Gene Therapy in the Management of Erectile Dysfunction (ED): Past, Present, and Future

In the past, many researchers considered viral vectors to be the most promising candidates to transfer genetic material into the corpora for the treatment of erectile dysfunction. However, at present, no viral vectors have progressed to human trials. In contrast, the use of naked gene therapy, a plasmid expressing the human Maxi-K potassium channel, is the only gene therapy treatment to be evaluated in clinical phase I trials to date. The success of these studies, proving the safety of this treatment, has paved the way for the development of future gene transfer techniques based on similar transfer methods, as well as novel treatment vectors, such as stem cell transfer.


As indicated in the
legend, the use of naked DNA, while having the advantage of safety because of its lack of chromosomal integration, also has the disadvantage of being highly susceptible to breakdown by endonucleases that render it ineffective. Thus, one of the primary goals for the use of naked DNA in both FIGURE 1. The differences in the cellular effect of viral vs. naked DNA vectors. Note that after transfer with a lentivirus vector, the gene is permanently integrated into a chromosome and is replicated. Another viral vector proposed for gene therapy is the adeno-associated virus, which integrates genetic material specifically into chromosome 19. When the adenoviral vector is used for gene therapy, the genetic material from the virus is not integrated, but resides in the nucleoplasm and is replicated. After naked DNA transfer, the plasmid resides in the nucleoplasm, but is not replicated.
preclinical trials in animal models as well as human trials is to determine the duration of expression (in animal models) and of the physiological effect in the human trials.
Erection is an event that requires proper function of arterial inflow, relaxation of corporal smooth muscle, and occlusion of out-flowing blood to achieve a sustained, long-lasting, rigid erection sufficient for sexual satisfaction [10,11,12]. The most commonly used therapy for the treatment of most cases of ED is with oral phosphodiesterase inhibitors.
Although the current generations of oral phosphodiesterase-5 (PDE-5) inhibitors for the treatment of ED are now a standard first-line therapy for the treatment of ED, there are problems with their use. Cost is a major issue for many men, particularly during this time of fiscal crisis and insurance company cutbacks that have diminished, or totally eliminated, the number of pills paid for per month. The PDE-5s are least effective in men who have more severe ED, including men who have diabetes, and are contraindicated in association with some families of drugs or medical conditions. The need for on-demand ingestion of the drug limits the spontaneity of the sexual act, leading to the need for "planned sex". There is also a welldescribed number of side effects, compounded by effects on the absorption and pharmacokinetics due to diet.
Several investigative laboratories have approached the potential use of gene therapy for ED by employing a variety of vectors to express target genes in the corporal bodies, primarily in rodent models of ED (Table 1). Interestingly, the majority of these studies demonstrated improved erections and cavernous pressures in response to cavernous nerve neurostimulation in the rat, demonstrating the plasticity of the erectile apparatus. However, the inherent immunogenicity of the viral vectors has limited the improvement of erectile function to weeks in the rodent model, and the use of viral vectors in humans has risks of a lethally pathologic immune response. Of greatest importance in a phase 1 clinical trial for a non-life-threatening illness is that the primary endpoint is safety not efficacy. The approach that we have taken at the Urology Laboratory at the Albert Einstein College of Medicine and through its academic-based company, Ion Channel Innovations, LLC, has been to use a plasmid-based vector for therapy of two smooth muscle disorders, ED and OAB, each of which has smooth muscle cell-heightened tone as an important aspect of the disease [5,9,27,28,29,30,31]. One of the genes that we have chosen to use for preclinical and clinical trials is KCNMA1, also referred to as hSlo, which encodes the alpha subunit of the potassium, large-conductance, calcium-activated channel, on chromosome 10; location 10q22.3. The channels are also known as Maxi-K and the product name we have chosen for our studies is hMaxi-K. Although the mature channel consists of a tetramer of alpha associated with beta subunits, hMaxi-K expresses only the alpha subunit. The alpha subunit forms a pore through which potassium ions can traverse the cytoplasmic membrane, whereas the beta subunit regulates the passage of potassium ions in response to intracellular calcium (Fig. 2).

FIGURE 2.
A representative smooth muscle cell that contains a native Maxi-K channel with natural expression of both an alpha and beta subunit, as well as a channel derived from hMaxi-K that expresses channels with only an alpha subunit. Both channels, when open, hyperpolarize the cell membrane and result in reduced calcium influx and reduction in smooth muscle cell tone.
In preclinical experiments, we have shown extensively that the physiological result of expressing the alpha subunit causes cell hyperpolarization, and smooth muscle cell and organ relaxation [7,27,30,32]. However, overexpressing only the alpha subunit channel appears to function within the complex network of pathways regulating smooth muscle tone as shown in Fig. 3.

PRESENT
To date, we have completed a phase 1a and 1b clinical trial with hMaxi-K. The trial was an open-label, sequential, single-dose, intracavernous instillation of seven doses of hMaxi-K, 500-16,000 µg, in 21 men with moderate to severe ED as defined by International Index of Erectile Function (IIEF) score [9,28,29]. The specifics of the entire trial are presented elsewhere, but the most pertinent findings are listed below.
1. There were no clinically relevant, transfer-related, serious adverse events in any participant at any time up to 4 years after transfer. 2. There was no plasmid evident in the semen of the participants. 3. There were no clinically relevant changes in any of the physical or chemical parameters measured, including EKG.
4. In the men who responded with improved erections after transfer, the response lasted for greater than 6 months. This supports the preclinical trials in aged or diabetic male rats where efficacy was noted for periods of at least 6 months FUTURE At the present time, with the exception of the trial noted above, there are no other FDA-approved, open ED trials that use gene transfer for the treatment of ED. Ion Channel Innovations, LLC, based on the positive results of the phase I trial, is planning to extend to a phase 2 trial that will include a placebo arm and an increased number of participants. Ion Channel Innovations, LLC-sponsored research has resulted in two published [30,33], preclinical papers in which the structure of the backbone of the vector of the parent plasmid has been modified so that the Maxi-K gene is expressed specifically in smooth muscle cells. Studies -one in aging rats and the other in atherosclerotic, crab-eating monkeys with ED -were successful in normalizing erectile function. The advantage of a smooth muscle-specific promoter of gene expression is an enhanced safety profile, limiting expression to the target cells (smooth muscle cells) even when there is unintended biodistribution into potentially susceptible organs.
Further advances in gene transfer with either of the plasmid vectors will be directed to increase the transfection efficiency and duration of response following transfer [34,35,36,37,38].
In order to regain acceptance of viral vectors for clinical application, it will be necessary to improve their safety profiles. Investigators are working to reduce the immunogenicity and replication of adenoviral vectors, and prevent the possibility of insertional mutagenesis (potentially activating oncogenes) by retroviral vectors [37,39]. Another potential line of therapy is the use of small interfering RNA (siRNA), using either naked plasmid or retroviral vectors for reduction of gene expression [24,40].
An alternative to gene therapy, which is presently being evaluated by several research groups, is the use of adult stem cells derived from bone marrow, blood, or organ of interest, and instilled into various organs. The goal would be to replace defective or missing cells from the organ of interest. Thus, the stem cells would transdifferentiate into the cell of interest (see http://stemcells.nih.gov/info/basics4.asp for a stem cell primer). For example, Song et al. transferred immortalized human fetus-derived mesenchymal stem cells into rat corpora, which differentiated into both endothelial and smooth muscle cells [41]. Another future possibility, if the high expectations for stem cell utilization are found to be successful, is to combine gene(s) of interest and stem cells so that the gene of choice would be engineered ex vivo into the stem cell for targeted protein expression in the organs of choice [42,43,44,45,46,47]. The endothelium as the tissue of choice for therapy of ED has been explored by Bivalacqua et al. [42]. In those experiments, in aged rats over a short duration (21 days), the stem cells with and without the addition of an adenoviral vector containing bovine eNOS gene under control of a Rous sarcoma virus improved erectile function with the production of both calcium-dependent NOS activity and cGMP levels. Of course, the endothelium is only one potential tissue that can be altered with aging and disease. Smooth muscle cells, neurons, and small arteries each are viable and potential targets for future research. Despite the present obstacles, gene transfer for treatment of ED represents an exciting potential leap for mankind into the next generation of modern medical treatment of the afflictions of aging and chronic disease in which urological laboratories are at the forefront of exploration.