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

doi:10.1016/j.isci.2019.04.036

CRISPR/Cas9 Genome Engineering in Engraftable Human Brain-Derived Neural Stem Cells

iScience. 2019 May 31:15:524-535. doi: 10.1016/j.isci.2019.04.036. Epub 2019 May 4.

Authors

Daniel P Dever¹, Samantha G Scharenberg¹, Joab Camarena¹, Eric J Kildebeck¹, Joseph T Clark¹, Renata M Martin¹, Rasmus O Bak¹, Yuming Tang², Monika Dohse², Johannes A Birgmeier³, Karthik A Jagadeesh³, Gill Bejerano⁴, Ann Tsukamoto⁵, Natalia Gomez-Ospina¹, Nobuko Uchida⁶, Matthew H Porteus⁷

Affiliations

¹ Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
² StemCells Inc, Newark, CA 94560, USA.
³ Department of Developmental Biology, Stanford University, Stanford, CA 94305, USA.
⁴ 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.
⁵ ReGen Med Division, BOCO Silicon Valley, Palo Alto, CA 94303, USA.
⁶ ReGen Med Division, BOCO Silicon Valley, Palo Alto, CA 94303, USA. Electronic address: nobuko.uchida@bocosiliconvalley.com.
⁷ Department of Pediatrics, Stanford University, Stanford, CA 94305, USA. Electronic address: mporteus@stanford.edu.

Abstract

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.

Keywords: Bioengineering; Biological Sciences Research Methodologies; Biotechnology; Cell Biology; Molecular Biology; Neuroscience.

Abstract

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