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Haberland, Annekathrin; Knaus, Thomas; Зайцев, С. В.; Buchberger, Bernd; Lun, Andreas; Haller, Hermann; Böttger, Michael

doi:10.1023/a:1007581700996

Cited by 33 publications

(7 citation statements)

References 18 publications

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“…It has been previously reported that the presence of serum may have a significant influence on the transfection efficiency of nonviral gene delivery vehicles (55–57). To determine the effect of serum on gene expression, A549 cells were transfected with optimized TAT-Ca/pDNA (0.3 M CaCl 2 ) or PEI/pDNA complexes in the absence and presence of 10% FBS.…”

Section: Resultsmentioning

confidence: 99%

“Soft” Calcium Crosslinks Enable Highly Efficient Gene Transfection Using TAT Peptide

et al. 2009

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Purpose Typically, low molecular weight cationic peptides or polymers exhibit poor transfection efficiency due to an inability to condense plasmid DNA into small nanoparticles. Here, efficient gene delivery was attained using TAT/pDNA complexes containing calcium crosslinks. Methods Electrostatic complexes of pDNA with TAT or PEI were studied with increasing calcium concentration. Gel electrophoresis was used to determine DNA condensation. The morphology of the complexes was probed by transmission electron microscopy. Transfection efficiency was assessed using a luciferase reporter plasmid. The accessibility of phosphate and amine groups within complexes was evaluated to determine the effect of calcium on structure. Results TAT/pDNA complexes were condensed into small, 50–100 nm particles by optimizing the concentration of calcium. Complexes optimized for small size also exhibited higher transfection efficiency than PEI polyplexes in A549 cells. TAT and TAT complexes displayed negligible cytotoxicity up to 5 mg/mL, while PEI exhibited high cytotoxicity, as expected. Probing the TAT-Ca/pDNA structure suggested that calcium interacted with both phosphate and amine groups to compact the complexes; however, these “soft” crosslinks could be competitively disrupted to facilitate DNA release. Conclusion Small and stable TAT-Ca/pDNA complexes were obtained via “soft” calcium crosslinks leading to sustained gene expression levels higher than observed for control PEI gene vectors. TAT-Ca/pDNA complexes were stable, maintaining particle size and transfection efficiency even in the presence of 10% of FBS. TAT-Ca complexes offer an effective vehicle offering potential for translatable gene delivery.

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Section: Resultsmentioning

confidence: 99%

“Soft” Calcium Crosslinks Enable Highly Efficient Gene Transfection Using TAT Peptide

et al. 2009

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“…Peptides corresponding to consecutive regions of charged amino acids peptides 10, 12, 13, and 16 were synthesized, and peptide 10, corresponding to residues 1-36 of histone H2A, was found to be active. Peptides that subdivided this N-terminal region (peptides [11][12][13][14] or fused it with the C terminus (peptide 17) had background levels of transfection activity. Similarly, peptide chimeras of the Nterminal portion of peptide 10 fused with sequences from H2B, H3, and H4 (peptides 27-29) lacked significant transfection activity.…”

Section: Resultsmentioning

confidence: 99%

“…It was not observed with other histones or cationic peptides (5,6). Previous studies have demonstrated that histones H1 (7)(8)(9)(10)(11)(12), H2A (5,6,13,14), and H3 and H4 (15) are effective mediators of transfection. The postulated mechanisms by which histone H1 increases gene transfection are through DNA condensation, DNase protection, and the mediation of nuclear import.…”

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confidence: 99%

Structure and function correlation in histone H2A peptide-mediated gene transfer

Balicki

Putnam

Scaria

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Histone H2A has been found to be efficient in DNA delivery into a number of cell lines. We have reasoned that this DNA-delivery activity is mediated by two mechanisms: (i) electrostatically driven DNA binding and condensation by histone and (ii) nuclear import of these histone H2A⅐DNA polyplexes via nuclear localization signals in the protein. We have identified a 37-aa N-terminal peptide of histone H2A that is active in in vitro gene transfer. This peptide can function as a nuclear localization signal and can bind DNA. Amino acid substitutions that replace positively charged residues and͞or DNA-binding residues of this peptide obliterate transfection activity. The introduction of a proline in the first turn of an ␣-helix of this 37-mer obliterates transfection activity, suggesting that the integrity of the ␣-helical structure of the N-terminal region of histone H2A is related to its transfection activity.transfection ͉ nuclear localization signal ͉ DNA binding G ene transfer into eukaryotic cells has the potential to treat a large number of incurable diseases. The goal of nonviral gene therapy is to mimic the successful viral mechanisms for overcoming cellular barriers that block efficient expression of the target gene while minimizing the toxicities associated with gene delivery (1). The capabilities of a synthetic nonviral vector could include specific binding to the cell surface, entry, endosomal escape, translocation to the nucleus, and in some cases stable integration into the target cell genome. The rate-limiting step of current nonviral gene delivery techniques is the transfer of encapsulated plasmids from the endosomes to the nucleus (2). The potential advantages of protein͞peptide gene transfer include ease of use, production, purity, homogeneity, ability to target nucleic acids to specific cell types, the potential for cost-effective large-scale manufacture, modular attachment of targeting ligands, and the lack of limitation on the size or type of the nucleic acid that can be delivered (1). The critical first step for efficient gene delivery is the formation of the polyplex, or complex between DNA and protein͞peptide (1-3).Eukaryotic DNA within the nucleus is packaged by wrapping around a histone octamer consisting of two molecules each of histone H2A, H2B, H3, and H4, and this packaging has dramatic effects on DNA metabolism and gene regulation (reviewed by Van Holde in ref. 4). We determined that histone octamers mediate transfection into a number of cell lines and that this activity is localized to H2A. It was not observed with other histones or cationic peptides (5, 6). Previous studies have demonstrated that histones H1 (7-12), H2A (5, 6, 13, 14), and H3 and H4 (15) are effective mediators of transfection. The postulated mechanisms by which histone H1 increases gene transfection are through DNA condensation, DNase protection, and the mediation of nuclear import. In these studies, histone H1 was considered to be the subclass of histones that mediates efficient gene transfer in the presence of chlo...

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“…The presence of 2 mM Cat" is capable to counteract the inhibition of transfection by serum when the DNA is already inside the cell (37). This effect may be explained because the serum seems to inhibit the escape of the DNA from the endosomal DNA-CLC rather than the translocation through the plasma membrane (37).…”

Section: Bombardementmentioning

confidence: 99%

“…This effect may be explained because the serum seems to inhibit the escape of the DNA from the endosomal DNA-CLC rather than the translocation through the plasma membrane (37). It is possible that this posttransfectional actiVity of Ca.'"…”

Section: Bombardementmentioning

confidence: 99%

Improvement of DNA transfection with cationic liposomes

Rocha

Ruiz

Coll

2002

J. Physiol. Biochem.

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The increasing use of cationic liposomes as vectors for DNA transfection of eukaryotic cells is due to its high efficiency and reproducibility. After the interaction of the DNA cationic-liposome complexes (DNA-CLC) with the plasma membrane, the entry into the cells delivers the DNA-CLC to the endosome-lysosome pathway where some of the DNA-CLC are degraded. The non-degraded DNA that escapes to the cytoplasm, still has to transverse the nuclear membrane to be transcribed and then translated. To improve the efficiency of the whole process, we can manipulate the DNA (sequences, promoters, enhancers, nuclear localisation signals, etc), the DNA-CLC (lipids) or the plasmatic, endosomal and/or nuclear cellular membranes (ultrasound, electroporation, Ca++, pH of the endosomes, mitosis, fusogenic peptides, nuclear localisation signals, etc). Most of these methods have been generally used individually but in combination, may greatly improve the efficiency and reproducibility of in vitro transfection. While much of this work remains yet to be done and present results further explored, the application of these efforts is essential to the future development of new gene therapy strategies.

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Untitled

Cited by 33 publications

References 18 publications

“Soft” Calcium Crosslinks Enable Highly Efficient Gene Transfection Using TAT Peptide

“Soft” Calcium Crosslinks Enable Highly Efficient Gene Transfection Using TAT Peptide

Structure and function correlation in histone H2A peptide-mediated gene transfer

Improvement of DNA transfection with cationic liposomes

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