WO1997042965A1 - Amelioration of erectile dysfunction - Google Patents

Abstract

Methods of treatment of erectile dysfunction are disclosed. In these methods, a medicament is introduced into the penile tissue of a patient. This medicament preferably is one which induces cavernosal smooth muscle relaxation, and/or produces an increase in the level of nitric oxide synthetase (NOS) in tissue. This treatment includes the transfer of cDNAs to the penis, wherein these cDNAs encode proteins that induce the relaxation of the corpora cavernosa smooth muscle. An isolated sequence encoding human penile inducible nitric oxide synthetase is also disclosed.

Description

AMELIORATION OF ERECTILE DYSFUNCTION

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention concerns a treatment for erectile dysfunction. More

particularly, the present invention relates to a treatment for erectile dysfunction wherein a

medicament is introduced into the penile tissue of a patient. This medicament preferably is

one which induces cavemosal smooth muscle relaxation, and/or produces an increase in the

level of nitric oxide synthetase (NOS) in tissue. This treatment includes the transfer of

cDNAs to the penis, wherein these cDNAs encode proteins that induce the relaxation of the

corpora cavemosa smooth muscle.

Description of the Related Art

Impotence, a condition afflicting 10-15 million men in the USA, is predominantly due

to inadequate relaxation of the penile corpora cavemosal smooth muscle after sexual

stimulation (1,2). The main risk factors for impotence are the neuropathies and

vasculopathies associated with diabetes, atherosclerosis, hypertension, aging, smoking, and

pelvic surgery (1-3). Current pharmacological treatment is based on the injection into the

corpora cavemosa of vasoactive substances that relax the corporal smooth muscle, e.g.,

papaverine, a cGMP phosphodiesterase inhibitor; prostaglandin E, an adenylate cyclase

stimulator; and phentolamine, an alpha-adrenergic blocker (2, 4, 5).

The discovery of nitric oxide (NO) as the main mediator of penile erection (6-9) has

opened new avenues of impotence therapy based on the potential manipulation of local NO

synthesis in the penis by biological mediation of the enzyme responsible for this process, NOS. In this organ, NO is released from the non-adrenergic noncholinergic nerve terminals

and possibly from the endothelium lining the cavemosal cistemae and blood vessels,

stimulating guanyl cyclase and cGMP synthesis in the penile smooth muscle (10-12). This

reduces intracellular Ca²⁺ and causes relaxation of this tissue, leading to enlargement and

entrapment of the blood in the corporal sinusoids, resulting in tumescence of the penis.

Two of the three known NOS isoforms catalyzing NO synthesis by the conversion of

L-arginine to L-citrulline (13, 14) have been detected in the penis. These are neuronal NOS

(nNOS) and endothelial NOS (eNOS). Both have been identified in the rat penile cytosol by

their respective antibodies as typical 155- and 140-kDa bands (12, 15-19). In the case of

erectile dysfunction in the rat associated with aging (20), diabetes (17), smoking (18), and

androgen depletion (15, 20, 21), penile NOS activity is reduced, accompanied in some cases

(17, 18) by a decrease of nNOS content. Penile eNOS seems to be more refractory' to down

regulation (18, 20). Reduction of NOS activity correlates in most cases with a decrease of

penile reflexes (17) or of the erectile response to electrical field stimulation (EFS) of the

cavemosal nerve (15, 16, 18-21). Although the individual expression of each one of the NOS

isoforms has been blocked in NOS gene knockout mice without impairing reproduction (22-

26), this does not imply that physiological erection is not dependent on the NO cascade.

Most likely, the remaining NOS isoform(s) or non-NO-dependent pathways may act as

compensatory mechanisms for the erectile response in these animals (22).

It is assumed that erectile dysfunction results from a decrease in penile NO synthesis

in the nerve terminals or endothelium, and/or an impaired mechanical compliance of the

target cavemosal smooth muscle to the relaxation induced by NO. Reduced compliance, as

shown by a defective erectile response to external vasoactive agents bypassing the NO cascade, has been observed in aging rats (20). Therefore, a logical therapeutic approach to

erectile dysfunction is to attempt to stimulate endogenous NO synthesis in the penis, so as to

achieve a more stable and biologically controlled relaxation than that caused by vasoactive

drugs injected into the corpora cavemosa. In principle, this may be accomplished by

increasing NOS levels in the penis by either transcriptional induction of endogenous NOS

(27) or by gene therapy (28) of the corpora cavemosa with exogenous NOS cDNA. The

prerequisite for erectile function therapy is that the increased penile NOS protein would

remain at the low level of activity present in the flaccid state and be activated upon

stimulation only in the target organ. Although such a tissue-specific activation would

probably require a specific neural signal, the control may be enhanced if an organ-specific

NOS isoform could be used for gene transfer.

Among the three NOS isoforms, only inducible NOS (iNOS) has not yet been

detected in the penis or related to the mechanism of physiological erection (10-12, 28). These

features make iNOS a good candidate for induction or gene therapy, because it may be

assumed that, once it is expressed, its activity in the penis would be regulated differentially

from that of nNOS and eNOS, which are involved in neurotransmission and vascular tone,

respectively. It was previously found that iNOS can be induced in cultures of rat penile

smooth muscle cells (RPSMC) and that its pattern of expression and regulation of activity

differ from the rat vascular mouse macrophage, and human hepatocyte iNOS species (28).

SUMMARY OF THE INVENTION

The present invention is directed to a method of treatment of erectile dysfunction in a

patient comprising the steps of providing an agent capable of treating erectile dysfunction,

and introducing an effective amount of the agent into penile tissue of the patient, thereby

treating the erectile dysfunction. Preferably, the agent induces cavemosal smooth muscle

relaxation, and/or produces an increase in the level of NOS in tissue. Preferably, the NOS is

iNOS, and the agent is introduced into the corpora cavemosa of the penis. The agent is

advantageously introduced into the penile tissue in an intermittent, continuous, or time-

release basis, or is administered locally by continuous infusion or repeated injection, topical

application, intraurethral administration, or injection of microcapsules containing the agent.

The agent is preferably an NOS inducer, an NOS protein, a cDNA encoding a protein

(e.g., an NOS cDNA), or cDNA-transformed penile cells (e.g., NOS cDNA-transformed

penile cells). Ideally, the inducer is bacterial lipopolysaccharide, interferon-γ, tumor necrosis

factor-α, interleukin- lβ, or mixtures thereof, the NOS protein comprises the sequence of SEQ

ID No. 2, the cDNA is a cDNA encoding the protein comprising the sequence of SEQ ID No.

2 (e.g., a cDNA comprising the sequence of SEQ ID No. 1), and the cDNA-transformed

penile cells are corpora cavemosa cells.

The present invention is also directed to an isolated sequence encoding a NOS protein,

preferably the NOS protein comprising the sequence of SEQ ID No. 2. Ideally, the isolated

sequence comprises the sequence of SEQ ID No. 1. Additionally, the present invention is

directed to a NOS protein, preferably a NOS protein comprising the sequence of SEQ ID No.

2. BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendant advantages

thereof will be readily obtained as the same becomes better understood by reference to the

following detailed description when considered in connection with the accompanying

drawings.

Figure 1. Improvement of the NO-dependent erectile response to EFS in rats

receiving a continuous local infusion of iNOS inducers into the penis for 3 days. Left) MIP at

10 volts; Right) MIP value redetermined after intrafemoral artery injection of suboptimal

dose of L-NAME (2 mg/kg). Blank bars, untreated controls; hatched bars, inducer-treated

animals. *p<0.05: **p<0.0l.

Figure 2. In vivo and in vitro expression of iNOS in the rat corpora cavemosa upon

induction. A,B: NADPH diaphorase staining (x75) of frozen sections from 20-month old rat

penises (n = 2) from the group depicted on Figure 1. A) Untreated; B) Treated with iNOS

inducer; C) Western blot from penile cytosols (n=2) with iNOS antibody. Markers indicate

the 125-kDa iNOS bands on the x-rays. CC, corpora cavemosa; CE, cerebellum; UT,

controls; TR, treated. D,E) Western blots of cytosols obtained from incubations of corpora

cavemosa (CC) slices. IMM, Induced murine macrophages. Nitrite concentration in the

medium is shown below. D) Rat (RCC, rat CC). E) Human, (HCC, human CC).

Figure 3. Cloning of rat penile iNOS cDNA from corpora cavemosa smooth muscle

cells and sequence comparison with the iNOS from rat vascular smooth muscle cells. Size

and position of the two clones selected for complete bidirectional sequencing on a 4.1 -kDa

region spanning the entire iNOS coding region, and schematic representation of active sites

on the amino acid sequence. Figure 4. Sequencing of human penile iNOS cDNA fragments generated by RT-PCR

and homology with the iNOS from human hepatocytes. RT-PCR primer location on the

schematic representation of 4-kDa cDNA containing the iNOS coding region, and

representation of active sites on the amino acid sequence.

Figure 5. Improvement of the erectile response to EFS without affecting arterial

blood pressure in rats treated with cDNA constructs of the rat penile iNOS. MIP and MAP

values were determined on 5- and 20-month-old rats after 10 days of intracavernosal

injection. Blank bars, untreated rats; dotted hatched bars, rats treated with vehicle only;

hatched bars, rats treated with the iNOS construct. *p<0.05.

Figure 6. Presence and expression of recombinant iNOS cDNA in the corpora

cavemosa of old rats subjected to iNOS therapy. A) iNOS cDNA fragments amplified from

genomic DNA from the penis of old rats treated as described on Figure 5, using primers 5/6

and 7/8 spanning distant exons on the human iNOS gene. B) The same, with primers linking

the 5' end of iNOS with a sequence on the pcDNA3 vector. Top panels, ethidium bromide

staining of agarose gels; diagrams, position of PCR primers and expected cDNA fragment

sizes. AR, PCR controls will rat androgen receptor primers; M, markers; C, control; T,

treated; +C, fragment generated from pcDNA3RiNOS. C) Southern blot of penile poly(A)+

RNA from control (C) and treated (T) rats submitted to RT with iNOS primer 5 and to PCR

with primers 2 and 5, at nonsaturating number of cycles. Hybridization performed with a

probe generated from DNA by PCR using the same primers; S, PCR only of pcDNA3-

RPiNOS. D) Western blot with antibodies against iNOS on penile cytosol samples from old

rats concentrated 5-fold (from rats used for Figure 5). RP, rat penis; C, control; T, treated;

RPSMC/D, direct analysis (no concentration). Detailed Description of the Preferred Embodiment

The following example is set forth by way of illustration only and nothing therein

shall be taken as a limitation upon the overall scope of the invention.

EXAMPLE

Whether iNOS may be pharmacologically induced in vivo in the rat corpora cavemosa

smooth muscle in order to compensate for the aging-associated erectile dysfunction, and

whether a similar but more stable stimulation of the erectile response can be obtained by gene

therapy with penile iNOS cDNA was investigated. For that purpose, both rat and human

iNOS cDNAs from penile cells and tissue induced in vitro or iNOS expression were cloned

and sequenced.

Materials and Methods

Materials

NADPH, protease inhibitors, Nω-nitro L-arginine methyl ester (L-NAME), and other

reagents were from Sigma Chemical Co. (St. Louis, MO). The L-(2, 3, 4, 5-(³H)) arginine

mono hydrochloride (specific activity: 35-70 Ci/mmol) was purchased from Amersham Corp.

(Arlington Heights, IL) and was purified in our laboratory by column chromatography.

Cloning vectors were as follows: pBlueScript SK (+/-) and UniZap XR, from Stratagene (La

Jolla, CA); and pcDNA3, PCRI, and PCRII from Invitrogen (San Diego, CA). The

antimurine iNOS antibodies were from Upstate Biotechnology (Lake Placid, NY). The

polyacrylamide Ready gels, prestained SDS-PAGE standards, Mini-Protean II

Electrophoresis Cell, and Mini Trans-Blot Electrophoretic Transfer Cell were from Bio-Rad

Laboratories (Hercules, CA). The horse radish peroxidase-linked secondary antibodies (anti- rabbit and anti-mouse IgGs), the Hybond ECL Western membrane, and the ECL Western

blotting kit were from Amersham Corp. (Life Science Div., Arlington Heights, IL). The

BCA protein assay kit was from Pierce (Rockford, IL). The EFS equipment was composed

of an S48 square pulse stimulator (Grass Instrument Co., Quincy, MA) and an integrated data

acquisition system model MP100WS from Biopac System Inc. (Goleta, CA). The latter

allows for simultaneous recording of the intracavemosal and systemic blood pressures, and

further data analysis with the AcqKnowledge III program. The densitometric Scan Analysis

program for the immunoblots was from BioSoft (Cambridge, UK) and was applied to get

images generated with the OneScanner/Ofoto scanning system from Apple Computer Inc.

(Cupertino, CA).

Screening and Sequencing of cDNA Clones for Rat Penile iNOS fRPiNOSI

Cultures of RPSMC were induced for iNOS expression at the 10-20th passage, for 48

h, with 10 μg/ml bacterial lipopolysaccharide (LPS)/250 U/ml recombinant rat τ-interferon (τ

INF) (27), designated "mix A." Induction of iNOS was followed by measurement of nitrites

in the medium with the Griess reaction. Poly(A)⁺ RNA was isolated (27) and used for the

construction of a cDN A library into the UniZap XR vector. Screening was carried out with

32-P cDNA probes generated from the induced RPSMC RNA, by reverse transcriptase (RT)

polymerase chain reaction ((PCR); see below) with 20 mer primers based on previous data

(27) and on the rat vascular iNOS sequences (29-31). They included the RPSMC-iNOS350,

from nucleotides (1590 to 1923, and two other fragments from nucleotides 123 to 404 and

from 509 to 1415. Five positive clones were isolated and partially sequenced with an automatic sequenator to detect their location on the coding region. Two of the clones,

designated #1 and #5, were sequenced in both directions on their entire length.

RPiNOS cDNA Constructs

Clones 1 and 5 were used to make a construct of the entire RPSMC iNOS coding

region in vector pBlueScript SK (+/-). Sticky ends were created in clone 1 with restriction

enzymes Kpn I and Nhe I and in clone 5 with Nhe I and Xho I. Both fragments were ligated,

and the dimer was separated by agarose electrophoresis and inserted into the Kpn \IXho I-

digested vector. The clone was designated pBS RPiNOS. The entire iNOS coding region

was then cleaved from pBS RPiNOS using Kpn I and Not I and cloned into these cloning sites

in the eukaryotic expression vector pcDNA3. This vector contains the CMV promoter and

SV40-driven neomycin gene conferring resistance to the antibiotic G418. The new construct

was designated pcDNA3-RPiNOS.

Sequencing of RT-PCR cDNA Fragments of Human Penile iNOS (ΗPiNOSI

Human penile smooth muscle cells (HPSMC) were cultured from pieces of surgically

discarded corpora cavemosa from impotent patients undergoing prosthesis implantation.

Tissue was removed with informed consent. At the 6- 12th passage, cells were transferred to 0.1% human serum in Dulbecco's Minimum Eagle Medium (DMEM: 24 h); this was

followed by replacement with fresh medium and induction (48 h) with LPS (10 μg/ml),

recombinant human τINF (500 U/ml), recombinant human tumor necrosis factor (TNFα: 200

U/ml), and recombinant human interleukin (IL) βl (4 ng/ml), designated "mix B." In another

experiment, human corpora cavemosa was cut in very small slices and incubated under

similar conditions for 4 days. Poly(A)⁺ RNA was isolated from the two different sources and submitted to RT (27) (see below) with antisense primers designed on the sequence of the

human hepatocyte iNOS (32, 33). The cDNA fragments were amplified by PCR with the

corresponding pairs of sense and antisense primers, as follows (nucleotide positions in

brackets); 1/2 (11-898), 3/4 (762-1167), 5/6 (1018-1851), 778' (1998-2366), 9/10 (1998- 2817), 11/12 (2694-3375), 13/14 (3267-4112), and 15716" (1651-2366). Odd numbers

indicate antisense primers, and even numbers are for sense primers. 8-Mer sequences are

present at the 5' end of the 20 mer iNOS sequence in each primer. These tails code for restriction enzyme sites intended for sequential fragment ligation: 1 (Xba I); 2,3 (BamHl); 4,5

(EcoRI); 6,7 (Ava I); 8,9 (Bel I); 10,11 (Bgl I); 12,13 (Sph I); and 14 (Hinϊ I). The PCR

fragments were isolated and purified, and some were cloned in PCRII. All fragments from

HPSMC and some from human corpora cavemosa were sequenced in both directions.

RT-PCR Procedure

RT was performed on 0.5 μg rat or human poly(A)⁺ RNA by a standard procedure

using MMLV reverse transcriptase (100 U) in the presence of the antisense oligonucleotide

primer (0.5 μM). An aliquot of 1/5 of the RT mix was submitted to PCR in the presence of

the corresponding sense and antisense primers (0.25 μM) in a series of 30-35 cycles at 94°C

(45 sec), 60°C (30 sec), and 72°C (90 sec), using the "hot start" method. An aliquot of 1/7 of

the PCR mix was analyzed by gel electrophoresis with ethidium bromide staining, and the

size of the bands was determined by comparison against an adequate standard. The

remainder of the PCR mix was submitted to the same procedure, and the fragments were

eluted and purified by conventional methods. In certain cases, a nonsaturating number of

cycles (10-20) was applied for PCR in order to maintain the exponential phase of the reaction. Ethidium bromide staining was omitted and a Southern transfer was performed, hybridizing

with the appropriate probe. When regular PCR (30 cycles) was directly used for genuine rat

DNA (2 μg), conditions were similar to those for cDNA.

In Vivo Therapy of the Rat Penile Corpora Cavemosa to Increase Penile iNOS Levels

Two different procedures were applied as follows. 1) Induction: 5-month-old (adult),

20-month-old (old), and 30-month-old (very old) male Fischer 344 rats (n = 7 per group)

were anesthetized with an i.p. injection of thiopental (50 mg/kg)/ketamine (35 mg/kg) and

received s.c. implants of 100-μl Alzet (Alza Corp., Palo Alto, CA) osmotic pumps (1 μl/h)

connected with a catheter to the corpora cavemosa and containing 100 μl of LPS (1 μg/ml),

rat τINF (2500 U/ml), human IL-lβ (50 ng/ml) in saline ("mix C"). Control groups were

either left untreated or, in some cases (adult), received implants of pumps delivering vehicle

only. Rats were allowed to recover, and at 3 days the response to EFS of the cavemosal

nerve was measured. In certain cases, osmotic pumps delivering 200 ul over a 6-day period

were implanted, and at completion animals were used for EFS. 2) Gene transfer: Adult and

old rats (n = 8) were anesthetized as above, and the penis was exposed with a small incision,

dissected, and constricted at its base. One hundred microliters of 10 μg pcDNA3 -RPiNOS: 80

μl lipofectamine (1:8 ratio) was injected into the corpora cavemosa, maintaining this

constriction for 2 min. A similar group was treated with lipofectamine only, and a third

group was left untreated. Rats were allowed to recover, and at 4, 10, and 21 days the EFS

response was determined. Measurement of the Erectile Response to EFS

The procedure was essentially as described (15, 16, 18-21). Briefly, the cavemosal

nerve of rats anesthetized as above was first electrically stimulated at 10 volts to obtain a

preliminary assessment of the response, and then the stimulation was repeated at 2.5, 5, and

10 volts to obtain the complete voltage response curve. The intracavemosal pressure

(mm Hg) was recorded with a pressure transducer integrated into a computerized data

acquisition system response. The animals received additional injections every 45 min of 35

mg/kg of ketamine for the duration of the experiment (about 1-1.5 h). Each rat was submitted

to EFS at a frequency of 15 Hz for pulses of 30 sec, separated by no less than 5-min intervals.

The response at 10 volts was designated maximal intracavemosal pressure (MIP). The mean

arterial pressure (MAP) was measured during the experiment by intrafemoral artery

cannulation and recorded as above. At completion, the NOS inhibitor L-NAME was given

intrafemorally at suboptimal doses (2 mg/kg), and the 10-volt response was measured 15 min

later.

Detection and Measurement of Penile NOS

Penile NOS was detected and measured by three different procedures as follows.

1) NOS activity was measured in the penile cytosol from animals not subjected to EFS, by

determining the conversion of (³H)-L-arginine into L-citrulline as previously described (15-

22), in the presence or absence of L-NAME (2 mM). All values were expressed per

milligram soluble protein or per gram of original tissue. 2) NOS associated activity was

visualized in 10- μm frozen sections from skin-denuded shafts fixed in 4%

paraformaldehyde/0.2% triton X100 by NADPH diaphorase staining, as described (34, 35). 3) Inducible NOS protein content was measured in the penile cytosol from animals subjected

to EFS as described for nNOS and eNOS (15-19, 21). Briefly, equal amounts of protein

(80 μg) were run on 7.5% polyacrylamide SDS gels and transferred by electroblotting. In

certain cases, proteins were concentrated 1 :5 before SDS-PAGE, by precipitation on ice with

5% trichloroacetic acid followed by cold acetone extraction.

Immunodetection was carried out with an affinity chromatography-purified primary

antibody consisting of the rabbit polyclonal IgG against the murine iNOS from induced RAW

264.7 cells (1 :250 dilution) (Upstate Biotechnology). The secondary antibody, luminol

reaction, and x-ray exposure were as described (34, 35). Positive controls were the soluble

fractions from induced mouse macrophages and RPSMC. The negative control was a human

endothelial lysate.

Statistical Analysis

Experimental values were expressed as means ± SEM for the number of separate

animals indicated in each case. The normality distribution of the sample was established

using the Wilk-Shapiro normality test. For comparison of treated groups against the

respective control group, one-way ANOVA was determined for significance using the Fisher

test. The unpaired Student's /-test was used for calculating probabilities, and those less than 5% (p < 0.05) were considered significant.

Results

Effects of iNOS Induction In Vivo

The initial aim was to show that iNOS could be induced in vivo in penile tissue and

stimulate the erectile response to EFS, by using a delivery system that would minimize

hypotension and other undesirable side effects from generalized iNOS induction. Three

groups of rats— adult, old, and very old— were treated with a quaternary mix (27) previously

found to be the most efficient in inducing iNOS in vitro in RPSMC and in slices of human

corpora cavemosa. Direct i.p. or intracorporeal injection of the inducer failed to improve the

erectile response to EFS measured at 2- to 48-h periods and caused substantial hypotension.

Therefore, in the selected paradigm the inducer was continuously and very slowly infused via

a pump directly into the penile corpora cavemosa for 3 days. No sustained erection

(priapism), inflammation, inactivity, or other side effects were observed.

Preliminary experiments showed that implanting of saline-containing infusion pumps

into adult rats for 3 days did not affect the erectile response to EFS as compared to that in

untreated controls of the same age (data not shown), as measured by the maximal

intracavemosal pressure. Therefore, all subsequent controls remained untreated. Figure 1

shows that the local administration of iNOS inducers to the penis of 20-mo-old rats

significantly stimulated the MIP (by 50%) and increased it above the MIP in the 5-mo-old

control rats. This indicates that the aging-associated erectile dysfunction may be corrected by

continuous infusion of the penis with iNOS inducers. Even in the 5-mo-old rats, the erectile

response was stimulated significantly (18%) by this treatment but was only slightly above

that achieved in the treated 20-mo-old rats. Although there was an increase in MIP values in the 30-mo-old rats, this stimulation was not significant, probably because of poor compliance

(20) of the senescent corpora cavemosa in some of the animals.

The erectile response to EFS in all groups remained mainly as an NO-dependent

pathway, a suboptimal dose of L-NAME causing only a partial inhibition of the EFS response

(35-55% according to age; see (20)) was enough to inhibit it by 60-80% (Figure 1, right

panel). However, since the L-NAME blockade was intended to be only partial, these

experiments per se cannot rule out the contribution of an NO-independent pathway in the

stimulation of the erectile response observed in the rats treated with iNOS inducers.

No significant changes in MAP were noticed in any age group (data not shown).

Other modalities of treatment, such as implantation of pumps for 6 and 14 days, induced

nonsignificant increases in the MIP when the total population was considered, but there was a

significant stimulation in half of the rats, which were considered "responders" (data not

shown).

NADPH diaphorase staining is a technique widely used to detect NOS in tissue

sections (34, 35) because it recognizes the oxidoreductase activity of NOS isoforms. This

staining was observed in frozen sections of the penis from untreated old rats as scattered blue

granules presumably associated with the nerve terminals (see dark areas on Figure 2A). In

the peruses from rats treated with iNOS inducers (Figure 2B), the blue granules became more

intense and diffused throughout the corpora cavemosa smooth muscle. Other tissue sections

(data not shown) looked similar. This suggests a generalized stimulation of NOS activity in

the rats treated with iNOS inducers, detectable when the NADPH cofactor is added in situ for

the diaphorase reaction. To corroborate this assumption, the L-arginine/citrulline conversion

assay (16-20) was carried out in the presence of NADPH and other required factors using cytosol obtained from untreated and treated old rats. NOS activity was found to be increased

by iNOS induction by nearly 30%, from 0.54 ± 0.06 to 0.86 ± 0.12 nmol L-citrulline/min per

gram tissue, but since n = 3, no statistical comparison was made.

More specific evidence on NOS induction was obtained by Western blot assays

(Figure 2C) with a specific antibody against iNOS from induced mouse macrophages. This

antibody strongly reacts with a 125-kDa band in the cytosol from induced RPSMC that is

completely absent in untreated RPSMC and in the cerebellum from untreated rats. A faint

125-kDa band is visible in one of the untreated penile cytosols, suggesting that iNOS may be

occasionally expressed in the penis in the absence of induction, probably due to the

spontaneous release of endogenous cytokines. The intensity of the iNOS band is clearly

enhanced in the two treated penile cytosols. This demonstrates that continuous delivery of

the inducer mix to the penis results in an effective induction of penile iNOS that we assume is

responsible for the augmentation of the EFS-induced erectile response.

Although iNOS in the corpora cavemosa of treated rats is expressed at higher levels

than in the absence of inducers, the corresponding activity in vivo appears to remain under

control since no hypotension or other side effects were observed and no erection occurred

unless EFS was applied. This view was supported by in vitro experiments in which corpora

cavemosa slices were incubated for 48 h with inducer mix A in the presence of DMEM/10%

calf serum, and nitrites were measured in the medium as an indication of tissues NOS activity

(28). Figure 2D shows that in samples from two separate rats (#1, 3), no iNOS was expressed

in the absence of induction, whereas in one case (#2), a 125-kDa band was observed. This

resembles the situation found in vivo (section C). Upon induction, iNOS expression was

stimulated to the same extent in all penises, but very remarkably, there was very poor NO synthesis as measured by nitrites released to the medium and no correlation between these

levels and iNOS protein in the absence or presence of induction. The basal nitrite synthesis

probably arises from endogenous nNOS or eNOS rather than from induced iNOS. The

putative control of NOS activity observed both in vivo and in vitro is assumed to be

reversible, since, as shown in Figure 1, EFS triggered an augmented erectile response in the

rats submitted to penile iNOS induction.

Both iNOS induction and the control of its activity occur in vitro in the human

corpora cavemosa, as shown on Figure 2E. The iNOS antibodies detected strong 125-kDa

bands in the induced mouse macrophage lysate and the induced RPSMC cytosol used as

positive controls. In the non-incubated fresh tissue and in the penile slices incubated in the

absence of inducers, no iNOS band was seen. In the presence of iNOS inducers, an intense

125-kDa band appeared even though these antibodies cross-react weakly with human iNOS.

However, in the presence of an efficient iNOS protein induction, in vitro nitrite synthesis did

not correlate with the intensity of the iNOS bands. This agrees with the previous

observations on incubations of rat corpora cavemosa slices.

Cloning of the Rat and Human Penile iNOS cDNAs

The experiments described above suggested that the therapeutic induction of a

physiologically controlled penile iNOS in order to correct erectile dysfunction is feasible, and

led to the goal of increasing penile iNOS by exogenous iNOS cDNA constructs delivered to

the corpora cavemosa. This goal required the cloning of both the rat and human corpora

cavemosa iNOS cDNAs, to make sure that these constmcts would code for penile proteins in

case the iNOS isoforms were tissue-specific. Poly(A)⁺ RNA was isolated from induced RPSMC, and a cDNA library was prepared

and screened with a mix of iNOS probes. The two partially overlapping clones selected for sequencing and ligation are depicted on Figure 3. They cover the entire coding region for

iNOS. Sequencing in both directions of these clones and homology comparison with the

sequences of rat vascular iNOS obtained by two different groups (29, 30) showed 13

nucleotide and 6 amino acid differences. The number of amino acid residues that differ from

both reference sequences simultaneously is reduced to two (amino acids 270 and 591), in

regions placed upstream from the enzyme active groups. Table 1 shows the base and amino acid differences between RPSMC and rat vascular iNOS cDNA (REF 1 : (30); REF 2; (29)).

Table 1

* Amino acid change, as compared with only one of two published sequences. ** Amino acid change, as compared with both sequences.

The two selected RPSMC iNOS cDNA clones were cloned into a eukaryotic

expression vector containing the CMV promoter. The new constmct was designated

pcDNA3-RPiNOS and was sequenced in its entirety to confirm that the whole RPSMC iNOS

coding region was present. This construct was the one used for the in vivo gene therapy

experiments described below. In the case of human penile iNOS cloning, the preparation of a cDNA library from induced HPSMC poly(A)⁺ RNA was not considered an adequate strategy because of the poor

iNOS induction occurring in these cells, as opposed to RPSMC. This was demonstrated by

determination of nitrites in the medium and by Western blot with anti iNOS antibodies and

Northern blot with iNOS probes (data not shown). For that reason, an alternative procedure was applied, based on RT of the induced HPSMC mRNA with iNOS antisense primers combined with amplification by PCR of the resulting cDNA fragments. A series of primers,

based on the published sequence of the human hepatocyte iNOS cDNA (32, 33) and

encompassing a unique restriction site in each of the different fragments, was used (Figure 4).

This facilitated the subsequent cloning of the amplified fragments. Preliminary experiments showed that a cDNA library prepared from RNA isolated from human corpora cavemosa slices induced in vitro generates human iNOS cDNA fragments by RT-PCR that are

completely homologous to the ones found in HPSMC.

The sequencing of the 8 DNA fragments spanning the whole coding region of he HPiNOS showed that there are eight base changes leading to six amino acid variations,

clustered in a region between the flavin adenine dinucleotide (FAD) and NADPH binding

sites, and also before the calmodulin site. Table 2 shows the base and amino acid differences

between HPSMC and human hepatocyte iNOS cDNAs.

Table 2

*Amino acid change, as compared with one published sequence (32). All putative amino acid changes are indicated on Figure 4.

In both HPiNOS and RPiNOS, all the amino acid changes are outside of the enzyme

active sites. In addition, the 3' untranslated end in the sequenced HPSMC iNOS fragments showed four confirmed base differences at the following positions: 3802 (G to T), 3913 (A to T), 3925 (T to C), and 3973 (G to T). The nucleotide sequence of the HPiNOS gene is

presented in the Sequence Listing as SEQ ID No. 1. The amino acid sequence of the HPiNOS protein is presented in the Sequence Listing as SEQ ID No. 2.

Gene Therapy of the Rat Corpora Cavemosa with Rat Penile iNOS cDNA

The existence of amino acid changes in both the rat and human penile iNOS cDNA as

compared to iNOS cDNAs cloned from other organs, and the generation of a suitable

constmct of the rat penile iNOS (pcDNA3-RPiNOS) into a eukaryotic expression vector, justified the use of this plasmid for gene therapy of the corpora cavemosa in rats. The adult (5 mo old) and old (20 mo old) rat groups studied previously for the iNOS induction

experiments were selected for testing the efficacy of the rat penile iNOS cDNA constmct in

stimulating the erectile response by a single intracavemosal injection of a liposome-like preparation.

Figure 5 (top) shows the MIP generated by EFS of the cavemosal nerve after a 10-day

treatment of both the 5- and 20-mo-old rats. As in the case of the induction, a significant

stimulation of the EFS response was obtained in the old rats over both the respective

untreated animals (+71%) and the control group treated with a liposome preparation of the original plasmid vector without iNOS sequences (+46%). The erectile impairment occurring

in the aged rats as compared to the adult rats was corrected by iNOS cDNA therapy as shown by the mean MIP value in the 20-mo-old rats treated with the constmct which was above the

MIP in control 5-mo-old rats, both untreated and treated with vector alone. When the treatment of 20-mo-old-rats with the iNOS constmct was repeated and animals (n = 8) were

killed at longer (21 days) periods after injection, there was a lower stimulation (24%) over the

vector/liposome-treated controls (data not shown). However, no statistical significance was

achieved because of some nonresponding animals. The 10-day treatment appears therefore to

be an adequate period for the transient expression of the iNOS constmct in the penis of the 20-mo-old rats, at least with the type of liposome injection and the lipofectamine:DNA ratio

used in this case. The EFS erectile response of the iNOS-treated rats was inhibited to 60-

80%, as compared to 50-60% in the control rats, by systemically injecting the NOS inhibitor

L-NAME at a suboptimal dose (2 mg/kg). This indicates that the response continued to be

NO-dependent. The correction of the erectile dysfunction in the 20-mo-old rats by iNOS gene therapy

was not accompanied by the systemic hypotension that might result from a possible diffusion

of iNOS constmcts from the corpora cavemosa to the general circulation. Figure 5 (bottom)

shows that the mean arterial pressure (MAP) was not decreased in any of the rats treated with

the penile iNOS construct. As a result, the MIP:MAP ratios (data not shown) followed the

same pattern as the MIP values. No priapism, penile damage, or inflammation assessed

visually, or impairment of the alertness of the rat, was evident.

The presence of the exogenous iNOS in the penis of 20-mo-old rats treated for 10

days with the penile iNOS cDNA constmct was demonstrated by PCR of DNA isolated from

the penis of some of these rats, as compared to the DNA from rats injected with

vector/liposomes only. The initial strategy was based on using 1) DNA treated with RNase to

eliminate RNA that could generate cDNA through traces of transcriptase activity during PCR,

and 2) rat iNOS primers that correspond to regions well apart on the human iNOS gene (32,

33), so that only the plasmid iNOS cDNA could be amplified. Although no rat iNOS gene

map is available with the positioning of exons and introns, the exon spacing along the

selected gene span is assumed not to differ substantially from the human counterpart.

Figure 6 A shows two such rat iNOS primers: 1) 5/6, corresponding to exons 5 and 12 in the human iNOS gene (15-kDa span) and encompassing a 905-base pair (bp) RNA or

cDNA sequence; and b) 7/8, corresponding to exons 19 and 25 in the human iNOS gene

(8-kDa span). PCR amplification with these pairs of primers did not generate any fragment in

the penile DNA from rats treated with vector alone (C lanes) or in the reaction mix without

DNA (- lanes). However, the penile DNA from two old rats treated with the penile iNOS

cDNA constmct showed very clear bands of exactly the size predicted for exogenous DNA. An even more direct demonstration of the presence of the iNOS cDNA constmct in

the DNA fraction isolated from the treated old rats was obtained by an alternative PCR

strategy, using a sense primer for anchoring on the vector (pcDNA3) and an antisense primer

on the 5' end of iNOS. Figure 6B (left), shows that the expected 584-bp band plasmid/iNOS sequence is present in a positive control with the pcDNA3-RPiNOS and in the treated

samples, whereas it is absent in the control samples. The right side of the gel is the control,

showing that the quality of the DNA was adequate. A rat 424-bp androgen receptor cDNA

fragment present in exon 1 was amplified in exactly the same way in control and treated samples.

As opposed to the in vivo induction of iNOS, in which the continuous stimulation

caused a substantial increase of iNOS protein detectable by Western blots, the transfection of the corpora cavemosa with the iNOS constmct was expected to lead to a very low or even undetectable degree of expression. This is because the vector used in these experiments is not

specifically designed for stable transfection, and the method of delivery employed is

relatively inefficient. Therefore, two high-sensitivity procedures were used to detect penile

iNOS in this series. Figure 6C shows an assay to compare the relative level of iNOS mRNA

expression after and before treatment. This was done by RT of equal amounts of poly(A)^"1

RNA isolated from treated and control 20-mo-old rats, followed by PCR at a lower number of

cycles to allow for comparison of the relative levels of iNOS mRNA at the exponential stages

of amplification before the plateau is reached. The constmct (pcDN A3 -RPiNOS) was used as

standard for the PCR. Hybridization of the Southern blot shows that some iNOS mRNA was

detectable in this control sample but the mRNA from the treated corpora cavemosa had a

much higher level of iNOS mRNA. Internal controls for the RT-PCR were produced with β- actin primers that originated a fragment visible on the ethidium bromide stained gel at the

expected size. The fragment showed identical intensity on both the control and treated lanes

(data not shown).

The second approach was to concentrate, before Western blot analysis, the cytosol

from pcDNA3-RPiNOS-treated rats and from control rats treated with liposome/vector alone

in order to improve the assay sensitivity. Figure 6D shows that a band immunoreacting with

the iNOS antibodies is visible in the iNOS-treated samples, and its intensity is higher than on

the homologous band found in the control samples treated with vector/liposome alone. The

presence of basal iNOS in the penile cytosol from some control samples agrees with what was

observed in Figure 2 in the experiments on in vivo iNOS induction with the osmotic pumps,

and with the RT-PCR approach shown in Figure 6C. The slight retardation of the band in the

penile cytosol, as compared to the 125-kDa signal in induced RPSMC positive control, may

result from the concentration procedure. The iNOS antibody used in the present work did not

cross-react with nNOS or eNOS isoforms.

Discussion

Thus, it has been shown that it is possible, without noticeable adverse reactions, to

transiently mitigate the erectile dysfunction present in old rats by biological manipulation of

NOS levels in the penis. This was achieved either by selective moderate induction of

endogenous iNOS or by gene therapy with RPiNOS cDNA constructs. The latter may be

considered the preferred modality because it is a simpler procedure and leads to more

prolonged erectile stimulation effects with less risk of toxicity. In either case, the absence of

priapism and the experiments in vitro suggest that the increased penile iNOS remains under physiological control when no stimulation (such as EFS) occurs. This means that the higher

NOS levels continue to synthesize NO both in vitro and in vivo at the normal low levels

present in the constricted smooth muscle of the flaccid penis. It is believed that this is the

first report on gene transfer to the penis and on a therapy for erectile dysfunction that lasts

much longer that the transient corporal relaxation induced by vasoactive drugs (20). In this

respect, this procedure is consistent with the beneficial effects exerted by eNOS transfection

of the rat carotid artery, by compensating for decreased endogenous eNOS expression and in

this way preventing neointimal hyperplasia (35).

The slight nonsignificant stimulation of the erectile response in 20-mo-old rats treated

with liposomes (lipofectamine) and vector alone as compared with that in untreated rats may

be related to a liposome induced repair of endothelial cell function in the penis of some aged

rats. This effect has been described for aortic smooth muscle from spontaneously

hypertensive rats treated with phospholipid vesicles alone (36) and may have potential

usefulness for the treatment of erectile dysfunction (additive effect with iNOS cDNA

constmcts). Alternatively, the effect may be due to basal iNOS induction (see below).

The question of which cell types express recombinant iNOS in the penis of rats

submitted to induction or gene therapy remains open, although smooth muscle cells are likely

candidates because of their abundance in the corpora cavemosa. However, endothelial cells

may also produce iNOS upon induction or take up the transfected DNA. In the case of rats

treated with iNOS inducers, the NADPH diaphorase staining suggests that iNOS expression

occurred in the cavemosal smooth muscle, and this would agree with the ability of RPSMC to

express iNOS upon induction (27). Penile iNOS was not detectable in untreated rats or in rats

with implants of miniosmotic pumps without inducers. Therefore, the detection of basal levels of penile iNOS in some control rats receiving liposome preparations without iNOS

constmct suggests that lipofectamine may induce a low degree of iNOS induction in the

corporal smooth muscle.

Whatever the cell location, iNOS appears to be activated by cavemosal nerve

stimulation by an unknown mechanism, perhaps similar to the one occurring with

endogenous NOS in the penises of castrated rats (19). It is hypothesized that

neurotransmissions by nNOS in the nerve terminals would be the trigger of cavemosal

smooth muscle relaxation, and the activation of iNOS would simply amplify the signal in the

smooth muscle.

The putative control of augmented penile NOS activity occurs both in vivo, as

indicated by the requirement of a stimulus to elicit smooth muscle relaxation, and in vitro, as

shown by the inability of induced cavemosal slices to synthesize NO at levels compatible

with iNOS content. This process is also observed in incubations of rat pelvic plexus

(unpublished results) and contrasts with the situation in rat penile smooth muscle cells in

culture (27), suggesting that a paracrine mechanism is responsible for the control operating in

the tissue. NOS activity control is the key to achieving a successful NOS gene therapy

without undesirable side effects derived from physiologically unscheduled NO synthesis.

The fact that corporal iNOS activity in rats submitted to iNOS induction remained at a higher

level than in the control animals suggests that physiological NOS enzyme regulation is not as

tight as could be inferred from the in vitro experiments. The elevated basal penile NOS level

may result from a differential response of treated and control rats to repeated spontaneous

erectile stimuli (37). The existence of two amino acid differences and the additional nucleotide silent

changes may suggest that the RPiNOS sequence arises from a different gene than does the rat

vascular iNOS. The same occurs in the case of HPiNOS. However, since the possibility of

polymorphisms cannot be disregarded, the question of its biological significance remains

open. The first alternative is supported by the recent discovery of multiple iNOS gene

sequences in the human and primate genome, present even in separate chromosomes, 14 and

17 (38). Whether this is essential for other cloned iNOS cDNAs will work as effectively

remains to be determined.

In the case of the human penile tissue, the relative resistance of the cavemosal smooth

muscle cells in culture (HPSMC) to consistent induction of iNOS agrees with what occurs

with human macrophages and monocytes (31) and contrasts with the easier induction of

iNOS protein observed in corpora cavemosa slices. The fact that iNOS is expressed in whole

penile tissue but not in cultures of penile smooth muscle implies that either iNOS induction

takes place in nonsmooth muscle cells or , more probably, that positive modulation of the

smooth muscle response by paracrine factors triggers iNOS transcription. In any case, the

HPiNOS cDNA represents the penile isoform, and the cDNA fragments that have been

sequenced to provide the entire coding region can be ligated sequentially, because of their

engineered restriction sites, and cloned into an adequate construct.

This work opens up the field for extensive research on gene therapy for erectile

dysfunction in rat models by the use of iNOS or other NOS isoform constmcts in theory, any

truly penile-specific NOS cDNA should be an ideal candidate for gene therapy because the

encoded protein would most likely be subject to tissue-specific regulation of its activity. A

novel nNOS isoform from the rat corpora cavemosa that may exhibit such functional specificity has been detected and cloned (39). Therefore, the experiments conducted with the

iNOS constmct should be considered as the first stage in this approach, which may be

extended to other penile NOS isoforms. The main purpose of this work was to demonstrate

that an improvement of the NO-dependent erectile response is feasible by either NOS

induction or gene transfer, irrespective of whether the stimulation achieved may be

considered low and the effects transient. The continual advances in new vectors and methods

of delivery may allow easier ways to administer penile-targeted constmcts and more efficient

and stable expression of the NOS constructs.

Obviously, numerous modifications and variations of the present invention are

possible in light of the above teachings. It is therefore to be understood that within the scope

of the appended claims, the invention may be practiced otherwise than as specifically

described herein. This application is based upon U.S. Provisional Application Serial No.

60/017,373, entitled "CLONING OF RAT AND HUMAN INDUCIBLE PENILE NITRIC

OXIDE SYNTHASE - APPLICATION FOR GENE THERAPY OF ERECTILE

DYSFUNCTION" filed with the United States Patent and Trademark Office on May 10,

1996, the entire contents of which are herein incorporated by reference. Additionally, the

entire contents of U.S. Patent No. 5,594,032, entitled "AMELIORATION OF HUMAN

ERECTILE DYSFUNCTION BY TREATMENT WITH INOS, INDUCERS OF INOS OR

CDNA" which has a patent date of January 14, 1997, are herein incorporated by reference.

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in penis and lower urinary tract. Biochem Biophys Res Commun 1996; 226:145-151. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(1) APPLICANT: GONZALEZ-CADAVID, NESTOR F. RAJFER, JACOB

(11) TITLE OF INVENTION: AMELIORATION OF ERECTILE DYSFUNCTION

(m) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT,

P.C.

(B) STREET: 1755 S. JEFFERSON DAVIS HIGHWAY, SUITE 400

(C) CITY: ARLINGTON

(D) STATE: VA

(E) COUNTRY: USA

(F) ZIP: 22202

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/017373

(B) FILING DATE: 10-MAY-1996

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: OBLON, NORMAN F.

(B) REGISTRATION NUMBER: 24,618

(C) REFERENCE/DOCKET NUMBER: 013/PCT/CIP/US

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 703-413-3000

(B) TELEFAX: 703-413-2220

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4070 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ATCTCTCGGC CACCTTTGAT GAGGGGACTG GGCAGTTCTA GACAGTCCCG AAGTTCTCAA 60 GGCACAGGTC TCTTCCTGGT TTGACTGTCC TTACCCCGGG GAGGCAGTGC AGCCAGCTGC 120 AAGCCCCACA GTGAAGAACA TCTGAGCTCA AATCCAGATA AGTGACATAA GTGACCTGCT 180

TTGTAAAGCC ATAGAGATGG CCTGTCCTTG GAAATTTCTG TTCAAGACCA AATTCCACCA 240

GTATGCAATG AATGGGGAAA AAGACATCAA CAACAATGTG GAGAAAGCCC CCTGTGCCAC 300

CTCCAGTCCA GTGACACAGG ATGACCTTCA GTATCACAAC CTCAGCAAGC AGCAGAATGA 360

GTCCCCGCAG CCCCTCGTGG AGACGGGAAA GAAGTCTCCA GAATCTCTGG TCAAGCTGGA 420

TGCAACCCCA TTGTCCTCCC CACGGCATGT GAGGATCAAA AACTGGGGCA GCGGGATGAC 480

TTTCCAAGAC ACACTTCACC ATAAGGCCAA AGGGATTTTA ACTTGCAGGT CCAAATCTTG 540

CCTGGGGTCC ATTATGACTC CCAAAAGTTT GACCAGAGGA CCCAGGGACA AGCCTACCCC 600

TCCAGATGAG CTTCTACCTC AAGCTATCGA ATTTGTCAAC CAATATTACG GCTCCTTCAA 660

AGAGGCAAAA ATAGAGGAAC ATCTGGCCAG GGTGGAAGCG GTAACAAAGG AGATAGAAAC 720

AACAGGAACC TACCAACTGA CGGGAGATGA GCTCATCTTC GCCACCAAGC AGGCCTGGCG 780

CAATGCCCCA CGCTGCATTG GGAGGATCCA GTGGTCCAAC CTGCAGGTCT TCGATGCCCG 840

CAGCTGTTCC ACTGCCCGGG AAATGTTTGA ACACATCTGC AGACACGTGC GTTACTCCAC 900

CAACAATGGC AACATCAGGT CGGCCATCAC CGTGTTCCCC CAGCGGAGTG ATGGCAAGCA 960

CGACTTCCGG GTGTGGAATG CTCAGCTCAT CCGCTATGCT GGCTACCAGA TGCCAGATGG 1020

CAGCATCAGA GGGGACCCTG CCAACGTGGA ATTCACTCAG CTGTGCATCG ACCTGGGCTG 1080

GAAGCCCAAG TACGGCCGCT TCGATGTGGT CCCCCTGGTC CTGCAGGCCA ATGGCCGTGA 1140

CCCTGAGCTC TTCGAAATCC CACCTGACCT TGTGCTTGAG GTGGCCATGG AACATCCCAA 1200

ATACGAGTGG TTTCGGGAAC TGGAGCTAAA GTGGTACGCC CTGCCTGCAG TGGCCAACAT 1260

GCTGCTTGAG GTGGGCGGCC TGGAGTTCCC AGGGTGCCCC TTCAATGGCT GGTACATGGG 1320

CACAGAGATC GGAGTCCGGG ACTTCTGTGA CGTCCAGCGC TACAACATCC TGGAGGAAGT 1380

GGGCAGGAGA ATGGGCCTGG AAACGCACAA GCTGGCCTCG CTCTGGAAAG ACCAGGCTGT 1440

CGTTGAGATC AACACTGCTG TGCTCCATAG TTCTCAGAAG CAGAATGTGA CCATCATGGA 1500

CCACCACTCG GCTGCAGAAT CCTTCATGAA GTACATGCAG AATGAATACC GGTCCCGTGG 1560

GGGCTGCCCG GCAGACTGGA TTTGGCTGGT CCCTCCCATG TCTGGGAGCA TCACCCCCGT 1620

GTTTCACCAG GAGATGCTGA ACTACGTCCT GTCCCCTTTC TACTACTATC AGGTAGAGGC 1680

CTGGAAAACC CATGTCTGGC AGGACGAGAA GCGGAGACCC AAGAGAAGAG AGATTCCATT 1740

GAAAGTCTTG GTCAAAGCTG TGCTCTTTGC CTGTATGCTG ATGCGCAAGA CAATGGCGTC 1800

CCGAGTCAGA GTCACCATCC TCTTTGCGAC AGAGACAGGA AAATCAGAGG CGCTGGCCTG 1860

GGACCTGGGG GCCTTATTCA GCTGTGCCTT CAACCCCAAG GTTGTCTGCA TGGATAAGTA 1920

CAGGCTGAGC TGCCTGGAGG AGGAACGGCT GCTGTTGGTG GTGACCAGTA CGTTTGGCAA 1980 TGGAGACTGC CCTGGCAATG GAGAGAAACT GAAGAAATCG CTCTTCATGC TGAAAGAGCT 2040

CAACAACAAA TTCAGGTACG CTGTGTTTGG CCTCGGCTCC AGCATGTACC CTCGGTTCTG 2100

CGCCTTTGCT CATGACATTG ATCAGAAGCT GTCCCACCTG GGGGCCTCTC AGCTCACCCC 2160

GATGGGAGAA GGGGATGAGC TCAGTGGGCA GGAGGACGCC TTCCGCAGCT GGGCCGTGCA 2220

AACCTTCAAG GCAGCCTGTG AGACGTTTGA TGTCCGAGGC AAACAGCACA TTCAGATCCC 2280

CAAGCTCTAC ACCTCCAATG TGACCTGGGA CCCGCACCAC TACAGGCTCG TGCAGGACTC 2340

ACAGCCTTTG GACCTCAGCA AAGCCCTCAG CAGCATGCAT GCCAAGAACG TGTTCACCAT 2400

GAGGCTCAAA TCTCGGCAGA ATCTACAAAG TCCGACATCC AGCCGTGCCA CCATCCTGGT 2460

GGAACTCTCC TGTGAGGATG GCCAAGGCCT GAACTACCTG CCGGGGGAGC ACCTTGGGGT 2520

TTGCCCAGGC AACCAGCCGG CCCTGGTCCA AGGCATCCTG GAGCGAGTGG TGGATGGCCC 2580

CACACCCCAC CAGACAGTGC GCCTGGAGGC CCTGGATGAG AGTGGCAGCT ACTGGGTCAG 2640

TGACAAGAGG CTGCCCCCCT GCTCACTCAG CCAGGCCCTC ACCTACTCCC CGGACATCAC 2700

CACACCCCCA ACCCAGCTGC TGCTCCAAAA GCTGGCCCAG GTGGCCACAG AAGAGCCTGA 2760

GAGACAGAGG CTGGAGGCCC TGTGCCAGCC CTCAGAGTAC AGCAAGTGGA AGTTCACCAA 2820

CAGCCCCACA TTCCTGGAGG TGCTAGAGGA GTTCCCGTCC CTGCGGGTGT CTGCTGGCTT 2880

CCTGCTTTCC CAGCTCCCCA TTCTGAAGCC CAGGTTCTAC TCCATCAGCT CCTCCCGGGA 2940

TCACACGCCC ACGGAGATCC ACCTGACTGT GGCCGTGGTC ACCTACCACA CCCGAGATGG 3000

CCAGGGTCCC CTGCACCACG GCGTCTGCAG CACATGGCTC AACAGCCTGA AGCCCCAAGA 3060

CCCAGTGCCC TGCTTTGTGC GGAATGCCAG CGGCTTCCAC CTCCCCGAGG ATCCCTCCCA 3120

TCCTTGCATC CTCATCGGGC CTGGCACAGG CATCGCGCCC TTCCGCAGTT TCTGGCAGCA 3180

ACGGCTCCAT GACTCCCAGC ACAAGGGAGT GCGGGGAGGC CGCATGACCT TGGTGTTTGG 3240

GTGCCGCCGC CCAGATGAGG ACCACATCTA CCAGGAGGAG ATGCTGGAGA TGGCCCAGAA 3300

GGGGGTGCTG CATGCGGTGC ACACAGCCTA TTCCCGCCTG CCTGGCAAGC CCAAGGTCTA 3360

TGTTCAGGAC ATCCTGCGGC AGCAGCTGGC CAGCGAGGTG CTCCGTGTGC TCCACAAGGA 3420

GCCAGGCCAC CTCTATGTTT GCGGGGATGT GCGCATGGCC CGGGACGTGG CCCACACCCT 3480

GAAGCAGCTG GTGGCTGCCA AGCTGAAATT GAATGAGGAG CAGGTCGAGG ACTATTTCTT 3540

TCAGCTCAAG AGCCAGAAGC GCTATCACGA AGATATCTTC GGTGCTGTAT TTCCTTACGA 3600

GGCGAAGAAG GACAGGGTGG CGGTGCAGCC CAGCAGCCTG GAGATGTCAG CGCTCTGAGG 3660

GCCTACAGGA GGGGTTAAAG CTGCCGGCAC AGAACTTAAG GATGGAGCCA GCTCTGCATT 3720

ATCTGAGGTC ACAGGGCCTG GGGAGATGGA GGAAAGTGAT ATCCCCCAGC CTCAAGTCTT 3780

ATTTCCTCAA CTTTGCTCCC CATCAAGCCC TTTACTTGAC CTCCTAACAA GTAGCACCCT 3840 GGATTGATCG GAGCCTCCTC TCTCAAACTG GGGCCTCCCT GGTCCCTTGG AGACAAAATC 3900

TTTAATGCCA GGCCCGGCGA GTGGGTGAAA GATGGAACTT GCTGCTGAGT GCACCACTTC 3960

AATTGACCAC CAGGAGGTGC TATCGCACCA CTGTGTATTT AACTGCCTTG TGTACAGTTA 4020

TTTATGCCTC TGTATTTAAA AAACTAACAC CCAGTCTGTT CCCCATGGCC 4070 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1153 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ala Cys Pro Trp Lys Phe Leu Phe Lys Thr Lys Phe His Gin Tyr 1 5 10 15

Ala Met Asn Gly Glu Lys Asp lie Asn Asn Asn Val Glu Lys Ala Pro 20 25 30

Cys Ala Thr Ser Ser Pro Val Thr Gin Asp Asp Leu Gin Tyr His Asn 35 40 45

Leu Ser Lys Gin Gin Asn Glu Ser Pro Gin Pro Leu Val Glu Thr Gly 50 55 60

Lys Lys Ser Pro Glu Ser Leu Val Lys Leu Asp Ala Thr Pro Leu Ser 65 70 75 80

Ser Pro Arg His Val Arg lie Lys Asn Trp Gly Ser Gly Met Thr Phe 85 90 95

Gin Asp Thr Leu His His Lys Ala Lys Gly lie Leu Thr Cys Arg Ser 100 105 110

Lys Ser Cys Leu Gly Ser He Met Thr Pro Lys Ser Leu Thr Arg Gly 115 120 125

Pro Arg Asp Lys Pro Thr Pro Pro Asp Glu Leu Leu Pro Gin Ala He 130 135 140

Glu Phe Val Asn Gin Tyr Tyr Gly Ser Phe Lys Glu Ala Lys He Glu 145 150 155 160

Glu His Leu Ala Arg Val Glu Ala Val Thr Lys Glu He Glu Thr Thr 165 170 175

Gly Thr Tyr Gin Leu Thr Gly Asp Glu Leu He Phe Ala Thr Lys Gin 180 185 190

Ala Trp Arg Asn Ala Pro Arg Cys He Gly Arg He Gin Trp Ser Asn 195 200 205

Leu Gin Val Phe Asp Ala Arg Ser Cys Ser Thr Ala Arg Glu Met Phe 210 215 220 Glu His He Cys Arg His Val Arg Tyr Ser Thr Asn Asn Gly Asn He 225 230 235 240

Arg Ser Ala He Thr Val Phe Pro Gin Arg Ser Asp Gly Lys His Asp 245 250 255

Phe Arg Val Trp Asn Ala Gin Leu He Arg Tyr Ala Gly Tyr Gin Met 260 265 270

Pro Asp Gly Ser He Arg Gly Asp Pro Ala Asn Val Glu Phe Thr Gin 275 280 285

Leu Cys He Asp Leu Gly Trp Lys Pro Lys Tyr Gly Arg Phe Asp Val 290 295 300

Val Pro Leu Val Leu Gin Ala Asn Gly Arg Asp Pro Glu Leu Phe Glu 305 310 315 320

He Pro Pro Asp Leu Val Leu Glu Val Ala Met Glu His Pro Lys Tyr 325 330 335

Glu Trp Phe Arg Glu Leu Glu Leu Lys Trp Tyr Ala Leu Pro Ala Val 340 345 350

Ala Asn Met Leu Leu Glu Val Gly Gly Leu Glu Phe Pro Gly Cys Pro 355 360 365

Phe Asn Gly Trp Tyr Met Gly Thr Glu He Gly Val Arg Asp Phe Cys 370 375 380

Asp Val Gin Arg Tyr Asn He Leu Glu Glu Val Gly Arg Arg Met Gly 385 390 395 400

Leu Glu Thr His Lys Leu Ala Ser Leu Trp Lys Asp Gin Ala Val Val 405 410 415

Glu He Asn Thr Ala Val Leu His Ser Phe Gin Lys Gin Asn Val Thr 420 425 430

He Met Asp His His Ser Ala Ala Glu Ser Phe Met Lys Tyr Met Gin 435 440 445

Asn Glu Tyr Arg Ser Arg Gly Gly Cys Pro Ala Asp Trp He Trp Leu 450 455 460

Val Pro Pro Met Ser Gly Ser He Thr Pro Val Phe His Gin Glu Met 465 470 475 480

Leu Asn Tyr Val Leu Ser Pro Phe Tyr Tyr Tyr Gin Val Glu Ala Trp 485 490 495

Lys Thr His Val Trp Gin Asp Glu Lys Arg Arg Pro Lys Arg Arg Glu 500 505 510

He Pro Leu Lys Val Leu Val Lys Ala Val Leu Phe Ala Cys Met Leu 515 520 525 Met Arg Lys Thr Met Ala Ser Arg Val Arg Val Thr He Leu Phe Ala 530 535 540

Thr Glu Thr Gly Lys Ser Glu Ala Leu Ala Trp Asp Leu Gly Ala Leu 545 550 555 560

Phe Ser Cys Ala Phe Asn Pro Lys Val Val Cys Met Asp Lys Tyr Arg 565 570 575

Leu Ser Cys Leu Glu Glu Glu Arg Leu Leu Leu Val Val Thr Ser Thr 580 585 590

Phe Gly Asn Gly Asp Cys Pro Gly Asn Gly Glu Lys Leu Lys Lys Ser 595 600 605

Leu Phe Met Leu Lys Glu Leu Asn Asn Lys Phe Arg Tyr Ala Val Phe 610 615 620

Gly Leu Gly Ser Ser Met Tyr Pro Arg Phe Cys Ala Phe Ala His Asp 625 630 635 640

He Asp Gin Lys Leu Ser His Leu Gly Ala Ser Gin Leu Thr Pro Met 645 650 655

Gly Glu Gly Asp Glu Leu Ser Gly Gin Glu Asp Ala Phe Arg Ser Trp 660 665 670

Ala Val Gin Thr Phe Lys Ala Ala Cys Glu Thr Phe Asp Val Arg Gly 675 680 685

Lys Gin His He Gin He Pro Lys Leu Tyr Thr Ser Asn Val Thr Trp 690 695 700

Asp Pro His His Tyr Arg Leu Val Gin Asp Ser Gin Pro Leu Asp Leu 705 710 715 720

Ser Lys Ala Leu Ser Ser Met His Ala Lys Asn Val Phe Thr Met Arg 725 730 735

Leu Lys Ser Arg Gin Asn Leu Gin Ser Pro Thr Ser Ser Arg Ala Thr 740 745 750

He Leu Val Glu Leu Ser Cys Glu Asp Gly Gin Gly Leu Asn Tyr Leu 755 760 765

Pro Gly Glu His Leu Gly Val Cys Pro Gly Asn Gin Pro Ala Leu Val 770 775 780

Gin Gly He Leu Glu Arg Val Val Asp Gly Pro Thr Pro His Gin Thr 785 790 795 800

Val Arg Leu Glu Ala Leu Asp Glu Ser Gly Ser Tyr Trp Val Ser Asp 805 810 815

Lys Arg Leu Pro Pro Cys Ser Leu Ser Gin Ala Leu Thr Tyr Ser Pro 820 825 830

Asp He Thr Thr Pro Pro Thr Gin Leu Leu Leu Gin Lys Leu Ala Gin 835 840 845 Val Ala Thr Glu Glu Pro Glu Arg Gin Arg Leu Glu Ala Leu Cys Gin 850 855 860

Pro Ser Glu Tyr Ser Lys Trp Lys Phe Thr Asn Ser Pro Thr Phe Leu 865 870 875 880

Glu Val Leu Glu Glu Phe Pro Ser Leu Arg Val Ser Ala Gly Phe Leu 885 890 895

Leu Ser Gin Leu Pro He Leu Lys Pro Arg Phe Tyr Ser He Ser Ser 900 905 910

Ser Arg Asp His Thr Pro Thr Glu He His Leu Thr Val Ala Val Val 915 920 925

Thr Tyr His Thr Arg Asp Gly Gin Gly Pro Leu His His Gly Val Cys 930 935 940

Ser Thr Trp Leu Asn Ser Leu Lys Pro Gin Asp Pro Val Pro Cys Phe 945 950 955 960

Val Arg Asn Ala Ser Gly Phe His Leu Pro Glu Asp Pro Ser His Pro 965 970 975

Cys He Leu He Gly Pro Gly Thr Gly He Ala Pro Phe Arg Ser Phe 980 985 990

Trp Gin Gin Arg Leu His Asp Ser Gin His Lys Gly Val Arg Gly Gly 995 1000 1005

Arg Met Thr Leu Val Phe Gly Cys Arg Arg Pro Asp Glu Asp His He 1010 1015 1020

Tyr Gin Glu Glu Met Leu Glu Met Ala Gin Lys Gly Val Leu His Ala 1025 1030 1035 1040

Val His Thr Ala Tyr Ser Arg Leu Pro Gly Lys Pro Lys Val Tyr Val 1045 1050 1055

Gin Asp He Leu Arg Gin Gin Leu Ala Ser Glu Val Leu Arg Val Leu 1060 1065 1070

His Lys Glu Pro Gly His Leu Tyr Val Cys Gly Asp Val Arg Met Ala 1075 1080 1085

Arg Asp Val Ala His Thr Leu Lys Gin Leu Val Ala Ala Lys Leu Lys 1090 1095 1100

Leu Asn Glu Glu Gin Val Glu Asp Tyr Phe Phe Gin Leu Lys Ser Gin 1105 1110 1115 1120

Lys Arg Tyr His Glu Asp He Phe Gly Ala Val Phe Pro Tyr Glu Ala 1125 1130 1135

Lys Lys Asp Arg Val Ala Val Gin Pro Ser Ser Leu Glu Met Ser Ala 1140 1145 1150

Leu

Claims

WE CLAIM:

1. A method of treatment of erectile dysfunction in a patient comprising the steps

of:

(a) providing an agent capable of treating erectile dysfunction;

(b) introducing an effective amount of the agent into penile tissue of the patient, thereby treating the erectile dysfunction.

2. The method of claim 1, wherein the agent induces cavemosal smooth muscle

relaxation.

3. The method of claim 2, wherein the agent is a cDNA encoding a protein which induces cavemosal smooth muscle relaxation.

4. The method of claim 1, wherein the agent produces an increase in the level of

NOS in tissue.

5. The method of claim 4, wherein the NOS is iNOS.

6. The method of claim 1, wherein the agent is introduced into the corpora

cavemosa.

7. The method of claim 1 , wherein the agent is introduced into the penile tissue

in an intermittent, continuous, or time-release basis.

8. The method of claim 1, wherein the agent is administered locally by

continuous infusion or repeated injection, topical application, intraurethral administration, or

injection of microcapsules containing the agent.

9. The method of claim 1 , wherein the agent is a cDNA.

10. The method of claim 1, wherein the agent is selected from the group consisting

of an NOS inducer, an NOS protein, an NOS cDNA, and NOS cDNA-transformed penile

cells.

1 1. The method of claim 1 , wherein the agent is an NOS inducer selected from the

group consisting of bacterial lipopolysaccharide, interferon-γ, tumor necrosis factor-α,

interleukin-1 β, and mixtures thereof.

12. The method of claim 1 , wherein the agent is a protein comprising the sequence

of SEQ ID No. 2.

13. The method of claim 1 , wherein the agent is a cDNA encoding a protein

comprising the sequence of SEQ ID No. 2.

14. The method of claim 13, wherein the cDNA comprises the sequence of SEQ

ID No. 1.

15. The method of claim 1 , wherein the agent is cDN A-transformed corpora

cavemosa cells.

16. The method of claim 15, wherein the cDNA encodes a protein which induces

cavemosal smooth muscle relaxation.

17. An isolated sequence encoding an NOS protein.

18. The isolated sequence of claim 17, wherein the isolated sequence encodes the

protein comprising the sequence of SEQ ID No. 2.

19. The isolated sequence of claim 18, wherein the isolated sequence comprises

the sequence of SEQ ID No. 1.

20. An isolated NOS protein.

21. The protein of claim 20, wherein the protein comprises the sequence of SEQ

ID No. 2.