Development of a Generic Pharmacodynamic Reporter Model for Assessing in vivo Activity and Selectivity of Targeted siRNA

Short interfering RNAs (siRNAs) can reduce the expression of disease-related genes by binding to and mediating the degradation of mRNA (the intermediate of DNA and protein) to which they are complementary. However therapeutic siRNA must be delivered to appropriate parts of the body which is a significant challenge. If the organs are not directly accessible i.e. skin, eyes, muscles, then siRNA must be delivered via the blood stream. Currently there are no methods to sensitively determine both the quantity and efficacy of siRNA reaching the desired organ in a living animal. We propose to create a mouse that will express light (detected by a camera) in organs where siRNA has been delivered. Mouse models will be created by introducing two pieces of foreign DNA into every cell in the mouse, one that makes light emitting protein and one that makes a repressor protein that regulates light expression. When siRNA that specifically recognises the repressor mRNA enters cells the amount of repressor protein will fall and light will be generated. By combining target specificity and biological functionality this model will permit testing of various siRNA delivery mechanisms leading to improved design and effectiveness.

The major obstacle for development of short interfering RNA (siRNA) as a therapeutic agent is effective and selective delivery to the target site. Currently there are no models that jointly assess pharmacokinetic profiles and biological activity in living animals making it impossible to assess target selectivity and identify spatially off target effects. The ideal tool to facilitate development of delivery strategies would be a non-invasive in vivo model capable of giving time-resolved pharmacodynamic readout following siRNA administration. We propose to develop a transgenic mouse model carrying the lac operator-repressor system controlling luciferase expression in every tissue. Successful delivery of siRNA targeting lac repressor mRNA will down regulate lacI expression leading to derepression of the luciferase reporter gene and a positive readout detectable by CCD camera in real time. An easily detectable signal showing activity of siRNA in target and non-target tissues, will enable assessment of selectivity and potential toxicity of any siRNA targeting strategy. To achieve this aim we will generate founder lines expressing either lac I repressor protein or luciferase, downstream of lac operator elements, which will be crossed to generate progeny with a functional lac operon-repressor system. Using this model it will be possible to test novel and existing formulations of siRNA vectors for tissue targeted delivery.