Identification of novel chromatin opening elements that facilitate stable gene expression
The sales of therapeutic recombinant proteins exceeded US$125 billion in 2012. A leading priority for the manufacturers of biopharmaceuticals is reducing the extensive time and effort required to identify mammalian cell lines that express recombinant genes at a high level for long periods. Recombinant genes tend to be become silenced when integrated in the genome of the host cell line as most of the genome is repressive to transcription at any given time. Most biopharma companies have developed high throughput strategies to identify recombinant cell lines where the transgene is expressed at a high level, often because it has integrated into an active gene locus. This processes is time consuming and expensive and offers little guarantee that the cell line will continue to stably produce the recombinant protein over the long periods required for full scale production. We have a project with UCB Pharma, where we are taking a synthetic biology approach to engineer mammalian cells to maximise the production of therapeutic antibodies. In particular, we are testing novel chromatin opening elements that facilitate stable gene expression. Incorporating chromatin opening elements into transgene designs will greatly reduce the effort required to develop highly productive cell lines, ultimately bringing down the cost of the latest therapies.
TALE and CRISPR strategies for genetic and epigenetic modification of gene expression
Controlled genome modification in mammals had been restricted to mouse embryonic stem cells and their derivatives. Recent advances in synthetic biology allow us to make any genetic or epigenetic modification in a rapid and controlled manner in most cell types. The Transcription Activator Like Effector (TALE) proteins are transcription factors with a unique modular DNA-recognition mechanism. TALE proteins can be readily engineered to bind to any DNA sequence of choice. The fusion of an effector domain to TALE-DNA binding domains can be employed to develop novel gene regulatory events. For example, we fuse short transcription factor domains that mediate the recruitment of transcription co-activator or co-repressor complexes to the TALE effector target locus. TALE repressors, for example can direct chromatin and DNA modifications that result in heritable target gene silencing.
We also design and construct engineered TALE nucleases (TALENs) with designed sequence specificity in our laboratory. These enzymes facilitate specific genetic alterations in cultured mammalian cells. In addition to gene knock-out and knock-in strategies, we are using genome engineering to study the function of gene regulatory elements in their endogenous chromosomal contexts. We are also developing enzymes that facilitate accurate genetic correction of mutant gene loci responsible for human genetic disorders. CRISPR strategies are also being employed, where short RNAs guide the Cas9 endonuclease, bypassing the need to develop engineered proteins.