Nuclear delivery of plasmid DNA determines the efficiency of gene expression

As a cationic non‐viral gene delivery vector, poly(agmatine/ N, N′‐cystamine‐bis‐acrylamide) (AGM‐CBA) showed significantly higher plasmid DNA (pDNA) transfection ability than polyethylenimine (PEI) in NIH/3T3 cells. The transfection expression of AGM‐CBA/pDNA polyplexes was found to have a non‐linear relationship with AGM‐CBA/pDNA weight ratios. To further investigate the mechanism involved in the transfection process of poly(AGM‐CBA), we used pGL3‐control luciferase reporter gene (pLUC) as a reporter pDNA in this study. The distribution of pLUC in NIH/3T3 cells and nuclei after AGM‐CBA/pLUC and PEI/pLUC transfection were determined by quantitative polymerase chain reaction (qPCR) analysis. The intracellular trafficking of the polyplexes was evaluated by cellular uptake and nuclei delivery of pLUC, and the intracellular availability was evaluated by the ratio of transfection expression to the numbers of pLUC delivered in nuclei. It was found that pLUC intracellular trafficking did not have any correlation with the transfection expression, while an excellent correlation was found between the nuclei pLUC availability and transfection expression. These results suggested that the intracellular availability of pLUC in nuclei was the rate‐limiting step for pLUC transfection expression. Further optimization of the non‐viral gene delivery system can be focused on the improvement of gene intracellular availability.     

Cationic lipids derived from glycine betaine promote efficient and non-toxic gene transfection in cultured hepatocytes

Background

The low efficiency and toxicity of transfection in a primary culture of hepatocytes using cationic lipids remains a limiting step to the study of gene function and the setting up of non‐viral gene therapy.

Methods

A novel class of cationic lipids (GBs) derived from natural glycine betaine compounds covalently linked to acyl chains by enzymatically hydrolysable peptide and ester bonds, a structure designed to reduce cytotoxicity, was used to improve transfection efficiency in a primary culture of rat hepatocytes. The relationship between lipid structure, lipoplex formulation and transfection efficiency was studied using six GBs (12‐14‐16, 22‐24‐26) varying in their spacer and acyl chains.

Results

GB12, characterized by short [(CH₂)₁₀] acyl chains and spacer, allowed plasmid uptake in all cells and reporter gene expression in up to 40% of hepatocytes with a low cytotoxicity, a much higher efficiency compared with transfections using other reagents including Fugene6^? and Lipofectin^?. We also showed that numerous cells accumulated high amounts of plasmids demonstrating that GB12 promoted a very efficient DNA transfer through plasma membrane leading to an increase in nuclear plasmid translocation, allowing a much higher gene expression. Moreover, GB12‐transfected hepatocytes survived to injection in normal livers and were found to express the LacZ reporter gene.

Conclusions

The non‐toxic GB12 formulation is a powerful vehicle for plasmid delivery in cultured hepatocytes with relevance in liver gene therapy. Copyright © 2002 John Wiley & Sons, Ltd.

     

Up to 100-Fold Increase of Apparent Gene Expression in the Presence of Epstein-Barr Virus oriP Sequences and EBNA1: Implications of the Nuclear Import of Plasmids

A 100-fold increase in luciferase activity was observed in 293 cells, stably expressing Epstein-Barr nuclear antigen 1 (EBNA1; 293-EBNA1 cells), that had been transiently transfected with plasmids carrying Epstein-Barr virus (EBV) oriP sequences. This increase was observed in comparison to reporter gene activity obtained after transfection with a plasmid carrying no oriP sequences. The luciferase gene on these plasmids was under the control of either the cytomegalovirus immediate-early 1 gene enhancer-promoter (CMV IE1) or the Rous sarcoma virus promoter. The increase of reporter gene activity was not due to plasmid replication, since a similar enhancement was observed in the presence of aphidicolin, an inhibitor of replicative DNA synthesis, or after deletion of the dyad symmetry (DS) element within oriP. Luciferase production was not increased in the presence of only the DS element. Microinjection of plasmids carrying the CMV IE1 promoter-driven luciferase gene with or without oriP sequences into the nuclei of 293-EBNA1 cells resulted in a 17-fold increase in luciferase activity. Cytoplasmic injection of these plasmids led to an enhancement of luciferase activity of up to 100-fold. This difference in the factor of activation after nuclear or cytoplasmic injection could be ascribed to increased transport of plasmids carrying oriP from the cytoplasm to the nucleus in the presence of EBNA1. These data suggest the possibility of substantially increasing the apparent expression of a gene under the control of a strong constitutive promoter in the presence of oriP sequences and EBNA1. This improvement in expression is due to intranuclear enhancement of gene expression. oriP-specific transport of plasmid DNA from the cytoplasm of 293-EBNA1 cells to the nucleus seems to contribute to the observed effect.     

Immediate transfection of patient-derived leukemia: a novel source for generating cell-based vaccines

Background

The production of cell-based cancer vaccines by gene vectors encoding proteins that stimulate the immune system has advanced rapidly in model systems. We sought to develop non-viral transfection methods that could transform patient tumor cells into cancer vaccines, paving the way for rapid production of autologous cell-based vaccines.

Methods

As the extended culture and expansion of most patient tumor cells is not possible, we sought to first evaluate a new technology that combines electroporation and chemical transfection in order to determine if plasmid-based gene vectors could be instantaneously delivered to the nucleus, and to determine if gene expression was possible in a cell-cycle independent manner. We tested cultured cell lines, a primary murine tumor, and primary human leukemia cells from diagnostic work-up for transgene expression, using both RFP and CD137L expression vectors.

Results

Combined electroporation-transfection directly delivered plasmid DNA to the nucleus of transfected cells, as demonstrated by confocal microscopy and real-time PCR analysis of isolated nuclei. Expression of protein from plasmid vectors could be detected as early as two hours post transfection. However, the kinetics of gene expression from plasmid-based vectors in tumor cell lines indicated that optimal gene expression was still dependent on cell division. We then tested to see if pediatric acute lymphocytic leukemia (ALL) would also display the rapid gene expression kinetics of tumor cells lines, determining gene expression 24 hours after transfection. Six of 12 specimens showed greater than 17% transgene expression, and all samples showed at least some transgene expression.

Conclusion

Given that transgene expression could be detected in a majority of primary tumor samples analyzed within hours, direct electroporation-based transfection of primary leukemia holds the potential to generate patient-specific cancer vaccines. Plasmid-based gene therapy represents a simple means to generate cell-based cancer vaccines and does not require the extensive infrastructure of a virus-based vector system.     

Investigation of polyethylenimine‐grafted‐triamcinolone acetonide as nucleus‐targeting gene delivery systems

Background

Nuclear membrane is one of the main barriers in polymer mediated intracellular gene delivery. To improve the transgenic activity and safety of nonviral vector, triamcinolone acetonide (TA) as a nuclear localization signal was conjugated with different molecular weight polyethylenimine (PEI).

Methods

Different molecular weight PEI [600, 1800, 25 000 (25k)] was conjugated with TA to synthesize PEI‐TA by two‐step reaction. Their physicochemical characteristics, in vitro cytotoxicity and transfection efficiency were evaluated. To investigate the difference of transfection efficiency of various molecular weight PEI‐TA, their transfection mechanism was further investigated by confocal microscopy and competition assay. Transgenic expression in vivo was evaluated by injection into hepatic portal vein of mice.

Results

All PEI‐TA could form nanosize polyplexes with DNA and their physicochemical properties resemble each other. Their cytotoxicities were negligible compared to PEI 25k. The order of transfection efficiency was PEI 1800‐TA > PEI 600‐TA > PEI 25k‐TA. A transfection mechanism study displayed that TA could inhibit considerably the transgenic activity of PEI 1800‐TA and PEI 600‐TA, but that of PEI 25k‐TA was not inhibited. It was suggested that PEI 1800‐TA and PEI 600‐TA might translocate into the nucleus. Confocal microscopy investigation verified this suggestion. The data strongly suggested that the transfection efficiency of PEI 1800‐TA in vivo was much higher than that of PEI 25k, which was consistent with the results obtained in vitro.

Conclusions

Low molecular weight PEI‐TA could translocate into the nucleus efficiently. PEI 1800‐TA presented higher transgenic activity and it has a great potential for gene therapy as a nonviral carrier. Copyright © 2010 John Wiley & Sons, Ltd.     

Cross-linked polyethylenimine as potential DNA vector for gene delivery with high efficiency and low cytotoxicity

Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationiccharge potential.High transfection efficiency of PEI,along with its cytotoxicity,strongly depends on itsmolecular weight.To enhance its gene delivery efficiency and minimize cytotoxicity,we have synthesizedsmall cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro.Inthis study,branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate[1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h.The efficiencies of thecross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein(EGFP) reporter gene were assessed in melanoma B 16F10 cell line and other cell lines.Flow cytometrywas used to quantify the cellular entry efficiency of plasmid and the transgene expression level.Thecytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay.EGDMA-PEI 800-4h,atypical cross-linked PEI reported here,mediated a more efficient expression of reporter gene than thecommercially available 25-kDa branched PEI control,and resulted in a 9-fold increase in gene deliveryin B16F10 cells and a 16-fold increase in 293T cells,while no cytotoxicity was found at the optimizedcondition for gene delivery.Furthermore,the transfection activity of polyplexes was preserved in thepresence of serum proteins.     

Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties

Background

Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA.

Results

We have coupled a 62-aminoacid peptide derived from hSRP1α importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl)-6-maleimidyl hexanamide (TFPAM-6). When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin β, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction.

Conclusions

The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer.

     

Predictive modeling of non‐viral gene transfer

In non‐viral gene delivery, the variance of transgenic expression stems from the low number of plasmids successfully transferred. Here, we experimentally determine Lipofectamine‐ and PEI‐mediated exogenous gene expression distributions from single cell time‐lapse analysis. Broad Poisson‐like distributions of steady state expression are observed for both transfection agents, when used with synchronized cell lines. At the same time, co‐transfection analysis with YFP‐ and CFP‐coding plasmids shows that multiple plasmids are simultaneously expressed, suggesting that plasmids are delivered in correlated units (complexes). We present a mathematical model of transfection, where a stochastic, two‐step process is assumed, with the first being the low‐probability entry step of complexes into the nucleus, followed by the subsequent release and activation of a small number of plasmids from a delivered complex. This conceptually simple model consistently predicts the observed fraction of transfected cells, the cotransfection ratio and the expression level distribution. It yields the number of efficient plasmids per complex and elucidates the origin of the associated noise, consequently providing a platform for evaluating and improving non‐viral vectors. Biotechnol. Bioeng. 2010. 105: 805–813. © 2009 Wiley Periodicals, Inc.     

Differential intracellular distribution of DNA complexed with polyethylenimine (PEI) and PEI-polyarginine PTD influences exogenous gene expression within live COS-7 cells

Background

Polyethylenimine (PEI) is one of the most efficient and versatile non-viral vectors available for gene delivery. Despite many advantages over viral vectors, PEI is still limited by lower transfection efficiency compared to its viral counterparts. Considerable investigation is devoted to the modification of PEI to incorporate virus-like properties to improve its efficacy, including the incorporation of the protein transduction domain (PTD) polyarginine (Arg); itself demonstrated to facilitate membrane translocation of molecular cargo. There is, however, limited understanding of the underlying mechanisms of gene delivery facilitated by both PEI and PEI-bioconjugates such as PEI-polyarginine (PEI-Arg) within live cells, which once elucidated will provide valuable insights into the development of more efficient non-viral gene delivery vectors.

Methods

PEI and PEI-Arg were investigated for their ability to facilitate DNA internalization and gene expression within live COS-7 cells, in terms of the percentage of cells transfected and the relative amount of gene expression per cell. Intracellular trafficking of vectors was investigated using fluorescent microscopy during the first 5 h post transfection. Finally, nocodazole and aphidicolin were used to investigate the role of microtubules and mitosis, respectively, and their impact on PEI and PEI-Arg mediated gene delivery and expression.

Results

PEI-Arg maintained a high cellular DNA uptake efficiency, and facilitated as much as 2-fold more DNA internalization compared to PEI alone. PEI, but not PEI-Arg, displayed microtubule-facilitated trafficking, and was found to accumulate within close proximity to the nucleus. Only PEI facilitated significant gene expression, whereas PEI-Arg conferred negligible expression. Finally, while not exclusively dependant, microtubule trafficking and, to a greater extent, mitotic events significantly contributed to PEI facilitated gene expression.

Conclusion

PEI polyplexes are trafficked by an indirect association with microtubules, following endosomal entrapment. PEI facilitated expression is significantly influenced by a mitotic event, which is increased by microtubule organization center (MTOC)-associated localization of PEI polyplexes. PEI-Arg, although enhancing DNA internalization per cell, did not improve gene expression, highlighting the importance of microtubule trafficking for PEI vectors and the impact of the Arg peptide to intracellular trafficking. This study emphasizes the importance of a holistic approach to investigate the mechanisms of novel gene delivery vectors.     

Functional correction of episomal mutations with short DNA fragments and RNA-DNA oligonucleotides

Background

Gene correction is an alternative approach to replacement gene therapy. By correcting mutations within the genome, some of the barriers to effective gene therapy are avoided. Homologous nucleic acid sequences can correct mutations by inducing recombination or mismatch repair. Recently, encouraging data have been presented using both short DNAfragments (SDFs) and RNA–DNA oligonucleotides (RDOs) in experimental strategies to realize clinical gene correction.

Methods

The delivery of labelled SDFs and RDOs to a variety of cell lines was tested using both FACS analysis and confocal microscopy. A GFP‐based reporter system was constructed, containing a nonsense mutation, to allow quantitation of gene correction in living cells. This reporter was used to compare efficiencies of functional gene correction using SDFs and RDOs in arange of mammalian cell lines.

Results

The delivery experiments highlight the inefficient delivery of SDFs and RDOs to the nucleus using polyethylenimine (PEI) transfection. This study compared the episomal correction efficiency of the reporter plasmid mediated by SDFs and RDOs within different cell types; low levels of functional correction were detected in cell culture.

Conclusions

Whilst delivery of PEI‐complexed SDFs or RDOs to the cell is highly effective, nuclear entry appears to be a limiting factor. SDFs elicited episomal GFP correction across a range of cell lines, whereas RDOs only corrected the reporter in a cell line that overexpresses RAD51. Copyright © 2002 John Wiley & Sons, Ltd.

     

Expression of HIV-1 antigens in plants as potential subunit vaccines

Background

Efforts to improve the efficiency of non-viral gene delivery require a better understanding of delivery kinetics of DNA molecules into clinically relevant cells. Towards this goal, three DNA molecules were employed to investigate the effects of DNA properties on cellular delivery: a circular plasmid DNA (c-DNA), a linearized plasmid DNA (l-DNA) formulated by single-site digestion of c-DNA, and smaller linear gene cassette generated by PCR (pcr-DNA). Four non-viral gene carriers were investigated for DNA delivery: polyethyleneimine (PEI), poly-L-Lysine (PLL), palmitic acid-grafted PLL (PLL-PA), and Lipofectamine-2000?. Particle formation, binding and dissociation characteristics, and DNA uptake by rat bone marrow stromal cells were investigated.

Results

For individual carriers, there was no discernible difference in the morphology of particles formed as a result of carrier complexation with different DNA molecules. With PEI and PLL carriers, no difference was observed in the binding interaction, dissociation characteristics, and DNA uptake among the three DNA molecules. The presence of serum in cell culture media did not significantly affect the DNA delivery by the polymeric carriers, unlike other lipophilic carriers. Using PEI as the carrier, c-DNA was more effective for transgene expression as compared to its linear equivalent (l-DNA) by using the reporter gene for Enhanced Green Fluorescent Protein. pcr-DNA was the least effective despite being delivered into the cells to the same extent.

Conclusion

We conclude that the nature of gene carriers was the primary determinant of cellular delivery of DNA molecules, and circular form of the DNA was more effectively processed for transgene expression.     

Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells

We constructed multimers of the TAT-(47-57) peptide. This polycationic peptide is known to be a protein and particle transduction domain and at the same time to comprise a nuclear localization function. Here we show that oligomers of the TAT-(47-57) peptide compact plasmid DNA to nanometric particles and stabilize DNA toward nuclease degradation. At optimized vector compositions, these peptides mediated gene delivery to cells in culture 6-8-fold more efficiently than poly-L-arginine or the mutant TAT(2)-M1. When DNA was precompacted with TAT peptides and polyethyleneimine (PEI), Superfect, or LipofectAMINE was added, transfection efficiency was enhanced up to 390-fold compared with the standard vectors. As early as after 4 h of transfection, reporter gene expression mediated by TAT-containing complexes was higher than the 24-h transfection level achieved with a standard PEI transfection. When cells were cell cycle-arrested by serum starvation or aphidicolin, TAT-mediated transfection was 3-fold more efficient than a standard PEI transfection in proliferating cells. In primary nasal epithelial cells and upon intratracheal instillation in vivo, TAT-containing complexes were superior to standard PEI vectors. These data together with confocal imaging of TAT-DNA complexes in cells support the hypothesis that the TAT nuclear localization sequence function is involved in enhancing gene transfer.     

Nonviral vector loaded collagen sponges for sustained gene delivery in vitro and in vivo

Background

Naked DNA and standard vectors have previously been used for gene delivery from implantable carrier matrices with great potential for gene therapeutic assistance of wound healing or tissue engineering. We have previously developed copolymer‐protected gene vectors which are inert towards opsonization. Here we examine their potency in carrier‐mediated gene delivery in comparison to standard vectors using a vector‐loaded collagen sponge model.

Methods

Equine collagen type I sponges were loaded by a lyophilization method with naked DNA, polyethylenimine (PEI)‐DNA, DOTAP/cholesterol‐DNA and copolymer‐protected PEI‐DNA. These preparations were characterized in terms of vector‐release, cell growth on the matrices and reporter gene expression by cells colonizing the sponges in vitro and in vivo. Subcutaneous implantation of sponges in rats served as an in vivo model.

Results

At the chosen low vector dose, the loading efficiency was at least 86%. Naked DNA‐loaded collagen matrices lost 77% of the DNA dose in an initial burst in aqueous buffer in vitro. The other preparations examined displayed a sustained vector release. There was no difference in cell growth and invasion of the sponges between vector‐loaded and untreated collagen grafts. Reporter gene expression from cells colonizing the sponges in vitro was observed for not more than 7 days with naked DNA, whereas the lipoplex and polyplex preparations yielded long‐term expression throughout the experimental period of up to 56 days. The highest expression levels were achieved with the PEI‐DNA‐PROCOP (protective copolymer) formulation. Upon subcutaneous implantation in rats, no luciferase expression was detected with naked DNA preparations. DOTAP/cholesterol‐DNA and PEI‐DNA‐loaded implants lead to reporter gene expression for at least 3 days, but with poor reproducibility. PEI‐DNA‐PROCOP collagen matrices yielded consistently the highest reporter gene expression levels for at least 7 days with good reproducibility.

Conclusions

With the preparation method chosen, lipoplex‐ and polyplex‐loaded collagen sponges are superior in mediating sustained gene delivery in vitro and local transfection in vivo as compared to naked DNA‐loaded sponges. Protective copolymers are particularly advantageous in promoting the tranfection capacity of polyplex‐loaded sponges upon subcutaneous implantation, likely due to their stabilizing and opsonization‐inhibiting properties. Copyright © 2002 John Wiley & Sons, Ltd.

     

The use of low-molecular-weight PEIs as gene carriers in the monkey fibroblastoma and rabbit smooth muscle cell cultures

Background

Polyethylenimines (PEIs) and cationic polymers have been used successfully in gene delivery. In earlier reports, only large PEIs (MW>10 000) have shown significant transfection efficiency. In the present study, the roles of small PEIs (MW 700 and 2000) were studied as additional compounds to see if they can improve gene delivery with cationic liposomes.

Methods

The TKBPVlacZ expression plasmid was transfected in the CV1‐P (monkey fibroblastoma) and SMC (rabbit smooth muscle) cell lines using various combinations of PEIs (MW 700, 2000, and 25 000) and Dosper liposomes. The transfection efficiency was determined with the fluorometric ONPG (o‐nitrophenol‐β‐D ‐galactopyranoside) assay and histochemical X‐gal staining. The toxicity of the transfection reagents was estimated by the MTT [3‐(4,5‐dimethylthiazolyl‐2)‐2,5‐diphenyl tetrazolium bromide] assay.

Results

Transfection of TKBPVlacZ plasmid by the small PEIs (MW 700 and 2000) combined with Dosper liposomes was associated with high expression of the lacZ reporter gene in the CV1‐P and SMC cell lines. The transfection efficiencies of the low‐molecular‐weight PEI/liposome combinations were several fold higher than those of PEIs or liposomes alone. PEI/liposome combinations had no toxicity on the cell lines tested.

Conclusions

The low‐molecular‐weight PEIs could be used successfully for gene delivery when combined with the cationic liposomes, resulting in a synergistic increase of the transfection efficiency in both cell lines studied. Copyright © 2002 John Wiley & Sons, Ltd.

     

Noncovalently linked nuclear localization peptides for enhanced calcium phosphate transfection

The generation of cell lines stably expressing recombinant material is a lengthy process and there has thus been much interest in the use of transient expression systems to rapidly produce recombinant material. To achieve this, the DNA of interest must be delivered into the nucleus of the target cell. The mechanisms by which this process occurs are poorly understood and the efficiency of various methods differs widely. Recently, nuclear localization signals (NLSs) have been investigated to target entry of DNA into the nucleus of mammalian cells. We have used NLSs from the SV40 and Tat antigens mixed with our model luciferase reporter gene plasmid for the transfection of Chinese hamster ovary (CHO) cells using calcium phosphate and FuGNE 6 transfection technology. The nocovalent complexation of NLSs with plasmid DNA before calcium phosphate-mediated transfection resulted in enhanced reporter gene expression with increasing ratios of NLS to plasmid until reaching a mximum. At higher ratios than maximum expression, the expression levels decreased. On the other hand, when using FuGENE 6 reagent NLSs did not enhance reporter gene expression. Cell cycle arrest in G₂/M phase obliterated the effect of the NLS on reporter gene expression when using the calcium phosphate transfection method.     

Effect of chitosan‐alginate nanoparticles and ultrasound on the efficiency of gene transfection of human cancer cells

Background

Gene therapy has been used to treat a variety of health problems, but transfection inefficiency and the lack of safe vectors have limited clinical progress. Fabrication of a vector that is safe and has high transfection efficiency is crucial for the development of successful gene therapy. The present study aimed to synthesize chitosan‐alginate nanoparticles that can be used as carriers of the pAcGFP1‐C1 plasmid and to use these nanoparticles with an ultrasound protocol to achieve high efficiency gene transfection.

Methods

Chitosan was complexed with alginate and the pAcGFP1‐C1 plasmid at different charge ratios to create chitosan‐alginate‐DNA nanoparticles (CADNs). The average particle size and loading efficiency were measured. Plasmid DNA retardation and integrity were analysed on 1% agarose gels. The effect of CADNs and ultrasound on the efficiency of transfection of cells and subcutaneous tumors was evaluated.

Results

In the CADNs, the average size of incorporated plasmid DNA was 600–650 nm and the loading efficiency was greater than 90%. On the basis of the results of the plasmid DNA protection test, CADNs could protect the transgene from DNase I degradation. The transgene product expression could be enhanced efficiently if cells or tumor tissues were first given CADNs and then treated with ultrasound.

Conclusions

The use of CADNs combined with an ultrasound regimen is a promising method for safe and effective gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.