Engineering neural stem cells using homologous recombination
Inventors
Rao, Mahendra S. • MALIK, Nasir • FUNAHASHI, Raymond • ZOU, Jizhong
Assignees
US Department of Health and Human Services
Publication Number
US-9951353-B2
Publication Date
2018-04-24
Expiration Date
2034-11-14
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Abstract
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.
Core Innovation
The invention provides recombinant polynucleotide-binding polypeptides, recombinant fusion proteins containing these polypeptides, and methods to use these proteins to modify genomic DNA of cells, including creation of recombinant cells. Specifically, methods for genetically modifying neuronal stem cells (NSCs) are disclosed, including introducing polynucleotides into safe harbor loci within the NSC genome. The polynucleotides may encode factors sufficient to differentiate NSCs toward neuronal or glial lineages.
The background identifies the problem that, although gene editing techniques such as ZFN, TALEN, and CRISPR have been used effectively in pluripotent stem cells, multiplexed knock-in or transfer of large DNA fragments into human pluripotent or multipotent stem cells has not been reported thus far. The lack of efficient one-step modification of multiple loci in human stem cells limits their application in multi-lineage labeling, drug-screening, and gene therapy. Furthermore, although NSCs are accessible from various sources and have therapeutic potential for neurodegenerative disorders, further genomic engineering of NSCs is necessary to enhance their versatility in therapeutic applications.
Claims Coverage
The patent contains one independent claim that covers a method involving a pair of specific TALENs targeting an endogenous CLYBL gene to introduce a double-stranded break.
Method of creating a double-stranded break in the CLYBL gene using TALENs
Provides a method of making a double stranded break in an endogenous CLYBL gene in an isolated human cell by introducing into the cell both (a) an upstream TALEN comprising an upstream DNA-binding domain linked to a DNA cleavage domain and (b) a downstream TALEN comprising a downstream DNA-binding domain linked to a DNA cleavage domain, thereby causing the double stranded break.
The claim discloses the inventive feature of using a pair of specifically designed TALENs with DNA-binding domains linked to cleavage domains to target the endogenous CLYBL gene for double-stranded cleavage in human cells.
Stated Advantages
Achieves efficient targeted knock-in of various transgenes up to 8 kb into safe harbor loci in human iPSCs and NSCs with nearly 100% targeted integration in some cases.
Allows robust, long-term, and proportional expression of inserted transgenes, with the CLYBL locus providing 5 to 10-fold higher expression than the AAVS1 safe harbor locus.
Permits multiplexed simultaneous targeting of two safe harbor loci resulting in dual expression of reporter genes without silencing during extended culture.
The targeted integration causes minimal impact on genome-wide expression, preserving cellular properties after gene editing.
Provides rapid and efficient genetic modification of NSCs, enabling their use in therapeutic applications while maintaining self-renewal and multipotency.
Documented Applications
Production of genetically modified neuronal stem cells and differentiated neuronal or glial cells for use in treatment of neurodegenerative disorders, stroke, spinal cord injury, and peripheral nerve injury.
Engineering stem cells for research purposes such as multicolor labeling, drug screening, and evaluation of gene function through insertion or excision of sequences in safe harbor loci.
Generation of induced pluripotent stem cells (iPSCs) from somatic cells through targeted insertion of pluripotency factors.
Screening pharmaceutical agents to identify compounds that affect neuronal stem cell differentiation or physiology.
Use of modified NSCs or differentiated neurons for cell transplantation therapies, including treatment of Parkinson's disease and other CNS disorders.
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