Antisense and short hairpin RNA (shRNA) constructs targeting PIN (Protein Inhibitor of NOS) ameliorate aging-related erectile dysfunction in the rat.
BACKGROUND
Over-expression of penile neuronal nitric oxide synthase (PnNOS) from a plasmid ameliorates aging-related erectile dysfunction (ED), whereas over-expression of the protein inhibitor of NOS (PIN), that binds to nNOS, increases ED.


OBJECTIVE
To improve this form of gene therapy for ED by comparing the electrical field response of short hairpin RNA (shRNA) for PIN with that of antisense PIN RNA.


METHODS
Both shRNA and antisense RNA gene therapy vectors increased intracavernosal pressure in aged rats.


METHODS
PIN small interfering RNA (siRNA), and plasmid constructs for cytomegalovirus promoter plasmid vector (pCMV-PIN), pCMV-PIN antisense RNA, pSilencer2.1-U6-PIN-shRNA; and pSilencer2.1-U6-randomer-shRNA were prepared and validated by transfection into HEK293 cells, determining the effects on PIN expression by Western blot. Plasmid constructs were then injected, followed by electroporation, into the penile corpora cavernosa of aged (20-month-old) Fisher 344 rats and, 1 month later, the erectile response was measured by intracavernosal pressure increase following electrical field stimulation (EFS) of the cavernosal nerve. PIN was estimated in penile tissue by Western blot and real-time reverse transcriptase-polymerase chain reaction. Cyclic guanosine monophosphate (cGMP) measurements were conducted by competitive enzyme immunoassay (EIA). Immunohistofluorescence detected PIN in corporal tissue sections.


RESULTS
In cell culture, PIN siRNA and plasmid-expressed pU6-PIN-shRNA effectively reduced PIN expression from pCMV-PIN. pSilencer2.1-U6-PIN-shRNA corrected the impaired erectile response to EFS in aged rats and raised it above the value for young rats, more efficiently than pCMV-PIN antisense RNA. PIN mRNA expression in the penis was decreased by >70% by the shRNA but remained unaffected by the antisense RNA, whereas PIN protein expression was reduced in both cases, particularly in the dorsal nerve. PIN antisense increased cGMP concentration in treated tissue by twofold.


CONCLUSIONS
pSilencer2.1-U6-PIN-shRNA gene therapy was more effective than the antisense PIN mRNA in ameliorating ED in the aged rat, thereby suggesting that PIN is indeed a physiological inhibitor of nNOS and nitrergic neurotransmission in the penis.
Magee
Thomas R
TR
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA;; Department of Urology, David Geffen School of Medicine at University of California-Los Angeles, Los Angeles, CA, USA;. Electronic address: tmagee@labiomed.org.
Kovanecz
Istvan
I
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA.
Davila
Hugo H
HH
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA.
Ferrini
Monica G
MG
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA;; Department of Urology, David Geffen School of Medicine at University of California-Los Angeles, Los Angeles, CA, USA.
Cantini
Liliana
L
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA.
Vernet
Dolores
D
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA.
Zuniga
Freddi I
FI
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA.
Rajfer
Jacob
J
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA;; Department of Urology, David Geffen School of Medicine at University of California-Los Angeles, Los Angeles, CA, USA;; Division of Urology, Harbor-UCLA Medical Center, Torrance, CA, USA.
Gonzalez-Cadavid
Nestor F
NF
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA;; Department of Urology, David Geffen School of Medicine at University of California-Los Angeles, Los Angeles, CA, USA;; Division of Urology, Harbor-UCLA Medical Center, Torrance, CA, USA.
D000375:Aging / Q000235:genetics*; D000818:Animals; D015153:Blotting, Western; D004195:Disease Models, Animal; D007172:Erectile Dysfunction / Q000188:drug therapy* / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D008297:Male; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics*; D052248:Nitric Oxide Synthase Type I; D010410:Penile Erection / Q000187:drug effects / Q000502:physiology; D010413:Penis / Q000201:enzymology / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D051381:Rats; D011916:Rats, Inbred F344
D034741:RNA, Small Interfering; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat