Intracellular trafficking and transgene expression of viral and non-viral gene vectors

Kamiya, Hiroyuki; Tsuchiya, Hiromichi; Yamazaki, Jumpei; Harashima, Hideyoshi

doi:10.1016/s0169-409x(01)00216-2

Cited by 101 publications

(72 citation statements)

References 62 publications

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“…Studies on Burkitt's lymphoma cells show that protracted expression of the transgene requires continuous selection by G418, arguing against integration. The observed instability of the transgene in B lymphocytes after spontaneous transgenesis in the absence of selective pressure is consistent with an extrachromosomal localization of the plasmid DNA 56,57 so that at each round of mitosis only one of the two daughter cells inherits the transgene causing its Transgenesis of human B lymphocytes G Filaci et al rapid dilution. Theoretical considerations also argue that integration is a remote eventuality.…”

Section: Transgenesis Of Human B Lymphocytes G Filaci Et Alsupporting

confidence: 56%

Spontaneous transgenesis of human B lymphocytes

et al. 2003

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DNA can cross the cell membrane by natural means, but the functional relevance of this phenomenon has not been fully elucidated. Here, we analyzed spontaneous transgenesis of human B cells using plasmid DNA coding for a functional immunoglobulin (Ig) heavy chain gene under the control of a B-cell-specific promoter. Using polymerase chain reaction (PCR), reverse transcriptase-PCR, and flow cytometry in combination, spontaneous transgenesis was documented in Burkitt's lymphoma cell lines, Epstein-Barr virus-transformed cell lines, and peripheral blood B lymphocytes of the mature naïve phenotype (IgM þ /IgD þ /CD27 À ). By immunoelectron microscopy, the internalized DNA was seen in the lysosomes/late endosomes and in the cytosol proximal to the nucleus. Importantly, spontaneously transgenic B cells processed and presented to major histocompatibility complex (MHC)-restricted T lymphocytes a peptide expressed in the transgenic product. This is the first demonstration that primary B lymphocytes possess a program for the spontaneous internalization of DNA, which in turn imparts the cell with new immunological functions. As spontaneous transgenesis is obtained using a nonviral vector, does not require prior cell activation, and is not associated with chromosomal integration, the findings reported here open new possibilities for genetic manipulations of mature naïve B lymphocytes for therapy and vaccination.

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Section: Transgenesis Of Human B Lymphocytes G Filaci Et Alsupporting

confidence: 56%

Spontaneous transgenesis of human B lymphocytes

et al. 2003

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“…Moreover, the intranuclear disposition, after the nuclear DNA entry, is also a key factor for efficient and prolonged transgene expression [4]. For example, transgene expression with non-viral vectors is generally transient [2]. This would be a consequence of the degradation/excretion of the exogenous DNA.…”

Section: Introductionmentioning

confidence: 99%

Intranuclear disposition of exogenous DNA in vivo: Silencing, methylation and fragmentation

2006

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The intranuclear disposition of exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was delivered into mouse liver by a hydrodynamics-based injection, and the amounts of intranuclear plasmid DNA, luciferase, and its mRNA were quantitated at various time points. Methylation of the promoter of the luciferase gene was also analyzed. Expression efficiency from one copy of the exogenous DNA dramatically decreased over time, and the DNA was methylated and degraded into fragments. Unexpectedly, methylation of the intact plasmid DNA was low and did not increase over time. Rather, the fragmented DNA was methylated more frequently than the intact plasmid. These results suggest that the CpG methylation and the degradation of exogenous DNA, and its 'silencing', occurred in parallel in the nucleus.

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“…Nonviral vectors are quite attractive, due to their excellent safety profile [1][2][3][4][5]. However, their low transgene expression efficiency, in comparison to that of viral vectors, is a major problem.…”

Section: (Introduction)mentioning

confidence: 99%

The location of the left-handedly curved DNA sequence affects exogenous DNA expression in vivo

Kamiya

Fukunaga

Ohyama

et al. 2007

Archives of Biochemistry and Biophysics

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The intranuclear disposition of a plasmid is extremely important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were . They also showed that histones bound to the left-handedly curved DNA, and that the TATA box was exposed out of the nucleosome core when the curved DNA was located at appropriate positions [9]. This finding prompted us to examine the effects of this left-handedly curved sequence in vivo. In this study, we investigated the in vivo effects of this sequence on plasmids when delivered in a naked form into mouse liver. We found that the position of the left-handedly curved sequence affected the transgene expression by one order of magnitude, without altering the amount of the exogenous DNAs, suggesting the different accessibility of transcriptional factors to the plasmids in vivo. Similar results were observed when plasmids complexed with cationic lipids were delivered into mouse liver. These results indicate that the left-handedly curved sequence on plasmids also determined 5 transgene expression in vivo, and that controlled interactions between the plasmid and the histones are important for the intranuclear disposition. Materials and Methods MaterialsOligodeoxyribonucleotides were purchased from Sigma Genosys Japan (Ishikari, Japan) in purified forms. The pLHC4/TLN-6, pLHC4/TLN-16, pLHC4/TLN+47, and pST0/TLN-7 plasmids, containing the thymidine kinase (tk) promoter and the luciferase gene (Fig. 1) [9], were amplified in Escherichia coli strain DH5α and purified with a Qiagen (Hilden, Germany) EndoFree Plasmid Mega kit. Hydrodynamics-based injectionPlasmid DNA (20 µg in 2 ml of saline) was injected into the tail vein of male six week-old Balb/c mice within 5 sec [10,11]. The livers were harvested from the injected mice at various time points, and the luciferase activity and the amount of the exogenous DNA were measured, as described below. Luciferase activity 6Livers were minced with scissors and homogenized completely in Lysis Buffer (100 mM Tris-HCl, 2 mM EDTA, 0.1% Triton X-100, pH 7.8). After centrifugation at 13,000 g for 10 min at 4°C, the supernatant was examined for luciferase activity, using a Luciferase Assay Systems kit (Promega, Madison, Wisconsin, USA). Isolation of nuclear DNA and Quantitative PCRLivers were homogenized in phosphate-buffered saline (PBS).After centrifugation at 2,500 g for 5 min at 4°C, the pellet was washed three times with PBS. The pellet was resuspended in DNA Lysis Buffer (100 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl 2 , 0.5% (w/v) IGEPAL-CA630, pH 7.4) [12]. After centrifugation at 1,400 g for 5 min at 4°C, the pellet was washed three times with DNA Lysis Buffer. The intranuclear DNA was extracted with the SepaGene reagent (Sanko Jun-yaku, Tokyo, Japan).Quantitative PCR (Q-PCR) was performed using an ABI 7500 real time PCR system, and SYBR-Green...

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Intracellular trafficking and transgene expression of viral and non-viral gene vectors

Cited by 101 publications

References 62 publications

Spontaneous transgenesis of human B lymphocytes

Spontaneous transgenesis of human B lymphocytes

Intranuclear disposition of exogenous DNA in vivo: Silencing, methylation and fragmentation

The location of the left-handedly curved DNA sequence affects exogenous DNA expression in vivo

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