Development of self-delivering RNAi to evaluate PTEN as a therapeutic target to p

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Project Summary Innovative drug/therapy combinations directed at multiple and proven therapeutic targets have the potential to dramatically improve outcomes after SCI. Studies on axon regeneration and recovery in rodents have revealed that the spinal cord is not hard wired and "learning" can occur in spinal cord circuits. Compounds that elicit axonal regeneration and sprouting help plasticity in the spinal cord and dramatically improve recovery from traumatic injury, at least in rodents. Many different compounds/therapies tested in rodents promote regeneration. Almost without exception, compounds that promote regeneration also improve functional recovery after spinal cord injury. Recently, very impressive axon regeneration was obtained from deletion of PTEN. The goal of this application is to simultaneously evaluate PTEN as target for therapeutic treatment of spinal cord injury and a novel RNAi delivery technology to provide prolonged in vivo gene knockdown effect. We will create "self-delivering" small interfering RNAs (sdRNA) targeting PTEN. The chemical modification introduced into sdRNA molecules makes them cell- permeable. This technology has been demonstrated to work in vivo in several applications, including delivery to the retina. As the first step we will synthetize a panel of sdRNA sequences and evaluate them in mammalian cell cultures to select lead candidate(s) efficient in PTEN knockdown. Next, we will test the compounds in neuron cell cultures, then with primary neurons. We will also examine effects on morphology and proliferation of primary glial cells and human glioblastoma cells. Once we determine lead candidate(s) that promote robust neurite growth, we will test the compounds for efficacy of knockdown in vivo. We will confirm ability to promote regeneration using an optic nerve model because retinal ganglion cells are known to respond to PTEN knockdown. Safety will be assessed by following clinical signs and clinical chemistry.