Modulation of microRNA as a therapeutic strategy for stroke

Stroke is the 3rd leading cause of death in the UK and the leading cause of longterm disability. With a growing elderly population the socioeconomic burden attributed to stroke is going to continue to increase in coming years. There is currently only one clinically approved treatment for stroke and due to tight safety constraints only 5% of sufferers receive this intervention. Clearly, there is a pressing and urgent need to identify novel intervention strategies and better define the mechanisms of stroke lesion progression and accompanying neurological deficit. microRNAs (miRNAs) are small molecules which can alter the expression of many targets involved in the divergent and converging pathways implicated in stroke lesion progression. By manipulating the expression of these miRNAs I postulate that improvments in outcome following stroke can be achieved. Uniquely, by altering the expression of one single miRNA effects on many pathways involved in disease progression can be achieved. This polytherapy approach has been shown to be successful for the management of diseases such as high blood pressure through multi-drug prescribing. To improve the clinical translation of our studies we will study these effects in an animal model which reflects many of the risk factors those likely to suffer a stroke have (e.g. high blood pressure, altered metabolic handling) and look to alter miRNA expression in the days following experimental stroke - a time window which would be attractive from a clinical standpoint. Alteration of miRNA will be achieved using novel gene delivery sytems based on a non-replicating virus which has been shown to efficiently target neurons in the brain. This virus is safe and doesn t cause an immune response in humans (unlike some gene delivery vectors). Together, the approach outlined here will help define the validity of gene delivery to alter miRNA expression in a robust experimental model of stroke and so could have important implications for future intervention strategies in the clinic.

This project aims to determine the effect of microRNA (miRNA) modulation as a novel intervention strategy in a robust pre-clinical model of stroke. Given that one miRNA can alter the expression of many both divergent and converging genes in multiple pathways, through a single modulation a significant effect on those pathways implicated in lesion progression or the promotion of endogenous repair will be achieved. Initially, the expression of miRNA which have been previously published to be altered following cerebral ischaemia will be validated in our experimental model of transient middle cerebral artery occlusion (tMCAO) in the spontaneously hypertensive stroke prone rat (SHRSP) and an in vitro model of hypoxia/reoxygenation. The in vitro model will allow the effect of specific miRNA modulation by lentivirus infection (over-expression or knockdown) to be determined through a number of functional assays prior to translation to the in vivo model. On identification of the most promising therapeutic miRNA candidates for modulation from the in vitro characterisation studies a canine adenovirus-2 (CAV-2) vector will be generated. CAV-2 has been shown to be highly neurotrophic in the SHRSP and other animal models of neurodegenerative diseases. Its marked efficiency of neuronal transduction, retrograde transport from the injection site resulting in a significant area of transduction and lack of immune response due to a lack of existing immunity make it an ideal and novel vector for study in the current proposal. CAV-2 will be administered by stereotaxic injection 24 hours post-tMCAO in the SHRSP and the effect of miRNA modulation (up to 4 therapeutic groups vs control tMCAO and sham controls) determined. To fully characterise the effect of such intervention animals will be taken out to 21 days post-tMCAO to monitor neurological recovery (using the tapered beam test and assessment of neurological score through a battery of 10 tests), infarct volume (by H&E staining) and functional assays (IHC for specific proteins, western blotting, biochemical assays, pressure myography). This will provide a comprehensive follow-up to ascertain the effect of specific miRNA in the setting of experimental stroke. Through the studies outlined here, I aim to demonstrate the therapeutic potential of miRNAs for stroke using a novel gene delivery vector in a robust and clinically relevant experimental model at a timepoint which would be clinically significant.