Sleeping Beauty transposase variants with increased safety due to reduced half-life

Technology Description

SB transposase, including the currently most widely used hyperactive variant SB100x, is a DNA modifying enzyme that localizes and acts in the nucleus and has a half-life of more than 30 h, posing an inherent risk for genotoxicity and genomic instability. To address these issues, the SB100x gene was modified to mediate the N-terminal fusion of an optimized nuclear export signal (NES) and a chaperone-mediated autophagy (CMA) signal to facilitate nuclear export and routing to the lysosomal degradation pathway, respectively. The resulting fusion protein (NES-CMA-SB100x) showed the intended reduction of half-life, but at the same time, also an undesired loss of transfection efficiency (see Figure). To restore the latter, extensive profiling and optimization of SB100x mRNA was performed, as mRNA represents the preferred mode of delivery of the transposase for gene therapy applications. Profiling included (i) codon optimization, (ii) modification of untranslated regions, (iii) variation of the length of the poly(A) tail, and (iv) introduction of modified nucleotides, and resulted in NES-CMA-SB100x variants with the desired properties and safety profile for clinical applications. The variants are currently combined and tested with other recently identified SB100x mutants to further improve the safety (see TO 03-00541) and efficacy (see TO 03-00542) of the SB100x system.