ENGINEERING NEURAL STEM CELLS USING HOMOLOGOUS RECOMBINATION

摘要

Described herein are recombinant polynucleotide-binding polypeptides, recombinant fusion proteins made with the described polynucleotide-binding polypeptides, methods of using the described recombinant polynucleotide-binding polypeptides and recombinant fusion proteins to modify genomic DNA of cells and, in some embodiments, create recombinant cells. Methods are also provided herein for genetically modifying a neuronal stem cell. Methods are also provided for treating a neurological disorder in a subject that include the administration of genetically modified neuronal stem cells produced by the methods disclosed herein.

主权项

1. A method for introducing a polynucleotide of interest into a safe harbor locus in a genome of a neuronal stem cell, comprising
introducing into the neuronal stem cell (a) an upstream transcription activator-like effector nuclease (TALEN) comprising an upstream DNA-binding domain linked to a DNA cleavage domain, wherein the upstream DNA binding domain specifically binds to the safe-harbor locus at a site upstream of a genomic insertion site in the genome of the neuronal stem cell, (b) a downstream transcription activator-like effector nuclease (TALEN) comprising a downstream DNA-binding domain linked to a DNA cleavage domain, wherein the downstream DNA binding domain specifically binds to the safe-harbor locus at a site downstream of the genomic insertion site in the genome of the neuronal stem cell, and (c) a single or double-stranded donor polynucleotide comprising sense and/or antisense strand polynucleotide overhangs that are complementary to corresponding polynucleotide overhangs of cleaved the genomic DNA when cleaved at the genomic insertion site, wherein the complementary overhangs facilitate homologous recombination of the donor polynucleotide with the cleaved genomic DNA, thereby introducing the donor polynucleotide into the genomic insertion site within the safe harbor locus in the genome of the neuronal stem cell.