21. Peptide-Targeted Ad19p-Based Adenoviral Vectors for Renal Gene Delivery
Authors: Laura Denby, Lorraine M. Work, Dan J. Von Seggern, Eugene Wu, Ela Hunt, Stuart A. Nicklin, Andrew H. Baker
Journal: Molecular Therapy (2006)

Systemic administration of gene delivery vectors resulting in renalselective transduction would have significant implications for therapy of kidney diseases. To identify kidney-targeting peptides we used in vivo phage display with intravenous administration of a M13 phage library into 12 week old, male Wistar Kyoto (WKY) rats. Following 3 rounds of in vivo biopanning 307 phage inserts were sequenced and 3 consensus peptides identified –APASLYN (APA), HITSLLS (HIT) and HTTHREP (HTT). Homogeneous phage populations were amplified and infused into rats and the level of phage accumulation in the kidney determined. Each peptide demonstrated increased accumulation in the kidney compared to a control, insertless phage (APA 255-fold increase; HIT 152-fold increase; HTT 43-fold increase vs control insertless phage). Our previous studies identified an adenovirus serotype 5 capsid pseudotyped with serotype 19p fibers (Ad19p) as a candidate platform vector for re-targeting due to its lack of native hepatic tropism (Denby et al, 2004). Each candidate kidney-targeting peptide (APA, HIT, HTT) was genetically incorporated into the HI loop of the fiber of Ad19p between amino acids 331 and 332. Initially, transduction with the peptide modified vectors was assessed in vitro in rat cell lines. Peptide-modified Ad19p (APA, HIT and HTT) produced a significant increase (8- to 24-fold increase versus unmodified Ad19p; p<0.05) in transduction of rat glomerular endothelial cells in a dose-dependent manner. Transduction was restricted to the renal cell line with no elevation in gene delivery evident in either rat hepatocytes (Arl-6) or rat prostate endothelial cells (Y-PEN) compared to non-modified Ad19p. In vivo transduction was assessed in 8 week old, male WKY rats administered 3.5 × 1011 viral particles of peptide-modified or control Ad19p and sacrificed 5 days post-virus delivery. Immunohistochemical staining of tissues demonstrated gene expression in the kidney of those animals infused with the peptide-modified Ad19p but not the unmodified control.Furthermore, each vector had a different distribution pattern with the HTT vector transducing collecting duct cells and the APA and HIT vectors predominantly transducing glomerular endothelial cells. Combining phage display targeting peptides with the novel Ad19p platform vector resulted in renal-targeting viral vectors via systemic administration. These studies have important implications for genetherapy based intervention in the treatment of renal disease.Denby et al (2004) - Adenoviral serotype 5 vectors pseudotyped with fibers from subgroup D show modified tropism in vitro and in vivo. Human Gene Therapy, 15: 1054–1064. Systemic administration of gene delivery vectors resulting in renalselective transduction would have significant implications for therapy of kidney diseases. To identify kidney-targeting peptides we used in vivo phage display with intravenous administration of a M13 phage library into 12 week old, male Wistar Kyoto (WKY) rats. Following 3 rounds of in vivo biopanning 307 phage inserts were sequenced and 3 consensus peptides identified –APASLYN (APA), HITSLLS (HIT) and HTTHREP (HTT). Homogeneous phage populations were amplified and infused into rats and the level of phage accumulation in the kidney determined. Each peptide demonstrated increased accumulation in the kidney compared to a control, insertless phage (APA 255-fold increase; HIT 152-fold increase; HTT 43-fold increase vs control insertless phage). Our previous studies identified an adenovirus serotype 5 capsid pseudotyped with serotype 19p fibers (Ad19p) as a candidate platform vector for re-targeting due to its lack of native hepatic tropism (Denby et al, 2004). Each candidate kidney-targeting peptide (APA, HIT, HTT) was genetically incorporated into the HI loop of the fiber of Ad19p between amino acids 331 and 332. Initially, transduction with the peptide modified vectors was assessed in vitro in rat cell lines. Peptide-modified Ad19p (APA, HIT and HTT) produced a significant increase (8- to 24-fold increase versus unmodified Ad19p; p<0.05) in transduction of rat glomerular endothelial cells in a dose-dependent manner. Transduction was restricted to the renal cell line with no elevation in gene delivery evident in either rat hepatocytes (Arl-6) or rat prostate endothelial cells (Y-PEN) compared to non-modified Ad19p. In vivo transduction was assessed in 8 week old, male WKY rats administered 3.5 × 1011 viral particles of peptide-modified or control Ad19p and sacrificed 5 days post-virus delivery. Immunohistochemical staining of tissues demonstrated gene expression in the kidney of those animals infused with the peptide-modified Ad19p but not the unmodified control.Furthermore, each vector had a different distribution pattern with the HTT vector transducing collecting duct cells and the APA and HIT vectors predominantly transducing glomerular endothelial cells. Combining phage display targeting peptides with the novel Ad19p platform vector resulted in renal-targeting viral vectors via systemic administration. These studies have important implications for genetherapy based intervention in the treatment of renal disease. Denby et al (2004) - Adenoviral serotype 5 vectors pseudotyped with fibers from subgroup D show modified tropism in vitro and in vivo. Human Gene Therapy, 15: 1054–1064.