Volume 9, Issue 2, Pages 218-230 (February 2004) Ablating CAR and Integrin Binding in Adenovirus Vectors Reduces Nontarget Organ Transduction and Permits Sustained Bloodstream Persistence Following Intraperitoneal Administration  Masaki Akiyama, Stephen Thorne, David Kirn, Peter W Roelvink, David A Einfeld, C.Richter King, Thomas J Wickham  Molecular Therapy  Volume 9, Issue 2, Pages 218-230 (February 2004) DOI: 10.1016/j.ymthe.2003.10.010 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Schematic diagram of Ad vectors with altered cell surface interactions. CAR and integrins mediate cellular transduction by adenovirus vectors. The single-ablated vector (Ad.F*) has a modified fiber in which binding to CAR is abolished. In addition to the modification of the fiber, the double-ablated vector (Ad.PB*F*) has a modified penton base in which the integrin-binding RGD sequence is deleted. The HA tag, which is recognized by the artificial receptor (αHA), was inserted into the HI loop of the fiber knob of both ablated vectors. Molecular Therapy 2004 9, 218-230DOI: (10.1016/j.ymthe.2003.10.010) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Noninvasive imaging of luciferase expression. After the intraperitoneal administration of (A) conventional AdL or (B) double-ablated AdL.PB*F* at a dose of 1 × 1011 pu, photons emitted from the abdominal side were collected with an ultrasensitive photon detector (exposure time: 30 s). Luciferin was administered into the peritoneal cavity just before the image acquisition. (C) Digitalization of photons around the lower abdominal area was performed on the images acquired at indicated time points (▴, AdL; ▪, AdL.PB*F*). Luciferin was administered intraperitoneally just before the image acquisition at each time point. Data show averages ±standard deviations (n = 5). FOI, field of interest. Molecular Therapy 2004 9, 218-230DOI: (10.1016/j.ymthe.2003.10.010) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Green fluorescent protein (GFP) expression followed by intraperitoneal administration. Adf or Adf.PB*F* GFP-expression vectors were injected into the peritoneal cavity of CD-1 nude mice (2 × 1011 pu/0.4-ml dose, n = 3). Three days after the administration, tissues were resected for image acquisition. Pictures are the GFP expression on the surface of tissues (peritoneum, liver, spleen, and kidney) acquired using a fluorescence stereomicroscope. Representative images are shown. Molecular Therapy 2004 9, 218-230DOI: (10.1016/j.ymthe.2003.10.010) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Luciferase expression and Ad DNA content in tissues. Conventional (AdL), single-ablated (AdL.F*), or double-ablated (AdL.PB*F*) vectors were administered intraperitoneally at 1 × 1010 and 1 × 1011 doses into C57Bl/6 mice (0.5-ml dose, n = 5). One day after administration, harvested tissues were snap-frozen and ground into powder. Aliquoted powder was lysed with CCLR (Promega), and the supernatant was assayed for luciferase activities with the Luciferase Assay System (Promega). Protein content in supernatant was used for normalization. Black column shows luciferase activities (RLU/mg protein, left axis). DNA was isolated from aliquoted powder using the DNeasy Tissue Kit (Qiagen). Ad genome content was analyzed by quantitative PCR (ABI Prism 7700), using primers to the pIX region of adenovirus as described under Materials and Methods. Normalization was performed using the endogenous ribosomal RNA genome content in each preparation originating from host cells (Applied Biosystems). Gray column shows Ad genome contents (copies/μg DNA, right axis). Left side of graph, 1 × 1010-pu dose; right side of graph, 1 × 1011-pu dose. Each column shows the geometric mean ± standard deviation (n = 5). Molecular Therapy 2004 9, 218-230DOI: (10.1016/j.ymthe.2003.10.010) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Analysis of vector kinetics in the bloodstream. Luciferase-expressing vectors were injected into the peritoneal cavity of C57Bl/6 mice (1 × 1010 or 1 × 1011 pu/0.5 ml). After 30, 90, and 180 min of administration, blood was collected by retro-orbital bleed using heparinized capillary tubes. (A) Adenoviral content in the plasma was determined by reporter gene expression on A232-HA cells as described under Materials and Methods. (AdL, ▪; AdL.F*, ▴; AdL.PB*F*, ▪; solid line, 1 × 1011 dose; dashed line, 1 × 1010 dose.) Results are shown using the geometric means + standard deviations (n = 4). Three of four plasma specimens collected at 180 min post-conventional vector (1 × 1010) administration showed less expression than the detection limit. (B) Area under the curve between 30 and 180 min was calculated applying the trapezoidal rule, multiplying the particle concentration by its retention time. (Gray column, 1 × 1010 pu/0.5 ml dose; black column, 1 × 1011 pu/0.5 ml dose.) Each column shows the geometric mean ± standard deviation (n = 4). Molecular Therapy 2004 9, 218-230DOI: (10.1016/j.ymthe.2003.10.010) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Transduction of mesothelium and/or parenchyma. β-Galactosidase-expressing vectors were administered into the peritoneal cavity of C57Bl/6 mice (1 × 1011 pu/0.5 ml). At 3 days postadministration, the liver and spleen were resected. To show the transduction of the mesothelium (exterior area), whole tissues were stained with X-gal. For the staining of parenchyma (interior area), tissues were sliced with a razor blade and immersed into X-gal solution. After the staining, images were acquired using a SPOT-RT digital camera connected to a fluorescence stereomicroscope under bright field. Representative images are shown. (Bar, 200 μm.) Note: In the surface staining procedure for visualizing transduced mesothelial cells, the X-gal stain would penetrate into and stain parenchymal tissues within these organs. This staining is observed in the surface-stained samples for AdZ.F*. However, comparison of the surface-stained AdZ.F* samples with the surface-stained AdZ samples clearly shows that mesothelial cells are not transduced by AdZ.F*. Molecular Therapy 2004 9, 218-230DOI: (10.1016/j.ymthe.2003.10.010) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 7 Targeting to an artificial receptor in vivo. The feasibility of retargeting the Adf.PB*F* vector to cells in the peritoneal cavity was tested in vivo. (A) The pseudo-receptor expression vector, Adα(HA), or control vector, Ad.null, was injected at the indicated doses (0.5 ml) into the peritoneal cavity of C57Bl/6 mice. The administration of Adα(HA) was expected to cause the artificial receptor expression on mesothelial cells. At 2 days postadministration, 1 × 1011 pu of double-ablated GFP-expression vector, Adf.PB*F*, was administered intraperitoneally. Three days later, GFP expression on the surface of abdominal tissues (liver, peritoneum, and spleen) was captured using a fluorescence stereomicroscope. (Bar, 200 μm.) (B) AE25-HA tumor cells were injected into the peritoneal cavity of nude mice. Adf.PB*F* or Adf was injected intraperitoneally 30 min later at a dose of 1 × 1011 pu. Two days later, GFP expression in tumor foci and on the surface of abdominal tissues was captured using a fluorescence stereomicroscope. Representative images are shown. (Bar, 500 μm.) Molecular Therapy 2004 9, 218-230DOI: (10.1016/j.ymthe.2003.10.010) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 8 Model for two pathways of transduction following intraperitoneal administration. Schema of adenoviral biodistribution after ip administration (peritoneum, gray frame; blood circulation, red line; transduction, black arrow; viral uptake into circulation, gray arrow; green, transduced cells). (A) Conventional vector transduces mesothelium of abdominal tissues in CAR-dependent manner. Uptake of viral particles into bloodstream is lower than that of ablated vectors. (B) CAR-binding ablated vector does not transduce the mesothelium and exudes into bloodstream. Parenchyma is transduced by integrin interaction. (C) Double-ablated vector exudes to bloodstream as well as single-ablated vector; however, it does not transduce on parenchyma. (D) Transduction mediated by specific receptor–ligand system. Pseudo-receptor expression on mesothelia redirects doubly ablated vector to mesothelium. Molecular Therapy 2004 9, 218-230DOI: (10.1016/j.ymthe.2003.10.010) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions