Intravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugates.
The present study investigated the potential of intravesical instillation for localized reduction of NGF (nerve growth factor) expression in the urinary bladder. Overexpression of NGF has been linked to the pathogenesis of interstitial cystitis (IC). A minimum free energy algorithm was used to predict suitable regions in mRNA of rat betaNGF, which can be targeted for an antisense approach. The candidate antisense oligos were evaluated for their ability to reduce NGF expression in vitro by cotransfecting HEK293 cells with NGF cDNA. A single oligonucleotide ODN sequence was chosen for testing in an acute cystitis model in rat induced by cyclophosphamide. Overexpression of NGF is known to mediate inflammation of bladder in this model. For improved stability, antisense ODN was replaced with antisense peptide nucleic acid (PNA) and its penetration into bladder was facilitated by tethering TAT peptide sequence. Rat bladders were instilled with either antisense or its scrambled control prior to cystitis induction. Cystometrograms performed on rats under urethane anaesthesia exhibited bladder contraction frequency that was significantly decreased in the antisense treated rats than rats treated with the control. NGF immunoreactivity was also decreased in the urothelium of the antisense treated bladders. Our findings demonstrate the feasibility of using TAT-PNA conjugates for intravesical antisense therapy.
Tyagi
Pradeep
P
Department of Urology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
Banerjee
Rajkumar
R
Basu
Soumitra
S
Yoshimura
Naoki
N
Chancellor
Michael
M
Huang
Leaf
L
D000283:Administration, Intravesical; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003520:Cyclophosphamide / Q000633:toxicity; D003556:Cystitis / Q000139:chemically induced / Q000235:genetics / Q000628:therapy*; D016373:DNA, Antisense / Q000008:administration & dosage / Q000235:genetics; D015696:Gene Products, tat / Q000008:administration & dosage* / Q000235:genetics*; D015316:Genetic Therapy; D006678:HIV / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D020932:Nerve Growth Factor / Q000235:genetics; D009690:Nucleic Acid Conformation; D020135:Peptide Nucleic Acids / Q000008:administration & dosage* / Q000235:genetics*; D012313:RNA / Q000737:chemistry / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D016373:DNA, Antisense; D015696:Gene Products, tat; D020135:Peptide Nucleic Acids; D054322:tat Gene Products, Human Immunodeficiency Virus; D012313:RNA; D003520:Cyclophosphamide; D020932:Nerve Growth Factor