Enhanced non-viral gene delivery by coordinated endosomal release and inhibition of β-tubulin deactylase

Ho, Yew Kee; Zhou, Li; Tam, Kam Chiu; Too, Heng Phon

doi:10.1093/nar/gkw1143

Cited by 24 publications

(30 citation statements)

References 53 publications

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“…12,13 Many DNMT inhibitors such as 5-Aza-2'-deoxycytidine and zebularine have been used to prevent methylation of DNA. 14,15 Several inhibitors of histone deacetylases (HDACs) have also been shown to increase transgene expression, including Entinostat (a HDAC 1/3 inhibitor) 16 , Tubacin (HDAC6i) 17 , Trichostatin A (HDAC6i) [18][19][20] , and Vorinostat (pan-HDACi) [21][22][23] . However, it is important to note that some of these HDAC inhibitors enhance transgene expression by influencing cytoplasmic transport instead of directly influencing histone modifications.…”

Section: Introductionmentioning

confidence: 99%

Enhancement of Transgene Expression by NF-Y and CTCF

Zimmerman

Patel

Hall

et al. 2018

Preprint

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If a transgene is effectively delivered to a cell, its expression may still be limited by epigenetic mechanisms that silence the transgene. Indeed, once the transgene reaches the nucleus, it may be bound by histone proteins and condensed into heterochromatin or associated with repressor proteins that block transcription. In this study, we sought to enhance transgene expression by adding binding motifs for several different epigenetic enzymes either upstream or downstream of two promoters (CMV and EF1α). Screening these plasmids revealed that luciferase expression was enhanced 10-fold by the addition of a CCAAT box just upstream of the EF1α promoter to recruit nuclear transcription factor Y (NF-Y), while inserting a CCCTCbinding factor (CTCF) motif downstream of the EF1α promoter enhanced expression 14-fold (14.03 ± 6.54). ChIP assays confirmed that NF-Y and CTCF bound to the motifs that were added to each plasmid, but the presence of NF-Y and CTCF did not significantly affect the levels of histone acetylation (H3K9ac). Overall, these result show that transgene expression from the EF1α promoter can be significantly increased with motifs that recruit NF-Y or CTCF.

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Section: Introductionmentioning

confidence: 99%

Enhancement of Transgene Expression by NF-Y and CTCF

Zimmerman

Patel

Hall

et al. 2018

Preprint

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“…Removal of excess polymers via size-exclusion chromatography (SEC) greatly improved the toxic profile of PEI polyplexes [ 26 ]. On the other hand, mild centrifugation at 200–280 g for 5 min was shown to facilitate the deposition of polyplexes [ 27 , 28 ]. Instead of SEC, we explored the feasibility of mild centrifugation in depositing LG100 polyplexes onto the cell surface and subsequently removing excess polymer.…”

mentioning

confidence: 99%

“…We have previously explored the effects of various enhancers in improving gene delivery including chloroquine [ 31 ], PLUS reagent™, and fusogenic peptides [ 32 ]. In all cases, these reagents did not significantly improve the transfection efficiencies in differentiated neuronal cells [ 33 ]. Our previous study in unraveling the mechanisms in intracellular trafficking of polyplexes resulted in the development of a transfection enhancing method [ 33 ].…”

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confidence: 99%

Enhanced transfection of a macromolecular lignin-based DNA complex with low cellular toxicity

Kai

et al. 2018

Bioscience Reports

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Cationic polymers remain attractive tools for non-viral gene transfer. The effectiveness of these vectors rely on the ability to deliver plasmid DNA (pDNA) into the nucleus of cells. While we have previously demonstrated the potential of Lignin-PGEA-PEGMA as a non-viral gene delivery vector, alterations of cellular phenotype and cytotoxicity were observed post transfection. The present study aims to explore transfection conditions for high efficiency and low toxicity of the Lignin-PGEA-PEGMA based gene delivery system. Cellular toxicity was significantly reduced by using the centrifugation protocol, which enables rapid deposition of DNA complexes. Replacement of media post centrifugation resulted in minimal exposure of cells to excess polymers, which were toxic to cells. At an optimized DNA amount (500–750 ng) and molar ratios of nitrogen (N) in polymer to phosphate (P) in pDNA (N/P = 30–40), with the use of a novel transfection enhancer that facilitates endosomal escape and nuclear trafficking, the efficiency of gene delivery was increased significantly 24 h post transfection. The present study demonstrated an appropriately optimized protocol that enabled the utility of a novel cationic polymer blend with a mixture of fusogenic lipids and a histone deacetylate inhibitor in non-viral transfection, thereby providing an attractive alternative to costly commercial gene carriers.

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“…24,25 Nonviral gene delivery could be highly efficient in ex vivo, including that asso-ciated with plasmid DNA and mRNA. 26 CRISPR/Cas9 delivery by the nonviral approach has some advantages and could partly avoid various concerns associated with viral-based delivery. Hence, in this study, liposome nanoparticles were used for the nonviral delivery of the CRISPR/Cas9 E6E7-KO plasmid.…”

Section: Discussionmentioning

confidence: 99%

Gene Targeting of HPV18 E6 and E7 Synchronously by Nonviral Transfection of CRISPR/Cas9 System in Cervical Cancer

Ling

Yang

et al. 2020

Human Gene Therapy

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High-risk human papillomavirus (HPV) E6 and E7 genes display vital oncogenic properties in cervical cancer. Eliminating HPV driver gene or loss of function by the clustered regularly interspaced short palindromic repeat (CRISPR)/ Cas9 system is a promising treatment for the HPV-associated cancer. Thus, this study designed a CRISPR/Cas9 system to target the E6 and E7 genes at once, to detect whether it have efficacy in vitro and in vivo. Meanwhile, CRISPR/Cas9 system was measured after transfection with liposomes but virus. Cervical cancer lines (HeLa and SiHa) were used in this study. Sanger sequencing confirmed that the single CRISPR/Cas9 vector [termed E6E7-knockout (KO)] containing guide RNAs could targeting both HPV18 E6 and E7 genes in vitro. In addition, double-targeting E6 and E7 increased p53 protein expression significantly while compared with E6 or E7 targeting, respectively. Mice with xenografts were divided into four groups: three doses of experimental groups (20, 40, and 60 lg) and one control group. The E6E7-KO through liposome delivery was injected into tumors. Tumor growth was measured and protein expression was observed through immunohistochemistry. The toxic side effects in vivo were also evaluated. E6E7-KO induced cell apoptosis and inhibited cell proliferation markedly in vitro. E6E7-KO downregulated the messenger RNA and protein expression of E6 and E7, whereas p53 and p21 protein levels were upregulated accordingly. Notably, E6E7-KO delivery by liposome exhibited an effect in vivo. Tumor growth was inhibited in the E6E7-KO groups, which was accompanied by decreased E6/E7 protein expression and increased p53/p21 protein expression, especially the level of p53 protein expression. Therefore, E6E7-KO could have synergy efficient by p53 pathway. Furthermore, local injection with CRISPR/Cas9 by nonviral delivery may be regarded as a potential therapy for cervical cancer in the future.

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Enhanced non-viral gene delivery by coordinated endosomal release and inhibition of β-tubulin deactylase

Cited by 24 publications

References 53 publications

Enhancement of Transgene Expression by NF-Y and CTCF

Enhancement of Transgene Expression by NF-Y and CTCF

Enhanced transfection of a macromolecular lignin-based DNA complex with low cellular toxicity

Gene Targeting of HPV18 E6 and E7 Synchronously by Nonviral Transfection of CRISPR/Cas9 System in Cervical Cancer

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