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Order Tymovirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Kim et al. (2018) demonstrated a clear potential of modified TYMV as an efficient system for therapeutic cargo delivery to mammalian cells. The authors provided TYMV with Tat, a cell penetrating peptide, and demonstrated ability of such particles to enter animal cells, namely, baby hamster kidney (BHK) cells. The Tat peptide was chemically attached to the surface lysine residues of TYMV using hydrazone chemistry. The Tat conjugation was more efficient than lipofectamine in allowing TYMV to enter the cells, and the Tat-assisted transfection was associated with less loss of cell viability than lipofection. Among the cell-penetrating peptides tested (Tat, R8, Pep-1 and Pen), the R8 and Pen were also effective while Pep-1 was not. The internal space of TYMV was loaded with fluorescein dye as a model cargo, after viral RNA was removed by freezing and thawing. When the resultant empty particles were reacted with fluorescein-5-maleimide using interior sulfhydryl groups as conjugation sites, about 145 fluorescein molecules were added per particle. The fluorescein-loaded TYMV particles were conjugated with Tat and introduced into BHK cells, again with higher transfection efficiency compared to lipofection (Kim et al. 2018).
Functional Study of Lysosomal Nutrient Transporters
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Xavier Leray, Corinne Sagné, Bruno Gasnier
Another technical aspect to consider is the method used to express recombinant proteins. Lipofection should be avoided because endocytosis of DNA/lipofectant complexes induces a long-lasting lysosomal stress, resulting in a strong nuclear translocation of TFEB even 72 h after transfection. In contrast, electroporation promotes DNA entry through transient holes in the plasma membrane. Although nuclear translocation of TFEB also occurs on the first day, this effect fully reverses after 48 h if no other lysosomal stress (ester treatment) is delivered. Another advantage of electroporation over lipofection is the lower level of expression, yielding better lysosomal targeting of the recombinant transporter.
Nucleic Acids
Published in Danilo D. Lasic, LIPOSOMES in GENE DELIVERY, 2019
With current developments larger and larger quantities of plasmids are required. Several purification recipes have been improved and allow large-scale preparation. The purity of the plasmid is very important not only for transfection but also for complexation with cationic species. Very often contamination with RNA, proteins, short fragments of degraded DNA and chromosomal DNA occurs which adversely (and irreproducibly) affects the properties of DNA, and complexes as well as transfection experiments. Some researchers have told me that none of the commercially available DNA preparation kits can purify DNA adequately and that they do additional extractions and precipitations. For in vivo applications special care must be taken to ensure minimal levels of endotoxins. Their role in transfection is not known while in the in vitro experiments it was shown that increasing levels decreased gene expression (Weber et al., 1995). Briefly, gene expression decreases rapidly with increasing endotoxin. At 100 units of lipopolysaccharide per microgram of plasmid, DNA gene expression halves lipofection while Ca phosphate transfection is ten times more sensitive. Endotoxin-contaminated preparations cause death in laboratory animals which can be observed by the pathology of the digestive tract and swollen spleen and liver.
In vivo dynamics and anti-tumor effects of EpCAM-directed CAR T-cells against brain metastases from lung cancer
Published in OncoImmunology, 2023
Tao Xu, Philipp Karschnia, Bruno Loureiro Cadilha, Sertac Dede, Michael Lorenz, Niklas Seewaldt, Elene Nikolaishvili, Katharina Müller, Jens Blobner, Nico Teske, Julika J. Herold, Kai Rejeski, Sigrid Langer, Hannah Obeck, Theo Lorenzini, Matthias Mulazzani, Wenlong Zhang, Hellen Ishikawa-Ankerhold, Veit R. Buchholz, Marion Subklewe, Niklas Thon, Andreas Straube, Joerg-Christian Tonn, Sebastian Kobold, Louisa von Baumgarten
A PCR product containing the sequence of tdTomato (vector ptdTomato; #63-2531, TaKaRa Clontech) was cloned into the lentiviral expression vector pLVX-IRES-neo (LentiX-Bicistronic Expression System; #63-2181, TaKaRa Clontech) to generate a pLVX-tdTomato-IRES-Neo construct. Notably, a resistance-sequence for G418-sulfate is contained in the lentiviral expression vector. The resulting nucleotide pLVX-tdTomato-IRES-Neo was verified by Sanger sequencing and restriction enzyme digestion.12 LL/2 was transfected with pLVX-tdTomato-IRES-Neo using lipofection (Lipofectamine 3000; Thermo Fisher Scientific). tdtLL/2 was enriched by cultivation in selection medium containing G418-sulfate (#A2912; Biochrom) and by repetitive FACS sorting. As previously described in detail,13 tdtLL/2 cells were stably transduced with a pMXs vector containing the full-length murine EpCAM (UNIPROT entry: #Q99JW5) cDNA to generate the EpCAM-overexpressing cell line EpCAM/tdtLL/2.
Lipoplex-based therapeutics for effective oligonucleotide delivery: a compendious review
Published in Journal of Liposome Research, 2020
Pirthi Pal Singh, Veena Vithalapuram, Sunita Metre, Ravinder Kodipyaka
The deliberate introduction of naked NAs into the eukaryotic cell is termed as transfection and the process utilizing liposomes for this purpose is known as lipofection. The liposomes transport the genetic material into a cell by means of merging with the cell membrane as shown in Figure 4. The trials on the improvement of the lipofection strategy are still largely based on empirical approaches towards the formulation of optimal lipids, especially in the case of new and distinct classes of lipofecting compounds. The efficient in-vivo lipofection was reported to be the result of cross-talk between lipoplex composition, interaction with serum, hemodynamics, and target tissue susceptibility to transfection (Simberg et al.2003).
Physical transfection technologies for macrophages and dendritic cells in immunotherapy
Published in Expert Opinion on Drug Delivery, 2021
Aranit Harizaj, Stefaan C. De Smedt, Ine Lentacker, Kevin Braeckmans
Alternatively, transfection technologies making use of non-viral nanocarriers have been used for the cytosolic delivery of various compounds into macrophages and DCs. Several types of nanocarriers have been shown to protect antigens against degradation and increase intracellular delivery of the antigens compared to spontaneous uptake of the antigens by itself [28]. The primary intended use of nanocarriers is for treatment of APCs in vivo, which is quite attractive as well as it would reduce the labor-intensive isolation and ex vivo modification of APCs and the associated costs. However, at present this remains a great challenge and many steps still need to be taken, including specific in vivo targeting of APCs. Cationic nanocarriers such as liposomal formulations have been frequently explored to deliver pDNA, mRNA and siRNA into APCs [28–30]. However, lipofection experiments have shown variable results with quite low transfection efficiencies between 0–10% for pDNA [31–34] and 0–30% for mRNA [32,34–37]. For other cationic nanocarriers it was shown that delivery of model antigens (e.g., ovalbumin) into APCs an antigen specific immune response could be obtained which outperformed the response induced by soluble antigens alone [38,39]. A particular reported disadvantage of nanocarriers, especially for ex vivo modification of cells, is the potential induction of cellular toxicity [34,35,40–42]. Several nanocarriers were reported to influence some important functions of APCs like maturation, migration and antigen presentation [43]. Moreover, since nanocarriers are often charged, they can induce the activation of the NLRP3 response resulting in increased immunogenicity [44].