Gene therapy applications in the lower urinary tract

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The lower urinary tract is ideally suited for minimally invasive therapy. All of the lower urinary tract can be reached either percutaneously or through endoscopy. Using MDSCs to deliver growth factor genes, an ex viv approach, could treat such disabling and prevalent conditions as urinary incontinence, interstitial cystitis (IC), and erectile dysfunction (ED) and limit the risk of systemic side effects. Figure 2 depicts MDSC-based tissue engineering in the lower urinary tract. This section focuses on gene therapy strategies that use both viral and nonviral approaches in the lower urinary tract.

5.1. Urinary Incontinence

Urinary incontinence is a serious and prevalent condition worldwide. The pharmaceutical industry has recently realized the significance of this disability and the potential market size that urinary incontinence represents. In the United States alone, an estimated 17 million men and women suffer from bladder control problems (36). The Agency for Health Care Policy and Research and the World Health Organization have estimated the total economic costs of urinary incontinence as $26 billion a year (37). As the population within developed countries continues to age, the economic costs will continue to soar. Thus, the economic impact of urinary incontinence is staggering.

The three main types of urinary incontinence are stress, urge, and overflow incontinence (38). Stress and urge incontinence each account for 45% of all incontinence cases, whereas overflow incontinence accounts for approx 5% of cases. Stress incontinence occurs when the urethral sphincter muscle is weak and cannot prevent urine leakage during activities that put stress on the abdomen, such as coughing, sneezing, or jumping. Urge incontinence is a condition characterized by urinary urgency and frequency associated with uncontrollable urine leakage. Overflow incontinence is a devastating condition in which patients cannot urinate because of damage to the nerves innervating the bladder. One of the most common causes of overflow incontinence is diabetes mellitus, which produces bladder neuropathy.

5.1.1. Stress Incontinence

There are three approaches to the treatment of stress incontinence: exercise, bladder suspension surgery, or injection of bulking agents (38). Intrinsic sphincteric deficiency (ISD) is the most severe type of stress incontinence. It is usually the result of prior surgery that damaged the sphincter muscle or pudendal nerve. The injection of bulking agents, such as collagen, into the urethra at the level of the urinary sphincter serves to produce a functional obstruction that will help to correct urine leakage. The injection of collagen into the urinary sphincter is a quick outpatient procedure that gives the patient little pain or risk; however, the long-term success of this procedure is limited by several disadvantages (39). Collagen is often reabsorbed, which adversely affects a successful outcome and requires repeat injections in the majority of patients. An average of three collagen injections is needed to achieve partial or total improvement. Also, 5% of patients are allergic to bovine collagen, and in most injected with collagen, antibodies develop to bovine antigens.

Greater persistence of injected MIDCvs bovine collagcn

Autologous MDC injection may be preferred substance for stress incontinence Inwiallargflnlc and long-term persistence)

Fig. 3. A histologic comparison of collagen and MDSC injected into the rat urethra. A and D, d 3 after collagen injection. B and E, d 30 after collagen injection. C and F, d 30 after MDSC injection. A, B, and C reduced from x40. D, E, and F reduced from x100. The following were noted: (1) greater persistence of injected MDSCs vs bovine collagen; (2) autologous MDSC infection may be preferred for stress incontinence (nonallergenic and long-term persistence). (See color plate 4 in the insert following page 82.)

In one study, the feasibility of injecting MDSCs into the urethra was studied in rats and compared to bovine collagen injection (40). A large number of the cells, transduced with the LacZ reporter gene, expressed -galactosidase 3 and 30 d after autologous MDSC injection. The persistence of the MDSCs at 3 d was similar to that of collagen; however, at 30 d, 88% of the cells survived, compared to only a scant amount of collagen. Figure 3 shows the histologic comparison between collagen and MDSCs in this study. In addition, the injection of the MDSCs into the urethral wall had no adverse effects. Thus, autologous MDSC injection may be more desirable than collagen injection for treating stress incontinence because the MDSCs are nonallergenic, they persist long term, and they directly correct the underlying pathophysiology by regenerating damaged urethral muscle. Also, it can be speculated that the cost of MDSC injection would be much less than the cost of many collagen injections because the MDSCs are obtained from the host patient.

In another study, stem cell tissue engineering was used to restore deficient urethral sphincter muscle in a rat model (41). In the study, MDSCs were isolated from normal rats, transduced with LacZ for labeling, and injected into the proximal urethra of rats with denervated urinary sphincters.

Fig. 3. A histologic comparison of collagen and MDSC injected into the rat urethra. A and D, d 3 after collagen injection. B and E, d 30 after collagen injection. C and F, d 30 after MDSC injection. A, B, and C reduced from x40. D, E, and F reduced from x100. The following were noted: (1) greater persistence of injected MDSCs vs bovine collagen; (2) autologous MDSC infection may be preferred for stress incontinence (nonallergenic and long-term persistence). (See color plate 4 in the insert following page 82.)

After 2 wk, strips of urethra were tested from normal, denervated, and den-ervated-MDSC-injected rats. Fast twitch muscle contractions were recorded after electrical field stimulation. The amplitude of fast twitch muscle contractions was decreased in denervated sphincters and was improved by 88% in denervated sphincters injected with MDSCs. Histological examination revealed the formation of new skeletal muscle fibers at the injection sites of the urethral sphincter. This study lays the foundation for further investigation into the use of stem cells to treat urinary incontinence.

5.1.2. Urge Incontinence

The standard therapy for urge incontinence involves anticholinergic drugs, which work to reduce the involuntary bladder contractions, and behavioral therapy (42). Gene therapy strategies for overactive bladders might include suppression of bladder muscle activity or neural pathways that trigger the micturition reflex. Christ et al. studied K+ channel gene therapy as a treatment for urge incontinence in rats (43). The intravesical inoculation of naked hSlo/pcDNA suppressed bladder hyperactivity in rats with partial urethral obstruction. They postulated that the overexpression of K+ channels in the bladder might inhibit the overactive bladder.

5.1.3. Overflow Incontinence

Preliminary work has been carried out using HSV to treat overflow incontinence caused by diabetic neurogenic bladder dysfunction. Diabetic animals exhibit a decrease in nerve growth factor (NGF) production in target tissues (44). There is evidence that the neuronal gene targets for NGF are understimulated (45). Apfel et al. reported that exogenously administered NGF is capable of preventing the behavioral and biochemical manifestations of diabetic sensory neuropathy in the streptozotocin (STZ)-induced diabetic rat model (46). In one study, HSV-1 vectors carrying NGF were injected into the bladder walls of rats 6 wk after the induction of diabetes by STZ (47). Bladder function was compared between STZ-induced diabetic rats injected with the NGF expressed vector or rats injected with the control vector at 4 wk following HSV injection. The NGF-treated diabetic rats exhibited a 50% decrease in volume each void without significant changes in total urine output when compared with control diabetic rats. These results indicate that NGF expression via HSV vectors in the bladder afferent pathways improved bladder function in diabetic rats.

5.2. Interstitial Cystitis

Interstitial cystitis (IC) is a voiding dysfunction that affects nearly a million people in the United States (48). IC is characterized by chronic pelvic pain associated with bladder symptoms of urinary frequency and urgency. A

Fig. 4. Extensive P-galactosidase staining in the rat bladder. (See color plate 5 in the insert following page 82.)

possible gene therapy approach involves the delivery of preproenkephalin to the peripheral nerves of the bladder. This method delivers low, but therapeutic, quantities of enkephalin only to sensory nerves that innervate the organ in pain, but not to the whole animal. In one study, the preproen-kephalin gene was transferred and maintained in the bladders and bladder afferent nerves of rats using the HSV-1 vector (49). Also, this study concluded that the increased expression of enkephalin in bladder afferent pathways suppressed nociceptive responses induced by bladder irritation. Figure 4 depicts gene therapy for such lower urinary tract dysfunction as overflow incontinence and interstitial cystitis. This technique of gene transfer may be useful for treating IC and other types of visceral pain syndromes.

5.3. Erectile Dysfunction

Several studies of nitric oxide synthase (NOS) gene therapy for erectile dysfunction have been reported. Champion et al. demonstrated the feasibility of gene transfer of endothelial NOS (eNOS) augmenting erectile responses in the aged rat (50). They administered a recombinant adenovirus containing the eNOS gene into the corpora cavernosa of the aged rat. An increase in cavernosal pressure with cavernosal nerve stimulation was enhanced in animals transfected with eNOS. In another study, Tirney et al. assessed inducible NOS (iNOS) gene therapy into the corpus cavernosum of adult rats (51). They compared injections of plasmid, adenovirus, or aden-ovirus-transduced muscle cells. Muscle cell-mediated gene therapy was more successful for delivering iNOS into the corpus cavernosum than direct adenovirus or plasmid transfection methods. Gene therapy of NOS, using MDSCs, could open new avenues of treatment for erectile dysfunction. Control of NOS expression would be necessary to prevent prolonged erection. Finally, Wessells and Williams demonstrated endothelial cell-based ex vivo gene therapy for erectile dysfunction in rats (52).

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