9 Genome Engineering in Engraftable Human Brain-Derived Neural Stem Cells
Daniel P. Dever,
Samantha G. Scharenberg,
Joab Camarena,
Eric J. Kildebeck,
Joseph T. Clark,
Renata M. Martin,
Rasmus O. Bak,
Yuming Tang,
Monika Dohse,
Johannes A. Birgmeier,
Karthik A. Jagadeesh,
Gill Bejerano,
Ann Tsukamoto,
Natalia Gomez-Ospina,
Nobuko Uchida,
Matthew H. Porteus
Affiliations
Daniel P. Dever
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
Samantha G. Scharenberg
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
Joab Camarena
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
Eric J. Kildebeck
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
Joseph T. Clark
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
Renata M. Martin
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
Rasmus O. Bak
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
Yuming Tang
StemCells Inc, Newark, CA 94560, USA
Monika Dohse
StemCells Inc, Newark, CA 94560, USA
Johannes A. Birgmeier
Department of Developmental Biology, Stanford University, Stanford, CA 94305, USA
Karthik A. Jagadeesh
Department of Developmental Biology, Stanford University, Stanford, CA 94305, USA
Gill Bejerano
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA; Department of Developmental Biology, Stanford University, Stanford, CA 94305, USA; Department of Computer Science, Stanford University, Stanford, CA 94305, USA
Ann Tsukamoto
ReGen Med Division, BOCO Silicon Valley, Palo Alto, CA 94303, USA
Natalia Gomez-Ospina
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
Nobuko Uchida
ReGen Med Division, BOCO Silicon Valley, Palo Alto, CA 94303, USA; Corresponding author
Matthew H. Porteus
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA; Corresponding author
Summary: Human 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. : Molecular Biology; Neuroscience; Bioengineering; Biotechnology; Cell Biology; Biological Sciences Research Methodologies Subject Areas: Molecular Biology, Neuroscience, Bioengineering, Biotechnology, Cell Biology, Biological Sciences Research Methodologies
