High transfection efficiency, gene expression, and viability of monocyte-derived human dendritic cells after nonviral gene transfer

The interest in the use of human dendritic cells in cancer immunotherapy calls for efficient ex vivo methods of dendritic cell education. To extend the range of methods available, we generated phenotypically characteristic dendritic cells from peripheral blood monocytes incubated with granulocyte-macrophage colony-stimulating factor and interleukin-4 and infected them with an adenovirus containing a humanized version of green fluorescent protein as a marker of gene expression. The levels of expressed protein were high, but they were further increased in combination with cationic liposomes. In comparison to transfection efficiency of the homologous expression plasmid, adenovirus-mediated gene transfer was substantially more efficient. With the aid of liposome-mediated infection, gene transfer into CD83+ dendritic cells was highly effective, resulting in more than 90% of the cells expressing the transgene.

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High Efficiency Adenovirus-Mediated Gene Transfer to Human Dendritic Cells

Blood ◽

10.1182/blood.v91.2.392 ◽

1998 ◽

Vol 91 (2) ◽

pp. 392-398 ◽

Cited By ~ 152

Author(s):

Allan B. Dietz ◽

Stanimir Vuk-Pavlovic

Keyword(s):

Dendritic Cells ◽

Gene Transfer ◽

High Efficiency ◽

Fluorescent Protein ◽

Ex Vivo ◽

Transfection Efficiency ◽

Interleukin 4 ◽

Blood Monocytes ◽

Homologous Expression ◽

Human Dendritic Cells

Abstract The interest in the use of human dendritic cells in cancer immunotherapy calls for efficient ex vivo methods of dendritic cell education. To extend the range of methods available, we generated phenotypically characteristic dendritic cells from peripheral blood monocytes incubated with granulocyte-macrophage colony-stimulating factor and interleukin-4 and infected them with an adenovirus containing a humanized version of green fluorescent protein as a marker of gene expression. The levels of expressed protein were high, but they were further increased in combination with cationic liposomes. In comparison to transfection efficiency of the homologous expression plasmid, adenovirus-mediated gene transfer was substantially more efficient. With the aid of liposome-mediated infection, gene transfer into CD83+ dendritic cells was highly effective, resulting in more than 90% of the cells expressing the transgene.

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Nanosphere Induced Gene Expression in Human Dendritic Cells

Nano Letters ◽

10.1021/nl050541s ◽

2005 ◽

Vol 5 (11) ◽

pp. 2168-2173 ◽

Cited By ~ 15

Author(s):

Michiya Matsusaki ◽

Kristina Larsson ◽

Takani Akagi ◽

Malin Lindstedt ◽

Mitsuru Akashi ◽

...

Keyword(s):

Gene Expression ◽

Dendritic Cells ◽

Human Dendritic Cells

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Gene activation precedes DNA demethylation in response to infection in human dendritic cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814700116 ◽

2019 ◽

Vol 116 (14) ◽

pp. 6938-6943 ◽

Cited By ~ 39

Author(s):

Alain Pacis ◽

Florence Mailhot-Léonard ◽

Ludovic Tailleux ◽

Haley E. Randolph ◽

Vania Yotova ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Dendritic Cells ◽

Transcriptional Activation ◽

Time Course ◽

Gene Activation ◽

Dna Demethylation ◽

Epigenetic Mark ◽

Distal Enhancer ◽

Human Dendritic Cells

DNA methylation is considered to be a relatively stable epigenetic mark. However, a growing body of evidence indicates that DNA methylation levels can change rapidly; for example, in innate immune cells facing an infectious agent. Nevertheless, the causal relationship between changes in DNA methylation and gene expression during infection remains to be elucidated. Here, we generated time-course data on DNA methylation, gene expression, and chromatin accessibility patterns during infection of human dendritic cells withMycobacterium tuberculosis. We found that the immune response to infection is accompanied by active demethylation of thousands of CpG sites overlapping distal enhancer elements. However, virtually all changes in gene expression in response to infection occur before detectable changes in DNA methylation, indicating that the observed losses in methylation are a downstream consequence of transcriptional activation. Footprinting analysis revealed that immune-related transcription factors (TFs), such as NF-κB/Rel, are recruited to enhancer elements before the observed losses in methylation, suggesting that DNA demethylation is mediated by TF binding to cis-acting elements. Collectively, our results show that DNA demethylation plays a limited role to the establishment of the core regulatory program engaged upon infection.

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Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle

Gene Therapy ◽

10.1038/sj.gt.3301678 ◽

2002 ◽

Vol 9 (6) ◽

pp. 372-380 ◽

Cited By ~ 312

Author(s):

Y Taniyama ◽

K Tachibana ◽

K Hiraoka ◽

M Aoki ◽

S Yamamoto ◽

...

Keyword(s):

Skeletal Muscle ◽

Gene Transfer ◽

Plasmid Dna ◽

Transfection Efficiency ◽

Nonviral Gene Transfer ◽

Naked Plasmid ◽

Ultrasound Enhancement ◽

Naked Plasmid Dna

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Faculty Opinions recommendation of Tumor-derived immuno-modulators induce overlapping pro-tolerogenic gene expression signatures in human dendritic cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726757992.793550880 ◽

2018 ◽

Author(s):

Xiaojing Ma

Keyword(s):

Gene Expression ◽

Dendritic Cells ◽

Gene Expression Signatures ◽

Human Dendritic Cells

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Reduction of Inhibitory Anti-FVIII Antibody Titer by Using a B Domain Variant FVIII/N6 cDNA for Nonviral Gene Therapy in Hemophilia a Mice

Blood ◽

10.1182/blood.v112.11.3537.3537 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3537-3537 ◽

Cited By ~ 2

Author(s):

Dominika Jirovska ◽

Peiqing Ye ◽

Steven W. Pipe ◽

Carol H. Miao

Keyword(s):

Gene Expression ◽

Gene Therapy ◽

Gene Transfer ◽

Hemophilia A ◽

High Titer ◽

Specific Gene ◽

Nonviral Gene Transfer ◽

Inhibitory Antibodies ◽

Domain Variant

Abstract Due to the large size of FVIII, a B-domain deleted FVIII (BDD-FVIII) cDNA is usually used for developing gene therapy protocols for treating hemophilia A. Inefficient transcription of wild- type FVIII cDNA can be overcome by deletion of the heavily glycosylated B-domain encoding portion of the gene. BDD-FVIII is as clinically efficacious and not more immunogenic than full-length recombinant FVIII. More recently, it was demonstrated that a partial deletion of the B-domain leaving an N-terminal 226 amino acid stretch containing 6 putative asparagine-linked glycosylation sites intact (FVIII/N6) was able to increase in vitro and in vivo secretion of FVIII by 10–15 fold. We have inserted this B domain variant FVIII/N6 cDNA into our liver-specific gene expression vector. The resulting construct, FVIII/N6 plasmid was delivered into the hemophilia A mouse liver by the hydrodynamic method. In control mice treated with BDD-FVIII plasmid (n=5/group), FVIII expression dropped to undetectable levels at 2 weeks post injection and high-titer anti-FVIII antibodies were generated in all the plasmid-treated mice. However, in mice treated with FVIII/N6 plasmid (n=5/group), one out of five mice never developed inhibitory antibodies and still had some FVIII gene expression (~10%) at 8 weeks post gene transfer. Three FVIII/N6 plasmid-treated mice developed anti-FVIII antibodies with significantly reduced inhibitor titer and only one mouse developed high-titer inhibitory antibodies. The CD4+ T cells isolated from the spleen of mice injected with FVIII/N6 constructs proliferated less in response to FVIII stimulation than those from mice injected with BDD-FVIII. These results indicate that FVIII/N6 protein is less immunogenic than BDD-FVIII. Interestingly, both BDD-FVIII and FVIII/N6 constructs produced similar levels of FVIII gene expression (100–300%) initially following nonviral gene transfer. However this could be due to saturation of the ER to Golgi transport apparatus for FVIII by the initial high-level gene expression. Gene expression levels produced by using reduced dosages of BDD-FVIII and FVIII/N6 plasmids are currently being evaluated and compared. These findings suggest that use of a FVIII/N6 construct decreases transgene-specific immune responses following nonviral gene transfer and facilitates long-term gene expression.

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