Nuclear-targeted minicircle to enhance gene transfer with non-viral vectorsin vitro andin vivo (original) (raw)
Related papers
DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery
Pharmaceutical Research, 2011
Purpose To evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA. Methods A set of DTS was identified and cloned into EGFPreporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry. Results Neither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNFα, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.
Journal of Biological Chemistry, 2003
The ultimate destination for most gene therapy vectors is the nucleus and nuclear import of potentially therapeutic DNA is one of the major barriers for nonviral vectors. We have developed a novel approach of attaching a nuclear localization sequence (NLS) peptide to DNA in a non-essential position, by generating a fusion between the tetracycline repressor protein TetR and the SV40-derived NLS peptide. The high affinity and specificity of TetR for the short DNA sequence tetO was used in these studies to bind the NLS to DNA as demonstrated by the reduced electrophoretic mobility of the TetR⅐tetO-DNA complexes. The protein TetR-NLS, but not control protein TetR, specifically enhances gene expression from lipofected tetO-containing DNA between 4-and 16-fold. The specific enhancement is observed in a variety of cell types, including primary and growth-arrested cells. Intracellular trafficking studies demonstrate an increased accumulation of fluorescence labeled DNA in the nucleus after TetR-NLS binding. In comparison, binding studies using the similar fusion of peptide nucleic acid (PNA) with NLS peptide, demonstrate specific binding of PNA to plasmid DNA. However, although we observed a 2-8.5-fold increase in plasmid-mediated luciferase activity with bis-PNA-NLS, control bis-PNA without an NLS sequence gave a similar increase, suggesting that the effect may not be because of a specific bis-PNA-NLS-mediated enhancement of nuclear transfer of the plasmid. Overall, we found TetR-NLS-enhanced plasmid-mediated transgene expression at a similar level to that by bis-PNA-NLS or bis-PNA alone but specific to nuclear uptake and significantly more reliable and reproducible.
Bioconjugate Chemistry, 2004
Nonviral gene delivery is limited by inefficient transfer of DNA from the cytoplasm to the nucleus. Nuclear localization sequence (NLS) peptides have been widely used to exploit intracellular transport mechanisms and promote nuclear uptake of DNA. However, the exact conditions to successfully utilize the properties of NLS peptides are still unclear. In the present study a panel of NLS peptides that bind different transport receptors were compared for their ability to enhance nonviral gene transfer. Several factors such as method of incorporating the NLS peptide, type of NLS peptide, DNA morphology, and proper characterization of NLS peptide/DNA conjugates were identified as important considerations in utilizing NLS peptides to enhance gene transfer. In particular, it was shown that a peptide derived from human T cell leukaemia virus type 1 (HTLV) was able to effectively condense DNA into discrete particles and mediate levels of transgene expression up to 32-fold greater than polylysine-based polyplexes. This is the first study to demonstrate efficient transfection mediated by an importin -binding peptide based on the HTLV sequence. Promising results were also achieved with a 7-fold increase in gene expression using a NLS peptide/DNA conjugate formed by site-specific linkage of an extended SV40 peptide via a peptide nucleic acid (PNA) clamp. Altogether, the results from this study should help to define the requirements for successful NLS-enhanced transfection.
A new DNA vehicle for nonviral gene delivery: supercoiled minicircle
Gene Therapy, 1997
Plasmids currently used for nonviral gene transfer have the excised in vivo after thermoinduction of the integrase gene disadvantage of carrying a bacterial origin of replication leading to the formation of two supercoiled molecules: the and an antibiotic resistance gene. There is, therefore, a minicircle and the starting plasmid lacking the expression risk of uncontrolled dissemination of the therapeutic gene cassette. In various cell lines, purified minicircles exhibited and the antibiotic resistance gene. Minicircles are new a two-to 10-fold higher luciferase reporter gene activity DNA delivery vehicles which do not have such elements than the unrecombined plasmid. This could be due to either and are consequently safer as they exhibit a high level of the removal of unnecessary plasmid sequences, which biological containment. They are obtained in E. coli by att could affect gene expression, or the smaller size of minisite-specific recombination mediated by the phage inte-circle which may confer better extracellular and intracellular grase. The desired eukaryotic expression cassette bioavailability and result in improved gene delivery bounded by the attP and attB sites was cloned on a properties. recombinant plasmid. The expression cassette was
Using nuclear targeting signals to enhance non-viral gene transfer
Immunology and Cell Biology, 2002
Gene therapy involves the introduction of DNA-encoding therapeutic gene products into appropriate cells of an affected individual. The limitations of the approach relate largely to the poor efficiency of the delivery of the therapeutic DNA to the nucleus. This review examines recent work in the area of non-viral gene transfer, building on developments in the field of nuclear protein import and their application in the field of non-viral gene transfer. In particular, advances in the area of enhancing DNA targeting to the nucleus are discussed, including the use of modular nuclear targeting signals recognised by the cellular nuclear import machinery and DNA condensing agents to facilitate passage through the nuclear pore. Optimising nuclear DNA delivery through these and other strategies should assist greatly in rendering gene therapy a viable and realistic possibility for treating disease.
A novel in vitro system for intracellular delivery of nonviral DNA
Journal of Orthopaedic Translation, 2014
Articular chondrocytes are the main cell population in cartilage, and damage to them is a key step in osteoarthritis. There are chondrocyte-based strategies to treat osteoarthritis, among which includes nonviral gene therapy. However, so far there is no ideal way to achieve this because chondrocyte cells are very difficult to transfect. Also, an effective tracking system to evaluate exogenous DNA delivery in chondrocytes is a necessary part of this strategy. Here, we show our development of a novel tracking system by labelling cell membranes, nuclei, and plasmids, without disturbing their expression, to view the intracellular behaviour of plasmids before, after, and during the entire transfection process. We applied this system to compare the intracellular behaviour of exogenous DNA in chondrocytes and cancer cells. We also used this system to compare the intracellular behaviour of exogenous DNA which is transfected by liposomes or polymers. Finally, we discovered that when transfected by liposomes, exogenous DNA has a quicker cell entry and nucleus entry in cancer cells than in chondrocytes, and the transfection efficiency is higher in cancer cells than in chondrocytes. When the cells are transfected by polymers, exogenous DNA has the same quick cell entry and nucleus entry in both types of cells. However, the transfection efficiency was higher in cancer cells than in chondrocytes. Applying this system has proved to be simple, easy to operate, repeatable, and stable and it enables tracking of the behaviour of plasmid DNA before and after expression. Meanwhile, the rate-limiting zones of exogenous plasmid DNA in living cells and a comprehensive overview of the expression and transfection efficiency of DNA can also be
Targeted delivery of DNA for gene therapy via receptors
Trends in Biotechnology, 1993
The general concept of targeted delivery of therapeutic agents was first recognized by Paul Ehrlich at the turn of the century I. Only recently has this strategy been applied to the delivery of DNA-protein complexes to cells 2. Most of the research on DNA delivery by this approach has been performed by targeting the liver-specific asialoglycoprotein receptor (ASGPr) (Refs 3-10) and the relatively ubiquitous transferrin receptor,1 19. DNA delivery to hepatocytes has also been effected using insulin-polylysine conjugates 2°, and to antigen-bearing cells using an antibody 21. The majority of these experiments use a covalently crosslinked receptor ligand-polycation conjugate to bind DNA in an electrostatic complex 5 . Receptormediated endocytosis of the ligand carries the bound DNA into a cell, allowing subsequent expression of the foreign DNA ). This technique, which has been demonstrated in animals using the ASGPr (Refs 5-8), is promising for the delivery of therapeutic DNAs as well as antisense oligonucleotides2L
Human Gene Therapy, 2005
We have shown previously that minicircle DNA vectors free of plasmid bacterial DNA sequences are capable of persistent high level of transgene expression in vivo. The minicircle is generated in bacteria from a parental plasmid containing an inducible phage C31 integrase gene and a therapeutic expression cassette flanked with attB and attP sites. The C31-mediated intramolecular recombination between attB and attP results in the formation of two circular DNA molecules, one containing the eukaryotic expression cassette (minicircle), and the other the plasmid bacterial DNA backbone (BB). Previously, the minicircle was purified away from the plasmid BB by a restriction enzyme digestion step and ultracentrifugation in cesium chloride. We have now included the endonuclease I-SceI gene together with its recognition site in the minicircle-producing plasmid to allow the linearization and degradation of the plasmid BB in bacteria. The minicircle can then be isolated by routine plasmid purification procedures such as a one-step affinity column. With additional modifications to our previous strategy, we can prepare a minicircle encoding a 4-kb human factor IX expression cassette, up to 1.8 mg of minicircle with 97% purity was prepared from a 1 liter bacterial culture. The high yield, simple purification, and robust and persistent transgene expression make these vectors viable for gene therapy applications. OVERVIEW SUMMARY Minicircle DNAs devoid of plasmid bacterial sequences are superior to standard plasmid as a nonviral DNA vector because: (1) of its relative safety due to the reduction of the inflammatory unmethylated CpG motifs; (2) more efficient transgene expression due to a reduced size; and (3) more robust and persistent transgene expression. The development of a one-step purification technology to isolate minicircle directly from bacterial lysate using commercially available affinity column allows production of this robust DNA vector capable of meeting clinical requirements, making it useful for clinical applications.
ChemBioChem, 2003
The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3b- [N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-a-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-b-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30 ± 45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. ™Stealth∫ systems obtained by post-coating LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol % fluorescein-PEG 5000 -N-hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.