Gene therapy for spinal code protection

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Spinal cord injury after successful aortic surgical operations is an unacceptable complication for patients, and new spinal cord protection method is required. Oxygen-regulated protein 150kD (ORP150) is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. And it was reported that cultured neurons overexpressing ORP150 were resistant to hypoxemic stress.We hypothesized that overexpression of Oxygen-regulated protein 150kD(ORP150) in spinal cord would reduced motor neuron death after ischemia. ORP150 cDNA was obtained from RT-PCR products of Rat brain, and was inserted into expressing plasmid driven by CAG promoter. Expression of ORP150 was confirmed by using Western blotting method. Though in vivo lipofection of ORP150 or LacZ expression plasmid were tried to rabbit spinal cord, transduced gene were not detected at all. Neurological score after lipofecton reduced as compared to sham control. ORP150 expressing adenovirus vector was constructed by using circular form of the adenoviral genome cloned in a cosmid and the Cre-loxP recombination system. However, enough amount of adenoviral particles could not be obtained. As CAG promoter, an actin-based hybrid promoter, has very strong activities in most cells, excess expression of ORP150 was considered to have toxicity against 293 cells. Though two different gene delivery methods were planed for spinal protection, results were limited. These findings suggested that excess expression of ORP150 potentially also had toxicity against nerve cells, and to manage the expression levels of ORP150 is considered to be important when we apply this method to aortic surgery.