The role of RNA in the response to cellular stress

Recently, a new gene delivery system has been developed, to specifically provide diseased cells with a particular protein to correct cell function. This method using messenger RNA molecules as a delivery vehicle. By changing the RNA molecules different proteins can be encoded on these RNA molecules and different human disease can be treated. However, our lack of understanding of these molecules has resulted in safety issue, which need to be resolved before they can be used to treat a large range of human diseases. Our laboratory has been investigating how RNAs are regulated and we have discovered that two particular RNA binding proteins associate with translationally repressed mRNAs. Using methods developed in our laboratory we can now identify RNA repression mechanisms operating in different tissues and thus tailor these RNA molecules for specific delivery to different tissue types, reducing toxic side effects. We will determine the applicability of this approach to identify the repertoire of repressive mechanisms operating within a cell, in order to engineer therapeutic-mRNAs for restricted on-target expression in particular tissues/cell types and disease states. This will facilitate the development of the next generation of mRNA-based therapeutics which will be specifically designed to minimise potential off-target adverse effects.

Modulation of gene expression via the delivery of nucleotide-based therapeutics has great potential for the treatment of a large range of human diseases. Major challenges still exist in the development of these next generation drugs due to limitations in our knowledge of RNA biology and delivery methodologies. In recent years, most attention with regard to gene-based therapies has focused on DNA and viral systems; however, their use is limited due to low efficacy, genetic transfer risk and poor control over dosage. More recently, the incorporation of modified nucleotides has meant that in vitro transcribed mRNA molecules have been successfully used for the delivery of genetic information without risk of genomic integration. These mRNAs are produced using modified nucleotides that mimic endogenous mRNA molecules and thus avoid activating the innate immune response. The relatively short half-life of RNA compared to DNA also allows precise control over final protein dosage.

In terms of the safety of these synthetic molecules, there are major challenges that need to be addressed before the full potential of mRNA-based therapeutics can be realised. These issues mainly revolve around our current lack of understanding of how these RNA molecules are controlled. Our laboratory has been investigating how mRNAs are regulated and we have discovered that two RNA DEAD-box helicases associate with translationally repressed mRNAs. The association of mRNAs with these DEAD-box proteins has allowed us to identify multiple repressive mechanisms imposed by the major cellular mRNA silencing apparatus - the Ccr4-NOT complex. Until now, identification of mRNAs controlled by the variety of mechanisms that the Ccr4-NOT complex plays a role in would have been extremely labour-intensive, requiring a number of different techniques and would not have given sufficient resolution for mRNAs with low cellular abundance. In contrast, our methodology is a relatively straightforward approach, which allows for simultaneous identification of mRNAs repressed by the Ccr4-NOT1 complex in a given cell type or tissue. These repressive characteristics can be used to build a signature of the regulatory mechanisms operating within a particular cell type. We will determine the applicability of this approach to identify the repertoire of repressive mechanisms operating within a cell, in order to engineer therapeutic mRNAs for restricted on-target expression in particular tissues/cell types and disease states. This will facilitate the development of the next generation of mRNA-based therapeutics which will be specifically designed to minimise potential off-target adverse effects.