Volume 15, Issue 9, Pages 1647-1654 (September 2007) Development of Renal-targeted Vectors Through Combined In Vivo Phage Display and Capsid Engineering of Adenoviral Fibers From Serotype 19p  Laura Denby, Lorraine M Work, Dan J Von Seggern, Eugene Wu, John H McVey, Stuart A Nicklin, Andrew H Baker  Molecular Therapy  Volume 15, Issue 9, Pages 1647-1654 (September 2007) DOI: 10.1038/sj.mt.6300214 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Assessment of coagulation factor binding by modified viruses. (a) HepG2 cells were distributed in the plates 24 hours prior to infection. Plated cells were washed in phosphate-buffered saline (PBS) and 50 μl of serum free media with physiological levels (1 IU/ml) of either factor IX (FIX) or FX added. This was followed by infection with the appropriate Ad at 10,000 virus particles (VP)/cell. Cells were incubated for 3 hours at 37°C before being washed and the media replaced. Cells were incubated for 72 hours before quantification of transgene expression. *P < 0.05 versus transduction in the absence of clotting factors. (b) CHO-K1 (wild-type) and CHO-pgsA745 (heparan sulphate proteoglycan-deficient)24 cells were used for elucidating the effects and mechanisms of coagulation factors on Ad19p-modified vector transduction. Plated cells were washed in PBS and 50 μl of serum free media with physiological levels (1 IU/ml) of either FIX or FX added. This was followed by infection with 20,000 VP/cell of the appropriate Ad. Cells were incubated for 3 hours at 37°C before being washed and the media replaced. Cells were incubated for 48 hours before quantification of transgene expression. *P < 0.05 versus transduction in the absence of clotting factors. (c, d) Ad5 or Ad19p vectors at various concentrations (×1011 VP/ml) were perfused over FX immobilised onto a CM-5 sensor chip in 50 mmol/l Tris pH 7.4; 150 mmol/l NaCl; 5 mmol/l CaCl2; 0.005% Tween 20 at a flow rate of 20 μl/min at 25°C. Depicted are sensorgrams of c FX (showing specific binding) of Ad5 and, to a lesser extent, Ad19p-Eco47. (d) The steady state change (δRU) in respiratory unit (RU) on binding of the virus to the FX was plotted against virus concentration. CHO, Chinese hamster ovary; RLU, relative light units. Molecular Therapy 2007 15, 1647-1654DOI: (10.1038/sj.mt.6300214) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 In vivo phage display. (a) Schematic representation of in vivo phage display in the rat, so as to identify renal targeting peptides. Recovered and amplified phage from the kidneys 5 minutes after injection were subjected to a total of three rounds of in vivo phage display, followed by sequence analysis after round 2 and 3. (b) Phage recoveries over sequential rounds of phage display. *indicates P < 0.05, NS, nonsignificant. IV, intravenous. Molecular Therapy 2007 15, 1647-1654DOI: (10.1038/sj.mt.6300214) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Analysis of selected peptide-expressing phage. (a) Demonstration of saturation kinetics of M13 phage in the liver, kidney, lung, and heart at increasing doses. *indicates P < 0.05 versus control phage. (b) Phage recoveries from kidney and liver (plaque forming units (PFU)/g tissue) from animals infused with 2 × 1010 PFU per animal of either control phage or targeted phage (as indicated) following 2 × 1011 PFU per animal control phage to saturate non-specific binding. *P < 0.05 versus control phage. (c) Phage recoveries expressed as a percentage of phage in the liver. Individual values shown above each bar. NS, non significant. Molecular Therapy 2007 15, 1647-1654DOI: (10.1038/sj.mt.6300214) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Engineering of Ad19p fibers for targeting peptide insertion. (a) Schematic illustrating the protocol followed for producing the modified vectors (refer to Materials and Methods). (b) Model of the predicted structure of the Ad19p fiber with and without insertion of the restriction site to allow cloning. (c) In vitro comparison of Ad19p and Ad19p-Eco47. Rat glomerular endothelial cells were infected with increasing doses of either virus for 3 hours at 37°C. They were then washed and the media was replaced. Seventy-two hours after infection, cells were harvested and β-galactosidase was measured and normalized to protein. (d) Model of the predicted structure of the Ad19p fiber with each peptide inserted into the HI loop. (e) Western blot of fiber monomer. Ten μg of viral protein was loaded, and the membrane was probed with the anti-fiber antibody 4D2 (Neomarkers, Fremont, CA) at 1:1,000. Ad, adenovirus; PCR, polymerase chain reaction; RLU, relative light units. Molecular Therapy 2007 15, 1647-1654DOI: (10.1038/sj.mt.6300214) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Analysis of kidney targeting in vivo. Eight-week old male Wistar Kyoto rats were infused with 3.5 × 1011 virus particles per rat of each modified vector or phosphate-buffered saline (PBS) and killed 5 days later. (a) Immunohistochemistry performed in kidney sections. Black staining indicates β-galactosidase activity. Representative sections are shown (n = 6 rats per group). Scale bar = 100 μm. (b) Quantitative analysis of transgene-expressing cells *P < 0.05 versus Ad19p-Eco47-injected rats and Ad5-injected rats. NS, not significant. Ad, adenovirus. Molecular Therapy 2007 15, 1647-1654DOI: (10.1038/sj.mt.6300214) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Analysis of non-renal tissue for reporter gene activity. (a) Eight-week old male Wistar Kyoto rats were infused with 3.5 × 1011 virus particles per rat of each modified vector or phosphate-buffered saline and killed 5 days later. Immunohistochemistry was performed on the liver, spleen, and heart. Black staining indicates β-galactosidase activity. Representative sections are shown (n= 6 rats per group). Scale bar = 100 μm. (b) Quantitative analysis of transgene-expressing cells from liver sections of animals infused using a pre-dose strategy. Ad, adenovirus. Molecular Therapy 2007 15, 1647-1654DOI: (10.1038/sj.mt.6300214) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 Assessment of early particle delivery to liver and kidney. Eight-week old male Wistar Kyoto rats were infused with 3.5 × 1011 virus particles per rat of each modified vector or phosphate-buffered saline and killed following perfusion at 1 hour after injection. Virion quantification was carried out by means of Taqman Data Analysis software (Applied Biosystems), using SYBR green. DNA was extracted from the kidney and 200 ng total DNA was amplified using LacZ primers, and the products were quantified using Taqman. *P < 0.05 versus Ad19p-Eco47. Ad, adenovirus. Molecular Therapy 2007 15, 1647-1654DOI: (10.1038/sj.mt.6300214) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions