WO1997030066A9 - NOVEL PENILE NEURONAL NITRIC OXIDE SYNTHASE (PnNOS) AND APPLICATIONS FOR DIAGNOSIS AND TREATMENT OF UROGENITAL DISORDERS - Google Patents

Abstract

A rat tissue nitric oxide synthase designated RPnNOS and the cDNA are disclosed. It is expressed in pelvic plexus, bladder, skeletal muscle, liver, cerebellum as well as penile tissue.

Description

NOVEL PENILE NEURONAL NITRIC OXIDE SYNTHASE (PnNOS) AND APPLICATIONS FOR DIAGNOSIS AND TREATMENT OF UROGENITAL

DISORDERS

DESCRIPTION

CROSS-REFERENCE TO RELATED APPLICATIONS

This regular application is related tc the provisional application entitled NOVEL PENILE NEURONAL NITRIC OXIDE SYNTHASE

(PnNOS) . APPLICATIONS FOR DIAGNOSIS AND TREATMENT OF UROGENITAL DISORDERS, SN 60/031,550, filed by us on December 03, 1996,

(5435-007-32 PROV) to the provisional application entitled PENILE nNOS AND IMPOTENCE IMPLICATIONS FOR NEUROSCIENCE, SN 60/011,707, filed by us on February 15, 1996 (5435-006-32 PROV) , and to the provisional application entitled CLONING OF NOVEL NEURONAL, NITRIC OXIDE ΞYNTΞASES EXPRESSED IN PENIS AND LOWER URINARY TRACT, SN 60/017,371, filed by us on May 10, 1996 (5435-002-32

PROV) , the priority dates of which are hereby claimed under 35 U.S.C. §119 (e), the disclosures of which are hereby incorporated by reference.

TECHNICAL FIELD:

A novel neuronal nitric oxide synthase (nNOS) , and more specifically penile nNOS (PnNOS) encoded by a novel cDNA sequence, process for preparation of various nNOS isoforms, and diagnostic and therapeutic uses therefore.

BACKGROUND ART:

Evidence accumulated in the last seven years has shown that nitric oxide (NO) is the main mediator of penile erection in men and in experimental animals such as dogs , rabbits , cats , and rats . NO is synthesized in tissues by the enzyme nitric oxide synthase (NOS), which exists as three different isoforms: the constitutive neuronal and endothelial NOS (nNOS or NOS 1, and eNOS or NOS 2 , respectively), and the inducible NOS { iNOS or NOS 2) . 3oth nNOS and eNOS have been identified in the penis, and nNOS has been located in the nerve terminals of the corpora cavernosa .

The dependence of the erectile mechanism on NO synthesis was initially demonstrated in vitro on pre-constricted penile corpora cavernosa strips . This was shown by the induction of relaxation by nitrodonors, and the blockade by NOS inhibitors of the relaxation resulting from electrical field stimulation (EFS) . This dependence was also directly confirmed in vivo by the effect of nitrodonors on penile erection and the inhibition of the erectile response to EFS of the cavernosal nerve by NOS inhibitors. NO stimulates guanylcyclase in the cavernosal smooth muscle, increasing cGMP and thus decreasing intraceliular Ca²⁺ . This triggers smooth muscle relaxation and blood inflow into the cavernosal cisternae.

The rat model of penile erection has been used extensively to characterize several conditions that mimic risk factors for erectile dysfunction in man. In vivo approaches are based on: a) the assessment of erectile response by measuring the maximum intracavernosal pressure (MIP) elicited by cavernosal EFS or vasodilators, and/or by determining erectile reflexes (cups and flips) ; b) penile NOS activity by the arginine/citrulline conversion assay; c) penile NOS content by quantitative western blot procedures; d) penile NOS localization by immunocytochemistry and in situ hybridization. The risk factors so far characterized in this respect include aging, androgen deficiency, diabetes, smoking, and hypertension. The gonadal/hypothalamic/ pituitary/adrenal control of penile NOS and erectile function has also been studied.

Recent results have indicated that penile erection in the Fischer 344 rat is androgen-dependent . Although castration decreases the erectile response to EFS by only 50%, when it is combined with the androgen blocker flutamide, the inhibition is nearly complete. DHT appears to be the active androgen in maiππaininσ erectile function, but pituitary and adrenal factors are also essential. Hypopnysectomy, administration of a GnRH antagonist, or adrenalectomy impair erection to different extents. The combination of castration and adrenalectomy blocks the erectile response completely but can be partially prevented by replacement androgen and/or corticosteroid therapy.

As to risk factors, aging in the rat model, decreases the erectile response to EFS and vasodilators, and this effect is blocked by long-term androgen administration. Diabetes types I and II in the BB rat are accompanied by the abolition of penile reflexes, but not of the erectile response to EFS, thus suggesting a peripheral neuropathy in the penis without a marked cavernosal nerve compromise . Both aging and diabetes are characterized by a severe reduction of serum testosterone.

The response of penile NOS follows two different patterns. In the case of androgen depletion, hypopnysectomy, GnRHA, or adrenalectomy, NOS activity is considerably depleted without decreasing significantly nNOS content, by an as yet unidentified mechanism of enzyme activity control. The corresponding replacement therapy prevents this reduction. In the case of aging, there is also a decrease of penile NOS activity. The control of penile NOS activity can directly be demonstrated in castrated rats by a short electrical stimulation of the cavernosal nerve that is able by itself to stimulate NOS activity, thus resembling the activation that snouid occur upon the sexual stimulus .

In contrast, two chronic conditions: diabetes and passive smoking m the rat model, are accompanied by a severe depletion of both penile nNOS content and NOS activity, without changes in eNOS . This suggests a loss of penile nerve terminals which agrees with the present models for the peripheral neuropathies present in these conditions. In most cases the decrease of penile NOS is m agreement with the impairment of erectile response. The exception s m rats submitted to chronic passive smoking where ancillary pathways may take over the defective NO cascade, or the mechanical effect of a moderate hypertension may compensate for the impaired smooth muscle relaxation.

A series of experiments in vitro have confirmed the role of NO, NOS, and specifically of nNOS, in penile erection. These approaches are based on the determination of NOS expression in cultures of rat penile smooth muscle cells (RPSMC) and in incubations of slices of rat corpora cavernosa by measuring RNA levels (northern blots and reverse transcription/PCR) , NOS protein content (immunoblot) , and NOS activity (arginine/citrulline assay and nitrite release) . It has been shown that no eNOS or nNOS appears to be present in RPSMC, but that iNOS can be induced in a process that may occur under certain conditions in the penis, although it is unlikely that it participates in the normal physiological erection.

Our previous US patent demonstrated that iNOS can be induced in vivo in the rat penis by direct local administration of inducers and that this treatment corrects the impairment of penile erection occurring during aging. The cDNAs for both the rat and human penile iNOS were cloned, sequenced, and shown to differ in some nucleotides from the iNOS mRNA expressed in other organs. An RPSMC iNOS cDNA construct has been applied for gene therapy of erectile dysfunction in the aging rat model and shown to correct the erectile response to EFS stimulating it to values found in adult rats. Currently, the .medical therapy for erectile dysfunction is based on the use of vasodilators and smooth muscle relaxants injected directly into the corpora cavernosa, or administered intra urethrally.

The role of NO in the regulation of smooth muscle tone in the urogenital system has been supported by a series of approaches similar to the ones described for the penile corpora cavernosa. nNOS and NOS activity have been detected by immunocytochemistry, NADPH diaphorase, arginine/citrulline conversion assay and western blot in the human and rat bladder, urethra and prostate. The in vitro relaxation of tissue strips from bladder, urethra, and prostate has been shown to be partially NO dependent, and the administration of NOS inhibitors to rats impairs urethral outlet activity during micturition. NO is considered to be the nonadrenergic- noncholinergic mediator of in vivo relaxation in the rat and human urogenital tract.

The urinary obstruction present in men with benign prostatic hyperplasia (BPH) is at least partially derived from an increase of the tone of the prostate and is medically treated with anti-alpha adrenergic receptor agents. Other obstructive symptoms may be caused by an increase in the tone of the internal urethral sphincter. These conditions lead to both bladder instability and hypertrophy.

Bladder instability (detrusor hyperactivity) leading to increases in micturition frequency is also associated in the rat with a stimulation of the in vitro contractile response of bladder strips and may occur as a compensatory mechanism for a mild outlet obstruction. Bladder enlargement in diabetes causes increases in voiding frequency and urinary retention, but in contrast to the former condition this overflow incontinence is associated with impairment of the detrusor muscle. Current medical treatment is based on anticholinergic agents.

The cerebellar-type nNOS has been identified in nerves along the urogenital system by both immuno cytochemical detection, and by NADPH diaphorase staining and has been detected by western blot assays as a 155-160 kD protein in the soluble fraction of tissue homogenate . In these respects, it is indistinguishable from the nNOS cloned originally from the cerebellum of the rat, human, and mouse, and it is assumed that only one gene exists in these species. However, due to the particular organization of its promoter, alternative start sites are possible, giving rise to the possibility of nNOS proteins of different sizes arising by differential promoter usage. A differential tissue splicing has been described for the mouse and human nNOS mRNA.

Additional background may be gleaned from the references cited in Appendix B.

DISCLOSURE OF INVENTION

_It is among the objects and advantages of the present invention to demonstrate the existence of a novel neuronal nitric oxide synthase in rat and human tissues. Other objects and advantages include : to provide for molecular cloning and characterization of this novel neuronal nitric oxide synthase from the rat penis; to isolate a cDNA clone for rat penile neuronal nitric oxide synthase; to demonstrate the presence of and to isolate penile neuronal nitric oxide synthase mRNA as the only or main neuronal nitric oxide synthase mRNA in the penis, urethra, prostate, and skeletal muscle; to demonstrate the presence of and to isolate penile neuronal nitric oxide synthase mRNA as an abundant neuronal nitric oxide synthase in the bladder and pelvic plexus ; to demonstrate the presence of and to isolate the penile neuronal nitric oxide synthase mRNA as, and as a minor neuronal nitric oxide synthase in the cerebellum and liver; to demonstrate the presence of and to isolate penile neuronal nitric oxide synthase in the human penis; to identify and compare a functional region of penile neuronal nitric oxide synthase in relation to the known gene map of the cerebellar neuronal nitric oxide synthase; to employ penile nitric oxide synthase in the regulation of the tone of the smooth muscle in the urogenital system, and of muscle tissue in general; to use DNA probes, and antibodies specific for the penile neuronal nitric oxide synthase, in diagnosis, therapy and research of urogenital disease and other conditions; and to use DNA constructs for penile neuronal nitric oxide synthase, and modulators of its activity and expression, for the therapy of urogenital disease and other conditions.

The present invention comprises a cDNA clone for rat penile neuronal nitric oxide synthase, designated RPnNOS, a process for preparing the same, a demonstration of its presence in the human penis and in several tissues of the rat, and the equivalence thereof with human penile neuronal NOS (HPnNos) and uses in diagnosis, therapy, and research.

More specifically, the process for preparing the RPnNOS clone includes: a) isolating total RNA and mRNA from rat penile smooth muscle cells (RPSMC) and rat corpora cavernosa tissue; b) demonstrating the absence of nNOS mRNA in RPSMC mRNA by reverse transcription (RT) /polymerase chain reaction (PCR), northern blot assay, and sequencing of the RT/PCR cDNA fragments; c) demonstrating the expression of nNOS protein in the whole corpora cavernosa homogenate by western blot, and the presence of a novel nNOS mRNA (RPnNOS) different from the nNOS expressed in the cerebellum (RCnNOS) , by RT/PCR followed by sequencing of the RT/PCR cDNA fragments; d) preparing and cloning a cDNA fragment of 102 nucleotides (designated RPnNOS102) present in RPnNOS and absent in RCnNOS; e) preparing a cDNA library from rat corpora cavernosa mRNA, screening it with RPnNOS102, selecting positive clones, isolating the DNA, and sequencing them; f) generating an RPnNOS cDNA corresponding to the 5" end region from the same cDNA library, and sequencing it; g) ligating cDNA regions from these fragments into a plasmid to generate a construct with the entire coding region of RPnNOS. The process for demonstrating the expression of sequences homologous to RPnNOS102 in different tissues includes: a) isolating total RNA and mRNA from human corpora cavernosa and from rat organs; b) applying RT/PCR for visualizing by agarose gel electrophoresis and southern blot both RPnNOS and RCnNOS sequences originating from the same RNA sample. The process for mapping the RPnNOS102 region on genomic DNA includes: a) DNA isolation, restriction fraction digestion and southern blotting; b) PCR with primers spanning intron regions, followed by sequencing.

BRIEF DESCRIPTION OF DRAWINGS AND TABLES:

The invention is illustrated in the drawings in which: Fig. 1. shows the sequence and position on the rat cerebellar nNOS cDNA of the primers used for amplification of selected nNOS-related DNA regions from rat RNA and DNA;

Fig. 2. shows the comparative size of cDNA fragments generated by RT/PCR from total RNA isolated from penile smooth muscle cells (RPSMC and HPSMC) , using nNOS primers based on the published sequence of rat cerebellar nNOS; Fig. 3. is a Northern blot analysis of RPSMC mRNA using a cDNA probe generated from RPSMC RNA with nNOS primers (RPSMC1/2) ;

Fig. 4. illustrates the sequence of cloned probe

RPSMC1/2. A. Reverse primer; B. NOl primer;

Fig. 5. illustrates the expression of nNOS protein in the urogenital organs, as detected by western blot analysis;

Fig. 6. illustrates the presence of a unique nNOS mRNA (RPnNOS) in the rat penile corpora cavernosa revealed by RT/PCR;

Fig. 7. shows the automated sequencing of penile nNOS probe RPnNOSl/2 showing the presence of a 102 bp insert. A. NOl primer; B) N06 primer;

Fig. 8. is the sequence and position on the published nNOS cDNA sequence of a 102 bp insert (RPnNOS102) found in RPnNOS mRNA. Demonstration of its presence as a minor species in rat cerebellum mRNA;

Fig. 9. shows the strategy used for sequencing a clone representing the 3' end region (3.5 kb) of RPnNOS cDNA;

Fig. 10. illustrates the strategy used for sequencing the 5' end region of RPnNOS cDNA and completing the RPnNOS sequencing;

Fig. 11. shows a comparison of nucleotide and amino acid sequences between RPnNOS and RCnNOS;

Fig. 12. illustrates the extended restriction enzyme pattern of RPnNOS102 and localization on the RPnNOS gene;

Fig. 13. shows the comparative expression of RPnNOS mRNA along the urogenital system and other organs of the rat;

Fig. 14. illustrates detection of an RPnNOS homologous sequence in human DNA and comparison of sequences around the insertion, suggesting an HPnNOS gene different from HcnNOS; and

Fig. 15. is a comparison of the nNOS102 insert in both the rat and human nNOS sequences and demonstration that the HPnNOS mRNA is expressed in the human penis. Tables are shown in Appendix A wherein: Table 1 is a List of Abbreviations

Table 2 lists the Nucleotide Sequence of the Coding

Region of the Rat Penile Neuronal Nitric Oxide Synthase (RPnNOS) ; Table 3 lists the RPnNOS Amino Acid Sequence;

Table 4 lists the Nucleotide Sequence of the 3 ' End of the Intro 16 Region Fragment of Human PnNOS ; Table 5 lists the Nucleotide Sequence of the 5 ' End of the Intron 16 Region Fragment of Human PnNOS ; and Table 6 lists the Rat PnNOS Nucleotide Sequences of

Intron 16 Region, 3' and 5' End Fragments.

BEST MODE FOR CARRYING OUT THE INVENTION:

The following detailed description illustrates the invention by way of example, not by way of limitation of the principles of the invention. This description will clearly enable one skilled in the art to make and use the invention, and describes with the aid of the Glossary of Table 1 several embodiments, adaptations, variations, alternatives and uses of the invention, including what We presently believe is the best mode of carrying out the invention.

Our previous USA patent postulates that the synthesis of the mediator of penile erection, NO, is controlled in an organ-specific fashion in order to assure a fast modulation of NOS activity exclusively in the penis upon sexual stimulation. This may occur through the existence of penile specific NOS species with amino acid regions conferring differential functional properties as compared to the homologous isoforms in other organs . Penile iNOS was found to have some amino acid differences with the iNOS cloned from other organs.

In the present invention, novel peptide regions so far not described in the nNOS sequence modulate the conformation and activity of penile NOS and confer to this protein the ability to respond specifically to the requirements of the erectile process independently from the rest of the vascular tree. This is because of the fundamental role nNOS fulfills in triggering the erectile response through the release of NO as a neurotransmitter in the nerve terminals of the penis . Secondly the novel nNOS species of this invention is not restricted to the penis but distributed along the lower urogenital tract, controlling the tone of the smooth muscle in those organs. The existence of a different nNOS protein isoform to the one present in cerebellum supports the concept of a second nNOS gene with its own promoter exerting an additional control of the smooth muscle relaxation machinery at the transcriptional level.

We have discovered that the novel nNOS protein of the present invention designated PnNOS, and not the cerebellar nNOS, designated CnNOS, is the exclusive or main nNOS in the penis with the function of controlling NO synthesis specifically in this organ for triggering and maintaining penile erection without affecting peripheral vascular tone. The cDNA for PnNOS may be cloned from a rat penile library and shown to contain a 102 nucleotide insert encoding for 34 amino acids and other distinctive changes as compared to CnNOS. The insert and other variations confer specific regulatory features to PnNOS to enable differential diagnosis of possible causes of impotence and for its treatment. The procedure for cloning PnNOS from the rat penis is shown.

We have also discovered that PnNOS is present in the prostate and urethra as exclusive or main nNOS, and in the bladder coexists with CnNOS. Since nNOS is considered to play a fundamental role in the relaxation of these organs and the control of micturition, the role of PnNOS is proposed to extend to the control of the smooth muscle tone in the lower urogenital tract. Therefore PnNOS is applicable for the therapy of urinary voiding disorders and for other related diagnostic purposes.

Since PnNOS and not CnNOS is present in the penis, the cavernosal and dorsal nerves must contain exclusively PnNOS. In contrast, the presence of both PnNOS and CnNOS in the pelvic plexus implies that other efferent nerves innervating the lower urogenital system contain CnNOS. Accordingly, procedures that provide a differential detection of PnNOS as compared to CnNOS will discriminate those nerves and help to devise surgical and medical treatments focused on the control of the smooth muscle tone of prostate, bladder, and urethra.

We have also discovered that PnNOS is not restricted to the urogenital system but is present in the skeletal muscle as single nNOS species. Accordingly, PnNOS controls striated muscle tone and not just smooth muscle tone. Moreover, we have also found that PnNOS is expressed as a minor species in the cerebellum, thus indicating its presence in the central nervous system.

We have determined that the human penis contains PnNOS (HPnNOS) and that the 102 bp insert is identical to that in the rat PnNOS (RPnNOS) . The procedure for cloning the full length HPnNOS is shown. Since HPnNOS arises from a gene different from the cerebellar nNOS, and a feature of the process of our invention is to use a different promoter to control HPnNOS expression.

The amelioration of aging-associated erectile dysfunction achieved by raising NOS levels in the rat penis by direct gene therapy of the organ with iNOS cDNA constructs indicates that NOS therapy is viable. This invention proposes procedures for treatment of erectile and urinary dysfunction by gene therapy with PnNOS constructs or derivatives. In addition, the biological modulation of PnNOS activity is disclosed.

EXAMPLE 1 : ABSENCE OF nNOS mRNA IN THE RAT AND HUMAN PENILE SMOOTH MUSCLE

This example shows that nNOS is not expressed in the corpora cavernosa smooth muscle, a tissue that in vivo is the target for NO action responsible for the relaxation that causes penile erection. This agrees with the fact that nNOS is located exclusively in the nerve terminals of the penis, as shown by immunocytochemistry. Since the isolation of cavernosal nerves for RNA extraction is not technically feasible, whole corpora cavernosa tissue needs to be employed for preparing a cDNA library for cloning penile nNOS (see example 2) . This example discards the possibility that the smooth muscle would be the source of penile nNOS mRNA within the corpora cavernosa tissue.

Rat and human penile smooth muscle cells (RPSMC and HPSMC, respectively) were cultured by standard procedures, and total RNA was isolated as described in our previous US and international patent applications. The RNA was submitted to DNAse treatment to eliminate any traces of contaminating DNA and aliquots of 0.5 ug were reverse transcribed at 37 C for 45 min by a standard procedure, using MMLV reverse transcriptase (100 U) in the presence of antisense oligonucleotide primer N02 (0.5 uM) . An aliquot of 1/5 of the RT mix was heated at 65 C and submitted to PCR in the presence of the sense (NOl) and antisense (N02) primers (0.25 uM) described below. The first series of 36 cycles was performed under non-stringent conditions of annealing at 94 C (45 sec) , 55 C (30 sec) , and 72 C (2 min) , with 5 min for the last cycle. An aliquot of 1/25 was used for a second similar round of amplification, except that annealing temperature was 62 C.

The oligonucleotide primers used for RT/PCR were synthesized based on information from the published sequence of the rat cerebellar nNOS (RCnNOS) cDNA on the calmodulin/FMN region (Fig. 1). Two 29-mer primers, designated NOl and N02 , encompass a 601 bp DNA sequence (200 amino acids) , starting on amino acid 704, as previously described in the literature for RCnNOS. These primers contain Eco RI and BamHl sites at the 5" end to facilitate cloning. All the cDNA lengths are given on this figure without considering the 5 ' restriction site tails that are present in the PCR products. A list of abbreviations for primers, probes, and other sequences is given on Table 1 of Appendix A. Two other primers were designed by us and designated N05 and N06 , encompassing a smaller internal 146 bp fragment in the same region. Finally, our primers N07 and N08 encompass a 460 bp fragment in a region 5' to the N01/N02 sequence. Primer N05 is a 20mer, and primers N06-8 are similar to NOl and N02 in length. Odd and even numbers in the designations correspond to sense and antisense directions, respectively. The initiation and termination sites and length of each fragment are indicated on the figure. The combination of primers NOl and N06 , and N05 and N02, encompass 493 bp and 254 bp fragments, respectively.

An aliquot (1/7) of the PCR mix was fractionated by gel electrophoresis, stained with ethidium bromide, and the size of the bands determined by comparison against an adequate standard. The RNAs were run on a separate non-denaturing 1.5% Nusieve gel and stained similarly. Fig. 2 (gel A) shows the quality of both RPSMC (lane 1) and HPSMC (lane 2) RNAs, as evidenced by the 28 and 18 S bands, against the appropriate markers (lane 3) . Fig. 2 (gel B) shows two bands of approximately 590 and 410 bp in the RPSMC cDNA (lane 2) , and 540 and 360 bp in the HPSMC cDNA (lane 3) , as determined with the markers (lane 1⁾ .

The 590 bp fragment in RPSMCnNOSl/2 cDNA was slightly smaller than the expected size for RCnNOS including the primer 5' tails (619 bp) , but it was the only likely candidate to represent nNOS in the RPSMC cDNA. Therefore, the remainder of the PCR mix run on gel b, lane 2, was submitted to the same agarose fractionation and the top and bottom fragments were excised. The 590 bp band was labeled in situ in the gel with 32P-dCTP by random priming. This cDNA probe was then used for northern blot hybridization of total RNAs isolated from RPSMC and two other rat SMC cultures: aorta SMC and spleen SMC. Fig. 3 (gel A, lanes 1-3) shows that a 10.5 kb band is visible in the RNA from three separate confluent RPSMC cultures, and absent in the RNA from rat spleen SMC (lane 4) . The mRNA size is exactly the one expected for RCnNOS mRNA, and the strong β-actin reference signal validates the detection. The 10.5 kb signal is even more intense in three separate cultures of rat aorta SMC (gel B, lanes 1-3) , and absent in RNA isolated from cultured tumor Leydig cells (lanes 5,6), thus indicating that this mRNA is generally expressed in rat vascular SMC.

The similar sizes for both the RT/PCR fragment and the northern blot band obtained from the RPSMC RNA, as compared to RCnNOS, would suggest that nNOS mRNA is indeed present in the penile smooth muscle from the rat, and by extension presumably from the human. Alternatively, these results may arise from a circumstantial coincidence between totally unrelated genes. To distinguish between both possibilities, an additional RT/PCR sample from RPSMC RNA was fractionated and the 590 bp fragment eluted, directly cloned into the PCRI vector (Invitrogen) , and sequenced. Fig. 4 shows the sequence of the RPSMCnNOSl/2 cDNA fragment in both directions . Homology comparisons with the corresponding region on RCnNOS was below 40% (not shown) and it was concluded that RPSMCnNOSl/2 is an unrelated sequence, and that nNOS is not expressed in vitro, and likely not in vivo, in the rat penile smooth muscle. Based on this evidence, the HPSMC mRNA bands detected on Fig. 3 were considered to arise from genes unrelated to human nNOS and were not studied further.

EXAMPLE 2: EXPRESSION OF A NOVEL FORM OF nNOS mRNA IN THE RAT PENIS

This example demonstrates that: a) in contrast to cultures of penile smooth muscle cells, nNOS is expressed in vivo in the rat penis, as a protein of approximately the same size as the nNOS isoform cloned and characterized in the central nervous system; and b) the penile nNOS is however different from the cerebellar nNOS isoform and it is a novel NOS species .

In order to confirm the absence of nNOS expression in RPSMC as compared with the whole rat penile shaft (corpora cavernosa, corpus spongiosum, and os penis) and bulb, and to determine whether there is another more abundant source of penile-related nNOS, an immunodetection procedure was applied to several rat tissue extracts (5 month-old, male Fischer rats) . For this purpose, a post-metachondrial fraction was obtained at 12,500 g for 60 min by a procedure described in our previous patent applications, since cerebellar and penile nNOS are essentially cytosolic. The following sources were used: a) cerebellum; b) kidney; c) skin-denuded penis (shaft and bulb) ; d) RPSMC; f) pelvic plexus ganglion. Aliquots with the same protein input (80 ug) , except for the pelvic plexus (40 ug) , were run on 7.5% polyacrylamide gel electrophoresis (PAGE). Western blot assays were performed with a commercial rabbit antibody against the carboxy terminus of the human nNOS (Transduction Laboratories) . The detection was made by a secondary antibody against rabbit IgG and a luminol-based reaction, and bands were visualized by autoradiography .

Fig. 5 (gel A) shows the expected 155-160 kD nNOS band in the cerebellum (C) , and a nearly as intense band in the penis (P) , with practically no expression in the kidney (K) . Confirming the RT/PCR data, the nNOS band was completely absent from the RPSMC. In contrast (Gel B) , the pelvic plexus (PP) shows a band even more intense than that in the cerebellum (C) . This indicates that nNOS proteins in the penis and pelvic plexus have approximately the same size as the nNOS protein expressed in the cerebellum, and have common antigenic determinants.

In order to determine whether despite the protein size similarity or agarose gels, the penile and cerebellar nNOS mRNAs are different, total RNA was isolated as above from penile tissue (skin-denuded bulb plus corpora cavernosa) and cerebellum. The polyA÷ RNA was prepared by a conventional procedure and treated with DNase. The RT/PCR used above for RPSMC and HPSMC total RNA was applied, but extending it to other combinations of the primers listed above. The RT was always carried out with the respective antisense oligonucleotide from each pair of primers.

The products were separated on Nusieve gels and the gels were stained with ethidium bromide and hybridized with suitable probes. Fig. 6A shows the position of the three primers on a selected region of the nNOS cDNA. The bottom left panel shows that in the case of the rat cerebellum (C) , primers NOl/2 (lane 3) and 1/6 (lane 2) give the expected 619 bp and 511 bp bands, respectively. Lane 1 corresponds to the markers. However, the bands generated in the penis are larger: approximately 720 bp (lane 4) and 615 bp (lane 5) , respectively. Blank lanes are not numbered. A Southern blot was performed and the membrane was stored for hybridization with the cDNA probes generated below. The results obtained are shown on the central and right panels but discussed below, after Figs. 7 and 8, once the cDNA probes are analyzed.

The clear-cut size difference between the DNA fragments generated from the penile RNA as compared to the cerebellum RNA was reproduced in three independent RT/PCRs run separately (not shown) . No bands were seen in the negative controls (no RNA or no reverse transcriptase) . The same bands could be generated by just one round of 35 cycles amplification at the highest stringency (2nd round of the previous procedure) . The NOl/2 and NOl/6 fragments were eluted from the gels, purified, and submitted to sequencing.

Fig. 7 shows the automated sequencing that first demonstrated the presence of a considerable difference between RPnNOS and RCnNOS, based on the sequencing of the fragment generated from the penile RNA with-NOl/2, designated RPnNOSl/2, as compared to that for the homologous region from RCnNOS, designated nNOSl/2. Section A presents the sequencing obtained with primer N06 , that is the complementary (antisense) strand. The nNOS sequence flows normally until the point indicated by the arrow that indicates the presence of a 102 bp insert, designated RPnNOS102. The second arrow indicates where the nNOS sequence resumes. As expected, this insert accounts for the approximately 100 bp difference between RPnNOSl/2 and RCnNOSl/2. This insert interrupts the normal nNOS reading frame at position # 2865 in the numbering of the published sequence of RCnNOS. Since the insert corresponds to exactly 34 triplets, the coding region remains in frame after the 3' end of RPnNOS102. Comparison of the RPnNOSl/6 with its counterpart for the cerebellar cDNA confirmed the presence of the 102 bp insert (not shown) . Section B presents the sense sequencing of RPnNOSl/2 using primer NOl. Homology comparison shows that it corresponds to nNOS but the length is not sufficient to reach the insert position.

Based on this particular sequencing and numerous repetitions of PCR material, a consensus sequence for RPnNOS102 was deduced and two primers were designed, named RCI-1 and RCI-2, as shown on Fig. 8 (upper panel) . The position of this 102 bp insert in relation to the coding region of the rat nNOS gene is represented above, with the insert shown below.

The sequencing (middle panel) showed that the first nucleotide of the insert interrupts the triplet for amino acid 839 (lysine) without changing it. The last two nucleotides from the 102 bp insert precede the displaced G from the original CnNOS sequence and form a triplet coding for arginine . The next amino acid is the original # 840 (serine) .

Using only RCI-2 for RT and both primers for the subsequent PCR, the 102 bp fragment was generated from penile mRNA but not from cerebellar mRNA. When the RT was done with either N02 or N06 , and the subsequent PCR with NOl, the and 1/6 fragments were generated from both penile and cerebellar RNA. The bands originated by RT/PCR from RPSMC RNA were as in the experiment depicted on Fig. 2. When. aliquots of the reaction were PCR-amplified with both RCI-l and 2 and fractionated by nusieve agarose electrophoresis, the 102 bp fragment was generated from both the penile and cerebellar parent fragments, but remained undetectable in the RPSMC mixes .

The above evidence shows that: a) the nNOS isoform in the penis is different from the major cerebellar nNOS at the mRNA and protein levels and is the only nNOS species expressed in this organ; and b) the penile nNOS is expressed as a minor band in the cerebellum that can be detected by ethidium bromide by amplification from parent larger fragments generated by a first round of PCR. The penile isoform was designated RPnNOS to differentiate it from the rat cerebellar nNOS (RCnNOS) .

The PCR-generated RPnNOS102 was then cloned into vector PCR- 1 and the insert was sequenced again. The construct was designated pPCR-RP102 and used to generate RPnNOS102 as a probe, by PCR with RPI-1 and RPI-2 in the presence of 32-PdCTP. Another cDNA probe corresponds to the 5' end of RPnNOSl/2 excluding the 102 bp insert and was prepared by digestion of RPnNOSl/2 with Banl. This probe was designated RPnNOS330 and labeled with 32P by random priming.

Hybridization with RPnNOS330 performed on the Southern blot for the gel presented on Fig. 6A is shown on Fig. 6B. It is clear that the penile RNA generates only the larger fragments, whereas the cerebellar RNA originates the smaller fragments and also traces of the larger fragments. Hybridization therefore reveals what the ethidium bromide pattern failed to show on that gel, that is that in the cerebellum there is a minor expression of the RNA containing the 102 insert which is the only nNOS- related mRNA in the penis. This is confirmed by hybridization with RPnNOS102 (Fig. 6C) , which confirms that in the cerebellum nNOS cDNA there are minor species containing the 102 bp insert, in contrast with the penis where they are virtually the single species .

EXAMPLE 3: CLONING AND SEQUENCING OF A cDNA ENCODING A NOVEL nNOS ISOFORM

EXPRESSED IN THE RAT PENIS AS THE ONLY nNOS SPECIES

This example demonstrates the procedure for cloning and sequencing the cDNA encoding the novel rat nNOS protein.

In order to carry RPnNOS gene therapy or to apply its derived products in research or diagnosis, it is essential to have cDNA constructs with the RPnNOS complete coding region and to determine whether in addition to the 102 bp insert there are other nucleotide and amino acid sequence differences in comparison with the cerebellar nNOS. This requires the cloning of RPnNOS from a cDNA library prepared from the rat penis mRNA.

Total RNA was isolated from 10 penises (skin-denuded bulbs and shafts) obtained from 5 -month old Fischer 344 rats, and the polyA+RNA was prepared by conventional procedures. 8 ug of mRNA were used for the construction of a cDNA library, by reverse transcription with oligodT primers (Xhol site at end) , ligation with EcoRl adaptors, cleavage of the Xhol site, and cloning into the XhoI/EcoRI sites of σZap vector arms (UniZap XR, Stratagene) . This library was plated, amplified, and the lysed plaques were transferred in duplicate (replica plating) to nylon membranes. Hybridization for plaque detection was carried out initially with a RPnNOS5/6 probe generated by RT/PCR from rat penis mRNA with the N05 and N06 primers. A total of 9 positives were then submitted to secondary screening with the same probe, and only two plaques were finally selected for tertiary screening with separate replicas for RPnNOΞ5/6 and RPnNOS102.

At the last stage two clones were designated #1 and #2, the pBS SK- phagemid was excised from the lamda vector, and the DNA was isolated by conventional procedures . EcoRl and Xhol digestion was followed by Southern blotting and sequential hybridization with the previous two probes . A 3.1 kb insert was found in both clones, which suggested they were nearly identical. Only clone #1 was selected for sequencing, extending to the 3' direction with the help of successive sequencing primers synthesized on the basis of each previous sequence. It was found to span from 1684 to 4839 bp (3155 bp total) , containing the 102 bp insert not found in the rat cerebellar nNOS. Antisense primers allowed sequencing of complementary sequences. Fig. 9 shows the list of individual sequencings with their corresponding direction and the sequencing primers. Primers 1, 2, 5, 6 , 7, 8 , RCI1, and RCI2, are the ones used for RT/PCR and PCR. The other primers were synthesized for the sequencing.

The 5 ' end of RPnNOS was poorly represented in the cDNA library from where clone 1 was isolated, most likely because in order to generate this region the cDNA extension should proceed for up to 4.5 kb. Therefore, a 2.7 kb fragment was generated by "extended" PCR using a new primer for the 3' end of RPnNOS102 (RCI-4) and a primer on the 5' end of the published sequence of RCnNOS. The assumption was that if the 5' end of both RPnNOS and RCnNOS were not too different, the PCR fragment would correspond only to RPnNOS because of the specificity imposed by the 102 bp 3' end primer (Fig. 10) .

The 5' region of RPnNOS, 2708 bp from position 248 to 2956, was obtained by extra long PCR using primers RCnNOSATG and RCI4 (GeneAmp XL PCR kit, Perkin-Elmer , Branchburg, NJ) . 1 μg of total rat penis RNA was reverse transcribed with AMV reverse transcriptase (Promega Corp., Madison, WS) , and amplified with the GeneAmp XL PCR kit, using optimized conditions in conjunction with rTH DNA polymerase to amplify long regions of DNA. The PCR fragment was cloned by blunt end ligation into EcoRV cut pZero2.1 (Invitrogen, San Diego, CA) and designated pZRCnNOSATG-RCI4. It was sequenced in both directions.

In order to construct a full length cDNA, both pBSRPnNOSl and pZRCnNOSATG-RCI4 were cut with Bstll07I and Xhol restriction enzymes. The Bstll07I-XhoI 3' region of pBSRPnNOSl was separated by agarose gel electrophoresis and purified using a Qiaquick purification column (Qiagen, Chatsworth, CA) . The vector portion along with the 5' region of the RPnNOS of pZRCnNOSATG-RCI4 was purified as above. The Bstll07I-XhoI 3¹ region of pBSRPnNOSl is ligated into Bstll07I-XhoI cut pZRCnNOSATG-RCI4 to give the full length clone, pZRPnNOS .

For gene therapy use, RPnNOS was cloned into the multi cloning site of pCDNA3 (Invitrogen, San Diego, CA) by digesting both pZRPnNOS and pCDNA3 with EcoRl and Xhol restriction enzymes. pCDNA3 and the RPnNOS cDNA fragment were gel purified and ligated as before to give pCRPnNOS .

The complete nucleotide and amino acid sequences for the RPnNOS are presented on Tables 2 and 3. The differences between RPnNOS and RCnNOS are summarized on Fig. 11, and they consist of the following: a) presence of the 102 bp insert after lysine 839, coding for 34 amino acids; b) five additional amino acids changed as indicated; c) a non-coding 3' end shorter by 319 bp; d) addition of a doublet in the conserved non-coding 3' end. We propose that these changes have functional significance related to RPnNOS role in penile erection and in general in smooth muscle relaxation.

The approximate location of the 102 bp insert within intron 16 was inferred from a Southern blot of genomic DNA digested with a series of restriction enzymes and hybridized with two different probes. Fig. 12, top left, presents the distinctive pattern obtained with the RPnNOS specific probe, that is RPnNOΞ102, whereas on the top right the hybridization with nNOS-

E17 is shown. The latter is a fragment of exon 17, a sequence adjacent to RPnNOS102 at the RNA level, and identical in RPnNOS and RCnNOS.

Based on the fact that the insert is on intron 16 and on the physical map of the human CnNOS gene and its restriction pattern for three enzymes, ΞcoRi, BamHl, and Hindlll, it may be predicted that the hybridization signals for the two probes should coincide on the same fragment in each digestion. However, this is not the case in EcoRl and Hindlll, which would suggest that there are two different genes with nNOS sequences : the one coding for RPnNOS mRNA and the one transcribed into RCnNOS mRNA. Alternatively, the corresponding restriction sites in RCnNOS may differ from the homologous gene in the human. The absence of a published nNOS restriction mapping ^'for the other enzymes leaves the comparison open for future analysis. In any case, the approximate position of the insert is indicated on this map based on evidence discussed below, between small exons 16 (59 bp) and 17 (117 bp) , in the 5' side of the 5 kb intron # 16.

EXAMPLE 4 : EXPRESSION OF RPnNOS mRNA THROUGHOUT THE LOWER

UROGENITAL TRACT

AND OTHER ORGANS OF THE RAT

This example demonstrates that RPnNOS mRNA expression is not restricted to the penile corpora cavernosa and that it occurs in other organs of the lower urogenital tract, in the pelvic plexus and efferent nerves, in striated muscle, and to a much lower degree in cerebellum and liver, and that in some cases it is accompanied by RCnNOS mRNA expression.

Total RNA was isolated from the penile shaft/bulb, bladder, urethra, ventral prostate, pelvic plexus, skeletal muscle, liver, and cerebellum. 2 ug of total RNA from each tissue was reversed transcribed with MMLV reverse transcriptase (100 U) using N02 , N06 , or oligoDT as primer for first strand DNA synthesis. 1/5 of each reaction was used for PCR with primer combinations of NOl and N06 or N05 and N06.

Fig. 13, top shows the pattern obtained with different combinations of NO primers in the bladder and urethra, as compared with the controls (penis and cerebellum) . Fragments were separated on 1.5% Nusieve, and the gel was stained with ethidium bromide (left panel) and submitted to Southern blotting (central and right panels) . Sizes expected for RCnNOS are 511 bp (lanes 1,3), 155 bp (lanes 2,4,6), and 619 bp (lane 5). Each fragment is 102 bp longer for RPnNOS. The ethidium bromide stain confirms that the nNOS cDNA fragments in the penis are larger than in the cerebellum as expected, and that the urethral pattern is identical to the penis. The bladder exhibits a mix of penile-like and cerebellar-like fragments.

The presence of RPnNOS mRNA in bladder and urethra was confirmed directly by hybridization with the RPnNOS102 probe, showing intense expression of single bands in the urethra and lesser levels in the bladder. The existence of additional shorter bands in some lanes is probably the result of artifactual amplifications. Hybridization with the probe common to RPnNOS and RCnNOS, nNOS-E17, replicated the ethidium bromide staining.

The other urogenital related tissues, ventral prostate and pelvic plexus, were examined with the same approach, against the control battery of penis, cerebellum, and urethra. Two non- urological tissues, liver and skeletal muscle were included (Fig.

13, bottom) . Skeletal muscle has been found to express high levels of nNOS in humans . Hybridization with the RPnNOS102 probe shows the expected signals for penis and urethra and their absence in cerebellum. They are intense in the prostate and moderate in the pelvic plexus, although for unknown reasons the amplification/hybridization failed in some cases (e.g., lanes 1,3 for prostate, and 2,4 for pelvic plexus) . Very interestingly, the RPnNOS is expressed in the skeletal muscle and in the liver. The ethidium bromide pattern and the hybridization with the common nNOS-E17 probe showed that in the prostate and skeletal muscle only RPnNOS mRNA is expressed, whereas in pelvic plexus and liver both RPnNOS and RCnNOS mRNAs are expressed, the latter appearing to be the predominant form.

This example shows that PnNOS is the only nNOS species in the penis, prostate, and urethra, and a significant fraction of the nNOS present in the bladder and in the nerves (pelvic plexus) innervating the urogenital tract. Since NO is the noncholinergic-nonadrenergic mediator of smooth muscle relaxation in these organs, and as such elicits and maintains penile erection, We presently believe that PnNOS controls penile erection and in general the tone of the lower urogenital tract. In addition, We believe that PnNOS fulfils an important role in the control of striated muscle relaxation.

EXAMPLE 5 : EVIDENCE THAT PnNOS IS ENCODED BY A GENE DIFFERENT FROM CnNOS

IN BOTH RAT AND HUMAN DNA AND IT IS ALSO EXPRESSED IN THE

HUMAN PENIS. This example demonstrates that the novel penile nNOS protein is expressed in the human penis, and supports the conclusion that it is the product of a new gene different from the one identified so far in rat and human tissues (CnNOS) and not a splicing variant. Although the existence of a penile- specific nNOS protein justifies most of the proposed applications, the discovery of a new gene preferentially expressed in the nerves of lower urogenital organs and skeletal muscle would allow the search for a specific promoter. The latter would be extremely useful for targeting gene expression in the lower urogenital organs in gene therapy approaches (see Example 6) .

The following experiment addressed both points simultaneously, based on the examination of nNOS intron 16 and the adjacent exons 16 and 17 sequences in the rat and human DNA. The rationale was to amplify by "extended" PCR different sections of intron # 16, bridging the 102 bp insert with internal regions in exons 16 or 17. The sets of primers were either: a) antisense primer RCI-4 on the 102 bp insert and sense primer NO-E16F on exon 16; and b) antisense primer NO-E17R on exon 17 and sense primer RCI-3 on the 102 bp insert.

Human lung and rat liver genomic DNA were isolated with a DNA extraction kit (Easy DNA, Invitrogen, La Jolla) . 1 ug DNA was subjected to extra long PCR (XL GeneAmp kit, Perkin Elmer, CA) using primer sets described above. Fig. 14A depicts the ethidium bromide staining of the PCR products separated on a 1% agarose gel, showing that an approximately 1.1 kb fragment was generated by the RCI-4/N016F primers from rat DNA (lane R) , and that a similar fragment was obtained from human genomic DNA, accompanied with a smaller band of approximately 1.0 kb (lane H) . Fig. 14B shows that in the case of RPnNOS it was possible to span the whole intron # 16 and amplify an approximately 5 kb sequence by using primer pairs consisting of NO-E16F and NO-E17R. Since only a single band was obtained, this suggests that if two genes really exist in the rat, intron 16 must be very similar in size in both the RPnNOS and RCnNOS. The region between RCI-3 and NO- 17R can be calculated by substraction to be about 4 kb, and the band obtained is approximately this size. The two right lanes show a repetition of the reactions run on Fig. 14A, to assess reproducibility. PCR fragments were isolated from 0.8% agarose gels and purified DNA fragments were partially sequenced in both directions (500 bp each way) . The product from the RCI-4/N016F amplification of RPnNOS (Table 6) revealed differences with the published sequence of the exon 16/intron 16 junction region in the human. This is due to either RPnNOS being a different gene from HCnNOS, or because of species differences.

The support for the existence of a second nNOS gene that is transcribed into a novel nNOS mRNA was obtained from the sequencing of the two bands generated from human DNA with NO-E16F and RCI-4. Both bands contain the expected sequences of the 102 bp insert and are very similar (Tables 4 and 5) . Since the sequencing is incomplete, the difference in size cannot be explained. When the homology comparison was made with the published sequences for HCnNOS exon 16 and the exon/intron 16 junction, it was shown that the fragments generated from human DNA by using RCI-4 had many base differences with the corresponding human cerebellum nNOS (HCnNOS) sequences (Fig. 14 bottom) .

This evidence and the results from the southern blotting shown on Fig. 13 lead to the postulation that a second human penile nNOS gene different from HCnNOS exists, designated HPnNOS gene. It is proposed that is this gene that maintains normal penile erection and the function of the lower urogenital tract in transgenic mice where the HCnNOS gene has been knock out. It is further postulated that HPnNOS is represented on Fig. 14 top, panels A and B by the bottom band seen on the amplification of exon/intron 16 in human DNA, whereas HCnNOS corresponds to the top band. However, the existence of PnNOS as a functional protein in the lower urogenital tract and other organs is a fact, irrespective of whether it arises from a second nNOS gene or from alternative.

The demonstration of the existence of PnNOS in the human DNA was based on showing the 102 bp insert in the human genome. This was inferred from the use of a RPnNOS102 primer for generating the fragments on Fig. 14, and the positive hybridization with the RPnNOS102 probe. The confirmation was achieved by sequencing in both directions the bands presented on Fig. 14. A consensus sequence for the initial 96 nucleotides within human nNOS intron 16 nearly identical to the homologous region in RPnNOS 102 was obtained (Fig. 15 top) . Only two nucleotide changes (AGC to AAA, on amino acid #31 were found, denoting a change of serine to lysine. This gene is expressed in the human penile RNA, aa. shown by the fact that PCR amplification of lug DNA from a human penile cDNA library with primers RCI-3 and NO-6 generated the expected 207 bp fragment with a sequence identical to the one found in the DNA (Fig 15 bottom) .

EXAMPLE 6: APPLICATIONS OF PnNOS AND THEIR RELATED PRODUCTS FOR THE DIAGNOSIS

AND TREATMENT OF ERECTILE DYSFUNCTION, URINARY VOIDING DISORDERS,

AND OTHER CONDITIONS This example shows that the novel nNOS gene (PnNOS) , its specific coding and regulatory regions, its cDNA and related sequences, its recombinant DNA constructs, its host cells, its encoded protein and peptides, the respective antibodies, regulatory factors, and all other related products, have wide applications in: A) the diagnosis and treatment of human disorders such as: a) erectile dysfunction; b) urinary voiding disorders and other conditions related to the maintenance of smooth muscle tone; c) other conditions afflicting the lower urogenital tract; and B) biomedical research, mainly neurobiology .

A Diagnosis and treatment of human disorders a) Erectile dysfunction

The existence of a penile specific nNOS isoform ⁽PnNOS, irrespective of species) , most likely located in the penile nerve terminals and poorly represented in the central nervous system, indicates that the 34 amino acid insert confers a special role to this isozyme in the mechanism of penile erection. It is further postulated that the other amino acid differences enhance the specificity of PnNOS function.

Since none or very little CnNOS is present in the penis, We presently believe that PnNOS and not nNOS is responsible for the synthesis of NO during penile erection. In addition, the evidence here indicates that PnNOS is a gene different from the CnNOS gene and that this explains why knockout mice generated by blocking cerebellar nNOS using CnNOS-specific intron regions are potent (fertile) . Accordingly, in contrast, the PnNOS knockout should have a considerably decreased potency. The targeting construct used for the first nNOS knockout mouse published in the literature contained no cerebellar nNOS 5' exons, that are similar to the homologous region in PnNOS. The construct included only the intron sequences surrounding the deleted mouse nNOS exon 1 (exon 2 region in human nNOS) . These regions are apparently different in cerebellar nNOS and PnNOS, possibly because of variants arising from alternate splicing or parameter useage .

Considering that the PnNOS protein is expressed preferentially in the penis, and that a novel gene may encode it, the PnNOS gene is apparently subject to a transcriptional regulation in this organ different from the one operating with CnNOS elsewhere in the organism. In addition to this level of control, that there is an even more stringent and rapid regulation mechanism related to the role played by PnNOS in evoking the quick response required by the erectile process. This latter control would be exerted at the level of enzyme activity and it is likely that the 34 amino acids insert present in PnNOS are crucial for assuring this type of fast and fine tuning.

This invention has shown that PnNOS is present in the human, and therefore it is proposed that erectile dysfunction may be treated by PnNOS-based therapy (HPnNOS cDNA and recombinant protein) , by applying procedures similar to those described in our Patent 5,594,032 for iNOS gene-related therapy. The plasmid vector used for delivery may either have standard promoters or penile-specific promoters (see below) . The latter cDNA construct is the presently preferred modality. The HPnNOS cDNA constructs are intended for medium or long-term effects, whereas the HPnNOS recombinant protein is for immediate or short-term effects.

The administration procedures include single or multiple injections into the corpora cavernosa of HPnNOS constructs in liposomal complexes, or in suitable adenoviral or retroviral vectors, or in general in any formulation and route that would facilitate the HPnNOS cDNA uptake by the corpora cavernosa tissue. In the specific case of this nNOS isoform, because of the features of the HPnNOS protein that presumably makes its activity regulation highly penile specific, the cDNA construct may be also given parenterally or orally. If the PnNOS protein proves to be penile specific in its regulation at the enzyme level, it may also be administered systemically (e.g., orally) , in addition to the local delivery to the penis described for iNOS. Administration via urethra may also be employed.

The implantation of human penile smooth muscle cells (HPSMC) in vitro transfected with HPnNOS constructs may also be used as for iNOS constructs, if PnNOS is regulated in smooth muscle cells as it is in neural tissue. The transfection of human penile tissue with HPnNOS is assumed to direct some of the transferred gene to the penile nerve terminals . Pursuant to the invention substances that control the conformation, dimerization, phosphorylation, and in general enzyme activity of PnNOS are therapeutically applied or introduced to the penis, or introduced systemically, to upregulate NO synthesis and stimulate penile erection. Some of these substances include: a) co-factors, like tetrahydrobiopterine, its precursors, and genes coding for its synthesis; b) substrates, like L-arginine and analogs; c) associated regulatory proteins similar to the PINs described for CnNOS .

Probes derived from the 102 bp insert in PnNOS should be able to detect and quantitate specifically the amount of PnNOS RNA or protein in very small biopsies of penile corpora cavernosa, without cross-reactivity with the nNOS isoform. This will have diagnostic value for evaluating erectile dysfunction and help in determining whether erectile dysfunction is due to a decrease in penile NOS content . This should contribute to the clinical differentiation of neurogenic and vasculogenic impotence .

b) Urinary voiding disorders and other conditions related to the maintenance of smooth muscle tone

The expression of substantial levels of PnNOS in the pelvic plexus, and of basal levels in the central nervous system, are indicators that PnNOS exists in considerable amounts in nerves outside the penile nervous system. This invention proposes that PnNOS has a physiological role exceeding penile erection, namely to synthesize NO as a peripheral neurotransmitter in nerve terminals controlling endothelium-independent smooth muscle relaxation, through an isoform-specific modulation of NOS enzyme activity.

In the case of the lower urogenital system, PnNOS is expressed as the main or only nNOS species in prostate and urethra, and in combination with CnNOS in bladder. This invention predicts that the tone of these organs and other ones in the lower urogenital tract will be partially determined by PnNOS. Therefore, a PnNOS-based therapy and differential diagnosis will be feasible for disorders such as ^• bladder instability, stress incontinence, urinary obstruction, etc., associated with diabetes, benign prostatic hyperplasia, aging, and other conditions, in all or some of the approaches described those proposed for erectile dysfunction. This invention proposes that premature ejaculation be treated by applying one or several of those modalities focused on the control of prostatic tone for regulating the appropriate emission of seminal fluid.

The fact that RT/PCR detects some PnNOS expression in the cerebellum indicates that this protein may act in the central nervous system as well. It is predicted that certain neuronal populations contain mainly PnNOS, while others depend on cerebellar nNOS. The differentiation will be based on immunocytochemistry or in situ hybridization of tissue sections with probes related to the ones discussed in this invention.

The quantitative RT/PCR approach on fresh tissue RNA is proposed as a strategy to determine PnNOS/CnNOS ratios. The diagnostic application of RPnNOS102 may facilitate the direct examination of the pelvic plexus at the immunocytochemical level to differentiate fibres going to the penis, bladder, urethra, etc. (HPnNOS+) , from those containing mainly the cerebellar nNOS. This opens up applications in the therapy of conditions associated with excessive or defective NO production in those types of neurons. This concept applies to afferent and efferent nerves in other tracts where the control of the smooth muscle tone is fundamental for its function, such as the gastrointestinal tract.

c) Other conditions afflicting the lower urogenital tract

The existence of a specific PnNOS gene expressed as sole nNOS species in the penis, and also in the prostate and urethra, indicates that its promoter will be recognized mainly by transcription factors present in these organs and in certain nerve fibers innervating them. Systemic gene therapy driven by the PnNOS promoter will not assure specific targeting to the urogenital tract (since PnNOS is expressed in skeletal muscle) but it should restrict considerably the expression of the targeted gene in other organs .

This invention proposes that the HPnNOS promoter be detected and cloned in plasmid vectors and sequenced for the expression of cloned cDNAs for therapeutically useful proteins in the lower urogenital system. The NOS isoforms proposed for the treatment of erectile dysfunction (iNOS, PnNOS) may be linked to these PnNOS promoter-driven vectors. It is also proposed that this promoter be used in conjunction with any gene therapy for impotence based on other genes, such as the cDNAs coding for vasoactive intestinal polypeptide (VIP) , calcitonin gene related peptide (CGRP), and other vasoactive or neuroactive peptides. In this way expression of these constructs will be targeted to the penis. The applicability of the HPnNOS promoter may extend to the delivery of growth-related genes or to the antisense blockade of genes involved in penile tissue fibrosis, for the correction of conditions ranging from impotence to micropenis, hypospadias, ambiguous genitalia, etc.

The same principles can be applied to use PnNOS promoter-driven plasmids for the therapy of conditions affecting the bladder, urethra, etc., such as bladder cancer, cystitis, prostate growth in BPH, etc.

INDUSTRIAL APPLICABILITY :

Application of the composition and methods of this invention have direct application to the mitigation of penile erectile dysfunction in humans and in animals. Horticultural applications include extending the breeding capabilities of livestock. Products generated from this invention will have wide applicability in biomedical research connected to areas described above and other fields. These products include the PnNOS-related cDNAs, cDNA constructs, host cells, recombinant proteins, synthetic or natural peptides, antisera and purified antibodies, regulatory factors, and others, as specified above and in the claims section.

It should be understood that various modifications within the scope of this invention can be made by one of ordinary skill in the art without departing from the spirit thereof. We therefore wish our invention to be defined by the scope of the appended claims as broadly as the prior art will permit, and in view of the specification if need be. APPENDIX A Table 1

Glossary of Terms

All are listed for the rat, and have in common R as the first letter. For human material they are identical, except for H substituting for R.

A. mRNAs

RPSMC mRNA: mRNA isolated from rat penile smooth muscle cells RC mRNA: " " " " cerebellum

RP mRNA: " " " " penis

B. cDNAs

RCnNOS: nNOS isoform expressed in the rat cerebellum

RPnNOS : nNOS isoform expressed in the rat penis

PnNOS: nNOS isoform, irrespective of species clone 1 : 3.5 kD cDNA clone at the 3 ' end of RPnNOS fragment 2: RT/PCR cDNA for the 5' end of RPnNOS

C. Oligonucleotide primers

Odd numbers are sense, and even numbers are antisense

NOl, N02 , N05-8: primers based on the published sequence of

RCnNOS

NO-El6, NO-E17: primers for exons 16 and 17 (F : forward,^•

R: reverse) of both RCnNOS and RPnNOS

RC1-RC4: primers based on RPnNOS102 sequence

N09-20: sequencing primers for clone 1 of RPnNOS

D. PCR fragments and hybridization probes

RPnNOS102 : 102 bp insert present in RPnNOS and absent in RCnNOS RPnNOSl/2: RT/PCR fragment generated from RPnNOS mRNA with primers NOl and N02

RPnNOS5/6: ibid, with primers N05 and N06 RPnNOSl/6: ibid with primers NOl and N06 nNOS601: RT/PCR fragment generated from RCnNOS cDNA with primers NOl and N02 nNOS334: 3' end region of nNOS601, generated by Banl restriction digestion nNOSE17: exon 17 generated from RPnNOS clone 1 with primers

NO-E17F/R

RPSMC1/2 : RT/PCR fragment generated from RPSMC RNA with primers

NOl and N02 pPCR-RP102: RPnNOS102 cloned in PCRI vector

TABLE 2

RPnNOS DNA sequence Length: 4592

1 AGACAGAGGC GACAGAAACT CTGCAGCCAG TCTTGCCCC GAGGAGCTC

51 AGGTTCCTGC AGGAGTCATT TTAGCTTAGT CTTCTGAAGG ACACAGATAC

101 CATGGAAGAG AACACGTTTG GGGTTCAGCA GATCCAACCC AATGTAATTT

151 CTGTTCGTCT CTTCAAACGC AAAGTGGGAG GTCTGGGCTT CCTGGTGAAG

201 GAACGGGTCA GCAAGCCTCC CGTGATCATC TCAGACCTGA TTCGAGGAGG

251 TGCTGCGGAG CAGAGCGGCC TTATCCAAGC TGGAGACATC ATTCTCGCAG

301 TCAACGATCG GCCCTTGGTA GACCTCAGCT ATGACAGTGC CCTGGAGGTT

351 CTCAGGGGCA TTGCCTCTGA GACCCACGTG GTCCTCATTC TGAGGGGCCC

401 TGAGGGCTTC ACTACACATC TGGAGACCAC CTTCACAGGG GATGGAACCC

451 CCAAGACCAT CCGGGTGACC CAGCCCCTCG GTCCTCCCAC CAAAGCCGTC

501 GATCTGTCTC ACCAGCCTTC AGCCAGCAAA GACCAGTCAT TAGCAGTAGA

551 CAGAGTCACA GGTCTGGGTA ATGGCCCTCA GCATGCCCAA GGCCATGGGC

601 AGGGAGCTGG CTCAGTCTCC CAAGCTAATG GTGTGGCCAT TGACCCCACG

651 ATGAAAAGCA CCAAGGCCAA CCTCCAGGAC ATCGGGGAAC ATGATGAACT

701 GCTCAAAGAG ATAGAACCTG TGCTGAGCAT CCTCAACAGT GGGAGCAAAG

751 CCACCAACAG AGGGGGACCA GCCAAAGCAG AGATGAAAGA CACAGGAATC

801 CAGGTGGACA GAGACCTCGA TGGCAAATCG CACAAAGCTC CGCCCCTGGG

851 CGGGGACAAT GACCGCGTCT TCAATGACCT GTGGGGGAAG GACAACGTTC

901 CTGTGGTCCT TAACAACCCG TATTCAGAGA AGGAACAGTC CCCTACCTCG

951 GGGAAACAGT CTCCCACCAA GAACGGCAGC CCTTCCAGGT GCCCCCGTTT

1001 CCTCAAGGTC AAGAACTGGG AGACGGACGT GGTCCTCACC GACACCCTGC

1051 ACCTGAAGAG CACACTGGAA ACGGGGTGCA CAGAGCACAT TTGCATGGGC 1101 TCGATCATGC TGCCTTCCCA GCACACGCGG AAGCCAGAAG ATGTCCGCAC

1151 AAAGGACCAG CTCTTCCCTC TAGCCAAAGA ATTTCTCGAC CAATACTACT

1201 CATCCATTAA GAGATTTGGC TCCAAGGCCC ACATGGACAG GCTGGAGGAG

1251 GTGAACAAGG AGATTGAAAG CACCAGCACC TACCAGCTCA AGGACACCGA

1301 GCTCATCTAT GGCGCCAAGC ATGCCTGGCG GAACGCCTCT CGATGTGTGG

1351 GCAGGATCCA GTGGTCCAAG CTGCAGGTGT TCGATGCCCG AGACTGCACC

1401 ACAGCCCACG GCATGTTCAA CTACATCTGT AACCATGTCA AGTATGCCAC 1451 CAACAAAGGG AATCTCAGGT CGGCCATCAC GATATTCCCT CAGAGGACTG 1501 ACGGCAAACA TGACTTCCGA GTGTGGAACT CGCAGCTCAT CCGCTACGCG 1551 GGCTACAAGC AGCCAGATGG CTCTACCTTG GGGGATCCAG CCAATGTGCA 1601 GTTCACGGAG ATCTGTATAC AGCAGGGCTG GAAAGCCCCA AGAGGCCGCT 1651 TCGACGTGCT GCCTCTCCTG CTTCAGGCCA ATGGCAATGA CCCTGAGCTC 1701 TTCCAGATCC CCCCAGAGCT GGTGCTGGAA GTGCCCATCA GGCACCCCAA 1751 GTTCGACTGG TTTAAGGACC TGGGGCTCAA ATGGTATGGC CTCCCCGCTG 1801 TGTCCAACAT GCTGCTGGAG ATCGGGGGCC TGGAGTTCAG CGCCTGTCCC 1851 TTCAGCGGCT GGTACATGGG CACAGAGATC GGCGTCCGTG ACTACTGTGA 1901 CAACTCTCGA TACAACATCC TGGAGGAAGT AGCCAAGAAG ATGGATTTGG 1951 ACATGAGGAA GACCTCGTCC CTCTGGAAGG ACCAAGCACT GGTGGAGATC 2001 AACATTGCTG TTCTATATAG CTTCCAGAGT GACAAGGTGA CCATCGTTGA 2051 CCACCACTCT GCCACGGAGT CCTTCATCAA ACACATGGAG AATGAATACC 2101 GCTGCAGAGG GGGCTGCCCC GCCGACTGGG TGTGGATTGT GCCTCCCATG 2151 TCGGGCAGCA TCACCCCTGT CTTCCACCAG GAGATGCTCA ACTATAGACT 2201 CACCCCGTCC TTTGAATACC AGCCTGATCC ATGGAACACC CACGTGTGGA 2251 AGGGCACCAA CGGGACCCCC ACGAAGCGGC GAGCTATCGG CTTTAAGAAA 2301 TTGGCAGAGG CCGTCAAGTT CTCAGCCAAG CTAATGGGGC AGGCCATGGC 2351 CAAGAGGGTC AAGGCGACCA TTCTCTACGC CACAGAGACA GGCAAATCAC 2401 AAGCCTATGC CAAGACCCTG TGTGAGATCT TCAAGCACGC CTTCGATGCC 2451 AAGGCAATGT CCATGGAGGA GTATGACATC GTGCACCTGG AGCACGAAGC 2501 CCTGGTCTTG GTGGTCACCA GCACCTTTGG CAATGGAGAC CCCCCTGAGA 2551 ACGGGGAGAA ATTCGGCTGT GCTTTAATGG AGATGAGGCA CCCCAACTCT 2601 GTGCAGGAGG AGAGAAAGTA CCCGGAACCC TTGCGTTTCT TTCCCCGTAA 2651 AGGGCCTTCC CTCTCCCATG TTGACTCTGA AGCCCACAGT CTGGTTGCTG 2701 CCCGTGACAG CCAACACAGG AGCTACAAGG TCCGATTCAA CAGCGTCTCC 2751 TCCTATTCTG ACTCCCGAAA GTCATCGGGC GACGGACCCG ACCTCAGAGA 2801 CAACTTTGAA AGTACTGGAC CCCTGGCCAA TGTGAGGTTC TCAGTGTTCG 2851 GCCTCGGCTC TCGGGCGTAC CCCCACTTCT GTGCCTTTGG GCATGCGGTG 2901 GACACCCTCC TGGAGGAACT GGGAGGGGAG AGGATTCTGA AGATGAGGGA 2951 GGGGGATGAG CTTTGCGGAC AGGAAGAAGC TTTCAGGACC TGGGCCAAGA

3001 AAGTCTTCAA GGCAGCCTGT GATGTGTTCT GCGTGGGGGA TGACGTCAAC 3051 ATCGAGAAGG CGAACAACTC CCTCATTAGC AATGACCGAA GCTGGAAGAG 3101 GAACAAGTTC CGCCTCACGT ATGTGGCGGA AGCTCCAGAT CTGACCCAAG 3151 GTCTTTCCAA TGTTCACAAA AAACGAGTCT CGGCTGCTCG ACTCCTCAGC 3201 CGCCAAAACC TGCAAAGCCC TAAGTCCAGC CGATCGACCA TCTTCGTGCG 3251 TCTCCACACC AACGGGAATC AGGAGCTGCA GTACCAGCCA GGGGACCACC 3301 TGGGTGTCTT CCCCGGCAAC CACGAGGACC TCGTGAATGC ACTCATTGAA 3351 CGGCTGGAGG ATGCACCGCC TGCCAACCAC GTGGTGAAGG TGGAGATGCT 3401 GGAGGAGAGG AACACTGCTC TGGGTGTCAT CAGTAATTGG AAGGATGAAT 3451 CTCGCCTCCC ACCCTGCACC ATCTTCCAGG CCTTCAAGTA CTACCTGGAC 3501 ATCACCACGC CGCCCACGCC CCTGCAGCTG CAGCAGTTCG CCTCTCTGGC 3551 CACTAATGAG AAAGAGAAGC AGCGGTTGCT GGTCCTCAGC AAGGGGCTCC 3601 AGGAATATGA GGAGTGGAAG TGGGGCAAGA ACCCCACAAT GGTGGAGGTG 3651 CTGGAGGAGT TCCCGTCCAT CCAGATGCCG GCTACACTTC TCCTCACTCA 3701 GCTGTCGCTG CTGCAGCCTC GCTACTACTC CATCAGCTCC TCTCCAGACA 3751 TGTACCCCGA CGAGGTGCAC CTCACTGTGG CCATCGTCTC CTACCACACC 3801 CGAGACGGAG AAGGACCAGT CCACCACGGG GTGTGCTCCT CCTGGCTCAA 3851 CAGAATACAG GCTGACGATG TAGTCCCCTG CTTCGTGAGA GGTGCCCCTA 3901 GCTTCCACCT GCCTCGAAAC CCCCAGGTGC CTTGCATCCT GGTTGGCCCA

3951 GGCACTGGCA TCGCACCCTT CCGAAGCTTC TGGCAACAGC GACAATTTGA 4001 CATCCAACAC AAAGGAATGA ATCCGTGCCC CATGGTTCTG GTCTTCGGGT

4051 GTCGACAATC CAAGATAGAT CATATCTACA GAGAGGAGAC CCTGCAGGCC 4101 AAGAACAAGG GCGTCTTCAG AGAGCTGTAC ACTGTCTATT CCCGGGAACC 4151 GGACAGGCCA AAGAAATATG TACAGGACGT GCTGCAGGAA CAGCTGGCTG 4201 AGTCTGTGTA CCGCGCCCTG AAGGAGCAAG GAGGCCACAT TTATGTCTGT 4251 GGGGACGTTA CCATGGCCGC CGATGTCCTC AAAGCCATCC AGCGCATAAT 4301 GACCCAGCAG GGGAAACTCT CAGAGGAGGA CGCTGGTGTA TTCATCAGCA

4351 GGCTGAGGGA TGACAACCGG TACCACGAGG ACATCTTTGG AGTCACCCTC

4401 AGAACGTATG AAGTGACCAA CCGCCTTAGA TCTGAGTCCA TCGCCTTCAT 4451 CGAAGAGAGC AAAAAAGACG CAGATGAGGT TTTCAGCTCC TAACTGGATC 4501 CTCCTGCCCC CGTGCGTGCG ATGTGGCGGC TGCCCCAAGT GCCCAAGTAA 4551 GGGCGGCCGC AGGTTGACTA AATTCGGACA CACACGGCTG AA TABLE 3

RPnNOS Amino acid sequence

MEENTFGV_QQI_QPNVISVRLFKRKVGGLGFLVKERVSKPPVIISDLIRGGAAEQSGLIQAGD

IILAVN DRPLVDLSYDSALEVLRGIASETHWLILRGPEGFTTHLETTFTGDGTPKTIRVTQPLGPPT

KAVDLS HQPSASKDQSLAVDRVTGLGNGP_QHA_QGHG_QGAGSVSQANGVAIDPTMKSTKANLQDIGEHD

ELLKEI EPVLSILNSGSKATNRGGPAKAEMKDTGI_QVDRDLDGKSHKAPPLGGDNDRVFNDLWGKDNV

PWLNN PYSEKE_QSPTSGK_QSPTKNGSPSRCPRFLKVKNWETDWLTDTLHLKSTLETGCTEHICMGS

IMLPSQ HTRKPEDVRTKD_QLFPLAKEFLD_QYYSΞIKRFGSKAHMDRLEEVNKEIESTSTYQLKDTELI

YGAKHA WRNASRCVGRI_QWSKL_QVFDARDCTTAHGMFNYICNHVKYATNKGNLRSAITIFPQRTDGKH

DFRVWN SQLIRYAGYK_QPDGSTLGDPANV_QFTEICIQ_QGWKAPRGRFDVLPLLLQANGNDPELFQIPP

ELVLEV PIRHPKFDWFKDLGLKWYGLPAVSNMLLEIGGLEFSACPFSGWYMGTEIGVRDYCDNSRYNI

LEEVAK KMDLDMRKTSSLWKD_QALVEINIAVLYSF_QSDKVTIVDHHSATESFIKHMENEYRCRGGCPA

DWVWIV PPMSGSITPVFH_QEMLNYRLTPSFEY_QPDPWNTHVWKGTNGTPTKRRAIGFKKLAEAVKFSA

KLMGQA

MAKRVKATILYATETGKSQAYAKTLCEIFKHAFDAKAMSMEEYDIVHLEHEALVLWTSTFG

NGDPPE NGEKFGCALMEMRHPNSV_QEERKYPEPLRFFPRKGPSLSHVDSEAHSLVAARDSQHRSYKVR

FNSVSS YSDSRKSSGDGPDLRDNFESTGPLANVRFSVFGLGSRAYPHFCAFGHAVDTLLEELGGERIL

KMREGD ELCG_QEEAFRTWAKKVFKAACDVFCVGDDVNIEKANNSLIΞNDRSWKRNKFRLTYVAEAPDL

TQGLSN VHKKRVSAARLLSR_QNL_QSPKSSRSTIFVRLHTNGN_QEL_QY_QPGDHLGVFPGNHEDLVNALI ERLEDA PPANHWKVEMLEERNTALGVISNWKDESRLPPCTIFQAFKYYLDITTPPTPLQLQQFASLA

TNEKEK QRLLVLSKGLQEYEEWKWGKNPTMVEVLEEFPSIQMPATLLLTQLSLLQPRYYSISSSPDMY

PDEVHL TVAIVSYHTRDGEGPVHHGVCSSWLNRIQADDWPCFVRGAPSFHLPRNPQVPCILVGPGTG

IAPFRS FWQQRQFDIQHKGMNPCPMVLVFGCRQSKIDHIYREETLQAKNKGVFRELYTVYSREPDRPK

KYVQDV LQEQLAESVYRALKEQGGHIYVCGDVTMAADVLKAIQRIMTQQGKLSEEDAGVFISRLRDDN

RYHEDI FGVTLRTYEVTNRLRSESIAFIEESKKDADEVFSS

TABLE 4

Human PnNOS Intro 16 Region (3' end of fragment)

A. Top Band

Length: 270 bp

1 TTGGGGATAC CNAGNGNTCA GAGNGGGGAG CNCATCGGG GGNAGANNAA

A

51 AGGTCCCNNC GACGTNAAGN GTGTGGNCCA TAGACATCC GGNNCGTGTC

C

101 NNNTCCACTC NCAGCATCTC CCCCNTCCCN GCCTNNTNC NNNNNNNNNN

N

151 NNNNNNNANN NNNTNNNCNN NNNNNNGAAϋ CCNNAACCC TGCGTTTCTT

T_ 201 TCCCCGTAGA GGGCCTTCCC TCTCCCATGN TGACTCTGA GCCCACAGTC

A.

251 TGGTTGCTGC CCGTGA_.CAAA

E Bottom band

Length: 285 bp

1 TTCTGGGTAC CCAGAGATCA GAGCCATGAG CACCTCNCT GGCAGNGCCT

A

51 TGCTCCCCAC CACCTTAAGC ATGTAAGCCC TCAACCTCC CTTCCCTNTC

C

101 CTTTCCACTC ACAGCATCTC CCCCATGCCC TGCCTCCTT AGGGAGAGAT

C

151 NNNNNNCNNT NCNNNNNNNN NNNNTNNNAA ACCGCTCTA TGGTACCCGG

G

201 AACCCTTGCG TTTCTTTCCC CGTAAAGGGC CTTCCCTCT CCATGNTGΛC

251 TCTGAAGCCC ACAGTCTGGT TGCTGCCCGT QAC_A£

(Table 4 continued on next page) C. Homology between Top band (A) and Bottom band (B) (3' end)

3 T T G G G G A T A C C N A G N G N T C A G A G N G G G G A G C N C A T C G G G A G G N A G A N N A A 50

I I I I I I I I : I I : I : I I I I I I : I I I I : I I I : | | I : | I : :

1 T T C T G G G T A C C C A G A G A T C A G A G C C A T G A G C A C C T C N C T A G G C A G N G C C T 50

51 A G G T C C N N C G A C G T N A A G T G T G G N C A G A C A T C C C G G N N C 100

I I I I ' I I I I I I I I I

51 T G A C C T T A A G C A T G T A A G C C C T C A A C C T C C C N T C 100

101 N N N T C C A C T C N C A G C A T C T C C C C C N T . C C C N G C C T N N T N C N N N N N N N N N N 149

: : : I I I I I I I : I I I I I I I I I I I I I ' I I I I : I I I I : : I : I : : : : : : : : : : 101 C T T T C C A C T C A C A G C A T C T C C C C C A T G C C C T G C C T C C T T C A G G G A G A G A T 150 . . . . . ISO N N N N N N N N A N N N N N T N N N C N N N N N N N N G. A A E C. C {J N 184 I I I I I I : I : : I ' I I : I I I : I I I I I : I I : I | : | | : : 151 N N N N N N C N N T N C N N N N N N N N N N N N T N N N A A A C C G C T C T A G T G G T A C C C G G 200 ^° 185 A A C C C T T G C S X X T C T Ϊ l C C C C g T A g A S £ S C I 1 £ £ C X £ T £ £ £ A T 2 G A C 234 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 201 A A C C C T T G C G T X X C T X X C C C C G T A A A S S S ^c- C χ T C C C T £ I C £ C A X G N T G A C 250 235 X C X G A A G C C C A C A G χ C χ G G T T G. C T G. C C C G T G. A C A A A 270 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 251 T C T G A A S C C C A C A G T C T G G X T G £ X S £ £ £ 2 Ϊ G A £ A A 285

301 ACTCTTTGA TGCTGA C

(Table 5 continued on next page)

TABLE 5

Human PnNOS Intron 16 Region (5¹ end of fragment)

P. top band

Length: 315 bp

1 TNTGGAGAT AGGCACCCC ACTCTGTGC AGAGGAGAG AAGTAAGTGA G A A G

51 ATGTCTNTT CCACCACCC CACCACCAC CCTCCGATG CATAGGATGC A C C T

101 CCTGGGGAA NCCTTGGCC GTCTTGACA CTCGGATTC TGTGGAGGCC N A T A

151 AGGCCGTGG TGCCAGTCT TGGANATGG TTTTTAGCT GTGATCTGGG

T T A G

201 CTTCACCGT GCTGANGTT TGGTCTCTG CTCANATCA ACCTCTCCTC G C A C

251 TGTGCCTCT ATTCCTTAG GATCNTGCN AGATTATCC GTTTANCTGG G T N T

301 ACTCTNTGA TGCTGA C

E. Bottom band

Length: 316

1 GTTGGAGAT AGCACCCCA ACTCTGTGCA AGAGGAGAG AAGTAAGTG G G A

51 ATGTCTNTT CCACCACCC CACCACCACC CCTCCGATG CATAGGATG A C T C

101 CCTGGGGAA CCCTTGGCC GTCTTGACAT CTCGGATTC TGTGGAGGC A A A C

151 AGGCCGTGG TGCCAGTCT TGGAGATGGA TTTTTAGCT GTGATCTGG T T G

201 CTTCACCGT GCTGACGTT TGGTCTCTGA CTCAAATCA ACCTCTCCT G C C C

251 TGTGCCTCT ATTCCTTAG GATCATGCAG AGATTATCC GTTTACCTG G T T G F. Homoloσy Between Top Band (C) and Bottom Band (D) (5' end)

1 T N T G G A G A T G A G G c A C C c C A A C T c T G T G C A A G A G G A G A G G A A G T A A G T G A

1 1 1 1 1 1 | 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 | 1 1 | 1 1 1 1 1 1 | 1 1 1 1 1 | 1 1 I I I I I I I I I I I I I I I I I I

1 G T T G G A G A T G A G G c A C c c C A A C T C T G T G C A A G A G G A G A G G A A G T A A G T G A

51 A T G T C T N T T A c C A c C A c c C c c A C C A C C A c C C c T C C G A T G T C A T A G G A T G C 1 I 1 1 1 1 1 1 1 | 1 1 1 1 1 1 1 1 1 1 | 1 1 1 t 1 1 1 | 1 1 1 1 1 1 1 r 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I I I I I I I I I I I I I I I I I I

51 A T G T C T N T T A c c A c c A c c c c C A c c A c C A C C C c T C C G A T G T C A T A G G A T G C 1

101 C C T G G G G A A N N c C T T G σ c c A G T c T T G A C A T C T C G G A T T C A T G T G G A G G C C 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I I I I I I I i I I I I I I I I I I

101 C c T G G G G A A A c c c T T G G c c A G T C T T G A C A T c T C G G A T T C A T G T G G A G G C C 1

151 A G G C C G T G G T T G c c A c T c T T T G G A N A T G G A T T T T T A G C T G G T G A T C T G G G 2

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I I I I I I I I I I I I I I I I I I

151 A G G C C G T G G T T G c c A G T c T T T G G A G A T G G A T T T T T A G C T G G T G A T C T G G G 2

201 C T T C A C C G T G G C T G A N G T T c T G G T C T c T G A c T C A N A T C A C A C C T C T C C T C 2 t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I I I I I I I I I I I I I I I I I

201 C T T c A C c G T G G C T σ A C G T T c T G G T c T C T G A c T C A A A T C A C A C C T C T C C T C 2

251 T G T G C C T C T G A T T c C T T A G T G A T C N T G c N N A G A T T A T G T T T A N C T G G

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I I I I I I I I I I = I I I I

251 T G T G c c T c T G A T T c c T T A σ T G A T c A T G c A G A G A T T A T G T T T A C C T G G

301 A C T c T N T G A c T G c T G A 3

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 301 A c T c T T T G A c T G c T G A 3

TABLE 6 Rat PnNOS Intron 16 Region (5' end of fragment)

Length: 500 bp

1 TNCNTNNNNN CCTTNGGATG NNNCANATTA NTGAACATC CTGNCNGAT C G

51 TCATAGGATA CCCTTGGGAA CCCTTGACCA ATGTGTCTT ACACCTAAG G A

101 CTTGTGGGAA GACCGGGACG TGGTGAACAG TCTTTGGAA TAGATTTTT A A

151 GCTGGCAGCC CAGGCTCTAC CGTGGCTAAT GTTCTGGTC CTGACTCAA T T

201 CGTGCCTCTC CTCTCTGCCT CCAATTCCTT AGTTATCAA CCGAGATTG G T

251 ACTATTTACC TGGACCCTT GACTGCTGAG AACCTAACC GAAAAAAAA A

301 ACAAAGGTGG GANANTCTTT GGAAAGCTTA NTCCCTCCT TGCCTTTGA c A

351 ACCCACACTG GACTTCAGTG GGAGCCATTC TGTCCTTAN ACTGCCCCG A T

401 CTGGCGCTCA CTGTGGTTGT GAANCTAAGG TCNAGTTCA AGGGTAAAA A A

451 TAANATACCT GCGTTTCATA CACTCTTGAA GGGGTCAAC CCTGTTCTA T A

Rat PnNOS Intro 16 Region (3' end of fragment)

Length: 507 bp

1 TTTNGGAGAA GCCTGNAGTA CATGNGTTGA CCCNTTANAG NGCGGANGAA

51 ACGCAGGTAT AGANTATTTA CCGNTANAAC CAGACCCTNG TTGANGACCA

101 NAANNNGGGC CAGGGGGGGN AGGTGCAATN NCNGAAGGNG NCCCACAGAG

151 GTNNGGTTCG GGTAGAGCAG ATCGTGGGAG GTAAGGATTT ACGNGAGATT

201 CTNNNNCNGA GTGCAGTTAG NGGGTGGGNN TCCTCATGAG AGCACAGGAC

251 CAAACCCAGC TTATGGGTCG CCAGGGATCA TAGCCATGAG CACCTCCTAG

301 GCATTCCCCT GCTCCCCACC ACCTCGAGCA TCTTATCCCT CAATCTCCCC 351 ACCTTTCCCC TTTTCATTCA TAGCAGCTCC CCTACTCCTG CCTCCTTCAG

401 GGAGAGATAG GGCGCCATCC GTTCTCCGCT CTAGGTA££C GGAACCCTTG 451 CGTTTCTTTC CCCGTAANGG CTTCCNTCTC CNTAATNGGG NCGAACANGA 501 GNAANGN

APPENDIX B

List of References

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Bredt DS, Hwang PM, Glatt CE, Lowenstein C, Reed RR, Snyder SH (1991) Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase . Nature 351:714-718

Bredt DS, Snyder SH (1994) Nitric Oxide: A Physiologic Messenger Molecule. Annu Rev Biochem 63:175-195

Burnett AL (1995) Nitric oxide control of lower genitourinary tract functions: A review. Urology 45:1071-1083

Charles IG, Chubb A, Gill R, Clare J, Lowe PN, Holmes LS, Page M, Keeling JG, Moncada S, Riveros-Moreno V (1993) Cloning and expression of a rat neuronal nitric oxide synthase coding sequence in a baculovirus/insect cell system. Biochem Biophys Res Comm 196:1481-1489

Ehren I, Iversen H, Jansson O, Adolfsson J, Wiklund NP (1994) Localization of nitric oxide synthase activity in the human lower urinary tract and its correlation with neuroeffector responses. Urology 44:683-687

Garban H, Vernet D, Freedman A, Rajfer J, Gonzalez -Cadavid NF (1995) Effect of aging on nitric oxide-mediated penile erection in the rat. Amer J Physiol 268 :H467-H475

Garban H, Marquez D, Cai L, Rajfer J, Gonzalez -Cadavid NF (1995) Restoration of normal penile erectile response in aged rats by long-term treatment with androgens . Biol Reprod, 53 :1365-1372

Gonzalez-Cadavid NF, Rajfer J (1996) Nitric oxide and other neurotransmitters of the corpus cavernosum. In: "Textbook of

Andrology: Relevant Issues in Male Infertility and Sexual

Dysfunction" . ed. by WJG Hellstrom, Sp [ringer-Verlag, New York, in press.

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Hall AV, Antoniou H, Wang Y, Cheung AH, Arbus AM, Olson SL, Lu WC, Kau C-L, Marsden PA (1994) Structural organization of the human neuronal nitric oxide synthase gene (NOS1) . J Biol Ch^τη 269 :33082-33090

Huang PL, Dawson TM, Bredt DS, Snyder SH, Fishman MC (1993) Targeted disruption of the neuronal nitric oxide synthase gene. Cell 75:1273-1286

Hung A, Vernet D, Rajavashisth T, Rodriguez JA, Rajfer J, Gonzalez-Cadavid NF (1995) Expression of the inducible nitric oxide synthase in smooth muscle cells from the rat penile corpora cavernosa. J Androl . 16:469-481

Kishimoto J, Spurr N, Liao M, Lizhi L, Emson P, Xu W (1992) Localization of brain nitric oxide synthase (NOS) to human chromosome 12. Genomics 14:802-804

Lugg J, Rajfer J, Gonzalez-Cadavid NF (1995) Dihydrotestosterone is the active androgen in the maintenance of nitric oxide mediated penile erection in the rat. Endocrinology 136:1495-1501

Lugg J, Gonzalez-Cadavid NF, Rajfer J (1995) The role of nitric oxide in erectile function. J Androl 16:2-6

Lugg J, Ng Ch, Rajfer J, Gonzalez-Cadavid NF (1996)

Cavernosal nerve stimulation reverses castration- induced decrease in rat penile nitric oxide synthase activity. Am J Phvai.ol. 271: E 354-E 361.

McConnell JD (1991) The pathophysiology of benign prostatic hyperplasia. J Androl 12:356-363.

Ogura T, Yokoyama T, Fujisawa H, Kurashima Y, Esumi H (1993) Structural diversity of neuronal nitric oxide synthase mRNA in the nervous system. Biochem Biophys Res Comm 193:1014-1022 - Penson DF, Ng Ch, Cai L, Rajfer J, Gonzalez-Cadavid NF (1996) Androgen and pituitary control of nitric oxide synthase activity and erectile function in the rat penis. Biol Reprod, 55: 567- 574.

Penson DF, Ng Ch, Cai L, Rajfer J, Gonzalez-Cadavid NF (1996) Androgen dependence of neuronal nitric oxide synthase content and erectile function in the rat penis. In: The Biology of Nitric Oxide, ed by J Stamler, S Gross, S Moncada, AE Higgs, Portland Press, London, 245

Penson DF, Ng Ch, Rajfer J, Gonzalez-Cadavid NF (1996) Adrenal control of erectile function and nitric oxide synthase in the rat penis. Endocrinology, submitted.

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Claims

CLAIMSWe claim:

1. A cDNA molecule which encodes all or a portion of a mammalian neuronal nitric oxide synthase having the nucleotide sequence shown in Table 2, and designated for the rat as RPnNOS to differentiate it from the sequence of the cerebellar nNOS enzyme, designated for the rat as RCnNOS.

2. A cDNA molecule which encodes all or a portion of a 102 nucleotide sequence comprising the nucleotides sequence between nucleotides 2865 and 2967 in the rat sequence of Table 2, and the corresponding 102 bp sequence in the human homologous to the 102 bp rat sequence, which in both species is designated as PnNOS102.

3. A DNA vector molecule comprising said nucleotide sequence of claim 1.

4 A DNA vector molecule comprising said nucleotide sequence of claim 2.

5. A cDNA molecule of claim 1 which encodes the amino acid sequence shown in Table 3 for RPnNOS, and the homologous human cDNA consisting in the insertion of PnNOS102 in the human cerebellar nNOS sequence between amino acids 839 and 873, in the junction between exons 16 and 17.

6. A cDNA molecule of claim 2 wherein the amino acid sequence is encoded between amino acids 839 and 873 in the sequence of Table 3.

7, A cDNA molecule of claim 1 labeled with a detectable moiety .

8. A cDNA molecule of claim 2 labeled with a detectable moiety.

9. A cDNA molecule which hybridizes with the nucleotide sequence of claim 1.

10. A cDNA molecule which hybridizes with the nucleotide sequence of claim 2.

11. Recombinant host cells comprising the cDNA molecule of claim 1.

12. Recombinant host cells comprising the cDNA molecule of claim 2.

13. The recombinant or native protein encoded by the nucleotide sequence of Table 2, designated as PnNOS.

14. Peptides corresponding to 4 to 34 amino acids in the amino acid sequence encoded between amino acids 839 and 873 in the sequence of Table 3.

15. Antisera and purified antibodies elicited with natural or synthetic peptides encoded by the nucleotide sequence of Table 2.

16. A method or treating erectile dysfunction comprising the steps of : a) providing a nucleotide sequence selected from the nucleotide sequence as in Table 2 and the nucleotide sequence between nucleotides 2865 and 2967 in the rat sequence of Table 2 and the corresponding 102 bp sequence in the human homologous to the 102 bp rat sequence, which in both species is designated as PnNOS102; b) inserting said nucleotide sequence in a liposome, plasmid, adenoviral, retroviral, or other DNA vectors, alone or in combination; and c) administering said DNA vector directly into the penis via at least one of single or multiple injections, continuous infusion, topical application (lotions, creams, ointment, patches, or similar) or intraurethral delivery, systemic injection, infusion, topical application, and orally, with the purpose of gene therapy.

17. A method for treating erectile dysfunction comprising the steps of: a) transforming host cells with a nucleotide sequence selected from the nucleotide sequence in Table 2 and the nucleotide sequence between nucleotides 2865 and 2967 in the rat sequence of Table 2 and the corresponding 102 bp sequence in the human homologous to the 102 bp rat sequence, which in both species is designated as PnNOS102; b) administering said transformed cells into a penis by direct injection or implantation in the penis, with or without devices to confine them in a restricted area.

18. A method for treating erectile dysfunction comprising the steps of : a) providing an amino acid sequence selected from PnNOS and an amino acid sequence encoded between amino acids 839 and 873 in the sequence of Table 3; and b) administering said sequence to the penis for direct enzyme therapy.

19. A method for treating erectile dysfunction as in claim 14, further comprising the steps of: a) providing at least one of a protein inhibitor and enzyme cofactor, to regulate the expression or activity of said amino acid sequence.

20. A method for treating the urinary voiding dysfunction associated with benign prostatic hyperplasia comprising the steps of : a) providing a nucleotide sequence selected from the nucleotide sequences as in Table 2 and the nucleotide sequence between nucleotides 2865 and 2967 in the rat sequence of Table 2 and the corresponding 102 bp sequence in the human homologous to the 102 bp rat sequence, which in both species is designated as PnNOS102; b) inserting said nucleotide sequence in a liposome, plasmid, adenoviral, retroviral, or other DNA vectors, alone or in combination; and c) administering said DNA vector directly into the prostate via at least one of single or multiple injections, continuous infusion, topical application (lotions, creams, ointment, patches, or similar) or intraurethral delivery, systemic injection, infusion, topical application, and orally, with the purpose of gene therapy.

21. A method as in claim 17 for treating urinary voiding dysfunction conditions including incontinence, bladder instability, outlet obstruction, and related, wherein said nucleotide sequence is administered to the bladder or urethra.

22. A method as in claim 17 for treating premature ejaculation, wherein said nucleotide sequence is administered to the prostate.

23. Any diagnostic, research, or clinical method based on the detection, histological localization, or quantization of a nucleotide sequence selected from the nucleotide sequence of Table 2 and the nucleotide sequence between nucleotides 2865 and 2967 in the rat sequence of Table 2, and the corresponding 102 bp sequence in the human homologous to the 102 bp rat sequence, which in both species is designated as PnNOS102; and amino acid sequence of Table 3.

24. A method of treatment of erectile dysfunction in a patient comprising the steps of; a) providing an agent which produces an increase in in vivo tissue PnNOS or nNOS level; b) introducing an effective amount of a PnNOS or nNOS agent into penile tissue.

25. An erectile dysfunction treatment method as in claim

24 wherein: a) said agent is selected from the group consisting essentially of PnNOS or nNOS inducers, nNOS protein, nNOS cDNA, and nNOS cDNA- ransformed penile cells or nNOS cDNA-transformed tissue .

26. An erectile dysfunction treatment method as in claim

25 wherein said nNOS inducer is a penile nNOS inducer, said nNOS protein is a penile nNOS protein, said nNOS cDNA is penile nNOS cDNA, and said nNOS cDNA-transformed cells or tissue are penile cells or tissue.

27. An erectile dysfunction treatment method as in claim

24 wherein: a) said step of introduction includes introducing nNOS inducers directly into in vivo penis tissue in an intermittent, continuous or time release basis.

28. An erectile dysfunction treatment method as in claim

25 wherein: a) said agent is nNOS protein produced in vitro.

29. An erectile dysfunction treatment method as in claim 28 wherein said nNOS protein is penile nNOS protein.

30. An erectile dysfunction treatment method as in claim 28 wherein said nNOS protein is administered locally by continuous infusion or repeated injection, topical application, intraurethral administration, microcapsules , or related procedures .

31. An erectile dysfunction treatment method as in claim 25 wherein: a) said agent is penile or other nNOS cDNA administered locally by continuous infusion or repeated injection, topical application, intraurethral administration, microcapsules, or related procedures, as well as by systemic routes .

32. An erectile dysfunction treatment method as in claim 25 wherein: a) said agent is nNOS cDNA-transformed penile corpora cavernosa cells or tissue implanted directly into the corpora cavernosa or tissue implanted directly into the corpora cavernosa or retained in microcapsules, pellets or other procedures intended to limit the release of cells into the general circulation.

33. An erectile dysfunction treatment method as in claim 25 wherein: a) said agent is penile or other nNOS or eNOS cDNAs administered locally by continuous infusion or repeated injection, topical application, intraurethral administration, microcapsules, or related procedures.

34. An erectile dysfunction treatment method as in claim 25 wherein: a) said agent is nNOS or eNOS cDNA transferred penile corpora cavernosa cells or tissues implanted directly into the corpora cavernosa or tissue implanted directly into the corpora cavernosa or retained in microcapsules, pellets or other procedures intended to limit the release of cells into the general circulation.

35. An erectile dysfunction treatment method as in claim 25 wherein: a) said agent is a biological modulator of endogenous or exogenous penile NOS.

36. A cDNA expression construct comprising plasmid pBS RPnNOS .

37. A DNA fragment consisting essentially of a structural gene encoding a protein selected from the group consisting of eNOS , nNOS, eNOS and nNOS.

38. A DNA fragment as in claim 37 wherein said DNA fragment is cDNA.

39. A DNA fragment as in claim 38 wherein the DNA fragment is recombinant DNA.

40 A DNA fragment as in claim 39 operably linked to a promoter to express said gene.

41. A recombinant host expressing a gene of claim 37.

42. A DNA sequence coding for induced nitric oxide synthase enzyme .

43. The sequence of claim 42 wherein the DNA is a cDNA.

44. The sequence of claim 42 derived from a natural source .

45. The sequence of claim 44 derived from the group consisting of rat PSMC and human PSMC, said cDNA from rat PSMC having the DNA sequence, in a 5 ' to 3 ' direction, as shown in Table 2 , said cDNA from human PSMC having the DNA sequence in a 5 ' to 3 ' direction, as shown in Tables 4 and 5.

46. A cDNA expression construct which comprises: a) a promoter fragment corresponding to a PnNOS gel which functions in a host organism; b) a cDNA segment, said cDNA codes for an nNOS, a protein expressable in the penis, bladder or urethra; c) said cDNA segment being in an orientation with the promoter fragment such that it is expressed in the host to produce the correspondence, recombinant protein.

47. The expression vector of claim 46 wherein said host organisms are selected from animal, bacterial, yeast cells or PSMC.

498 A transfected host organism transformed by the expression construct of claim 46.

49. A transfected host organism comprising: a) a cell having disposed therein a DNA expression vector for coding penile nitric oxide synthase enzyme. b) said cell is a microbial or mammalian cell.

50. An improved tissue, the tissue relying on the production of nitric oxide for normal function, the improvement comprising introduction of transfected cells to the host tissue, said cells having disposed therein a DNA expression vector coding for nitric oxide synthase.

51. Nitric oxide synthase enzymes derived from PSMC cDNA, said nitric oxide synthase enzyme selected from the group consisting of rat nNOS and human nNOS, said rat nNOS having the amino acid sequence as shown in Table 3 , and said human nNOS having the amino acid sequence as shown in Appendix C .

52. A eukaryotic expression vector:

(a) pc DNA3 RPiNOΞ consisting essentially of pcDNA3 containing a penile eNOS coding region, a plasmid and a promoter region.