C11129: Site-specific and Permanent Modification of Genomes with Reduced CytotoxicityNovelty:
This technology comprises the generation of re-engineered obligate heterodimer FokI nuclease domain variants to create ZFNs with minimal cell toxicity for targeted modifications in plant, mammalian and even human genomes.
Value Proposition:
Zinc finger nucleases (ZFN)-mediated gene targeting yields high gene modification efficiencies, in a variety of cell type. Since the recognition specificities of ZFN complex can be easily manipulated they offer a general way for delivering a recombinogenic double-strand break (DSB) to the targeted chromosomal locus, using re-designed ZFNs. However, ZFNs fused to wild-type Fokl cleavage domains can form homodimers, which limits the efficacy and safety of the ZFNs by inducing off-target cleavage. The current technology discloses a method for delivering a targeted genomic DSB with more efficacy and efficiency using custom- designed ZFNs with minimal cytotoxicity.
• Potential to enable wider application of ZFN technology in human therapeutics
• Potential to increase viability of gene modified cells, especially the sensitive human primary cells
Technical Details:
Johns Hopkins researchers have identified ZFN-mediated gene targeting which yields in high gene modification with reduced toxicity. Generally ZFNs are formed by fusing zinc finger proteins (ZFPs) which bind to the adjacent cognate sites on DNA and FokI nuclease domain dimerize to form the active catalytic center for DSB induction. Two different approaches are available to reduce cytotoxicity of ZFNs. In this study, we have used structure-based redesign of FokI cleavage domain at dimer interface to further improve FokI obligate heterodimer variants. Attenuation of ZFN toxicity could also be achieved by small molecule regulation of ZFN protein levels in cells. This study shows that further improvements in the FokI cleavage domain to create customized ZFNs results in greatly minimized cellular toxicity critical for a cell’s viability.
Looking for Partners:
To develop and commercialize the technology as an efficient tool to deliver a targeted genomic DSB and modification to human cells, while leaving the rest of the genome unchanged.
Stage of Development:
Discovery
Data Availability:
Under CDA / NDA
Publications/Associated Cases:
J Mol Biol. 2011 January 21; 405(3): 630–641
