Stable Gene Transfer in Hard-to-Transfect Vertebrate Cells with New Hyperactive Sleeping Beauty Transposase
Referenznummer TO 03-00251
The Challenge
DNA transposons can be viewed as natural gene delivery vehicles that integrate into the host genome via a cut-and-paste mechanism. They provide the basis for long-term expression of transgenes in cells and organisms. The Sleeping Beauty (SB) transposon is the first transposon system used in a clinical trial. As its preferential insertional pattern lays outside of coding sequences, it could be the preferred transgene vector over viral systems. But yet, its efficiency of gene insertion shows strong limitations in hard-to-transfect cell types, including stem cell populations relevant in gene therapy. Generating hyperactive versions of the transposase could overcome these hurdles.
Technology
A hyperactive transposase (SB100X) has been engineered with ~100-fold enhancement in efficiency when compared to the first-generation transposase.
A transposon encoding human clotting factor IX (hFIX) was co-transfected by hydrodynamic gene delivery with different SB transposase versions. Mean values of plasma hFIX concentrations by SB100X (circle) and SB11 (square) are shown.
In hard-to-transfect cells SB100X supports:
- 35-50% stable gene transfer in human CD34+ hematopoietic stem cells (HSCs),
- long-term engraftment and hematopoietic reconstitution in immunodeficient mice after transplantation of gene-marked HSCs,
- sustained (>1 year) expression of physiological levels of hFIX upon transposition in the mouse liver in vivo.
Further applications were SB100X shows advantages:
- High-producer cell lines: transgenic cell lines often produce high levels of the protein-of-interest. High yield of proteins-of-interest would have direct influence on the production costs of e.g. therapeutic proteins.
- Generating animal models: Efficient transgenesis (40-70%) in rodents - compared to 10% with SB basic.
Commercial Opportunity
In-licensing of IP and Know-How. The scientist are very open to collaborate in future product development.
Patent Situation
Priority claiming patent application in 2007, National Phases in EP, US, JP, CA, AU.
Further Reading
Mátés L, et al., 2009, Nat Gen; Williams DA, et al., 2008, Mol Ther; Vigdal TJ, et al., 2002, J Mol Biol; Orban T, et al., 2009, Stem Cell; Xue X, et al., 2009, Blood; VandenDriessche T, et al., 2009, Blood.
