Erectile dysfunction (ED) affects over 15 million men and their partners in the USA. Aging is a major risk factor for ED, presumably due to an insufficient synthesis of nitric oxide (NO) in the penile nerves and an impaired compliance of the corpora cavernosa (CC) caused by a loss of smooth muscle cells (SMC). Failure to respond to PDE5 inhibitors may be related to these factors. The overall .goal of this proposal is to extend our previous grant findings towards a curative approach for aging-related ED based on: a) increasing the content and activity of penile neuronal nitric oxide synthase (PnNOS) in the CC, to release more NO upon sexual stimulation; and b) decreasing the levels of inducible NOS (iNOS) and reactive oxygen species (ROS) in the CC to reduce peroxynitrite formation and SMC apoptosis. We propose: Aim 1: to improve the duration of intracavernosal gene therapy with PnNOS cDNA, by: a) stimulating persistence of gene expression in the penis of adult rats using novel helper-dependent (HD) adenovirus (AdV) and tissue-specific promoters directing the expression of beta-galactosidase; b) determining whether hybrid AdV/adeno-associated virus (AdV-AAV) are more effective than HD-AdV; c) comparing electroporation and minipumps for construct delivery, and conducting safety tests, and d) applying the selected vectors or cells to express PnNOS and related genes in the aged CC, to correct the impaired erectile response to electrical stimulation of the cavernosal nerve; Aim 2: to characterize modulators of PnNOS activity that may affect the control of erection in the CC, by: a) stimulating PnNOS activity and erectile function through the activation of nNOS-associated NMDA receptors; and b) finding novel PnNOS binding protein factors modulating its activity and erectile function; Aim 3: to correct the aging-related loss of SMC by manipulating the NO/ROS ratio within the CC, by a) determining whether there is SMC replication and whether this is decreased with aging, and correlating with the increase in iNOS, ROS, peroxynitrite, and apoptosis; b) reducing NO (from iNOS) and ROS levels to reverse the loss of SMC and ameliorate aging-related ED; and c) determining whether obliteration of iNOS expression in the iNOS knock-out mouse leads to a reduction in the age-related loss of SMC.