1. Volume 21, Issue 1, Pages 139-148 (January 2013)
Infectivity-selective Oncolytic Adenovirus Developed by High-throughput Screening of Adenovirus-formatted Library 
Yoshiaki Miura, Satoshi Yamasaki, Julia Davydova, Eric Brown, Kazunori Aoki, Selwyn Vickers, Masato Yamamoto 
Molecular Therapy 
Volume 21, Issue 1, Pages (January 2013)
DOI: /mt
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Novel system for adenovirus (Ad) vector production. (a) Schematic presentation of the rescue virus system. The fiber-modified Ad library, the core technology for infectivity-selective oncolytic adenovirus (ISOAd), was produced with the newly developed rescue virus system by CRE-lox recombination in CRE-expressing, fiber-transcomplementing cell line (293CRE-69). The shuttle plasmid coding fiber library has a single loxP site in the upstream of the fiber coding region. The genetically fiberless rescue virus with one loxP was produced with fiber-transcomplementing producer cells for peudotyping. The cells were preinfected with the rescue virus 24 hours before library plasmid transfection, and the produced library viruses were harvested 48 hours after transfection. (b) Design of the rescue virus, plasmid constructs, and Ad producer cells. The rescue virus (AdMLΔF), generated with the shuttle plasmid pMLΔF, has a wild-type E1 gene, a single loxP site replacing the E3 gene, and a deletion of its fiber region (79.4–91.3 map unit (m.u.)). pMLABΔSK, starting plasmids for AB-loop mutants, has the 1.8 kb fragment of the fiber region (87.6–91.3 m.u.) deleted. The shuttle plasmids of the fiber library (pMLAB-lib) included a 76.1–100 m.u. of the adenoviral genome with a single loxP site and library sequences in the AB-loop region of the fiber in place of the E3 region deleted (79.4–84.8 m.u.).16 The reporter shuttle, pBΔCAR-GFP, was inserted a CMV-promoter-driven GFP expression cassette at the downstream of a loxP site in the E3 region, and the AB-loop of the fiber-knob region possesses four point-mutations for ablating CAR-binding. In 293CRE (fiber noncomplementing) and 293CRE-69 (Ad5/Ad3 fiber-transcomplementing) cells, the CRE recombinase was overexpressed by permanent transfection. CAR, Coxsackie adenovirus receptor; GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Efficiency of adenovirus (Ad) production with the rescue virus system. (a) After infection of 293CRE-69 with the fiber-pseudotyped AdMLΔF at 1 × 104 vp/cell, 5 µg of pBΔCAR-GFP was transfected into 293CRE-69 cells. Ad yield was assessed by analyzing 0.2% of the 1st crude viral lysate by qPCR for GFP-coding region. The data indicate that this system can generate 5 × 109 copies of Ad particles per single 6-cm dish. (b) Adenoviral cytopathic effect (cpe) was compared between the producer cells with and without fiber-transcomplementation after transfection with various shuttle plasmids (Supplementary Figure S1). CAR, Coxsackie adenovirus receptor; GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Application of the rescue virus system for library generation. (a) The AB-loop of the fiber library has seven amino acids in major CAR-binding domain replaced with the random seven amino acids. X: random amid acid. (Supplementary Figure S2) (b, c) To determine diversity, limit dilution experiments with the shuttle plasmid library mixed with the GFP-coding shuttle plasmid were performed. A small amount of pBΔCAR-GFP was mixed with pMLAB-lib at various ratios, and Ad libraries were generated from the mixtures, respectively. The viral DNA was extracted from the viral solution after treatment with DNaseI, and then recombinant viral copy numbers were determined by qPCR for GFP sequence. When 1/20 amount of the viral solution was assessed, the GFP sequence coding virus was detected in a 4 × 107 dilution (b). When total volumes (20 runs of 1/20 amount) of the viruses from further diluted plasmids were tested, the GFP-coding virus was detected in a dilution as low as 5 × 108 (c). The thick line (12 copies/6-cm dish) represents the highest number of the background. No GFP: no GFP-coding shuttle plasmid. CAR, Coxsackie adenovirus receptor; GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
High-throughput screening of the adenovirus library in the AB-loop for mesothelin (MSLN)-expressing cells. (a) Via the replication-based high-throughput screening, the ISOAd was isolated from a high-diversity Ad library of targeting motifs based on transduction and subsequent replication. First, the target cells were infected with the Ad library prepared from 10 dishes of 6 cm (5 × 109 diversity) at a low multiplicity of infection. After 5–7 days, the amplified Ads were recovered from the cells and subjected to a few more rounds of re-infection. The viral solution from each round was harvested and the sequences of the targeting motifs were analyzed. (b) The DNA sequences of the AB-loop region of the Ads screened with 293-MSLN cells were amplified by PCR and analyzed after cloning into a plasmid. Although the initial library sequence was completely diverse, screening after virus amplification in 293-MSLN cells started to show convergence of the targeting motif sequences as early as the first round of screening. After subsequent rounds of screening, the sequence further converged eventually to a single clone (VTINRSA).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Binding of the isolated adenovirus clone to mesothelin (MSLN). (a) Binding ability of AdML-VTIN correlated well with MSLN expression in various cell lines (293, 293-MSLN, Panc-1, A549, MiaPaCa-2, and AsPC-1). Binding of AdML-5WT (control Ad with a wild-type fiber) did not correspond to the MSLN level. The isolated total DNA was analyzed by the E4 qPCR to determine the adenoviral copy number bound to the surface of the cells. The level of MSLN expression was determined by flow-cytometry (shown below the graph). (b) Flow-cytometry of cell surface MSLN. Expression of MSLN was determined by flow-cytometry. Gray: without anti-MSLN antibody, Black-line; with anti-MSLN antibody. (c) Suppression of MSLN expression with the anti-MSLN siRNA eliminated AdML-VTIN binding to the target cells (**P < 0.01, ***P < 0.001). (d) Pretreatment with the anti-MSLN antibody (2 hours at 4 °C) significantly reduced binding of AdML-VTIN to the MSLN-positive target cells. The results of binding assay were shown as E4 copy number per cell (n = 3).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Characterization of the newly isolated adenovirus (Ad) AdML-VTIN. (a) The analysis of infectivity demonstrated that the MSLN-targeted AdML-VTIN outperformed not only the control Ad with a native fiber (AdML-5WT) but also the infectivity-enhanced Ad with an Ad5/Ad3-fiber (AdMG553) in MSLN strongly-positive Panc-1. In MSLN-intermediately positive A549 cells, the infectivity of AdML-VTIN was as high as that with AdMG553. However, its binding to MSLN-negative MiaPaCa-2 and AsPC-1 cells was significantly lower than other vectors. The result was shown as E4 copy number per cell (n = 3). (b) AdML-VTIN showed exponential amplification selectively in MSLN-positive cells (Panc-1 and A549), and the extent of virus burst corresponded with the MSLN level of each cell line. The result was shown as a virus burst size (vp/cell) (n = 3). Mesothelin expression: strong (2+), moderate (+), low (−). (c) Cytolytic effect of isolated ISOAd in vitro in pancreatic cancer cell lines. Panc-1, MiaPaCa-2, AsPC-1, and 293 cells were infected at 0.1 to 1,000 vp/cell with AdML-VTIN and AdML-5WT, with surviving cells stained by crystal violet. Staining were performed at day 5, 10, and 15 in Panc-1 and MiaPaCa-2, at day5 and 15 in AsPC-1, and 293. Panc-1: MSLN-positive pancreatic cancer cell line, MiaPaCa-2 and AsPC-1: MSLN-negative pancreatic cancer cell line, 293: MSLN-negative control cell line.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
In vivo antitumor effect and viral replication of the infectivity-selective oncolytic Ad. (a) The in vivo antitumor effect of the mesothelin (MSLN)-targeted AdML-VTIN was analyzed in Panc-1 (MSLN-positive) and MiaPaCa-2 (MSLN-negative) subcutaneous xenografts. AdML-VTIN showed a strong antitumor effect only in the MSLN-expressing Panc-1 tumors, while the effect of AdML-5WT was not selective. Each symbol represents the mean of tumor volumes ± s.e.m. (n = 4–8) (*P < 0.05, **P < 0.01). (b) Five days after intratumoral injection of the viruses, the expression of an adenoviral late gene product (hexon) was assessed by immunostaining with the antihexon polyclonal antibody (counterstained with Hoechst 33342). Staining and sections were performed in at least two independent experiments. Green: Ad hexon protein, Blue: nucleus (original magnification: ×100). (c) The viral copy numbers in the DNA isolated from tumor specimens at day 5 were analyzed by qPCR. The result is shown as the adenoviral copy number per 1 ng DNA. (***P < 0.005) MSLN expression: strong (2+), low (−). PBS, phospate buffered saline.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions