Jizhong Zou (NIAMS)
Mahendra Rao (NIAMS)
- In vitro data available
A novel method of targeted insertion of transgenes at CLYBL locus directly in human cells is disclosed. Also, methods and compositions for increasing targeted insertion of a transgene into a specific location within the cell or increasing the frequency of gene modification in a targeted locus are disclosed. Genome modification by precise gene targeting at specific sequence/locus has great advantages over conventional transient expression or random integration methodologies and, therefore, has tremendous therapeutic potential. NIH investigators identified CLYBL gene in Chromosome 13 as a potential safe harbor locus. To directly target CLYBL safe-harbor in human cells without pre-engineering, they identified a unique transcription activator-like effector nuclease (TALEN) target sequence at CLYBL locus. The CLYBL TALENs (also termed as C13 TALENs) constructed using pZT backbone showed high gene editing efficiency in human 293T cells measured by both T7E1 mismatch assay and targeted sequencing. The inventors have used TALENs to simultaneously knock-in multiple reporter genes at up to four alleles of PPP1R12C/AAVS1 and new CLYBL safe-harbors in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The engineered safe-harbor knock-in cell lines maintain robust transgene expression during iPSC/NSC self-renewal and differentiation, and CLYBL locus allowed 10-fold stronger transgene expression than other loci. NSC lines engineered by this methodology as well as constructs and protocols for evaluation are also available.
- Human stem cell-based gene therapy
- Drug screening
- CLYBL safe harbor on Chromosome 13 allows 5~10-fold stronger transgene expression than AAVS1 safe harbor, providing an alternative and potentially better solution for targeted gene transfer/knock-in and drug-screening, especially for weak promoter-driven transgenes.