The ability to precisely modulate a gene expression in human cells provides a means to address a variety of genetic diseases as well as enable patient-specific somatic cell reprogramming for stem cell-based therapy. Traditionally, modular Zinc Finger proteins (ZFPs) and transcription activator-like effector (TALE) proteins have been the platforms of choice to develop therapeutic transcription factors (TFs). The rational design of ZFN and TALE proteins based on pre-established DNA recognition rules often yields inactive or poorly performing variants. Hence, generation of ultra-deep combinatorial TF libraries and then selection of highly-active variants at a low cost has not been achieved. Towards this goal, we have developed a novel recombinase-based technology termed DIVERGE (Directed In Vivo divERsity GEneration). It includes functional diversification of modular repeat proteins in Escherichia coli in a highly directed manner. Applying this method to modular ZFPs and TALE based transcriptional activators allowed us to generate billions to trillions of transcription factors variants in vivo starting from a single master construct. The aim of the DIVERGE PoC project was to explore the commercial potential of this technology for the isolation of transcriptional activators for human gene activation therapy, and to establish a strategy for commercialisation of current and conceivable future implementations of this technology. Prior art searches were concluded and identified 2 patentable innovative steps although filing was postponed and is to coincide with a first publication. Market research concluded that applications of the technology beyond transcription factors possibly have similar if not more market potential and potential for impact. We set out to screen our ZFP and TALE based TF libraries to isolate functional variant that have an ability to bind in the human gene promoter sequences from target genes that are involved in genetic disease or are considered therapeutic. Our screening efforts in mammalian cells are ongoing and we have also initiated the screening using a yeast one-hybrid method to isolate functional variants.
