Efficient Generation of Recombinant Adenoviral Vectors by Cre-lox Recombination In Vitro

Aoki, Kazunori; Barker, Christopher S.; Danthinne, Xavier; Imperiale, Michael J.; Nabel, Gary J.

doi:10.1007/bf03402119

Cited by 111 publications

(77 citation statements)

References 27 publications

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“…The replicationincompetent adenovirus vectors expressing EGFP (AdDE1-EGFP) or no gene (AdDE1) were prepared as described. 30,31 The adenovirus vectors were quantified by optical absorbance. 32 The infectious units of the viruses were examined in 293-38.HissFv.rec cells, and the ratio of the viral particle to infectious unit for each virus was approximately 30.…”

Section: Cell Linesmentioning

confidence: 99%

Oncolytic virus therapy for pancreatic cancer using the adenovirus library displaying random peptides on the fiber knob

Nishimoto

Yoshida²,

Miura³

et al. 2009

Gene Ther

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A conditionally replicative adenovirus is a novel anticancer agent designed to replicate selectively in tumor cells. However, a leak of the virus into systemic circulation from the tumors often causes ectopic infection of various organs. Therefore, suppression of naive viral tropism and addition of tumor-targeting potential are necessary to secure patient safety and increase the therapeutic effect of an oncolytic adenovirus in the clinical setting. We have recently developed a direct selection method of targeted vector from a random peptide library displayed on an adenoviral fiber knob to overcome the limitation that many cell type-specific ligands for targeted adenovirus vectors are not known. Here we examined whether the addition of a tumor-targeting ligand to a replication-competent adenovirus ablated for naive tropism enhances its therapeutic index. First, a peptide-display adenovirus library was screened on a pancreatic cancer cell line (AsPC-1), and particular peptide sequences were selected. The replication-competent adenovirus displaying the selected ligand (AdDCAR-SYE) showed higher oncolytic potency in several other pancreatic caner cell lines as well as AsPC-1 compared with the untargeted adenovirus (AdDCAR). An intratumoral injection of AdDCAR-SYE significantly suppressed the growth of AsPC-1 subcutaneous tumors, and an analysis of adenovirus titer in the tumors revealed an effective replication of the virus in the tumors. Ectopic liver gene transduction following the intratumoral injection of AdDCAR-SYE was not increased compared with the AdDCAR. The results showed that a tumor-targeting strategy using an adenovirus library is promising for optimizing the safety and efficacy of oncolytic adenovirus therapy.

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Section: Cell Linesmentioning

confidence: 99%

Oncolytic virus therapy for pancreatic cancer using the adenovirus library displaying random peptides on the fiber knob

Nishimoto

Yoshida²,

Miura³

et al. 2009

Gene Ther

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“…24,25 Based on that technology, we have developed a novel system for producing adenoviral libraries displaying a variety of peptides on the HI-loop of the fiber knob ( Figure 1a), and used the libraries to select an adenoviral vector with the high infectivity in target cells (Figure 1b). Our system may allow the selection of targeted vectors to any cell type of interest, and may have broad implications for the development of targeted therapies.…”

Section: Introductionmentioning

confidence: 99%

Direct selection of targeted adenovirus vectors by random peptide display on the fiber knob

Miura¹,

Yoshida²,

Nishimoto³

et al. 2007

Gene Ther

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Targeting of gene transfer at the level of cell entry is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success because proper targeting ligand-receptor systems on the cells of interest are generally unknown. Systematic approaches to generate adenovirus vectors targeting any given cell type need to be developed to achieve this goal. Here, we constructed an adenovirus library that was generated by a Cre-lox-mediated in vitro recombination between an adenoviral fiber-modified plasmid library and genomic DNA to display random peptides on a fiber knob. As proof of concept, we screened the adenovirus display library on a glioma cell line and observed selection of several particular peptide sequences. The targeted vector carrying the most frequently isolated peptide significantly enhanced gene transduction in the glioma cell line but not in many other cell lines. Because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector, the selection of targeting peptides is highly useful in the context of the adenoviral capsid. This vector-screening system can facilitate the development of a targeted adenovirus vector for a variety of applications in medicine.

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“…A full-length E1-, E3-deleted recombinant adenovirus was then generated using in situ loxP recombination between the shuttle vector (linearized with PmeI) and the cAd5-deltaE3.LoxP cosmid containing the Ad5 backbone (linearized with ClaI) in the presence of purified Cre recombinase. 14 The resulting recombinant adenoviral DNA was then transfected into HER 911 cells by standard calcium phosphate precipitation methods. Recombinant clones were identified as plaques in soft agar culture and characterized by dideoxynucleotide sequencing to verify the orientation and identity of the hSLC cDNA prior to further amplification.…”

Section: Adenovirusesmentioning

confidence: 99%

Chemokine gene modification of human dendritic cell-based tumor vaccines using a recombinant adenoviral vector

2004

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Previous animal studies conducted in our laboratory have shown that tumor antigen-pulsed dendritic cells (TP-DC) can mediate antitumor effects in vivo. However, durable and complete regression of established tumors has been difficult to achieve through the administration of TP-DC alone. To better augment immune priming to tumors in vivo, we have hypothesized that it is necessary to achieve an increased number of host-derived, naïve T cells at the site of TP-DC vaccine injections. To accomplish this goal, we have embarked on a series of studies that utilize defined chemokines. One of these molecules, secondary lymphoid tissue chemokine (SLC), has been shown to be uniquely chemoattractant for naïve T cells and dendritic cells. We propose that gene modification of DC-based tumor vaccines to produce human SLC will enhance T-cell recruitment and immune priming to tumorassociated antigens, and thereby translate into improved antitumor vaccine efficacy in vivo. Utilizing an E1-, E3-deleted adenoviral vector containing the gene for human SLC, we have been able to transduce human DC to produce biologically active human SLC that chemoattracts human T cells in vitro. SLC production by transduced DC was markedly enhanced upon DC maturation. Additionally, these SLC-secreting DC were found to be viable to a large extent despite the cytopathic effect inherent in adenoviral gene transfer and, most importantly, functional as determined by their ability to prime autologous T cells to a known melanomaassociated antigen, MART-1. Based on these encouraging results, we plan to initiate Phase I clinical studies utilizing DC-SLC to treat patients with advanced solid tumors.

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Efficient Generation of Recombinant Adenoviral Vectors by Cre-lox Recombination In Vitro

Cited by 111 publications

References 27 publications

Oncolytic virus therapy for pancreatic cancer using the adenovirus library displaying random peptides on the fiber knob

Oncolytic virus therapy for pancreatic cancer using the adenovirus library displaying random peptides on the fiber knob

Direct selection of targeted adenovirus vectors by random peptide display on the fiber knob

Chemokine gene modification of human dendritic cell-based tumor vaccines using a recombinant adenoviral vector

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