Gene therapy for lower urinary tract dysfunction

Authors


Correspondence: Naoki Yoshimura M.D., Ph.D., Department of Urology, University of Pittsburgh School of Medicine, Suite 700, Kaufmann Medical Building, 3471 Fifth Avenue, Pittsburgh, PA 15206, USA. Email: nyos@pitt.edu

Abstract

Lower urinary tract dysfunction is caused by functional and pathophysiological alterations of the peripheral organs, including the urothelium and detrusor smooth muscle, as well as peripheral and central nervous systems. Recent research in this field has increased our understanding of the mechanisms of lower urinary tract dysfunction, and new drugs have been developed, leading to increased treatment options and changing medical care for lower urinary tract symptoms. Nevertheless, clinicians still often experience refractory and treatment-resistant cases against conventional therapeutic modalities. For such cases, gene therapy targeting for the lower urinary tract and its afferent pathway is anticipated to offer a new therapeutic approach. Therefore, in this article, we review the possibility and current status of gene therapy for lower urinary tract dysfunction.

Abbreviations & Acronyms
AAV1

adeno-associated virus type 1

BOO

bladder outlet obstruction

DRG

dorsal root ganglion

GABA

γ-aminobutyric acid

GAD

glutamic acid decarboxylase

HGF

hepatocyte growth factor

HSV

herpes simplex virus

IC/BPS

interstitial cystitis/bladder pain syndrome

IL

interleukin

LUT

lower urinary tract

LUTS

lower urinary tract symptoms

MnSOD

manganese superoxide dismutase

NGF

nerve growth factor

POMC

pro-opiomelanocortin

SERCA

sarco/endoplasmic reticulum calcium ATPase

SR

sarco/endoplasmic reticulum

SUI

stress urinary incontinence

TNFα

tumor necrosis factor α

TNFαsR

tumor necrosis factor α soluble receptor

Introduction

The American Society of Gene and Cell Therapy has defined gene therapy as the use of genetic material to modify a patient's cells for the treatment of disease. This new therapeutic approach is accompanied by risks and uncertainties regarding the long-term impact of the manipulation of genetic material on cell, tissue and organismal biology.[1] Thus, gene therapy has most commonly been applied to the treatment of life-threatening diseases, such as cancer and other acquired and hereditary disorders with few effective treatments. However, this concept is changing, as recent progress in gene therapy has increased its safety, expanding its potential indications to non life-threatening diseases, including rheumatoid arthritis,[2, 3] neuropathy,[4, 5] optic neuritis,[6] Parkinson's disease,[7, 8] and LUT dysfunction.[9]

According to the Wiley registration, since the first clinical trial of gene therapy in 1990, 1785 clinical trials have been carried out in total. Among them, 1155 trials were for cancer, 151 for monogenic diseases, 150 for cardiovascular diseases, 142 for infectious diseases, 36 for neurological diseases and 26 for ocular diseases. There is only one phase I study for LUT dysfunction, which plans to evaluate the effects of maxi-K channel gene transfer on overactive bladder in postmenopausal women (GeMCRIS protocol # 0604-774). Various kinds of vectors have been used to deliver the therapeutic gene into the cell in these trials: adenovirus in 424 trials, retrovirus in 365, naked/plasmid DNA in 337, vaccinia virus in 146, lipofection in 110, poxvirus in 95, adeno-associated virus in 86 and HSV in 58. Among them, 63 trials are in phase III and two trials are in phase IV.

Gene therapy for lower urinary tract dysfunction

LUTS are a common urological problem. The LUT has two contrasting functions: storage and then elimination of urine, which are controlled by complex peripheral and central neural pathways. Thus, LUT function is easily disturbed by functional and pathophysiological changes at various levels, such as the urothelium, detrusor smooth muscle, and peripheral and central nervous systems. Recent progress in research has shown the mechanisms of LUT dysfunction, and the development of new drugs has increased the treatment options, changing medical care for LUTS. Nevertheless, clinicians sometimes experience refractory or treatment-resistant cases. For such cases, gene therapy to the LUT and its afferent pathways offers the potential to provide a paradigm shift from palliative or symptomatic relief to potentially curative therapeutic solutions. In addition, the anatomy of the LUT enables some straightforward approaches for local gene delivery, such as intravesical administration and intradetrusor/sphincter injection. The possibility of gene therapy for LUT has been examined in many preclinical studies, in which the indications for gene therapy include, potentially, almost all urological disorders: overactive bladder, underactive bladder, decreased bladder sensation, bladder hyperalgesia, urinary incontinence and decreased bladder capacity (Table 1).

Table 1. Preclinical gene therapy studies for LUT dysfunction
Gene targetVectorRat modelEffect
β-NGF[15]HSVDiabetic neuropathyBladder capacity ↑, postvoid residual volume ↓
NGF antisense[24]TAT-PNA conjugateChemically-induced cystitisBladder overactivity↓
Preproenkephalin[40, 41]HSVBladder overactivity↓, nociceptive behavior↓
Pro-opiomelanocortin[42]Plasmid DNA injected via gene gunBladder overactivity↓
TNFαsR[55]HSVInflammation↓, bladder overactivity↓, nociceptive behavior↓
IL-4[60]HSVInflammation↓, bladder overactivity↓, nociceptive behavior↓
HGF[29]Liposome complexBOOFibrosis↓, bladder overactivity↓, bladder capacity↑
GAD[35, 36]HSVSpinal cord injuryBladder overactivity↓, detrusor-sphincter dyssynergia↓
MnSOD[65]Plasma DNA liposomeRadiation cystitisBladder contraction↑
hSlo (maxi K channel)[72]Naked DNA (pc DNA)BOOBladder overactivity↓
SERCA[74]AAV1SUI (vaginal distension)Muscle contraction↑

Growth factors

Nerve growth factor

NGF is produced in the bladder urothelium and smooth muscle, transported through afferent neuronal axons to DRG neurons, and regulates the differentiation and survival of several target neurons.[10] NGF is necessary for maintaining the normal function of mature sensory and sympathetic neurons,[11] and retrograde axonal transport of NGF from target organs decreases in diabetic autonomic neuropathy.[12] NGF supplement therapy is effective for the treatment of diabetic sensory neuropathy.[13, 14] Thus, it seems reasonable to assume that systemic administration of NGF might also be effective for recovery of bladder sensation. However, a systemic approach for the delivery of NGF might cause severe and sometimes even fatal side-effects involving various organs.

The efficacy of organ-specific local gene delivery using replication-deficient HSV encoding β-NGF has been reported in a rat model of diabetic neuropathy induced by streptozotocin.[15] The vector-mediated transgene expression was found not only at the site of vector injection within the bladder wall, but also in afferent neurons in lumbosacral DRG. Bladder capacity and postvoid residual volume, which are increased in control diabetic rats, were reduced without enhancing bladder nociceptive responses after HSV-NGF vector injection.

In contrast, increased levels of NGF have been proposed as an important pathophysiological basis of OAB.[16-23] For example, benign prostate hyperplasia patients with OAB symptoms have significantly increased NGF, which decreases to baseline after BOO relief.[16] Rats with BOO also showed increased mRNA expression of bladder NGF and detrusor overactivity.[17, 18] Furthermore, overexpression of NGF has been reported in patients with IC/BPS.[19, 20] It has also been reported that bladder overactivity can be induced by intravesical acute administration or intrathecal chronic administration of NGF in rats.[21-23] Thus, it is hypothesized that suppression of NGF in the bladder could be effective to reduce bladder overactivity. In this regard, a gene therapy approach using NGF antisense PNA conjugated with tethering TAT peptide, which is the PTD from the HIV-1 TAT protein, showed that intravesical instillation of NGF antisense PNA-TAT successfully reduced NGF immunoreactivity in the urothelium and significantly decreased bladder overactivity in rats with chemically-induced cystitis.[24]

Hepatocyte growth factor

Abnormal accumulations of extracellular matrix are frequently observed during the development of BOO secondary to benign prostatic hyperplasia. This deposition of collagenous proteins and other matrix constituents is a component of a normal tissue response to injury, but it can eventually develop into organ fibrosis. Such fibrosis can adversely affect smooth muscle contraction and eventually reduce bladder capacity. HGF shows mitogenic and morphogenic activities in a wide variety of cells[25] and antiapoptotic properties through angiogenicity,[26] which occur through the activation of a matrix degradation pathway.[27] Thus, HGF is a unique growth factor that has an anti-fibrotic effect.[28]

The injection of naked DNA in a HGF-liposome complex in partially obstructed rat bladders successfully increased HGF mRNA and protein expression levels in the bladder.[29] Then the increased HGF reduced the expression of transforming growth factor-β1 mRNA and protein, and decreased the collagen content of the bladder wall, leading to larger bladder capacity and lesser bladder overactivity, raising the possibility that the HGF gene therapy could be effective for the treatment of bladder fibrosis.

Inhibitory neurotransmitters

Glutamic acid decarboxylase

In the central nervous system, glutamate is a major excitatory neurotransmitter, whereas glycine and GABA are the most abundant inhibitory amino acid neurotransmitters.[30] GABA is synthesized from glutamate by GAD,[30] and HSV-mediated gene transfer of GAD delivered by subcutaneous inoculation to the foot pad can reduce neuropathic pain in spinal cord-hemisected or spinal nerve-ligated rats.[31, 32] GABA is also important in the inhibitory regulation of micturition in normal, spinal cord-intact rats.[33] It has also been shown that chronic spinal cord injury lowers GAD levels in the lumbosacral spinal cord and bladder afferent pathways. Animals with spinal cord injury also show detrusor overactivity as evidenced by non-voiding contraction during storage phase during cystometry, which is suppressed by intrathecal administration of GABA receptor agonists.[34]

HSV-mediated gene transfer of GAD increased GABA levels in bladder afferent pathways and increased the release of GABA in the spinal cord, preventing non-voiding contractions in rats with spinal cord injury.[35] In addition, HSV-mediated GAD gene therapy suppressed detrusor-sphincter dyssynergia by reducing urethral pressure increases during bladder contraction in spinal cord-injured rats.[36] Thus, these results suggest that HSV-based GAD gene transfer to the bladder and bladder afferent pathway might represent a novel approach for the treatment of neurogenic detrusor overactivity.

Opioids

In addition to GABA and glycine, opioids are also the abundantly-expressed inhibitory neurotransmitter. Patients with IC/BPS often have severe and refractory bladder pain that is resistant to non-steroidal anti-inflammatory drugs, requiring long-acting opioids.[37] However, the use of systemic opioid therapy, such as morphine or oxycodone, has been limited because of its untoward side-effects, tolerance and dependency. Enkephalins, which are a subfamily of endogenous opioids, are expressed in bladder afferent and efferent pathways to inhibit micturition.[38] Enkephalinergic mechanisms in the brain and spinal cord also have inhibitory effects on the micturition reflex, and exogenous enkephalins or opiate drugs applied to the sacral spinal cord can suppress micturition.[39]

Replication-deficient HSV vectors encoding preproenkephalin, one of three genes that encode endogenous opioid peptides, injected into the bladder wall showed reductions in bladder overactivity and nociceptive behavior induced by intravesical application of capsaicin, whereas vector-mediated expression of enkephalin did not affect normal voiding.[40, 41] These results provide the proof of concept for a new gene therapy approach to enhance endogenous opioid mechanisms and reduce systemic side-effects for the treatment of bladder hypersensitive disorders, such as IC/BPS.

Another study reported the anti-nociceptive effect of human POMC,[42] which encodes β-endorphin, another endogenous opioid peptide, which activates μ-opioid receptors on sensory neurons and inhibits pain in the inflamed tissue.[43, 44] Intravesical instillation of acetic acid during cystometrograms shortened the intercontraction interval in control animals, but not in rats treated with POMC genes delivered by gene gun into the bladder wall. The effects of the POMC gene gun injection were reversed by an intramuscular injection of the opiate receptor antagonist, naloxone, supporting an opioid receptor-mediated analgesic mechanism of action.[42]

Overall, opioid gene therapy targeting the bladder and bladder afferent pathways could be an effective modality for the treatment of intractable pain symptoms of bladder hypersensitive disorders, such as IC/BPS.

Cytokines

Although the etiology of IC/BPS is not fully understood, bladder inflammation associated with the production of inflammatory cytokines has been proposed as a potential cause of pathogenesis of the disease.[45, 46] In previous studies, cytokines and chemokines such as IL-2, IL-6, IL-8 and TNFα are significantly more increased in IC/BPS patients' bladder tissue and urine than in controls, suggesting that these cytokines might represent specific markers of IC/BPS.[46-48] TNFα is a pro-inflammatory mediator that initiates inflammatory reactions of the innate immune system: induction of other cytokine production, activation and expression of adhesion molecules, and stimulation and recruitment of inflammatory cells.[49] Additionally, it is essential in the development of nociception, not just in inflammatory pain, but also in neuropathic pain.[50] Many studies have reported that TNFα influences pain sensation in the peripheral tissue and the spinal cord.[51] When TNFα activity is neutralized using anti-TNFα antibody or TNFαsR, the development of nociception is suppressed, as has been observed in several rat pain models.[51-54]

Recently, gene therapy of TNFαsR using replication-deficient HSV vectors has been investigated for bladder pain and urinary frequency using a rat model of chemically-induced cystitis.[55] Although TNFα mRNA in the bladder was upregulated by the intravesical administration of resiniferatoxin, the increase in TNFα protein levels was suppressed in TNFαsR-expressing vector-treated rats, indicating that HSV vector-delivered TNFαsR neutralized TNFα proteins increased by bladder irritation. The suppression of TNFα protein leads to the downregulation of IL-1β and IL-6, as well as a reduction in MPO activity in the resiniferatoxin (a TRPV1 receptor agonist)-treated bladder. Moreover, TNFα blockade reduced pain sensation and bladder overactivity induced by intravesical instillation of resiniferatoxin.

Another approach investigated for bladder pain is the HSV vector-mediated delivery of IL-4 to the bladder and bladder afferent pathways. IL-4 is a prototypical anti-inflammatory cytokine, known to inhibit the secretion of inflammatory cytokines, such as IL-1β, TNFα and IL-6.[56, 57] In contrast to TNFα or IL-6, IL-4 is decreased in the urine of IC/BPS patients, and then increases after the treatment of suplatast tosilate, an anti-allergic drug.[58] In rats, IL-4 expression after HSV vector administration to the plantar foot surface reduced in nociceptive behaviors after painful insult.[59] The effect of IL-4 delivered by HSV vector on bladder overactivity and nociceptive behaviors has been evaluated previously.[60] The IL-4 protein level was increased in the bladder and L6 DRG in IL-4-expressing HSV-injected rats, and MPO activity and IL-1β were decreased in the bladder of the rat with chemically-induced cystitis, in which bladder overactivity and nociceptive behaviors were also suppressed.

Overall, gene therapy targeting cytokine production in the bladder could be a new modality for the treatment of bladder hypersensitive disorders, such as IC/BPS, which is associated with increased production of inflammatory cytokines.

Manganese superoxide dismutase

Radiation therapy to the pelvic organs often causes bladder overactivity, known as radiation cystitis. Several approaches for suppression of superoxide free radicals in the bladder have been tried, because they are thought to contribute to the major process of radiation-induced damage. One of these approaches is the use of the MnSOD transgene in cells to protect them from irradiation-induced damage.[61-63] Increased MnSOD activity removes superoxide free radicals produced in the mitochondria[64] and stabilizes the mitochondria, thereby preventing mitochondrial membrane depolarization, release of cytochrome c and activation of caspase 3, leading to preventing apoptosis.[63] Overexpression of the MnSOD transgene in the mouse lung and esophagus protected normal tissue from irradiation-induced damage by preventing pulmonary fibrosis and esophagitis.[62]

When phospholipid complexes, liposomes with plasmid DNA containing the MnSOD transgene, were injected directly into the bladder through a catheter, the MnSOD human transgene could be detected in transfected rats for up to 3 days after the injection and the MnSOD biochemical activity was increased for up to 48 h.[65] These were sufficient to preserve urothelial barrier function and integrity against bladder irradiation. Detrusor function after radiation was better even at 6 months in the MnSOD gene-treated rats. An implication is that physiological antagonism of ionizing radiation at the time of irradiation can sufficiently prevent/reduce apoptosis of the umbrella and basal cells of the urothelium to prevent the long-term deleterious consequences to bladder function.

Maxi-K channel

Overactive bladder associated with BOO is caused by neurogenic hyperexcitability[66, 67] and/or myogenic overactivity.[68, 69] Some studies have shown that the reduction of K+ channel activity is associated with bladder overactivity, as K+ channel activation usually suppress cell excitability of muscles and neurons.[68, 70] In particular, the maxi-K channel plays a pivotal role in modulating contraction and relaxation responses in physiologically diverse myocytes.[71] Maxi-K channels are activated during cellular activation, leading to enhanced efflux of K+ ions from the cells and hyperpolarization of the cells, which reduces the activity or open probability of the L-type voltage-dependent calcium channels. Then, the influx of calcium ions will also be decreased, resulting in decreased free intracellular Ca2+ concentrations.

Bladder instillation of human maxi-K channel cDNA hSlo inserted in a pcDNA vector functionally antagonized the increased contractility observed in a rat model of BOO induced by partial urethral obstruction, thereby ameliorating bladder overactivity without detectably affecting any other cystometric parameters.[72] Thus, maxi-K channel gene therapy has been proposed for the treatment of patients with overactive bladder.

Sarco/endoplasmic reticulum calcium ATPase

The regulation of intracellular Ca2+ is important for all living cells, especially for smooth muscle, as Ca2+ is a key messenger leading to muscle contraction. Mechanisms for the cellular clearance of Ca2+ form one side of the calcium balance for homeostasis and include SERCA, which actively transports Ca2+ into the SR and regulates the cytosolic Ca2+ concentration.[73] In a preliminary study, female rats received periurethral injection of adAAV1-expressing SERCA2a.[74] One-half of the animals underwent vaginal distension 3 weeks after AAV1/SERCA2a administration. Four weeks after the administration, leak point pressure was significantly higher in animals receiving AAV1/SERCA2a compared with the control group. These results suggest that AAV1/SERCA2a improves urethral sphincter muscle function after vaginal distension-induced injury and decreases stress urinary incontinence.

SERCA might also play a major role in the regulation of Ca2+ for detrusor contraction, and might have a role in the functional outcome after outlet obstruction and ischemia/reperfusion injury. Thus, the therapeutic transfer of the SERCA gene might become a potential approach for gene therapy of LUT pathology, especially for underactive bladder and/or stress urinary incontinence.

Gene delivery strategy

As described earlier, several delivery methods for therapeutic genes have been reported from preclinical studies. Each delivery system has its own advantages and disadvantages with regard to efficacy and safety. A major advantage of non-viral delivery systems is low immunogenicity compared with viral vectors, which all induce some level of host response to the vector itself. Additionally, non-viral systems generally show shorter-term vector-delivered transgene expression because of limited maintenance of the delivery vehicle in the transduced cells. In contrast, viral vector-based gene delivery can avoid the need for frequent administration of short half-life peptides, and the lack of off-target activity suggests that this type of delivery can avoid systemic side-effects that have also been observed using direct delivery of therapeutic protein(s).[75] The physiological barrier function of normal urothelial cells, which possess tight junctions and high epithelial resistance, can prevent effective passage and infection/transfer of genes;[76, 77] whereas, the efficiency of delivering a genetic payload to the target cell is higher compared with non-viral systems, because over millennia, viruses have acquired effective methods to deliver their own genetic material to cells to replicate their genomes and further propagate themselves. However there are potential risks with viral vectors, such as endogenous viral recombination, cancer development and immunological reactions.[78]

HSV vectors have several advantages over other viral vectors for the treatment of peripheral nervous system disorders.[79] Replication-defective recombinant vectors, which lack multiple essential gene functions and are non-toxic in vivo,[79-82] have been generated to increase the overall safety for clinical applications. These vectors can be prepared to high titer and purity without contamination by the wild-type form.[83-85] Furthermore, because of its natural ability to efficiently transduce primary afferent neurons, where these vectors rapidly establish a “latent-like” state in the infected cells without altering the normal neuronal cell,[86] gene therapy using replication-deficient HSV could be an effective and safe method of delivery of therapeutic genes to the cells of the peripheral nervous system.

Genetically modified muscle-derived cells can also be used as cellular vectors.[87, 88] This system not only delivers the gene of interest to the target organ, but also could provide bulk for, for example, increased mechanical resistance to correct a defective urethral sphincter when used in patients with urinary incontinence.[89, 90] If the injected cells differentiate into functional myotubes, they might improve impaired detrusor contractility, which might be therapeutic for patients with underactive bladder.

Conclusions

The possibility of gene therapy for LUT dysfunction has been explored over the past decade (Table 1). Gene therapy could be curative, normalizing bladder and urethral function. Although the application of gene therapy for benign diseases faces major hurdles because of concerns over safety, this situation is changing. A phase I human clinical trial using replication-defective HSV vectors encoding human enkephalin is underway in patients with cancer pain to examine the safety of locally-applied viral gene therapy in contrast to the pleotrophic effects of opioid treatment,[91-93] and one clinical study is evaluating maxi-K channel gene transfer for overactive bladder in postmenopausal women (GeMCRIS protocol # 0604-774). With the accumulation of safety data for gene therapy, the indications will expand to less fatal diseases/disorders. Therefore, gene therapy might be a viable treatment option for LUT dysfunction in the not too distant future.

Acknowledgments

The authors' research has been supported by NIH DK057267, DK088836 and P01 DK044935 and the Samuel Wilan Research Fund for Interstitial Cystitis.

Conflict of interest

None declared.

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