A system for the enhancement of adenovirus mediated gene transfer to uro-epithelium.
OBJECTIVE
Recombinant adenovirus has been used widely as an in vivo gene transfer vector, although its transfection efficiency in bladder tissue is limited. Several studies have indicated that the bladder surface glycosaminoglycan (GAG) layer functions as a nonspecific anti-adherence factor and possibly as a first line anti-infection defense mechanism. We determined whether recombinant adenovirus mediated gene transfer could be enhanced in intact bladders by HCl pretreatment and by alterations in the GAG layer.


METHODS
In vitro viral transfection efficiencies with and without the GAG analog pentosan polysulfate (Sigma Chemical Co., St. Louis, Missouri) were determined in bladder muscle and urothelial cells. Immunocytochemical studies and Western blot analysis were performed to determine whether urothelial cells possessed the Coxsackievirus and adenovirus receptor. Rat bladders were intravesically pretreated with HCl at various concentrations and for various periods. After 60 mM. HCl pretreatment for 10 minutes 2 x 109 pfu of recombinant adenovirus carrying the Escherichia coli LacZ gene were intravesically instilled into the bladders.


RESULTS
Adenoviral infection of urothelial cells was significantly reduced in the presence of pentosan polysulfate in vitro. Coxsackievirus and adenovirus receptor expression was detected in urothelial cells in vivo and in vitro. Bladders pretreated with HCl resulted in an alteration of the bladder GAG layers. After intravesical gene instillation reporter gene analyses using X-5-bromo-4-chloro-3-inodolyl beta-D-galactopyranoside (Sigma Chemical Co.) showed approximately 80% urothelial cell transfection efficiency in bladders pretreated with HCl. However, less than 10% of the urothelial cells expressed the transfected gene in control HCl untreated bladders.


CONCLUSIONS
Primary urothelial cells and bladder carcinoma cells can be efficiently transfected using an adenoviral vector with similar infectivity. In vitro viral infection shows that the efficiency of adenoviral transfection is significantly reduced in the presence of pentosan polysulfate, a GAG analog. Adenoviral mediated gene transfer to bladder urothelium is enhanced by HCl pretreatment.
Department of Urology, Children's Hospital and Harvard Medical School and Renal Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Lee-Fong
LF
Lin
Guoming
G
Zhu
James J
JJ
Yoo
Shay
S
Soker
Vikas P
VP
Sukhatme
Anthony
A
Atala
D000256:Adenoviridae / Q000235:genetics*; D000283:Administration, Intravesical; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006025:Glycosaminoglycans / Q000502:physiology; D006851:Hydrochloric Acid / Q000494:pharmacology; D007763:Lac Operon / Q000235:genetics; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D001743:Urinary Bladder / Q000187:drug effects / Q000473:pathology*
D006025:Glycosaminoglycans; D006851:Hydrochloric Acid