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

Area of Science:

• Neuroscience
• Stem Cell Biology
• Gene Therapy

Background:

• Human neural stem cells (NSCs) hold promise for treating neurodegenerative diseases and neural injuries.
• Gene editing can enhance the therapeutic capabilities of NSCs.

Purpose of the Study:

• To investigate the feasibility of gene editing in human NSCs.
• To assess the migratory, differentiation, and therapeutic potential of gene-edited NSCs (GE-NSCs) in vivo.
• To explore GE-NSCs for treating lysosomal storage disorders.

Main Methods:

• Utilized Cas9 mRNA with chemically modified guide RNAs and DNA donor templates for gene targeting in NSCs.
• Transplanted GE-NSCs into oligodendrocyte mutant shiverer-immunodeficient mice.
• Generated GE-NSCs overexpressing the GALC enzyme.

Main Results:

• NSCs were successfully gene-edited at multiple loci.
• Transplanted GE-NSCs demonstrated self-renewal, migration, and differentiation into neurons, astrocytes, and oligodendrocytes.
• GALC-overexpressing GE-NSCs corrected enzyme activity via the mannose-6-phosphate receptor pathway.

Conclusions:

• Gene editing is effective in human NSCs, creating GE-NSCs with retained stem cell properties.
• GE-NSCs exhibit therapeutic potential for central nervous system disorders and injuries.
• GE-NSCs offer a promising avenue for investigational cell and gene therapy, particularly for lysosomal storage disorders.