Ultrasound／Microbubble Enhances Foreign Gene Expression in ECV304 Cells and Murine Myocardium

The success of gene therapy is largely dependent onthe development of vectors or vehicles that can selectivelyand efficiently deliver a therapeutic gene to cells or targetissues with minimal toxicity. Viruses are efficient transducing vectors. However, the safety concerns regardingthe use of virus vector in human make nonviral deliverysystem an attractive focus. Nonviral vectors are particularly suitable with respect to the simplicity of use, possibility of large-scale production and lack of s…     

Gene transfer into adult rat spinal cord using naked plasmid DNA and ultrasound microbubbles

BACKGROUND: Although gene therapy might become a promising approach to treat spinal cord injury, the safety issue is a serious consideration in human gene therapy. Plasmid DNA transfer is safer than viral vectors, but the transfection efficiency is quite low. To overcome the problem, we applied the ultrasound microbubbles-mediated transfection method to the spinal cord in adult rats, since ultrasound microbubbles have been reported to be efficient to increase transfection efficiency in various tissues. METHODS: After exposing T9-10 spinal cord with a laminectomy, we injected a mixture of naked plasmid DNA and microbubbles into cerebrospinal fluid by lumbar puncture. Then, the T9-10 spinal cord was exposed to ultrasound. CONCLUSIONS: An ultrasound intensity of 0.4-0.5 W/cm2 significantly increased luciferase expression up to approximately 15-60-fold at the insonated level as compared to naked plasmid DNA alone. Luciferase activity could be detected at least up to 7 days after transfection, while the expression level was almost returned to undetectable level at 14 days after transfection. The transfected cells were mainly meningeal cells in the surface of insonated spinal cord. There was no obvious evidence of worsening of neurological deficits as compared to rats transfected with naked plasmid DNA alone or untransfected rats. Similarly, successful gene transfer was also achieved in the insonated T9-10 spinal cord after spinal cord injury. Overall, the present study demonstrated the feasibility of ultrasound microbubbles-mediated plasmid DNA transfer into the target level of the spinal cord.     

Ultrasound-Targeted Microbubble Destruction (UTMD) Assisted Delivery of shRNA against PHD2 into H9C2 Cells

Gene therapy has great potential for human diseases. Development of efficient delivery systems is critical to its clinical translation. Recent studies have shown that microbubbles in combination with ultrasound (US) can be used to facilitate gene delivery. An aim of this study is to investigate whether the combination of US-targeted microbubble destruction (UTMD) and polyethylenimine (PEI) (UTMD/PEI) can mediate even greater gene transfection efficiency than UTMD alone and to optimize ultrasonic irradiation parameters. Another aim of this study is to investigate the biological effects of PHD2-shRNA after its transfection into H9C2 cells. pEGFP-N1 or eukaryotic shPHD2-EGFP plasmid was mixed with albumin-coated microbubbles and PEI to form complexes for transfection. After these were added into H9C2 cells, the cells were exposed to US with various sets of parameters. The cells were then harvested and analyzed for gene expression. UTMD/PEI was shown to be highly efficient in gene transfection. An US intensity of 1.5 W/cm², a microbubble concentration of 300μl/ml, an exposure time of 45s, and a plasmid concentration of 15μg/ml were found to be optimal for transfection. UTMD/PEI-mediated PHD2-shRNA transfection in H9C2 cells significantly down regulated the expression of PHD2 and increased expression of HIF-1α and downstream angiogenesis factors VEGF, TGF-β and bFGF. UTMD/PEI, combined with albumin-coated microbubbles, warrants further investigation for therapeutic gene delivery.     

Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG2 cells

Ultrasound-targeted microbubble destruction had been employed in gene delivery and promised great potential. Liver has unique features that make it attractive for gene therapy. However, it poses formidable obstacles to hepatocyte-specific gene delivery. This study was designed to test the efficiency of therapeutic gene transfer and expression mediated by ultrasound/microbubble strategy in HepG2 cell line. Air-filled albumin microbubbles were prepared and mixed with plasmid DNA encoding low density lipoprotein receptor (LDLR) and green fluorescent protein. The mixture of the DNA and microbubbles was administer to cultured HepG2 cells under variable ultrasound conditions. Transfection rate of the transferred gene and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, Western blot analysis and Trypan blue staining. The result demonstrated that microbubbles with ultrasound irradiation can significantly elevate exogenous LDLR gene expression and the expressed LDLRs were functional and active to uptake their ligands. We conclude that ultrasound-targeted microbubble destruction has the potential to promote safe and efficient LDLR gene transfer into hepatocytes. With further refinement, it may represent an effective nonviral avenue of gene therapy for liver-involved genetic diseases.     

Ultrasound-mediated microbubble destruction facilitates gene transfection in rat C6 glioma cells

The goal of this study was to determine whether ultrasound (US) exposure combined with microbubble destruction could be used to enhance non-viral gene delivery in rat C6 glioma cells. Microbubbles were prepared and gently mixed with plasmid DNA. The mixture of the DNA and microbubbles was administered to cultured C6 cells under different US/microbubble conditions. Transfection efficiency and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, and Trypan blue staining. The results demonstrate that microbubble with US exposure could significantly enhance the reporter gene expression as compared with other groups. No statistical significant difference was observed in the glioma cell viability between different groups. Our in vitro findings suggest that US-mediated microbubble destruction has the potential to promote safe and efficient gene transfer into C6 cells. This non-invasive gene transfer method may be useful for safe clinical gene therapy of brain cancer without a viral vector system.     

Ultrasound enhances retrovirus-mediated gene transfer

Viral vector systems are efficient for transfection of foreign genes into many tissues. Especially, retrovirus based vectors integrate the transgene into the genome of the target cells, which can sustain long term expression. However, it has been demonstrated that the transduction efficiency using retrovirus is relatively lower than those of other viruses. Ultrasound was recently reported to increase gene expression using plasmid DNA, with or without, a delivery vehicle. However, there are no reports, which show an ultrasound effect to retrovirus-mediated gene transfer efficiency. Retrovirus-mediated gene transfer systems were used for transfection of 293T cells, bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and rat skeletal muscle myoblasts (L6 cells) with beta-galactosidase (beta-Gal) genes. Transduction efficiency and cell viability assay were performed on 293T cells that were exposed to varying durations (5 to 30 seconds) and power levels (1.0 watts/cm(2) to 4.0 watts/cm(2)) of ultrasound after being transduced by a retrovirus. Effects of ultrasound to the retrovirus itself was evaluated by transduction efficiency of 293T cells. After exposure to varying power levels of ultrasound to a retrovirus for 5 seconds, 293T cells were transduced by a retrovirus, and transduction efficiency was evaluated. Below 1.0 watts/cm(2) and 5 seconds exposure, ultrasound showed increased transduction efficiency and no cytotoxicity to 293T cells transduced by a retrovirus. Also, ultrasound showed no toxicity to the virus itself at the same condition. Exposure of 5 seconds at the power of 1.0 watts/cm(2) of an ultrasound resulted in significant increases in retrovirus-mediated gene expression in all four cell types tested in this experiment. Transduction efficiencies by ultrasound were enhanced 6.6-fold, 4.8-fold, 2.3-fold, and 3.2-fold in 293T cells, BAECs, RASMCs, and L6 cells, respectively. Furthermore, beta-Gal activities were also increased by the retrovirus with ultrasound exposure in these cells. Adjunctive ultrasound exposure was associated with enhanced retrovirus-mediated transgene expression in vitro. Ultrasound associated local gene therapy has potential for not only plasmid-DNA-, but also retrovirus-mediated gene transfer.     

DNA Transfection of Bone Marrow Stromal Cells Using Microbubble-Mediated Ultrasound and Polyethylenimine: An In Vitro Study

Non-viral vector transfection efficiency is an issue affecting the clinical application of stem cell gene therapy. This study makes use of the synergistic effect of combining ultrasound (US) with microbubbles (MB) and polyethylenimine (PEI) to increase DNA transfection efficiency, which will enhance the efficiency of gene transfer to bone marrow stromal cells (BMSCs). The optimal parameters for primary-cultured rat-BMSC DNA transfection were examined. The study was arranged based on uniform design. Using a construct containing hepatocyte growth factor (HGF) tagged with enhanced green fluorescent protein (pEGFP-HGF) as example, the mixture of BMSCs, MB, and PEI:DNA complex were exposed to US with frequency of 1 MHz and 10 % duty cycle pulses. Other factors such as acoustic intensity (Q), MB dosage, and total treatment time (T) were also tested. The results were analyzed by regression analysis. Using the best match of parameters, Q = 0.6 W/cm², MB = 10⁶/ml, T = 30 s, different groups were compared. The cooperativity of MB-mediated US and PEI enhanced the gene transfection efficiency by nearly 38-times compared to the DNA without US group. Furthermore, the expression of HGF protein was confirmed by Western blot. The eGFP could be not only seen mainly at the cytoplasm, but also seen in the nucleus in a small proportion of the cells (<10 %) for up to 7 observed days. The transfected BMSCs maintained their capability of multi-directional differentiation and reproductive activity. Our results provide useful information in establishing a novel non-viral transfection method, which may be applied to clinical application in stem cell gene therapy.     

Enhanced DNA transfer into fish bone cells using polyethylenimine

The use of in vitro cell culture systems to assess gene function largely depends on the successful transfer of DNA into target cells. Well developed in mammals, transfection methods are still to be optimized for non-mammalian cell culture systems, like fish. Here we describe a rapid, cost-efficient, and successful method to transfer DNA into a fish bone-derived cell line using polyethylenimine (PEI) as the DNA carrier. Using this method, DNA transfer was remarkably enhanced in comparison with commercially available reagents, as demonstrated by the increased activity of both luciferase and green fluorescent protein observed in the transfected cells. Its efficiency in transferring DNA intoa wide range of cell types, including non-mammalian and hard-to-transfect cells, in addition to a low cost, show that PEI is a reagent of choice for nonviral vector transfection.     

Ultrasound Enhances Retrovirus‐Mediated Gene Transfer

Abstract

Viral vector systems are efficient for transfection of foreign genes into many tissues. Especially, retrovirus based vectors integrate the transgene into the genome of the target cells, which can sustain long term expression. However, it has been demonstrated that the transduction efficiency using retrovirus is relatively lower than those of other viruses. Ultrasound was recently reported to increase gene expression using plasmid DNA, with or without, a delivery vehicle. However, there are no reports, which show an ultrasound effect to retrovirus‐mediated gene transfer efficiency.

Retrovirus‐mediated gene transfer systems were used for transfection of 293T cells, bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and rat skeletal muscle myoblasts (L6 cells) with β‐galactosidase (β‐Gal) genes. Transduction efficiency and cell viability assay were performed on 293T cells that were exposed to varying durations (5 to 30 seconds) and power levels (1.0 watts/cm² to 4.0 watts/cm²) of ultrasound after being transduced by a retrovirus. Effects of ultrasound to the retrovirus itself was evaluated by transduction efficiency of 293T cells. After exposure to varying power levels of ultrasound to a retrovirus for 5 seconds, 293T cells were transduced by a retrovirus, and transduction efficiency was evaluated.

Below 1.0 watts/cm² and 5 seconds exposure, ultrasound showed increased transduction efficiency and no cytotoxicity to 293T cells transduced by a retrovirus. Also, ultrasound showed no toxicity to the virus itself at the same condition. Exposure of 5 seconds at the power of 1.0 watts/cm² of an ultrasound resulted in significant increases in retrovirus‐mediated gene expression in all four cell types tested in this experiment. Transduction efficiencies by ultrasound were enhanced 6.6‐fold, 4.8‐fold, 2.3‐fold, and 3.2‐fold in 293T cells, BAECs, RASMCs, and L6 cells, respectively. Furthermore, β‐Gal activities were also increased by the retrovirus with ultrasound exposure in these cells.

Adjunctive ultrasound exposure was associated with enhanced retrovirus‐mediated transgene expression in vitro. Ultrasound associated local gene therapy has potential for not only plasmid‐DNA‐, but also retrovirus‐mediated gene transfer.     

Expression of beta-galactosidase and pig leptin gene in vitro by recombinant adenovirus

Adenovirus has been used in vivo and in vitro as a vector to carry a foreign gene for gene transfer. Two kinds of replication defective human recombinant adenovirus vectors were used in this study, the first containing beta-galactosidase reporter gene (AdCMVLac-Z) and the second carrying a gene for porcine leptin gene (AdCMVpLeptin). AdCMVLac-Z was tested for its ability to transfer DNA into pig kidney and pituitary cells. These cells expressed Lac-Z transiently 48 hours after the infection. In addition, when the pig kidney cells expressing the Lac-Z were replated with low density for the formation of colonies from each cell, colonies of blue cells expressing Lac-Z were observed. These results demonstrate that human recombinant adenovirus can be used as a transducing viral vector for inducing long-term expression in pig kidney cells. We also constructed a recombinant adenovirus (AdCMVpLeptin) which contained a pig leptin gene for the expression of pig leptin in vitro in the 293 human kidney cell line. 293 cells transfected with AdCMVpLeptin produced both a 15 KDa of a secretory form of porcine leptin and an 18 KDa long form containing signal peptide. Our study demonstrated that the recombinant adenovirus system offers a method for gene transfer and expression in pig cells.     

Ultrasound: mechanical gene transfer into plant cells by sonoporation

Development of nonviral gene transfer methods would be a valuable alternative of gene therapy or transformation. Ultrasound can produce a variety of nonthermal bioeffects via acoustic cavitation. Cavitation bubbles can induce cell death or transient membrane permeabilization (sonoporation) on cells. Application of sonoporation for gene transfer into cells or tissues develops quickly in recent years. Many studies have been performed in vitro exposure systems to a variety of cell lines transfected successfully. In vivo, cavitation initiation and control are more difficult, but can be enhanced by ultrasound contrast agents (microbubbles). The use of ultrasound for nonviral gene delivery has been applied for mammalian systems, which provides a fundamental basis and strong promise for development of new gene therapy methods for clinical medicine. In this paper, ultrasound applied to plant cell transformation or gene transfer is reviewed. Recently, most researches are focused on sonication-assisted Agrobacterium-mediated transformation (SAAT) in plant cells or tissues. Microbubbles are also proposed to apply to gene transfer in plant cells and tissues.     

脂质体DC-Chol/DOPE对HEK293FT细胞的高效转染及其在腺病毒制备中的应用

重组病毒载体系统因为具有高效的基因转移能力得到了广泛应用,而病毒包装细胞的转染是重组病毒制备过程中的关键步骤。优化了脂质体DC-Chol/DOPE介导的转染常用的病毒包装细胞系HEK293FT的实验条件,比较了DC-Chol/DOPE、Lipofectamine2000和磷酸钙共沉淀法转染细胞的效率,并且比较了用DC-Chol/DOPE和磷酸钙共沉淀法转染293FT细胞制备重组腺病毒的结果,发现DC-Chol/DOPE对293FT细胞的转染效率以及最终收获的病毒滴度都远高于磷酸钙共沉淀法转染。所以,利用DC-Chol/DOPE转染293FT细胞制备重组病毒是一种简单、高效、成本低廉的方法。     

Gene transfer of calcitonin gene-related peptide to cerebral arteries

Overexpression of calcitonin gene-related peptide (CGRP), an extremely potent vasodilator, to blood vessels is a possible strategy for prevention of vasospasm. We constructed an adenoviral vector that encodes prepro-CGRP (Adprepro-CGRP) and examined the effects of gene transfer on cultured cells and cerebral arteries. Transfection of Adprepro-CGRP to Cos-7 and NIH-3T3 cells increased CGRP-like immunoreactivity in media and produced an increase in cAMP in recipient cells. Five days after injection of Adprepro-CGRP into the cisterna magna of rabbits, the concentration of CGRP-like immunoreactivity increased by 93-fold in cerebrospinal fluid. In basilar artery, cAMP increased by 2.3-fold after Adprepro-CGRP compared with a control adenovirus. After transfection of Adprepro-CGRP, contraction of basilar artery in vitro to histamine and serotonin was attenuated, and relaxation to an inhibitor of cyclic nucleotide phosphodiesterase 3-isobutyl-1-methylxanthine was augmented compared with nontransduced arteries or arteries transfected with a control gene. Altered vascular responses were restored to normal by pretreatment with a CGRP(1) receptor antagonist CGRP-(8-37). Thus gene transfer of prepro-CGRP in vivo overexpresses CGRP in cerebrospinal fluid and perivascular tissues and modulates vascular tone. We speculate that this approach may be useful in prevention of vasospasm after subarachnoid hemorrhage.     

Targeted retroviral gene delivery using ultrasound

BACKGROUND: Achieving specificity of delivery represents a major problem limiting the clinical application of retroviral vectors for gene therapy, whilst lack of efficiency and longevity of gene expression limit non-viral techniques. Ultrasound and microbubble contrast agents can be used to effect plasmid DNA delivery. We therefore sought to evaluate the potential for ultrasound/microbubble-mediated retroviral gene delivery. METHODS: An envelope-deficient retroviral vector, inherently incapable of target cell entry, was combined with cationic microbubbles and added to target cells. The cells were exposed to pulsed 1 MHz ultrasound for 5 s and subsequently analysed for marker gene expression. The acoustic pressure profile of the ultrasound field, to which transduction efficiency was related, was determined using a needle hydrophone. RESULTS: Ultrasound-targeted gene delivery to a restricted area of cells was achieved using virus-loaded microbubbles. Gene delivery efficiency was up to 2% near the beam focus. Significant transduction was restricted to areas exposed to > or = 0.4 MPa peak-negative acoustic pressure, despite uniform application of the vector. An acoustic pressure-dependence was demonstrated that can be exploited for targeted retroviral transduction. The mechanism of entry likely involves membrane perturbation in the vicinity of oscillating microbubbles, facilitating fusion of the viral and cell membranes. CONCLUSIONS: We have established the basis of a novel retroviral vector technology incorporating favourable aspects of existing viral and non-viral gene delivery vectors. In particular, transduction can be controlled by means of ultrasound exposure. The technology is ideally suited to targeted delivery following systemic vector administration.     

Biocompatible polymer enhances the in vitro and in vivo transfection efficiency of HVJ envelope vector

BACKGROUND: Vector development is critical for the advancement of human gene therapy. However, the use of viral vectors raises many safety concerns and most non-viral methods are less efficient for gene transfer. One of the breakthroughs in vector technology is the combination of the vector with various polymers. METHODS: HVJ (hemagglutinating virus of Japan) envelope vector (HVJ-E) has been developed as a versatile gene transfer vector. In this study, we combined HVJ-E with cationized gelatin to make it a more powerful tool and assessed its transfection efficiency in vitro and in vivo. In addition, we investigated the mechanism of the gene transfer by means of the inhibition of fusion or endocytosis. RESULTS: The combination of both protamine sulfate and cationized gelatin with HVJ-E, referred to as PS-CG-HVJ-E, further enhanced the in vitro transfection efficiency. In CT26 cells, the luciferase gene expression of PS-CG-HVJ-E was approximately 10 times higher than that of the combination of protamine sulfate with HVJ-E or the combination of cationized gelatin with HVJ-E, referred to as PS-HVJ-E or CG-HVJ-E, respectively. Furthermore, the luciferase gene expression in liver mediated by intravenous administration of CG-HVJ-E was much higher than the luciferase gene expression mediated by PS-HVJ-E or PS-CG-HVJ-E and approximately 100 times higher than that mediated by HVJ-E alone. CONCLUSIONS: Cationized gelatin-conjugated HVJ-E enhanced gene transfection efficiency both in vitro and in vivo. These results suggest that low molecular weight cationized gelatin may be appropriate for complex formation with various envelope viruses, such as retrovirus, herpes virus and HIV.     

Protamine-modified DDAB lipid vesicles promote gene transfer in the presence of serum

Cationic lipid vesicle-mediated gene transfer has become common for in vitro gene delivery. However, the transfection efficiency is often impaired by serum. DDAB (dimethyldioctadecyl ammonium bromide) lipid vesicle-mediated gene transfer, which we previously reported, has the same problem. To overcome this obstacle, we here report a novel transfection vehicle using protamine-modified DDAB lipid vesicles. While free protamine was simply added to the DNA/lipid complex in the previous study, in the present method the protamine is chemically conjugated to stearic acid and incorporated into DDAB lipid vesicles. Gene transfer was not significantly inhibited in 10% serum-containing medium by this method for the transfection of cultured cells. Protamine-modified DDAB lipid vesicles also enhanced virus transduction efficiency in the presence of serum using a replication-defective retroviral vector. Furthermore, the vesicles allowed efficient gene transfer for avian embryos in vivo. These results indicate that the method is useful for the production of transgenic animals and gene therapy.     

Spatial and acoustic pressure dependence of microbubble-mediated gene delivery targeted using focused ultrasound

BACKGROUND: Ultrasound/microbubble-mediated gene delivery has the potential to be targeted to tissue deep in the body by directing the ultrasound beam following vector administration. Application of this technology would be minimally invasive and benefit from the widespread clinical experience of using ultrasound and microbubble contrast agents. In this study we evaluate the targeting ability and spatial distribution of gene delivery using focused ultrasound. METHODS: Using a custom-built exposure tank, Chinese hamster ovary cells in the presence of SonoVue microbubbles and plasmid encoding beta-galactosidase were exposed to ultrasound in the focal plane of a 1 MHz transducer. Gene delivery and cell viability were subsequently assessed. Characterisation of the acoustic field and high-resolution spatial analysis of transfection were used to examine the relationship between gene delivery efficiency and acoustic pressure. RESULTS: In contrast to that seen in the homogeneous field close to the transducer face, gene delivery in the focal plane was concentrated on the ultrasound beam axis. Above a minimum peak-to-peak value of 0.1 MPa, transfection efficiency increased as acoustic pressure increased towards the focus, reaching a maximum above 1 MPa. Delivery was microbubble-dependent and cell viability was maintained. CONCLUSIONS: Gene delivery can be targeted using focused ultrasound and microbubbles. Since delivery is dependent on acoustic pressure, the degree of targeting can be determined by appropriate transducer design to modify the ultrasound field. In contrast to other physical gene delivery approaches, the non-invasive targeting ability of ultrasound makes this technology an attractive option for clinical gene therapy.     

Persistence of recombinant adenovirus in vivo is not dependent on vector DNA replication.

Recombinant adenovirus vectors represent an efficient means of transferring genes into many different organs. The first-generation E1-deleted vector genome remains episomal and, in the absence of host immunity, persists long-term in quiescent tissues such as the liver. The mechanism(s) which allows for persistence has not been established; however, vector DNA replication may be important because replication has been shown to occur in tissue culture systems. We have utilized a site-specific methylation strategy to monitor the replicative fate of E1-deleted adenovirus vectors in vitro and in vivo. Methylation-marked adenovirus vectors were produced by the addition of a methyl group onto the N6 position of the adenine base of XhoI sites, CTCGAG, by propagation of vectors in 293 cells expressing the XhoI isoschizomer PaeR7 methyltransferase. The methylation did not affect vector production or transgene expression but did prevent cleavage by XhoI. Loss of methylation through viral replication restores XhoI cleavage and was observed by Southern analysis in a wide variety of, but not all, cell culture systems studied, including hepatoma and mouse and macaque primary hepatocyte cultures. In contrast, following liver-directed gene transfer of methylated vector in C57BL/6 mice, adenovirus vector DNA was not cleaved by XhoI and therefore did not replicate, even after a period of 3 weeks. Although replication may occur in some tissues, these results show that stabilization of the vector within the target tissue prior to clearance by host immunity is not dependent upon replication of the vector, demonstrating that the input transduced DNA genomes were the persistent molecules. This information will be useful for the design of optimal adenovirus vectors and perhaps nonviral episomal vectors for clinical gene therapy.