WO1997030066A1 - NOVEL PENILE NEURONAL NITRIC OXIDE SYNTHASE (PnNOS) AND APPLICATIONS FOR DIAGNOSIS AND TREATMENT OF UROGENITAL DISORDERS - Google Patents
NOVEL PENILE NEURONAL NITRIC OXIDE SYNTHASE (PnNOS) AND APPLICATIONS FOR DIAGNOSIS AND TREATMENT OF UROGENITAL DISORDERS Download PDFInfo
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- WO1997030066A1 WO1997030066A1 PCT/US1997/001565 US9701565W WO9730066A1 WO 1997030066 A1 WO1997030066 A1 WO 1997030066A1 US 9701565 W US9701565 W US 9701565W WO 9730066 A1 WO9730066 A1 WO 9730066A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
- C12N9/0075—Nitric-oxide synthase (1.14.13.39)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
Definitions
- a novel neuronal nitric oxide synthase f 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.
- PnNOS penile nNOS
- NO nitric oxide
- NOS nitric oxide synthase
- 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 m unocytocnemistry and in situ hybridization.
- MIP maximum intracavernosal pressure
- vasodilators vasodilators
- penile NOS activity by the arginine/citrulline conversion assay
- penile NOS content by quantitative western blot procedures
- penile NOS localization by m unocytoc
- the risk factors so far characterized m 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.
- 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.
- 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.
- NO 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 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.
- Bladder instability 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.
- 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
- 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.
- RPnNOS rat penile neuronal nitric oxide synthase
- 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
- 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.
- 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 RPSMC1/2.
- 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.
- 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;
- 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
- RPnNOS Rat Penile Neuronal Nitric Oxide Synthase
- 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
- 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.
- nNOS fulfills in triggering the erectile response through the release of NO as a neurotransmitter in the nerve terminals of the penis.
- 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.
- 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.
- 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.
- 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.
- HPnNOSJ human penis contains PnNOS
- 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.
- the biological modulation of PnNOS activity is disclosed.
- 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.
- Rat and human penile smooth muscle cells 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) .
- These primers contain Eco Rl 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.
- primers, probes, and other sequences 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.
- our primers N07 and N08 encompass a 460 bp fragment in a region 5' to the N01/N02 sequence.
- Primer NOS is a 20mer
- 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.
- 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.
- gel A 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 ⁇ -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 eydig cells (lanes 5,6) , thus indicating that this mRNA is generally expressed in rat vascular SMC.
- EXAMPLE 2 EXPRESSION OF A NOVEL FORM OF nNOS mRNA IN THE RAT PENIS
- nNOS 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 .
- 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 (Transduc ion 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 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.
- 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.
- 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.
- 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 -N01/2, 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.
- 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.
- the sequencing 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) .
- the 102 bp fragment was generated from penile mRNA but not from cerebellar mRNA.
- the RT was done with either N02 or N06, and the subsequent PCR with NOl, the X 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.
- the 102 bp fragment was generated from both the penile and cerebellar parent fragments, but remained undetectable in the RPSMC mixes.
- 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) .
- 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 BanI . 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 .
- This example demonstrates the procedure for cloning and sequencing the cDNA encoding the novel rat nNOS protein.
- Total RN A 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 EcoRI adaptors, cleavage of the Xhol site, and cloning into the XhoI/EcoRI sites of ⁇ Zap 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 RPnNOS5/6 and RPnNOS102.
- RPnNOS 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) .
- RPnNOS 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) .
- l ⁇ g 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.l (Invitrogen, San Diego, CA) and designated pZRCnNOSATG-RCI4. It was sequenced in both directions.
- both pBSRPnNOSl and pZRCnN0SATG-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 3' region of pBSRPnNOSl is ligated into Bstll07I-XhoI cut pZRCnNOSATG-RCI4 to give the full length clone, pZRPnNOS.
- RPnNOS was cloned into the multi cloning site of pCDNA3 (Invitrogen, San Diego, CA) by digesting both pZRPnNOS and pCDNA3 with EcoRI and Xhol restriction enzymes. pCDNA3 and the RPnNOS cDNA fragment were gel purified and ligated as before to give pCRPnNOS.
- Fig. 12 top left, presents the distinctive pattern obtained with the RPnNOS specific probe, that is RPnNOS102, 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.
- 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.
- 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.
- 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) .
- 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.
- 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.
- NO is the noncholinergic-nonadrenergic mediator of smooth muscle relaxation in these organs, and as such elicits and maintains penile erection.
- PnNOS controls penile erection and in general the tone of the lower urogenital tract.
- 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
- HUMAN PENIS 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.
- CnNOS rat and human tissues
- 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.
- 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. 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) .
- 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 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.
- HPnNOS gene 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.
- 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.
- EXAMPLE 6 APPLICATIONS OF PnNOS AND THEIR RELATED PRODUCTS FOR THE DIAGNOSIS
- nNOS novel nNOS gene
- PnNOS novel nNOS gene
- 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
- 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) bio edical research, mainly neurobiology.
- PnNOS penile specific nNOS isoform
- 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.
- the PnNOS gene is apparently subject to a transcriptional regulation in this organ different from the one operating with CnNOS elsewhere in the organism.
- 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.
- PnNOS-based therapy HPnNOS cDNA and recombinant protein
- 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.
- 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.
- HPSMC human penile smooth muscle cells
- 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 .
- 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 isofor -specific modulation of NOS enzyme activity.
- 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.
- 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. (HPnN0S+) , 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.
- 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 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.
- VIP vasoactive intestinal polypeptide
- CGRP calcitonin gene related peptide
- 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.
- PnNOS promoter-driven plasmids for the therapy of conditions affecting the bladder, urethra, etc., such as bladder cancer, cystitis, prostate growth in BPH, etc.
- compositions 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.
- RPSMC mRNA mRNA isolated from rat penile smooth muscle cells
- RC mRNA " " " " 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
- NO-E16, NO-E17 primers for exons 16 and 17 (F: forward;
- RC1-RC4 primers based on RPnNOS102 sequence
- 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
- RPnN0S5/6 ibid
- 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
- RPSMC1/2 RT/PCR fragment generated from RPSMC RNA with primers
- Dihydrotestosterone is the active androgen in the maintenance of nitric oxide mediated penile erection in the rat.
- 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. Hiol. Repro • 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.
- 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.
Abstract
Description
Claims
Priority Applications (1)
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AU18509/97A AU1850997A (en) | 1996-02-15 | 1997-02-13 | Novel penile neuronal nitric oxide synthase (pnnos) and applications for diagnosis and treatment of urogenital disorders |
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US1170796P | 1996-02-15 | 1996-02-15 | |
US60/011,707 | 1996-02-15 | ||
US1737196P | 1996-05-10 | 1996-05-10 | |
US60/017,371 | 1996-05-10 | ||
US3155096P | 1996-12-03 | 1996-12-03 | |
US60/031,550 | 1996-12-03 |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1379876A2 (en) * | 2001-04-18 | 2004-01-14 | Innodia | Novel method for screening inhibitors of the linkage between the neuronal nitric oxide synthase associated protein and the protein inhibiting neuronal nitric oxide synthase |
WO2005018620A2 (en) * | 2003-08-26 | 2005-03-03 | Cell Center Cologne Gmbh | Use of nitric oxide synthase (nos) cofactor for the treatment of sexual dysfunction |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5171217A (en) * | 1991-02-28 | 1992-12-15 | Indiana University Foundation | Method for delivery of smooth muscle cell inhibitors |
US5439938A (en) * | 1993-04-07 | 1995-08-08 | The Johns Hopkins University | Treatments for male sexual dysfunction |
US5594032A (en) * | 1994-11-10 | 1997-01-14 | Gonzalez-Cadavid; Nestor F. | Amelioration of human erectile dysfunction by treatment with iNOS, inducers of iNOS or iNOS cDNA |
-
1997
- 1997-02-13 AU AU18509/97A patent/AU1850997A/en not_active Abandoned
- 1997-02-13 WO PCT/US1997/001565 patent/WO1997030066A1/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5171217A (en) * | 1991-02-28 | 1992-12-15 | Indiana University Foundation | Method for delivery of smooth muscle cell inhibitors |
US5439938A (en) * | 1993-04-07 | 1995-08-08 | The Johns Hopkins University | Treatments for male sexual dysfunction |
US5594032A (en) * | 1994-11-10 | 1997-01-14 | Gonzalez-Cadavid; Nestor F. | Amelioration of human erectile dysfunction by treatment with iNOS, inducers of iNOS or iNOS cDNA |
Non-Patent Citations (7)
Title |
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BIOCHEM. BIOPHYS. RES. COMMUN., 04 September 1996, Vol. 226, No. 1, MAGEE et al., "Cloning of a Novel Neuronal Nitric Oxide Synthase Expressed in Penis and Lower Urinary Tract", pages 145-151. * |
BIOCHEM. BIOPHYS. RES. COMMUN., 30 June 1993, Vol. 193, No. 3, OGURA et al., "Structural Diversity of Neuronal Nitric Oxide Synthase mRNA in the Nervous System", pages 1014-1022. * |
FEBS LETT., January 1993, Vol. 316, No. 2, NAKANE et al., "Cloned Human Brain Nitric Oxide Synthase is Highly Expressed in Skeletal Muscle", pages 175-180. * |
J. ANDROL., November/December 1995, Vol. 16, No. 6, HUNG et al., "Expression of Inducible Nitric Oxide Synthase in Smooth Muscle Cells from Rat Penile Corpora Cavernosa", pages 469-481. * |
J. BIOL. CHEM., 30 December 1994, Vol. 269, No. 52, HALL et al., "Structural Organization of the Human Neuronal Nitric Oxide Synthase Gene (NOS1)", pages 33082-33090. * |
J. NEUROCHEM., July 1994, Vol. 63, No. 1, FUJISAWA et al., "Expression of Two Types of Nitric Oxide Synthase mRNA in Human Neuroblastoma Cell Lines", pages 140-145. * |
NATURE, 27 June 1991, Vol. 351, BREDT et al., "Cloned and Expressed Nitric Oxide Synthase Structurally Resembles Cytochrome P-450 Reductase", pages 714-718. * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1379876A2 (en) * | 2001-04-18 | 2004-01-14 | Innodia | Novel method for screening inhibitors of the linkage between the neuronal nitric oxide synthase associated protein and the protein inhibiting neuronal nitric oxide synthase |
WO2005018620A2 (en) * | 2003-08-26 | 2005-03-03 | Cell Center Cologne Gmbh | Use of nitric oxide synthase (nos) cofactor for the treatment of sexual dysfunction |
WO2005018620A3 (en) * | 2003-08-26 | 2005-07-28 | Cell Ct Cologne Gmbh | Use of nitric oxide synthase (nos) cofactor for the treatment of sexual dysfunction |
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