european urology 50 (2006) 1208–1222

available at www.sciencedirect.com journal homepage: www.europeanurology.com

Review – Sexual Medicine

Gene Therapy for Erectile Dysfunction: Fact or Fiction? Muammer Kendirci a, Patrick E. Teloken b, Hunter C. Champion c, Wayne J.G. Hellstrom d, Trinity J. Bivalacqua e,* a

Department of Urology, Sisli Etfal Training and Research Hospital, Istanbul, Turkey Departamento de Urologia, Fundacao Faculdade Federal de CIencias Medicas de Porto Alegre (FFFCMPA), Porto Alegre, RS, Brasil c Department of Medicine, Division of Cardiology, Johns Hopkins Hospital, Baltimore, MD, United States d Section of Andrology, Department of Urology, Tulane University Health Sciences Center, New Orleans, LA, United States e Brady Urological Institute, Johns Hopkins Hospital, Baltimore, MD, United States b

Article info

Abstract

Article history: Accepted August 2, 2006 Published online ahead of print on August 18, 2006

Objectives: Erectile dysfunction (ED) is a major health problem that seriously affects the quality of life of patients and their partners. Although all three selective phosphodiesterase type 5 inhibitors (PDE5-Is) are effective in the majority of ED cases, PDE5-I therapy is less efficacious in some hard-to-treat populations (diabetics, men after radical prostatectomy), prompting the development of new approaches, including gene therapy strategies for ED. Methods: Gene therapy approaches are discussed in terms of the possible role of gene therapy for the treatment of ED, potential targets for gene transfer, vectors to carry targeted genes, and gene strategies for ED in certain disease states, such as diabetes, ageing, arterial and venogenic insufficiency, and cavernous nerve injury. Results: The penis is a convenient tissue target for gene therapy because of its external location and accessibility, the ubiquity of endotheliallined spaces, and low level of blood flow, especially in the flaccid state. Gene therapy approaches have focused on a number of signaling pathways that are crucial for penile erection, such as nitric oxide/cyclic guanosine monophosphate, RhoA/Rho-kinase, growth factors, ion channels, peptides, and control of oxidative stress. Conclusions: The need for effective ED therapies in difficult-to-treat patients has encouraged investigators to seek novel modalities for the treatment of ED. Recent preclinical and clinical trials have demonstrated that gene therapy strategies may be feasible for these purposes.

Keywords: Ageing Cavernous nerve injury Diabetes Erectile dysfunction Gene therapy Growth factors K channel Nitric oxide RhoA/Rho-kinase Superoxide dismutase

# 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. Johns Hopkins Hospital, Brady Urological Institute, 600 North Wolfe Avenue, Marburg 143, Baltimore, MD 21287, United States. Tel. +1 410 955 0352; Fax: +1 410 614 3695. E-mail address: [email protected] (T.J. Bivalacqua).

0302-2838/$ – see back matter # 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved.

doi:10.1016/j.eururo.2006.08.007

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1.

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Introduction

Basic science research on erectile physiology has focused on the pathogenesis of erectile dysfunction (ED) and has provided convincing evidence that ED is predominately a disease of vascular etiology [1]. Oral selective phosphodiesterase type 5 inhibitors (PDE5Is) are the agents recommended as first-line therapy because of their convenience and high rate of efficacy in a diverse population of men with ED. Although all three selective PDE5-Is are effective in the majority of ED cases (>70% efficacy), these oral medications have failed (40–60% efficacy) in certain disease states, including diabetic ED, postprostatectomy ED, and severe vasculogenic and veno-occlusive dysfunction [2]. This has resulted in the development of new approaches including gene- and cell-based therapies for the treatment of ED for those patients who choose this form of therapy over other second-line therapies (pharmacologic injection therapy). This review discusses the possible role of gene therapy for the treatment of ED, potential targets for gene transfer, vectors to carry targeted genes, and gene strategies in ED in certain disease states, such as ED related to diabetes, ageing, arterial and venogenic insufficiency, and cavernous nerve injury.

2. Concept of gene therapy for ED: advantages and disadvantages With the recent advancements in our understanding of molecular mechanisms involved in human disease, we are better equipped to understand and diagnose disease processes. The long-term goal for the treatment of any disease process is to identify molecular correlates involved in the pathophysiology of disease and to use this information to develop novel and more effective therapeutics, either with pharmacologic agents or through a gene therapy approach. A major hurdle in this regard is ensuring that the molecular targets of interest are indeed relevant to the physiology or pathophysiology of the processes being studied. Additionally, we must determine if changes in cellular function are specific to the tissue/organ being investigated. Since the discovery that the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway is the principle signalling cascade responsible for corporal smooth muscle relaxation, the molecular mechanisms governing corporal smooth muscle tone have led to improved understanding, diagnosis, and treatment of ED [3–6]. Therefore, the goal of gene therapy for the treatment of ED must be the selection of candidate genes that are disease-related, mecha-

Fig. 1 – Schematic drawing of the principles of gene therapy. Therapeutic genes of interest or growth factors that influence cellular function can be placed in viral or nonviral vectors that enter a targeted cell to significantly alter its function. This figure demonstrates the administration of a desired gene and subsequent transportation into the nucleus and alteration of cellular function. More specific delineation of the mechanisms for vector incorporation of a specific gene into a targeted cell and the alteration in cellular function can be found in the text and reference list.

nism-based therapies for the restoration of penile vascular tone. Gene therapy has evolved to the point where treatment of any disease process can be theoretically accomplished as long as there is a therapeutic gene, which can either (1) effectively restore or supplement defective functions, or (2) antagonise the expression of a mutant gene (Fig. 1). The major advantages to the use of gene therapy for the treatment of ED are: 1. The penis is easily accessible and the desired gene can be administered directly into the corporal tissue, without entering the systemic circulation (a penile tourniquet may further prevent systemic exposure). 2. Vascular smooth muscle cells of the penis have a relatively low turnover rate, thus allowing a desired gene to be expressed for long periods of time. 3. Transduced genes to the penis may affect any aspect of the erectile process by selectively altering the expression of a given molecular target and thus target disease-specific gene products that may improve the erectile response. Gene therapy for ED would potentially restore the erectile process in the absence of any other form of

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Table 1 – Molecular targets used in gene therapy preclinical studies in animal models of various diseases Molecular targets Nitric oxide: NOS isoforms Ion channels: Maxi-K channel Neurotransmitters: CGRP, VIP Growth factors: VEGF, BDNF, NT3 RhoA/Rho-kinase Superoxide dismutase

Expected effects Restore endothelial-derived and neuronally derived NO—ageing, diabetes Increased hyperpolarization of corporal myocyte—ageing, diabetes Improve corporal cAMP synthesis—ageing, diabetes Increase eNOS, nNOS expression, improve neovascularization—ageing, diabetes, cavernous nerve injury, vasculogenic Inhibition reduces corporal vascular tone, improve eNOS expression/ activity—ageing, diabetes Antioxidant, improve endothelial-derived NO—ageing, diabetes

NOS = nitric oxide synthase; NO = nitric oxide; CGRP = calcitonin gene-related peptide; VIP = vasoactive intestinal peptide; cAMP = cyclic adenosine monophosphate; VEGF = vascular endothelial growth factor; BDNF = brain-derived nerve growth factor; NT3 = neurotrophin 3; eNOS = endothelial nitrous oxide synthase; nNOS = neuronal nitrous oxide synthase.

therapy [7]. Alternatively, the combination of local gene therapy with any of the currently available oral medications (i.e., cGMP-specific PDE5-Is) could synergistically improve the efficacy of both therapies. The inherent disadvantages for the use of local gene therapy approaches for the treatment of ED would be the selection of an ideal vector system that is safe for the patient, as well as representing a noninvasive therapeutic modality. Therefore, before any gene therapy approach is brought to clinical practice, it must be safe as well as efficacious [7]. Currently, PDE5-I therapy is highly efficacious with few side-effects; therefore, local gene transfer of candidate gene products would be reserved for hardto-treat population of patients such as severe diabetic- and vasculogenic-associated ED as well as ED occurring after radical prostatectomy. These patient populations are less likely to respond to current oral pharmacotherapies and, therefore, would benefit from a disease-specific molecular approach to the treatment of penile vasculopathy and neuropathy in these disorders.

3.

Molecular targets

Somatic gene therapy can be defined as the ability to introduce genetic material (RNA or DNA) into an appropriate cell type in vitro or in vivo, thus altering gene expression of that cell to produce a therapeutic effect. Gene therapy involves a number of finite sequences: the administration of a desired gene into the body, delivery of the gene to a targeted cell, which is subsequently transported into the nucleus, and then expression of the therapeutic product (Fig. 1). Gene therapy is an attractive therapeutic possibility for the treatment of ED. A simple concept for gene therapy for ED is

that only a very small alteration in the balance between contracting and relaxing stimuli can cause significant effects on cavernous and penile arterial vascular smooth muscle tone [8–11]. Thus, transfer of a therapeutic gene product into the penis may restore impaired erectile function. Gene therapy strategies have been divided into two categories: (1) to correct the erectile deficit by increasing the supply/strength of the erectile stimulus, increasing the expression of a relevant endogenous vasomodulator of the erectile function, and (2) to alter the tissue or end-organ demand for a given erectile stimulus, making the corporeal tissue more sensitive to relaxatory stimulus [12,13]. Thus, erectile function can be modulated and potentially restored by altering the physiologic supply and demand of the erectile apparatus in both theory and practice. Gene therapy for ED is a relatively straightforward concept that simply restores the normal balance between contracting and relaxing stimuli in the penile endothelial and smooth muscle cells (Table 1 and Fig. 2). Gene therapy strategies for ED have focused on the NO/sGS/cGMP pathway. All three NO synthase (NOS) isoforms, endothelial NOS (eNOS), neuronal NOS (nNOS; penile nNOS [PnNOS] is the penilespecific variant of nNOS), and inducible NOS (iNOS), have been used for gene therapy to modulate erectile response. The rationale for these targets is that overexpression of important endogenous corporal smooth muscle vasodilators improves diminished erectile response in ED. Newer approaches suggest that other means of genetic manipulation— nerve and vascular growth factors (e.g., brainderived nerve growth factor [BDNF], vascular endothelial growth factor [VEGF]), the cyclic adenosine monophosphate (cAMP) cascade (e.g., calcitonin gene-related peptide [CGRP] receptor), or

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Fig. 2 – (A) Potential molecular targets that cause corporal smooth muscle relaxation. Endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) gene therapies increase endothelial-derived NO and promote corporal vasodilation. Additionally, VEGF preserves corporal smooth muscle integrity. Extracellular superoxide dismutase (EC-SOD) gene therapy reduces penile superoxide anion levels thus improving NO bioavailability and corporal smooth muscle relaxation. Neuronal NOS (nNOS) and brain-derived neurotrophic factor (BDNF) gene therapies increase nNOS expression and neuronal-derived NO. Vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) gene therapies increase corporal cyclic adenosine monophosphate (cAMP) levels via activation of adenylate cyclase to increase corporal smooth muscle relaxation. Maxi-K channel (Ca2+-sensitive potassium channel) hyperpolarises corporal smooth muscle to promote vasodilation. (B) Inhibition of RhoA or Rho-kinase using dominant-negative mutants promotes myosin light-chain (MLC) phosphatase to an activated form (nonphosphorylated), thus catalysing the dephosphorylation of MLC and thereby increasing corporal smooth muscle relaxation. Ach = acetylcholine; M3 = muscarinic receptor; L-arg = L-arginine; NO = nitric oxide; IP3 = inositol 1,4,5-trisphosphate; AA = arachidonic acid; PGE2 = prostaglandin E2; PG = prostaglandin; GC = guanylate cyclase; AC = adenylate cyclase; PLC = phospholipase C; PKG = protein kinase G; PKA = protein kinase A; ET = endothelin; NE = norepinephrine; EP = prostaglandin receptor.

inhibition of the calcium sensitisation pathway (RhoA/Rho-kinase)—can improve erectile physiology (Fig. 2). 3.1.

eNOS

NO generated by eNOS is important in the maintenance of vasomotor tone, systemic blood pres-

sure, vascular remodeling, angiogenesis, and penile erection [14]. eNOS is localised in the endothelial layers of the dorsal arteries and veins of the penis and the cavernosal sinusoidal spaces [15]. eNOS is subject to multiple modes of regulation in addition to primary regulation through reversible calciumcalmodulin binding and activation. These include reversible phosphorylation and palmitoylation,

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substrate and cofactor availability, dimerisation of enzyme subunits, intracellular translocation, and protein–protein interactions [13]. eNOS expression/ activity in the penis is reduced in a number of disease states including ageing and diabetes [16,17]. Recently, Hurt et al. have demonstrated the vital role of the penile vascular endothelium in maintaining the erectile response [6]. Therefore, a method to enhance local penile endothelial-derived NO delivery is an important molecular target for the treatment of ED. 3.2.

nNOS

NO is synthesised from the substrate L-arginine by the catalytic activity of nNOS and mediates the relaxation of corpus cavernosum smooth muscle [3,5]. Previous studies have demonstrated that knockout mice that are devoid of nNOS expression still possess erectile function sufficient for copulation and reproduction [18,19]. This finding contradicts the notion that the primary source of NO involved in the erectile response is derived from nNOS [20,21] and suggests that non-nitrergic compensatory mechanisms can maintain erectile function in the absence of nNOS. The most likely source is the vascular endothelium. However, substantial evidence supports PnNOS as a potential candidate for gene transfer [21,22]. nNOS is present in the nerve terminals of the corpora cavernosa, in the pelvic ganglion, and in the hypothalamic and spinal cord regions involved in the control of erectile function [23]. Therefore, restored expression and production of neuronally derived NO represents a potential gene product for ED gene therapy. 3.3.

Growth and neurotrophic factors

3.3.1.

BDNF

BDNF was initially characterised as a protein present in brain extracts that is capable of increasing the survival of dorsal ganglia cells [24]. BDNF is a member of the neurotrophin family and plays a distinct role in neuron regeneration in the central and peripheral nervous system [25]. When axonal communication within the cell body is interrupted by injury, Schwann cells produce neurotropic factors. In vitro studies have shown that BDNF can enhance the survival and differentiation of several classes of neurons [26]. BDNF is not restricted to neuronal target fields and thus may act in autocrine and paracrine fashions on neurons [27]. Approaches using BDNF would be of specific interest to address neuropathic changes associated with diabetes, ageing, and cavernous nerve injury.

Thus, several investigators have examined the ability of BDNF gene transfer to restore ED due to cavernous nerve injury [28,29]. 3.3.2.

VEGF

The development and growth of the vascular system—angiogenesis and vasculogenesis—are regulated by angiogenic factors [30]. VEGF, a potent angiogenic stimulator, was initially purified from tumour cell lines as a specific mitogen for endothelial cells in vitro [31]. VEGF stimulates endothelial cell growth and angiogenesis. VEGF is believed to play a role in embryonic vasculogenesis, maintenance of vascular structures in adults, and formation of new blood vessels in adults in response to ischaemia and other pathologic states through mechanisms involving eNOS [32]. An insufficient vascular supply is thought to be responsible for a large component of ED [33]. Although sufficient cavernosal smooth muscle relaxation can overcome this deficit to a certain degree, there is a biologic rationale for increasing blood flow to the cavernosal tissue to promote recovery of erectile function. To contemplate the potential of VEGF therapy for ED, Burchardt et al. investigated the expression of VEGF in the mammalian penis [32]. This study characterised the expression of various VEGF isoforms in the corporal erectile tissue and concluded that VEGF may help maintain penile vascular homeostasis. 3.3.3.

Neurotrophic factor 3

Four types of neurotrophins have been characterised in mammals: nerve growth factor (NGF), BDNF, neurotrophin 3 (NT3), and neurotrophin 4 (NT4). Although similar in sequence and structure, different neurotrophins play distinctive roles during neuronal development and regeneration [34]. NT3 is a member of the neurotrophin gene family and it is trophic for sensory neurons as well as for motor neurons that may protect sensory and motor nerves from mechanical and metabolic insults [35]. 3.4.

Ion channels (K+-Ca+2 systems via hSlo)

Although at least four distinct types of potassium channels are present in human corporeal smooth muscle, the maxi-K channel (Ca2+-sensitive potassium channel or KCa channel) is the most important in corporal smooth muscle physiology. Alterations in the function and regulation of the maxi-K channel are likely to be important in the pathogenesis of organic ED [36–38]. Activation of the maxi-K channel in corporal smooth muscle represents an important and attractive mechanism for control of corporal smooth muscle function [36–38]. Given the central

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role of the maxi-K channel in modulating intracellular Ca2+ levels and transmembrane Ca2+ flux in corporal smooth muscle, modification of channel function is a logical target for molecular and pharmacologic intervention in the treatment of ED. 3.5.

RhoA/Rho-kinase

The contracted state of the penile vasculature is thought to be mediated by the release of norepinephrine, endothelin 1, and a host of other vasoconstrictors [39]. Vasoconstrictor agents elevate intracellular calcium and activate myosin lightchain kinase (MLCK), causing myosin phosphorylation and cross-bridge activation. In addition, calcium sensitisation is activated through agonist stimulation of heterotrimeric G protein-coupled receptors and activation of RhoA through the exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP). Activated RhoA, in turn, activates Rho-kinase, which inhibits myosin light-chain phosphatase (MLCP), resulting in a net increase in myosin phosphorylation and force at a constant calcium level [40,41]. Chitaley et al. examined the role of Rho-kinase on cavernosal tone based on the hypothesis that antagonism of Rho-kinase caused corporal smooth muscle relaxation, initiating the erectile response independent of NO [42]. The fact that Rho-kinase antagonism stimulates penile erection in rats independently from the NO pathway introduces a potential alternative avenue for the treatment of ED. 3.6.

Superoxide dismutase

Endothelial cells produce reactive oxygen species (ROS) in response to shear stress, endotheliumderived agonists including acetylcholine and bradykinin, and also in various vascular disease states [43]. Potential sources of ROS in endothelial cells include nicotine amide adenine dinucleotide phosphate (NADP) oxidase (generates superoxide anion), lipoxygenase, cyclooxygenase, peroxidases, cytochrome P-450s, xanthine oxidase, and eNOS [44]. The reaction of superoxide anions and NO in the vascular endothelium or smooth muscle cells triggers the formation of the highly toxic molecule, peroxynitrite [45]. Due to its toxic effects, peroxynitrite can cause direct tissue injury, alterations in vascular tone, oxidation of vascular proteins and lipids, and overall organ dysfunction [46]. The antioxidants superoxide dismutase (SOD), catalase, glutathione peroxidase, and reductase play a pivotal role at the cellular level in protecting against ROS [43]. Among the three types of SOD isoforms in the

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human body, Cu/Zn and extracellular SOD (EC-SOD) have been identified in the penis, predominantly in the endothelial and cavernosal smooth muscle cells [47]. Increased levels of superoxide anions in the endothelium and cavernosal smooth muscle cells contribute to ED by causing endothelial dysfunction and reducing cavernosal NO biosynthesis. Overall, increased oxidative stress and superoxide anion production alter penile vasculature homeostasis and impair endothelial-derived NO in the erectile tissues [14,48]. Therefore, overexpression of SOD in the penile vasculature can reduce superoxide anion and restore NO bioavailability, thus representing another potential molecular target. 3.7.

Peptides

3.7.1.

CGRP

CGRP is a potent vasodilator in a number of peripheral vascular beds, whereas in the penis, its proerectile effects occur by relaxation of corporal smooth muscle cells by hyperpolarisation via K channel opening and activation of adenylate cyclase, with subsequent increases in intracellular cAMP [49,50]. 3.7.2.

Vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP), which is a 28residue polypeptide originally isolated from porcine duodenum, is a potent vasodilator and smooth muscle relaxant [51]. Some studies suggest that NO and VIP act as neural comediators for penile erection [52]. After release into the corpus cavernosa, VIP stimulates the activity of adenylate cyclase, resulting in increased cAMP. The increased cAMP activates protein kinase A, with subsequent closure of Ca2+ channels and opening of K+ channels, thereby inducing corporal smooth cell relaxation with subsequent penile vasodilation [53,54]. VIP has been localised to the terminals of the major pelvic ganglia, penile arteries, and cavernosal smooth muscle cells [55]. Additionally, VIP-containing secretory vesicles found within cholinergic nerve endings in the penis undergo exocytosis during penile erection [56].

4.

Ideal vector system

The ideal vector for gene transfer is one that allows for efficient transduction and long-term stable transgene expression while resulting in few or no adverse effects, such as risk of infection, immunogenicity, or host-cell mutagenesis. Viral vectors (adenovirus, adeno-associated virus,

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adeno-myoblast, and retrovirus), nonviral vectors (naked DNA, plasmid DNA, liposomes, and myoblast-mediated), and cell-based therapies (stem cells) have been used to transfer genetic material to target cells or tissue [7]. Each has advantages and disadvantages for their safety, efficiency, and immunogenicity. Adenoviral vectors provide high cellular transduction efficiency in a variety of cell types and tissues [57]. Of importance, they transfect both dividing and nondividing cell types and do not insert into the cell’s genome [58]. Major disadvantages of adenoviral vectors are host inflammatory and immune responses due to the expression of viral proteins in infected cells. Recently, second-generation or ‘‘gutless’’ (helper-dependent) adenoviral vectors have been developed to reduce cellular toxicity and immune response, while increasing efficient gene expression [59,60]. Because gene treatment of ED necessitates repeated injections to the penis, gutless adenoviral vectors may provide long-term efficiency without immune responses. With the ability of being maintained in targeted cells as integrated proviruses, adeno-associated virus is suggested to be an attractive vector for gene therapy. Adeno-associated viruses have low immunogenicity, high efficiency, ability to infect a variety of cell types, and no known pathogenicity, and they infect nondividing cells as well as dividing cells [61]. The possibility of being coinfected with adenoviruses is the major disadvantage of adeno-associated virus vector, making it difficult to prepare large quantities of pure vectors. A potential problem with this vector is the possibility of promoting insertional mutagenesis [62]. Although having the ability to remove all viral genes and replace those with the therapeutic gene, retroviral vectors have major disadvantages that restrict their therapeutic applicability. In contrast to adenoviruses and adeno-associated viruses, it is difficult to produce high titres with retroviruses. Additionally, successful retroviral gene transfer necessitates cell proliferation [63], which potentially limits its use for ED gene therapy because the endothelial cells and smooth muscle of the penile corpus cavernosum do not actively proliferate. It also has the risk of insertional mutagenesis. Use of both naked and plasmid DNA vectors has been limited to low efficiencies of transduction in vivo [64,65]. To increase the transgene expression, DNA has been incorporated into liposomes that facilitate increased stability of the desired DNA and increase cellular entry by promoting fusion with the plasma membrane. Although this approach has been demonstrated to increase transgene expression in

vitro, in vivo applications were documented less successful in a number of peripheral vascular beds [66,67]. Of note, this gene transfer approach has been highly successful in introducing genetic material to the penis because the penile vascular bed has interconnected gap junctions that allow smooth muscle cells to communicate [8]. Therefore, a smaller number of penile endothelial or smooth muscle cells must be transfected with genes to cause physiologic relevant changes in erectile physiology [7,8]. Any vector, viral or nonviral, must be safe before it is used for the delivery of specific genes of interest to the penis of humans. Therefore, ED gene therapy clinical trials will use nonviral vectors, growth factors, or cell-based therapies because of fewer immunogenic adverse events and less insertional mutagenesis.

5.

Preclinical gene therapy

5.1.

Gene therapy for age-associated ED

The natural ageing process is known to alter endothelial and neuronal cell function, increase smooth muscle cell proliferation, and cause impaired erectile function. Reductions in NOS activity, impaired neurogenic- and endothelialdependent smooth muscle relaxation, and diminished NO bioavailability in the corporal tissue of humans and experimental animals during normal ageing documents the contribution of the NO/cGMP cascade in diminished erectile responses with normal ageing [16,47,68–70]. Therefore, both eNOS and nNOS represent ideal gene products for gene therapy for ED as it relates to ageing. Our laboratory has investigated the influence of in vivo gene transfer of eNOS to the corpus cavernosum of aged rats [68,71]. At 1–7 d after gene transfer of eNOS to the aged corpus cavernosum, restored eNOS activity, mRNA expression, and improvements in corporal cGMP levels resulted in significant improvements in cavernous nerve-stimulated erectile response and agonist-induced erectile response to the endothelium-dependent vasodilator acetylcholine and the PDE5-Is (zaprinast and sildenafil) in animals receiving this gene therapy approach [68,71]. The results of these studies demonstrate that gene transfer of eNOS to the aged corpus cavernosum induces molecular changes at the endorgan level that result in physiologically relevant changes in erectile function. With regards to the time course of expression of the transgene, the reporter gene b-galactosidase (bgal) was present in the corporal tissues for up to 30 d after injection with

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the AdRSVbgal adenoviral vector and 10 d after injection with AdCMVbgal, suggesting the in vivo erectile effects observed with eNOS will persist at later time points [68,71]. The PnNOS is a potential candidate for gene transfer because it is considered one of the NOS isoforms responsible for penile erection [22]. Magee et al. investigated whether the nNOS variant responsible for erection, PnNOS, can exert a similar effect, and whether the combination of electroporation with a helper-dependent adenovirus can improve gene transduction efficiency [72]. PnNOS and b-galactosidase cDNAs were cloned in plasmid (pCMV-PnNOS; pCMV-bgal) and ‘‘gutless’’ adenovirus (AdV-CMVPnNOS; AdV-CMV-bgal) vectors, and injected into the penis of adult (bgal) or aged (PnNOS) rats, with or without electroporation. Penile erection was measured at different times after PnNOS cDNA injection by electrical field stimulation of the cavernosal nerve. These investigators found that electroporation increased pCMV-bgal uptake, and its expression was detectable at 56 d. In the aged rats treated with pCMV-PnNOS and electroporation, the mean intercavernous pressure/mean arterial pressure (ICP/MAP) were elevated for 11 and 18 d when compared with those in controls. Electroporated AdV-CMVPnNOS was effective at 18 d in improving impaired erectile function in aged rats, without inducing the expression of cytotoxic genes. These data suggested that intracavernosal gene therapy with PnNOS cDNA corrected ageing-related ED when given by electroporation in a helper-dependent adnovirus. The first use of gene therapy for ED, which was reported in 1997 by Garban et al., demonstrated that gene transfer of a plasmid expressing iNOS was effective in correcting ED in the aged rat [73]. This proof of principle was based on the fact that because NO is the chemical mediator of penile erection, higher NOS levels would lead to a higher NO production and overcome the defective cavernosal smooth muscle compliance or a putative excessive contractile tone present in the aged penis [74]. ED observed in ageing rats when compared to young animals was ameliorated by treatment with iNOS gene therapy. Tirney et al. evaluated iNOS gene transfer using plasmids, adenoviruses, or adenovirus-transduced myoblast cells (adeno-myoblast) [75]. Cavernous nerve-mediated erectile responses showed a 2-fold increase in erectile responses after iNOS adeno-myoblast gene therapy. However, the inducible form of NOS is not an ideal gene candidate because iNOS produces 100–1000-fold more NO in any given tissue bed; therefore, although sustained production of NO via iNOS may, in the short term,

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restore erectile function, long-term production can be toxic to endothelial and smooth muscle cells of the penis [76]. The strategy underlying ion channel gene therapy is based on the tight link among K+-channel activity, transmembrane Ca2+ flux through voltagedependent Ca2+ channels, and corporal smooth muscle tone [12]. Christ et al. demonstrated that injection of hSlo cDNA, which encodes for the large conductance Ca2+-sensitive maxi-K channels, into the rat corpora cavernosa could increase gap– junction formation and enhance erectile responses to nerve stimulation in aged and diabetic rats [77,78]. Christ et al. reported that naked pcDNA/ hSlo was easily incorporated and the expression was sustained in rat corporal smooth muscle in vivo for at least 4–6 mo [79]. This prolonged expression of hSlo cDNA was capable of altering erectile responses. Thus, increasing the expression of maxi-K channels influences the overall function of cavernosal smooth muscle cells. Oxidative stress is quite prominent in certain chronic disease states, including diabetes, hypercholesterolaemia, and ageing, and is associated with significant changes in the endothelium and smooth muscle cells in the penis [80,81]. In ageing, increased production of peroxynitrite from excessive superoxide anion accelerates the degeneration of nerves and endothelial cells involved in the erectile process [47,82]. An imbalance in superoxide anion generation and inactivation in the penile vasculature causes impaired endothelial-dependent smooth muscle relaxation and ED [47]. EC-SOD gene therapy decreased superoxide anion and increased cGMP levels in the aged penile vasculature and was found to enhance erectile response to cavernosal nerve stimulation and endothelium-dependent erectile responses. These observations implicate EC-SOD as beneficial in limiting superoxide anion production and preventing endothelial dysfunction in age-related ED [47]. Increased RhoA/Rho-kinase activity has been ascribed to cause increased vascular tone and thus cause ED. A recent study suggested that the RhoA/ Rho-kinase–mediated calcium sensitising pathway may play a role in age-associated ED [83]. Jin et al. used gene transfer of a dominant-negative RhoA (T19NRhoA) mutant as a molecular tool to target specifically RhoA [84]. Gene transfer of adenoassociated virus encoding T19NRhoA to the penis of aged rats markedly improved erectile function. Importantly, Rho-kinase activity was significantly reduced after T19NRhoA gene therapy. Collectively, these data suggested that impaired erectile function during the ageing process involves increased RhoA/

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Rho-kinase signaling, and this pathway may be exploited for gene therapy of age-associated ED. When CGRP is administered intracavernosally in patients suffering from age-related ED, a doserelated increase occurs in penile arterial inflow and erection [50]. Recently, immunoreactive CGRP has been shown to be down-regulated in the penis of aged rats [85]. Bivalacqua et al. demonstrated a significant decline in CGRP levels in the aged penile vasculature [69]. Five days after adenoviral gene transfer of CGRP, aged rats had an increase in corporal CGRP and cAMP levels. CGRP gene therapy caused a significant increase in erectile responses to cavernous nerve stimulation. These data suggest that gene transfer of CGRP can physiologically improve erectile function in the aged rat through a mechanism unrelated to the NO/cGMP signalling pathway, suggesting that alternative molecular pathways may be exploited for gene therapy techniques for ED. 5.2.

Gene therapy for diabetes-associated ED

ED associated with diabetes mellitus is caused in part by disordered penile endothelial smooth muscle relaxation and a decrease in NOS activity [14]. Bivalacqua et al. investigated whether adenoviral gene transfer of eNOS to the penis of streptozotocin (STZ)-induced diabetic rats could improve impaired erectile response [17]. STZ-diabetic rats transfected with AdCMVeNOS had complete resolution of their erectile impairments as measured by cavernous nerve stimulation or intracavernous injection of acetylcholine [14,17]. This change in erectile and endothelial function was a result of eNOS overexpression with an increase in eNOS protein expression/activity as well as an increase in NO biosynthesis. Diabetes causes alterations in endothelial-derived NO release or bioactivity in the penis and eNOS gene therapy represents an ideal disease-based molecular target. In a follow-up study, Bivalacqua et al. investigated whether a combination of sildenafil and adenoviral gene therapy with eNOS could enhance the erectile response in diabetic rats [86]. STZ-diabetic rats transfected with AdCMVeNOS and administered sildenafil had a significant increase in total erectile response that was greater than eNOS gene therapy alone. These data suggest that eNOS gene therapy in combination with PDE5-I therapy can enhance erectile function in a hard-to-treat population of patients, that is, severe diabetics. Substantial evidence supports the involvement of ROS, in particular superoxide anion, in diabetic vascular dysfunction [87,88]. Bivalacqua et al. inves-

tigated whether EC-SOD gene therapy can reduce superoxide anion formation in the diabetic rat penis [89]. Two days after administration of AdCMVECSOD, superoxide anion levels were significantly lower in the penes of diabetic rats, whereas total SOD activity and cavernosal cGMP was increased in the penis, which caused significant improvements in neurogenic-mediated erectile responses. These data demonstrate that in vivo adenoviral gene transfer of EC-SOD can reduce corporal superoxide anion levels and raise cavernosal cGMP levels by increasing NO bioavailability thus restoring erectile function in the STZ-diabetic rats. Antioxidant-based gene therapy for both ageing and diabetes-related ED may be used to improve endothelial and corporal function in the penis. It has been previously found that the Rho-kinase protein expression was increased in diabetic rabbit cavernosal tissue, suggesting that RhoA/Rho-kinase signalling pathway may contribute to diabetesrelated ED [90]. Bivalacqua et al. demonstrated that inhibition of RhoA/Rho-kinase can improve eNOS enzyme activity and protein expression and help restore ED in diabetes [91]. These results demonstrate that RhoA/Rho-kinase is up-regulated in cavernosal tissue isolated from the penes of diabetic rats and identifies a physiologic role for RhoA/Rhokinase in modulating erectile function in vivo by reducing eNOS thus decreasing penile cGMP levels. Adeno-associated virus gene transfer of the dominant-negative RhoA mutant (T18NRhoA) to the diabetic penis decreased RhoA/Rho-kinase protein expression and restored erectile function in vivo through two distinct mechanisms, a reduction in the increase in penile vascular tone and improvements in endothelial-derived NO. These findings show how the RhoA/Rho-kinase pathway influences erectile function through the attenuation of endothelialderived NO formation in the penis. Studies have demonstrated that expression of VEGF and its receptors (Flk-1 and Flt-1) are markedly diminished with eNOS in penile tissues of rats with type 2 diabetes [92]. Any decrease in the activity of VEGF in diabetic rats could contribute to the loss of expression of eNOS and cavernosal smooth muscle cells by apoptosis. Recent data provide a molecular explanation for the stimulatory effects of VEGF on penile erection via phosphorylation of eNOS [6]. In this study, animals were injected intracavernosally with a replication-deficient adenovirus expressing human VEGF145 and thereby established a specific mechanism whereby VEGF promotes erectile function. In a recent study, Yamanaka et al. investigated whether intracavernous administration of VEGF would recover erectile function due to diabetes

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[93]. These investigators found that intracavernous delivery of VEGF restores ED through the inhibition of apoptosis in diabetic rats. These studies provide a molecular basis for VEGF gene therapy for ED. Recent evidence indicates that altered smooth muscle K channel function may contribute to the etiology of vascular complications of diabetes mellitus, including ED [94,95]. Christ et al. investigated the ability of gene transfer with the poreforming subunit of the human maxi-K channel hSlo to restore impaired erectile function in an STZ-diabetes model in Fischer-344 rats [38]. A 2-mo period of STZ-diabetes was induced before gene transfer, and erectile capacity was evaluated. These investigators found increased erectile responses to cavernous nerve stimulation at 1 and 2 mo after injection of 100 mg pcDNA-hSlo. A second series of experiments further examined the dose dependence and duration of hSlo gene transfer. The ICP responses to submaximal (0.5 mA) and maximal (10 mA) nerve stimulation were evaluated 3 or 4 mo after injection of a single dose (10–1000 mg) of pcDNA-hSlo. This resulted in hSlo overexpression associated with increased erectile response at all doses. Taken together, these observations suggest a fundamental diabetes-related change in corporal myocyte maxi-K channel regulation, expression, or function that may be corrected by expression of recombinant hSlo. Previous studies reported the involvement of the VIP/cAMP pathway in diabetes-related ED. These studies in animals and humans collectively demonstrated that VIP-related fibres in the penis are attenuated in diabetic ED [96,97]. Shen et al. demonstrated that the feasibility of VIP cDNA transfer into the corpus cavernosum can augment impaired erectile function in STZ-diabetic rats [98]. These authors documented the persistence of VIP cDNA for at least 2 wk after intracavernous delivery of pcDNA3/VIP cDNA. The transfer of VIP was also reported to augment diminished erectile function after gene delivery as measured by cavernous nerve stimulation in vivo. This study supports the rationale of gene transfer using VIP as potential treatment of diabetes-related ED. Studies have demonstrated that diabetes is associated with diffuse neuropathic changes that result in ED without confounding vasculopathy [99,100]. Reduced nerve regeneration in diabetes has been attributed at least in part to lower levels of neurotrophic factors [101]. Therefore, gene transfer strategies to facilitate nerve regeneration in diabetic neuropathy would be a rationale for diabetesrelated ED. Bennett et al. investigated the feasibility of direct transfer of a recombinant HSV vector encoding NT3 into the corpora cavernosa in STZ-

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induced diabetes in rats [102]. Four weeks after gene transfer of NT3 to the penis, the number of nNOS+ neurons per section in the NT3-injected group was significantly higher than that in the lacZ control group. Furthermore, maximal ICP induced by cavernous nerve stimulation in the NT3 group showed significantly higher values compared to control. Although nNOS activity in the penis was not evaluated after NT3 gene transfer, this study shows the feasibility of neurotrophin gene transfer into the penis to promote the recovery of erectile function. 5.3.

Gene therapy for arterial and venogenic ED

Penile vascular insufficiency is believed to be a common pathologic mechanism for ED [33]. Regardless of the etiology of organic ED, venous leakage is a common final condition resulting from smooth muscle atrophy. Previous studies have shown that all VEGF isoforms are abundantly expressed in rat and human penile tissues [32,83,103]. It has been hypothesised that increased blood flow will aid recovery of erectile function via VEGF gene transfer through mechanisms associated with increased vascularity of the penis. Direct intracavernosal injection of VEGF in a rat model of vascular insufficiency restored the veno-occlusive mechanism as documented by nerve-stimulated ICP response [104]. Additionally, local intracavernous delivery of VEGF isoforms restored ED induced by castration [105], traumatised iliac arteries [104], or hyperlipidaemia [103,106,107]. Rogers et al. demonstrated that intracavernous injection of VEGF prevented veno-occlusive dysfunction in castrated rats with venogenic ED [105]. Wang et al. demonstrated that the application of VEGF at earlier stages of ischaemia may restore the damaged endothelial cells of the corpus cavernosum and support tissue perfusion [108]. In rats with venous leakage, VEGF gene therapy reversed the cavernosometric findings of venous leakage. It has been suggested that intracavernous injection of either the VEGF protein or VEGF gene may become the preferred treatment to preserve erectile function in patients with venoocclusive dysfunction [105]. 5.4.

Gene therapy for cavernous nerve injury

The neurovascular bundles that originate along the posterolateral location of the prostate are vulnerable to injury during radical prostatectomy or cystoprostatectomy. Recovery of erectile function may depend in part on regrowth of nerves from the remaining neural tissues [70]. Gene therapy applications to treat neurogenic ED would require the

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expression of neurotrophic factors to effect survival and regeneration of the neurons regulating erectile function. The effect of BDNF on neural recovery and erectile function after cavernous nerve injury has been studied. Bakircioglu et al. investigated the effect of BDNF gene transfer in rats after cavernous nerve freezing [29]. After undergoing a bilateral cavernous nerve injury with nerve freezing, rats received an intracavernous injection of adeno-associated virus (AAV) vector containing the BDNF gene. At 4 and 8 wk after transfection, their results revealed AAVBDNF therapy improved recovery of erectile function, enhanced regeneration of the intracavernous and dorsal nerves, and prevented neuronal degeneration in the major pelvic ganglia. The authors hypothesised that the production of BDNF protein in penile tissue was transported in a retrograde manner to the major pelvic ganglia to prevent neuronal damage and preserve nNOS activity. Further experiments with BDNF are planned, namely, to extend the treatment period to allow for complete regeneration of cavernous nerves via BDNF in this animal model.

6. First human gene therapy clinical trial for ED: ion channels Ion channels participate in important regulatory mechanism of corporal smooth muscle tone and represent a convergence point for mediating the effects of endogenous neurotransmitters such as NO. Extensive preclinical data demonstrate the importance of ion channels, in particular the K channels, in erectile physiology [109]. Several K channel subtypes have been reported in corporal myocytes, for example, KATP, Kv, and the large conductance, calcium-sensitive K+ channel (i.e., maxi-K or BKCa). The latter, thus far, seems to be the most likely pharmacologic/gene therapy target. Therefore, Arnold Melman and George Christ have the first Food and Drug Administration-approved human gene therapy trial for studying the effects of gene transfer of the maxi-K channel for the treatment of ED. In an initial report by Melman et al., the safety of a single intracavernous injection of a plasmid vector (hMaxi-K) that expresses the hSlo gene, which encodes the a-subunit of the maxiK channel, was used in six men with severe ED [110]. The primary end point was safety; therefore, serum chemistries and semen analysis were evaluated and there were no alterations in serum chemistries or no detectable evidence of hMaxi-K in semen measured by polymerase chain reaction in any

participant at any time after transfer for the 6 mo of the trial. The final conclusions were that in a singledose escalation study, ion channel gene transfer with hMaxi-K can be administered safely to men with ED without adverse events. Of note, there was no evaluation of efficacy of maxi-K gene therapy on erectile function in these men. Further data demonstrating long-term efficacy of this ion-based gene therapy for ED is warranted and currently underway.

7.

Conclusions

The past decade has seen an explosion of new information on the physiology of penile erection, pathophysiology of ED, and development of new oral agents (e.g., three PDE5-Is) to manage ED. Failure to respond to PDE5-Is in severe cases of ED has caused efforts to develop new treatment alternatives. The application of gene therapy for ED represents an exciting new field. Although preclinical studies have highlighted the application of local gene therapy as a viable treatment option for ED in diverse pathologic conditions including diabetes, ageing, hypercholesterolaemia, and cavernous nerve injury, this therapeutic approach still requires more clinical studies in humans. Recent improvements in carrying genes or proteins into the corpora cavernosa that offer longer gene-transfer efficiency, higher levels of expression of the transduced gene, and little or no immunogenic reactions using plasmid vectors or cell-based therapies (i.e., stem cells) represent important therapeutic parameters in developing gene therapy for safe application of ED [91,111–113]. Recent clinical studies using the maxi-K channel for the improvement of erectile physiology shows great promise and we look forward to long-term efficacy data in current clinical trials using this molecular target. Gene therapy for ED represents a viable future treatment option for urologists treating ED and will be used in the near future for patients with severe ED unresponsive to current first-line therapies.

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