Site-Specific Integration of a Transgene Mediated by a Hybrid Adenovirus/Adeno-Associated Virus Vector Using the Cre/loxP-Expression-Switching System

Ueno, Takashi; Matsumura, Hajime; Tanaka, Keiji; Iwasaki, Tomoko; Ueno, Mitsuhiro; Fujinaga, Kei; Asada, Kiyozo; Kato, Ikunoshin

doi:10.1006/bbrc.2000.2972

Cited by 34 publications

(19 citation statements)

References 28 publications

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“…To address this problem, several groups have developed hybrid adenovirus/adeno-associated virus vectors, which have the ability to integrate viral genomes into the host genome. [43][44][45] Ad35 would become a promising framework vector for the development of these improved vectors for efficient gene transfer into HSCs.…”

Section: Efficient Gene Transfer Into Human Cd34 + Cells F Sakurai Et Almentioning

confidence: 99%

Efficient gene transfer into human CD34+ cells by an adenovirus type 35 vector

2003

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Efficient gene transfer into human hematopoietic stem cells (HSCs) is the most important requirement for gene therapy of hematopoietic disorders and for study of the hematopoietic system. An adenovirus (Ad) vector based on the Ad serotype 5 (Ad5) is known to transduce HSCs, including CD34 + cells, with very low efficiency because of low-level expression of its primary receptor, coxsackievirus and adenovirus receptor (CAR). In the present study, we developed a recombinant Ad vector composed of the whole Ad serotype 35 (Ad35), which recognizes an unidentified receptor different from CAR for its infection. A transduction study showed that the Ad35-based vectors exhibit a higher transduction efficiency in human CD34 + cells than the conventional Ad5 vectors and the Ad5F35 vectors, which are fiber-substituted Ad5 vectors containing Ad35 fiber proteins. The mean of fluorescence intensity in the CD34 + cells transduced with the Ad35 vectors was 12-76 and 1.4-3 times higher than that in the cells transduced with the Ad5 and Ad5F35 vectors, respectively. The percentages of green fluorescent protein (GFP)-positive CD34 + cells by transduction with Ad35, Ad5, and Ad5F35 vectors expressing GFP at 300 PFU/cell were 53%, 5%, and 52%, respectively, suggesting that Ad35 vectors mediate a more efficient gene transfer into human CD34 + cells than Ad5 and Ad5F35 vectors, although the percentage of transduced cells was similar between Ad35 and Ad5F35 vectors. The Ad vector based on Ad35 could be very useful in gene therapy for blood disorders and gene transfer experiments using HSCs.

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Section: Efficient Gene Transfer Into Human Cd34 + Cells F Sakurai Et Almentioning

confidence: 99%

Efficient gene transfer into human CD34+ cells by an adenovirus type 35 vector

2003

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“…Since then, this rAd has been used in many studies involving gene regulation in mammalian cells (5,16,18,29,33,35,39,43) and mice (1, 8, 15, 19-21, 27, 31, 38, 41, 45-47). This method has also been valuable for tissue-specific enhanced gene expression using rAd expressing Cre in which Cre expression is specifically regulated (34,36).…”

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confidence: 99%

Practical Range of Effective Dose for Cre Recombinase‐Expressing Recombinant Adenovirus without Cell Toxicity in Mammalian Cells

Baba

Nakano

Yamada

et al. 2005

Microbiology and Immunology

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Recently, the importance of regulating transgeneexpression in mammalian cells has been well recognized in the fields of basic research and future gene therapy. Site-specific recombinases, like Cre (6,30,32) and FLP (2-4, 7, 24, 26, 44), are useful tools for this purpose. Cre recombinase derived from the bacteriophage P1, in particular, has been utilized for efficient gene activation and inactivation in mammalian cells (9,37). Cre is a 38-kDa protein that mediates the excisional deletion of a DNA sequence flanked by a pair of loxP sites, a Cre-specific recognition sequence of 34-base pairs, with the same orientation. The gene activation Abstract: The site-specific recombinase Cre is valuable for regulation of gene expression not only in vitro but also in vivo. We previously reported that replication-deficient recombinant adenovirus (rAd) expressing Cre can mediate efficient and strict regulation in 100% of cultured cells. Recently, the constitutive-expression of Cre using retrovirus or lentivirus vector reportedly inhibited cell-growth, but the effect of transient Cre expression have not yet been examined. Here we showed that an excess amount of Cre produced from Cre-expressing rAd caused a deleterious effect in cells even when Cre was transiently expressed. We used three rAds carrying promoters with different activities: the SV40 early promoter (AxSVENCre), the SR␣ promoter (AxSRCre) and the CAG promoter (AxCANCre). Cell toxicity was clearly caused by Cre itself and was distinguishable from that caused by rAd virions when the cytopathic effects of these rAds were compared with that of a control virus lacking the Cre expression unit. Cre toxicity was strongly correlated with the expression level of Cre. Importantly, AxSRCre and AxCANCre gave a 60-fold range of effective MOIs ("effective range") sufficient for gene activation without causing cell toxicity from either the rAd particles or Cre itself, while AxSVENCre failed to give such a range because the expression level of Cre was too low. When Cre was tagged with a nuclear localization signal (NLS), not only its activity but also Cre toxicity was increased fourfold, and the effective range was unchanged. Therefore, AxSRNCre might be more useful to control cell toxicity from the rAd virions than AxSRCre. Cre-induced cell toxicity can be avoided by pre-examining the "effective range" using the purpose cell lines before starting experiments utilizing the experiment of Cre-expressing rAd.

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“…To address this limitation, several groups have developed hybrid Ad vectors such as Ad/adeno-associated virus vectors, which can integrate viral genome into the host genome. [50][51][52] The Ad35 vector would be a promising framework for the development of these improved vectors.…”

Section: Discussionmentioning

confidence: 99%

Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities

et al. 2005

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Adenoviral gene transfer to hematopoietic stem cells (HSCs)/ progenitors would provide a new approach to the treatment of hematopoietic diseases and study of the hematopoietic system. We have previously reported that an adenovirus (Ad) vector composed of whole Ad serotype 35 (Ad35), which belongs to subgroup B, shows efficient gene transfer into human bone marrow CD34 + cells. However, Ad35 vector-mediated transduction into human HSCs/progenitors has not yet been fully optimized. In the present study, we have systematically examined promoter activity in the context of Ad35 vectors in human bone marrow CD34 + cells and primitive CD34 + subsets to optimize the transduction efficiency in human hematopoietic stem/progenitor cells. In the first of the transduction experiments, the improved in vitro ligation method was applied to Ad35 vector construction to allow for simple and efficient production of an E1/E3-deleted Ad35 vector. Using this method, we constructed a series of Ad35 vectors encoding the enhanced green fluorescence protein (GFP) under the control of a variety of strong viral and cellular promoters. Of the six types of promoters tested, significantly higher transduction efficiencies were achieved with the human elongation factor 1a promoter (EF1a promoter), the human cytomegalovirus (CMV) immediateearly 1 gene enhancer/b-actin promoter with b-actin intron (CA promoter), and the CMV promoter/enhancer with the largest intron of CMV (intron A) (CMVi promoter) in the human CD34 + cells and the immature subsets (CD34 + CD38 low/À and CD34 + AC133 + subsets). In particular, the CA promoter was found to allow for the highest transduction efficiencies in both the whole human CD34 + cells and the immature hematopoietic subsets. Furthermore, the CA promoter-mediated GFP-expressing cells differentiated into progenitor cells of all lineages. These results indicate the construction of an optimized Ad35 vector backbone for efficient transduction into HSCs/progenitors.

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Site-Specific Integration of a Transgene Mediated by a Hybrid Adenovirus/Adeno-Associated Virus Vector Using the Cre/loxP-Expression-Switching System

Cited by 34 publications

References 28 publications

Efficient gene transfer into human CD34+ cells by an adenovirus type 35 vector

Efficient gene transfer into human CD34+ cells by an adenovirus type 35 vector

Practical Range of Effective Dose for Cre Recombinase‐Expressing Recombinant Adenovirus without Cell Toxicity in Mammalian Cells

Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities

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