Study in gene therapy for lower urinary tract dysfunction

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Background : Muscarinic M3(M3) receptor has been recognized as a major muscarinic receptor for smooth muscle contractions of the urinary bladder. Nitric oxide(NO) is one of the important relaxing factor in bladder. Under the hypothesis that overexpression of nNOS and M3 receptor in the bladder would inhibition and enhancement of bladder contractions, respectively. So, we have transferred the nNOS and M3 receptor gene into rat bladders using electroporation(EP) and evaluated the functional expression of the gene.Methods : Plasmids expressing luciferase, GFP, nNOS and M3 receptor were injected into the rat bladder and square-wave electric pulses were immediately applied. Two days after gene transfer, we analyzed gene expression. Immunohistochemical staining for nNOS and M3 receptors was performed and the contractile responses from isolated bladder strips, which were carbachol and electrical field stimulation(EFS), were evaluated. NO released from isolated bladder strips was also assessed … More using microdialysis and HPLC. M3 gene transfer also preformed into bladder of deneravation(detrusor underactivity) model rat.Results : The optimal conditions of electroporation were 8 pulses, 45 voltages, 50 millisec/pulses and 1 Hz. Under these conditions, luciferase gene expression was enhanced approximately 300-fold, compared to an injection of DNA only. Regarding immunohistochemistry with an anti-nNOS and anti-M3 receptor, increases in immunoactivity wer observed in the nNOS and M3 receptor gene transferred rat bladder, compared to the bladder of the control rat. In rats with the nNOS gene injected by electroporation there was marked nNOS immunoreactivity, and NOx released from bladder strips was significantly greater than in the control groups. In rats with the transferred M3 receptor gene, carbachol- and EFS-induced maximum responses of bladder smooth muscle strips significantly increased. In the M3 receptor transferred denervation rats, detrusor contractility for carbachol and EFS significantly increased, as compared with the control rats. The cystometric findings showed, decreased micturituion interval and increased in bladder pressure.Conclusions : These findings suggest that an in vivo EP procedure is a useful method for gene transfer into the bladder. nNOS gene transferred by this procedure functionally expresses and contributes to NO production, and that an overexpression of M3 receptor in the rat bladder enhances bladder contractility. This technique may become a new treatment modality for detrusor underactivity. Less