Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo

Zufferey, Romain; Nagy, Dea; Mandel, Ron; Naldini, Luigi; Trono, Didier

doi:10.1038/nbt0997-871

Cited by 1,847 publications

(1,459 citation statements)

References 38 publications

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“…HEK 293T cells HEK‐293T cells were transfected with equal amounts of p8.91 (Zufferey et al , 1997), pMD‐G (Naldini et al , 1996), and pHR‐U6P‐dsRed encoding the shRNA or transgene of interest using Genejuice (Novagen Merck) as per manufacturers’ recommendation. Viral supernatant was harvested 48–72 h post‐transfection and concentrated by ultracentrifugation.…”

Section: Methodsmentioning

confidence: 99%

Snapin promotes HIV ‐1 transmission from dendritic cells by dampening TLR 8 signaling

et al. 2017

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HIV‐1 traffics through dendritic cells (DCs) en route to establishing a productive infection in T lymphocytes but fails to induce an innate immune response. Within DC endosomes, HIV‐1 somehow evades detection by the pattern‐recognition receptor (PRR) Toll‐like receptor 8 (TLR8). Using a phosphoproteomic approach, we identified a robust and diverse signaling cascade triggered by HIV‐1 upon entry into human DCs. A secondary siRNA screen of the identified signaling factors revealed several new mediators of HIV‐1 trans‐infection of CD4+ T cells in DCs, including the dynein motor protein Snapin. Inhibition of Snapin enhanced localization of HIV‐1 with TLR8+ early endosomes, triggered a pro‐inflammatory response, and inhibited trans‐infection of CD4+ T cells. Snapin inhibited TLR8 signaling in the absence of HIV‐1 and is a general regulator of endosomal maturation. Thus, we identify a new mechanism of innate immune sensing by TLR8 in DCs, which is exploited by HIV‐1 to promote transmission.

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Section: Methodsmentioning

confidence: 99%

Snapin promotes HIV ‐1 transmission from dendritic cells by dampening TLR 8 signaling

et al. 2017

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“…Infected cells were selected with 1 mg/ml puromycin. Recombinant retroviruses encoding DNp73a, c-Myc or shRNAs were produced by transient transfection of 293T cells according to standard protocols (Zufferey et al, 1997). Briefly, subconfluent 293T cells were cotransfected with a plasmid vector, and the helper plasmids pCMV-R8.74, pMD2G and pRSV-rev by calcium phosphate precipitation.…”

Section: Viral Vectors and Infectionsmentioning

confidence: 99%

Regulation of telomerase activity by the p53 family member p73

Beitzinger

Oswald

Beinoraviciute-Kellner

et al. 2005

Oncogene

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The terminal ends of eukaryotic chromosomes, termed telomeres, progressively shorten during each round of cell division eventually leading cells into senescence. Tumor cells typically overcome this barrier to unlimited proliferation by activation of the human telomerase reverse transcriptase (hTERT) gene. In contrast, in most human somatic cells hTERT expression is tightly repressed by multiple tumor suppressors. Here, we studied the regulation of hTERT by the p53 family member p73. We show that forced expression of p73 or activation of endogenous p73 by E2F1 results in the downregulation of telomerase activity. Vice versa, siRNA-mediated knockdown of p73 induces hTERT expression. Responsiveness to p73 is conferred by Sp1 binding sites within the hTERT core promoter. In tumor cells, p73 isoforms lacking the transactivation domain (DNp73) are frequently overexpressed and believed to function as oncogenes. We show that DNp73 antagonizes the repressive effect of the proapoptotic p53 family members on hTERT expression and, in addition, induces hTERT expression in telomerase-negative cells by interfering with E2F-RB-mediated repression of the hTERT core promoter. These data provide evidence that the p73 gene functions as an important regulator of telomerase activity with implications for embryonic development, cellular differentiation and tumorigenesis.

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“…[31][32][33] These vectors proved highly efficient for in vivo delivery and stable expression of the transgene in several target tissues. 31,32,34 Currently, lentivirus-derived vectors are obtained through transient transfection protocols.…”

Section: Discussionmentioning

confidence: 99%

Transgene amplification and persistence after delivery of retroviral vector and packaging functions with E1/E4-deleted adenoviruses

et al. 2000

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Adenovirus DNA is rapidly lost in actively dividing cells. In addition, first-generation (E1-defective) vectors trigger a strong cytotoxicity that impairs the duration of transgene expression. To solve these issues, we have developed a chimeric vector system that uses E1/E4 doubly defective adenoviruses for efficient production of infectious retroviral vectors. The retroviral vector sequences and packaging functions were split into two E1/E3/E4-deleted adenoviral vectors: the Moloney murine leukemia virus gag-pol cistron was expressed from the human EF1␣ (elongation factor) promoter (AdGAG/POL), whereas the thymidine kinase transgene, embedded in a retroviral vector context, and an amphotropic retroviral envelope cassette were included within a second adenovirus (AdTK/ENV). This chimeric vector system was evaluated with a special emphasis on recombinant retrovirus production in vitro, as well as transgene amplification and persistence in vivo. Retrovirus titers of Ͼ10 5 infectious units/mL were routinely obtained in W162 cells coinfected with both recombinant adenoviruses. Long-term transgene persistence (up to 3 months) was demonstrated in vitro in two different cell lines coinfected with AdGAG/POL and AdTK/ENV, and correlated with the detection of specific provirus sequences. A 10-to 50-fold transgene amplification also was demonstrated in an in vivo tumor model infected with the Ad/Rt chimeric vector system. The chimeric vector system described herein combines the efficiency of gene delivery by recombinant adenoviruses with the integrative properties of infectious retroviral vectors. This versatile vector system may open up new avenues for efficient production of oncogenic, but also non-oncogenic, retroviruses from cells of non-murine origin.

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Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo

Cited by 1,847 publications

References 38 publications

Snapin promotes HIV ‐1 transmission from dendritic cells by dampening TLR 8 signaling

Snapin promotes HIV ‐1 transmission from dendritic cells by dampening TLR 8 signaling

Regulation of telomerase activity by the p53 family member p73

Transgene amplification and persistence after delivery of retroviral vector and packaging functions with E1/E4-deleted adenoviruses

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