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. 2011 Jan;22(1):77-83.
doi: 10.1089/hum.2010.122. Epub 2010 Dec 12.

Efficient transgene reconstitution with hybrid dual AAV vectors carrying the minimized bridging sequences

Arkasubhra Ghosh  1 , Yongping Yue, Dongsheng Duan
Affiliations

V体育ios版 - Efficient transgene reconstitution with hybrid dual AAV vectors carrying the minimized bridging sequences

"V体育官网" Arkasubhra Ghosh et al. Hum Gene Ther. 2011 Jan.

Abstract

A hybrid dual-vector system was developed recently as a universal platform to double the packaging capacity of recombinant adeno-associated virus (AAV). In this system, the expression cassette is split into two independent AAV vectors. A highly recombinogenic bridging DNA sequence is engineered in both vectors to mediate target gene-independent homologous recombination between the split vector genomes VSports手机版. In the prototype hybrid vectors, a 0. 87-kb DNA fragment from the middle portion of the human placental alkaline phosphatase (AP) gene was used as the bridging sequence. Here we report the development of the minimized bridging sequences. Five independent bridging sequences (0. 26 to 0. 44 kb) were evaluated in MO59K cells and/or murine skeletal muscle in the context of the AP overlapping vectors and/or the β-galactosidase (LacZ) hybrid vectors. Robust reconstitution comparable to that of the original hybrid vectors was achieved from a 0. 26-kb and a 0. 27-kb bridging sequence. These newly developed bridging sequences greatly expand the utility of the hybrid dual AAV vector system for delivering larger therapeutic genes/expression cassettes. .

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"V体育ios版" Figures

FIG. 1.
FIG. 1.
In vitro analysis of the AAV-6 AP overlapping vectors and the AAV-6 LacZ hybrid vectors that are based on the 0.43- and 0.44-kb bridging sequences. (A) Left panel: Schematic outline of the overlapping regions in the original and two new AP overlapping vectors. In the original AP overlapping vectors, the upstream and downstream vectors shared a 0.87-kb fragment (the middle part of the AP gene). In the 1/2 head and 1/2 tail overlapping vectors, the upstream and downstream vectors shared a 0.44-kb (the head half of the 0.87-kb fragment) and a 0.43-kb (the tail half of the 0.87-kb fragment), respectively. Right panel: Quantification of AP-positive cells after co-infection (500 vg particles/vector/cell). n = 5 per group. (B) Left panel: Schematic outline of the LacZ hybrid vectors that used the 0.87-kb (original LacZ hybrid vectors), 0.44-kb (1/2 head LacZ hybrid vectors), and 0.43-kb (1/2 tail LacZ hybrid vectors) regions of the AP gene as the bridging sequences. Right panel: Quantification of β-galactosidase activities in coinfected cells (10,000 vg particles/vector/cell). *Significantly higher than other groups. n = 3 for the original LacZ hybrid vectors, n = 6 per group for the 1/2 head and 1/2 tail LacZ hybrid vectors.
FIG. 2.
FIG. 2.
Evaluation of the AAV-6 AP overlapping vectors based on the 0.26-, 0.27-, and 0.34-kb overlapping sequences in MO59K cells. (A) Schematic outline of the overlapping regions in the original and new overlapping vectors. (B) Quantification of AP-positive cells after coinfection (500 vg particles/vector/cell). *Significantly lower than other groups. n = 5 per group.
FIG. 3.
FIG. 3.
Evaluation of the AAV-6 LacZ hybrid vectors based on the 0.26-, 0.27-, and 0.34-kb bridging sequences in MO59K cells. (A) Schematic outline of the LacZ hybrid vectors that used the 0.87-kb (original LacZ hybrid vectors), 0.26-kb (1/3 head LacZ hybrid vectors), 0.34-kb (1/3 body LacZ hybrid vectors), and 0.26-kb (1/3 tail LacZ hybrid vectors) regions of the AP gene as the bridging sequences. (B) Quantification of β-galactosidase activities in coinfected cells (10,000 vg particles/vector/cell). *Significantly lower than other groups. n = 3 for the original LacZ hybrid vectors, n = 6 per group for the other LacZ hybrid vectors.
FIG. 4.
FIG. 4.
In vivo performance of the AAV-6 LacZ hybrid vectors based on the minimized bridging sequences. (A) Representative LacZ histochemical staining of the TA muscle coinfected with different sets of the LacZ hybrid vectors (1 × 1010 vg particles/vector/muscle). Scale bar applies to all images. (B) Quantification of the transduction efficiency in muscle section (percentage of LacZ-positive cells; left panel) and muscle lysate (β-galactosidase activity; right panel). *Significantly lower than other groups. n = 4 per group. Color images available online at www.liebertonline.com/hum.
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References

    1. Allalunis-Turner M.J. Barron G.M. Day R.S., 3rd Dobler K.D. Mirzayans R. Isolation of two cell lines from a human malignant glioma specimen differing in sensitivity to radiation and chemotherapeutic drugs. Radiat. Res. 1993;134:349–354. - PubMed (V体育安卓版)
    1. Allen J.M. Halbert C.L. Miller A.D. Improved adeno-associated virus vector production with transfection of a single helper adenovirus gene, E4orf6. Mol. Ther. 2000;1:88–95. - PubMed
    1. Bostick B. Ghosh A. Yue Y. Long C. Duan D. Systemic AAV-9 transduction in mice is influenced by animal age but not by the route of administration. Gene Ther. 2007;14:1605–1609. - V体育ios版 - PubMed
    1. Dong B. Nakai H. Xiao W. Characterization of genome integrity for oversized recombinant AAV vector. Mol. Ther. 2010;18:87–92. - PMC (V体育官网入口) - PubMed
    1. Dong J.Y. Fan P.D. Frizzell R.A. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum. Gene Ther. 1996;7:2101–2112. - PubMed

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