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cDNA expression cloning is a powerful method for the identification of genes that are able to confer a selectable phenotype on specific cell types. An adenovirus vector is characterized by several advantages over plasmid DNA and retroviral vector-mediated gene transfer, such as broad host range and high infectivity. However, an expression cloning protocol using the adenovirus vector has not been reported. We describe here a simple and efficient method for constructing adenovirus cDNA expression libraries based on Cre-lox-mediated in vitro recombination between adenoviral shuttle plasmid cDNA libraries and adenoviral genomic DNA tagged with terminal protein. In a model experiment, EGFP clones present at the frequency of 0.003% in the shuttle plasmid library could be efficiently converted to adenoviral vector in a 6-cm dish under optimized conditions, indicating that high-complexity libraries harboring low-abundance cDNAs can be produced. The efficiency of this system was demonstrated by the isolation of cDNA for CD2 (frequency less than 1 in 0.3 × 104 transcripts in T cells) from the human T cells. This effective and versatile method can facilitate the functional identification of genes for a variety of purposes. [Copyright &y& Elsevier]

Irinotecan is a key drug for advanced gastric cancer (AGC). We previously reported that patients with UGT1A1 single heterozygous (SH) had significantly high frequency of severe hematological adverse events (AEs) compared to patients with UGT1A1 wild type (WT) in irinotecan monotherapy for AGC. However, the difference of initial dose and relative dose intensity (RDI) of irinotecan between UGT1A1 WT and SH might affect those results. Therefore, we compared between UGT1A1 SH and WT regarding the clinical outcomes of the fixed dose irinotecan monotherapy for AGC. We retrospectively analyzed the clinical data of patients who received initial fixed dose irinotecan (150mg/m2, bi-weekly) in the multi-institutional retrospective study. 106 eligible patients were registered from 8 centers in Japan. The number of patients with UGT1A1 WT/SH were 62 and 38, respectively. In WT/SH patients, performance status 0/1/≥2 was 20/40/2 and 11/23/4, treatment line 2nd/3rd or later was 27/35 and 18/20, HER2 positive/negative 17/45 and 8/30, respectively. In WT/SH patients, median PFS was 3.2 and 3.3 months (HR = 1.137, P = 0.543) and median OS was 10.4 and 7.3 months (HR = 0.734, P = 0.184). In WT/SH patients, median treatment cycle was 6 and 4 (P = 0.973), and RDI was 0.85 vs 0.84(P = 0.915), respectively. Hematological AEs were observed in 82.2% and 92.1% (P = 0.138), severe hematological AEs (≥G3) were 35.4% and 63.1%(P = 0.006). Non-hematological AEs were 88.7% and 86.8%(P = 0.507), severe non-hematological AEs (≥G3) were 6.6% and 15.8% (P = 0.122), respectively. Severe AEs in more than 5% patients were leukopenia (11.3% and 15.8%), neutropenia (14.5% and 31.6%), anemia (21.0% and 23.7%), and anorexia (1.6% and 10.5%). UGT1A1 SH patients who received initial fixed dose Irinotecan had high frequency of severe hematological AE compared to WT patients. However, there was no significant difference in efficacy of Irinotecan monotherapy in each group. [ABSTRACT FROM AUTHOR]

Recombinant adenovirus vectors have been widely used for gene delivery to a range of cell types and employed in a number of gene therapy approaches. However, a selective delivery of a therapeutic gene to target cells by adenovirus vectors is precluded by the widespread distribution of the primary cellular receptors for adenoviruses. Recently, retargeting adenovirus vectors has been achieved by direct genetic modifications of the capsid proteins: targeting ligands can be incorporated into the C-terminal and HI-loop of fiber proteins ablated for native tropism through loss of binding with CAR and av integrins, and these vectors provide an important platform for evaluating the targeting potential of selected peptide ligands. However, cell-type specific lligand-receptor systems for targeted adenovirus vectors are generally unknown, which impedes the wider application of fiber-modified adenovirus vectors for targeted therapies. Moreover, because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector as shown in this study, the selection of targeting peptides is highly useful in the context of the adenoviral capsid. We reported previously a simple and efficient method for constructing an adenovirus cDNA expression library by in vitro Cre-loxP recombination (K. Hatanaka, et al. Mol Ther 8;158, 2003). Based on that technology, here we developed a novel system for adenoviral libraries which display a wide variety of peptides on the HI-loop of the fiber knob. A plasmid-based library containing a GFP clone at an abundance of 0.01% was readily converted to the adenovirus library in a convenient 60-mm dish scale, suggesting that the complexity of the virus library could be more than 1 × 104 per a 60-mm dish by the present method. As proof-of-concept, we screened the 2 × 105 complexity level of adenovirus display library on a human gliomacell line and observed selection of a particular peptide sequence. The gene transduction efficiency of a targeted vector carrying the peptide was 4- to 5-fold higher than that of an untargeted adenovirus in the glioma cell line but not in other cell lines. A substantial competitive inhibition by the cognate peptide but not by a control unrelated peptide was observed, confirming that the enhanced transduction of the targeted vector in the cells is mediated by the insertion of the selected peptide. Our system may allow the selection of specific targeted vectors to any given cell type of interest, and may have broad implications for the development of targeted therapies.Molecular Therapy (2006) 13, S7–S7; doi: 10.1016/j.ymthe.2006.08.027 [ABSTRACT FROM AUTHOR]