Liposome-mediated Gene Transfer Research Articles

Liposome-Mediated Gene Transfer in Differentiated HepaRG™ Cells: Expression of Liver Specific Functions and Application to the Cytochrome P450 2D6 Expression

The goal of this study was to establish a procedure for gene delivery mediated by cationic liposomes in quiescent differentiated HepaRG™ human hepatoma cells. We first identified several cationic lipids promoting efficient gene transfer with low toxicity in actively dividing HepG2, HuH7, BC2 and progenitor HepaRG™ human hepatoma cells. The lipophosphoramidate Syn1-based nanovector, which allowed the highest transfection efficiencies of progenitor HepaRG™ cells, was next used to transfect differentiated HepaRG™ cells. Lipofection of these cells using Syn1-based liposome was poorly efficient most likely because the differentiated HepaRG™ cells are highly quiescent. Thus, we engineered the differentiated HepaRG™ Mitogenic medium supplement (ADD1001) that triggered robust proliferation of differentiated cells. Importantly, we characterized the phenotypical changes occurring during proliferation of differentiated HepaRG™ cells and demonstrated that mitogenic stimulation induced a partial and transient decrease in the expression levels of some liver specific functions followed by a fast recovery of the full differentiation status upon removal of the mitogens. Taking advantage of the proliferation of HepaRG™ cells, we defined lipofection conditions using Syn1-based liposomes allowing transient expression of the cytochrome P450 2D6, a phase I enzyme poorly expressed in HepaRG cells, which opens new means for drug metabolism studies in HepaRG™ cells.

Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos.

Simple SummaryLiposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9, without performing electroporation or microinjection, remains unclear. In this study, we demonstrated that lipofection treatment successfully induced mutation into zygotes during in vitro fertilization and in embryos at the 2- and 4-cell stages. Although liposome-mediated gene editing is a feasible system for use with zona-pellucida-free oocytes/embryos, several challenges must be overcome.Liposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, how this system functions in mammalian oocytes and embryos remains unclear. The present study was conducted to clarify whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9 for 5 h without using electroporation or microinjection. A mosaic mutation was observed in blastocysts derived from zona pellucida (ZP)-free oocytes following lipofection treatment, regardless of the target genes. When lipofection treatment was performed after in vitro fertilization (IVF), no significant differences in the mutation rates or mutation efficiency were found between blastocysts derived from embryos treated at 24 and 29 h from the start of IVF. Only blastocysts from embryos exposed to lipofection treatment at 29 h after IVF contained biallelic mutant. Furthermore, there were no significant differences in the mutation rates or mutation efficiency between blastocysts derived from embryos at the 2- and 4-cell stages. This suggests that lipofection-mediated gene editing can be performed in ZP-free oocytes and ZP-free embryos; however, other factors affecting the system efficiency should be further investigated.

Efficient transfection of Xenobiotic Responsive Element-biosensor plasmid using diether lipid and phosphatidylcholine liposomes in differentiated HepaRG cells

In this study, we evaluated cationic liposomes prepared from diether-NH2 and egg phosphatidylcholine (EPC) for in vitro gene delivery. The impact of the lipid composition, i.e. the EPC and Diether-NH2 molar ratio, on in vitro transfection efficiency and cytotoxicity was investigated using the human HEK293T and hepatoma HepaRG cells known to be permissive and poorly permissive cells for liposome-mediated gene transfer, respectively. Here, we report that EPC/Diether-NH2–based liposomes enabled a very efficient transfection with low cytotoxicity compared to commercial transfection reagents in both HEK293T and proliferating progenitor HepaRG cells. Taking advantage of these non-toxic EPC/Diether-NH2–based liposomes, we developed a method to efficiently transfect differentiated hepatocyte-like HepaRG cells and a biosensor plasmid containing a Xenobiotic Responsive Element and a minimal promoter driving the transcription of the luciferase reporter gene. We demonstrated that the luciferase activity was induced by a canonical inducer of cytochrome P450 genes, the benzo[a]pyrene, and two environmental contaminants, the fluoranthene, a polycyclic aromatic hydrocarbon, and the endosulfan, an organochlorine insecticide, known to induce toxicity and genotoxicity in differentiated HepaRG cells. In conclusion, we established a new efficient lipofection-mediated gene transfer in hepatocyte-like HepaRG cells opening new perspectives in drug evaluation relying on xenobiotic inducible biosensor plasmids.

Enhancement of liposome mediated gene transfer by adding cholesterol and cholesterol modulating drugs

Cholesterol is an important cell membrane component and has been used as co-lipid for cationic liposome to enhance gene delivery. However, the role of cholesterol in transfection efficiency has not been fully understood. In this study, transfection efficiency of liposome was measured after cholesterol was added to the cell culture medium. As a result, addition of cholesterol increased transfection efficiency of several liposomes consisting of different lipid components in various cells (AGS, CHO, COS7 and, MCF7). Furthermore, treatment of cells with cholesterol modulating drugs, imipramine and U18666A, also increased transfection efficiency of liposomes. To elucidate the role of added cholesterol in gene transfer, endocytotic mechanism was studied and also revealed that adding cholesterol in culture media induced participation of caveolae-mediated endocytosis and micropinocytosis in CHO cell. Therefore, the results of this work suggest that modulation of intracellular cholesterol can be an important method to enhance gene delivery.

Contribution of syndecans to lipoplex-mediated gene delivery

The long awaited breakthrough of gene therapy significantly depends on the in vivo efficiency of targeted intracellular delivery. Hidden details of cellular uptake present a great hurdle for non-viral gene delivery with liposomes. Growing scientific evidence supports the involvement of polyanionic cell surface carbohydrates in cellular internalization of cationic liposomes. Syndecans, a highly conserved family of transmembrane heparan sulfate proteoglycans serve attachment sites for great variety of cationic ligands including growth factors, cytokines and even parasites. In the present study we quantitatively measured the contribution of various syndecan isoforms to liposome-mediated gene transfer. The obtained data show the superiority of syndecan-4, the ubiquitously expressed isoform of the syndecan family, in cellular uptake of liposomes. Applied mutational analysis demonstrated that gene delivery could be abolished by mutating the glycosaminoglycan attachment site of syndecans, highlighting the importance of polyanionic heparan sulfate side chains in the attachment of cationic liposomes. Blocking sulfation of syndecans also diminished gene delivery, a finding that confirms the essential role of polyanionic charges in binding cationic liposomes. Mutating other parts of the syndecan extracellular domain, including the cell-binding domain, had clearly smaller effect on liposome internalization. Mutational analyses also revealed that superiority of syndecan-4 in liposome-mediated gene delivery is significantly influenced by its cytoplasmic domain that orchestrates signaling pathways leading to macropinocytosis. In summary our study present a mechanistic insight into syndecan-mediated macropinocytic uptake of lipoplexes and highlights syndecan-4 as a superior target for cationic liposomes.

Direct Head-To-Head Comparison of Cationic Liposome-Mediated Gene Delivery to Mesenchymal Stem/Stromal Cells of Different Human Sources: A Comprehensive Study

Nonviral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources-bone marrow (BM MSC), adipose tissue-derived cells (ASC), and umbilical cord matrix (UCM MSC). The results obtained using a green fluorescent protein (GFP)-encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP(+) cells 48 hr post-transfection, 58 ± 7.1% and 54 ± 3.8%, respectively, versus 33 ± 4.7%. For all cell sources, high cell recoveries (≈50%) and viabilities (>85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division, were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein, we were able to maximize transfection efficiencies to human MSC (maximum of 74% total GFP(+) cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene.

The therapeutic influence of epsilon3 isoform of apolipoprotein E on pathophysiological mechanisms of secondary brain injury evolution after experimental traumatic brain injury

Purpose. To test the therapeutic potential of apolipoprotein E isoform epsilon3, whose synthesis in nerve tissue was induced by means of gene therapy, in an experimental traumatic brain injury (TBI) model, and to substantiate its mechanism of therapeutic action based on current literature data.Methods. To determine structural and functional changes associated with severe diffuse TBI, adult male Wistar rats were subjected to weight drop impact acceleration injury and evaluated at day 10 after trauma. A mixture of DOTAP liposomes and 25 µg of recombinant plasmid pCMV-APOE3 cDNA was infused intraventicularly immediately after TBI using ALZET osmotic pumps. The hippocampal CA1 regions were analyzed by means of light and electron microscopy, morphometry, immunocytochemical and confocal analysis using markers selective for neurons (NeuN) and astrocytes (GFAP). The neurofunctional outcome was assessed in an open field test.Results. The research data have shown that cationic liposome-mediated APOE3 gene transfer has a beneficial effect on hippocampal structure and ultrastructure, diminishes neuronal loss and severity of diffuse axonal injury, gliosis and microglial reaction, as well as aids regression of posttraumatic stress and anxiety.Conclusion. According to literature data, possible mechanisms of apoE therapeutic action are (1) normalization of neural cellular lipid component, (2) its role in regulating reactive gliosis and inflammatory response of CNS to injury, (3) antioxidant effects of apolipoprotein E, (4) regulation of cell death mechanisms, (5) influence of apolipoprotein E on differential gene expression and regulation of genomic response of CNS cells to injury

Effects of LRIG1 expression on radiosensitivity of human glioma cells and the possible mechanism

Objective To investigate the effect of the expression of leucine-rich repeats and immunoglobulin-like domains 1 （LRIG1） on the radiosensitivity of human glioma cell line U251 cells and the possible mechanism. Methods The PEGFP-N1, PEGFP-LRIG1 plasmids were transfected into U251 cells with application of liposome-mediated gene transfer technology. And the cells stably transfected were selected by G418. The radiosensitivity of N1-U251 and LRIG1-U251 cells was analyzed by clonogenic assay. Western blotting demonstrated the expression of LRIG1 and RAD51. Repairment of double-strand （ds） DNA breaks was measured by comet assay. Results The LRIG1 protein level in LRIG1-U251 group （0. 352±0. 011 ） was significantly up-regulated compared to NI-U251 group （0. 071±0. 003 ） （ P 〈 0. 01 ）. Overexpression of LRIG1 up-regulated the radiosensitivity of U251 cells with the sensitive enhancement ratio （SER） being 1. 448. The RAD51 protein levels were significantly suppressed before （P 〈0.01 ） and after irradiation （P 〈 0.01） by up-regulating the LRIG1 expression. Comet assay demonstrated the overexpression of LRIG1 increased the Olive Tail Moment of U251 cells from 21.14±3.42 to 32. 81±5.61 after 8-h radiation （P 〈 0.05）. Conclusion Up-regulation of the LRIG1 expression can down-regulate the RAD51 expression, and then inhibit the repairment of dsDNA breaks, increase the radiosensitivity of U251 cells. Key words: Glioma; Leucine-rich repeats and immunoglobulin-like domains 1; RAD51; Radiosensitivity

Effects of intravesical liposome-mediated human beta-defensin-2 gene transfection in a mouse urinary tract infection model

The purpose of this study was to assess human β-defensin-2 (hBD-2) gene transfection in human bladder epithelial cells and its therapeutic efficacy in a rat urinary tract infection (UTI) model via liposome mediated gene transfer. A large amount of hBD2 production (36.5 ± 3.2 ng/10(6) cells) was demonstrated in transfected cells' supernatants. In addition, a detectable amount of hBD-2 was identified in rats' urine (4.77 ± 1.4 ng/mL) by ELISA. Expression of the transgene hBD-2 in transfected cells and rats' bladders was also confirmed by RT-PCR and Western blotting. Immunohistochemistry revealed that the transgene hBD-2 expressed in the entire epithelial layer of the transduced bladders. Numbers of bacterial colony-forming units in urine and bladders from hBD2 gene treated UTI rats were significantly lower than those from the UTI rats administered PBS at 24, 36, and 72 hr after infection (P < 0.05). In addition, in vivo expression of hBD-2 reduced mucosal damage, interstitial edema and inflammatory cell infiltration in UTI animals. The results indicate that successful inhibition of UTI progression can be produced by hBD2 gene therapy. The liposome-mediated hBD2 plasmid DNA transfection system appears to be a promising method for antimicrobial gene therapy of UTI.

IL‐23 gene therapy for mouse bladder tumour cell lines

• To evaluate the antitumour effects of IL-23 gene transfer into mouse bladder carcinoma (MBT2) cells. • To investigate the mechanisms underlying the subsequent constitutive secrection of IL-23 by the MBT2 cells • An expression vector containing IL-23 gene was introduced into MBT2 cells by liposome-mediated gene transfer, and secretion of IL-23 was confirmed by ELISA. • The in vivo antitumour effect of IL-23-secreting MBT2 cells (MBT2/IL-23) was examined by injecting the cells into syngeneic C3H mice. • A tumour vaccination study using mitomycin C (MMC)-treated IL-23-secreting MBT2 cells was carried out, and the usefulness of in vivo CD25 depletion for an additional vaccine effect was also investigated. • The mechanisms underlying the antitumour effects were investigated by antibody depletion of CD8 or CD4 T cells, or natural killer cells, and cells infiltrating the tumour sites in vivo were assessed using immunohistochemistry. • Stable transformants transduced with MBT2/IL-23 secreted IL-23 into the culture supernatant. • Genetically engineered IL-23-secreting MBT2 cells were rejected in syngeneic mice. • MBT2/IL-23-vaccinated mice inhibited the tumour growth of parental MBT2 cells injected at a distant site and this vaccine effect was enhanced by combination with in vivo CD25 depletion by an antibody. • The main effector cells for the direct antitumour effect of MBT2/IL-23 were CD8 T cells, which was shown by in vivo depletion and immunohistochemical study. • IL-23-secreting MBT2 cells were rejected in syngeneic mice by the activation of CD8 T cells. • MMC-treated MBT2/IL-23 can have a tumour vaccine effect for parental MBT2 cells, and this effect was enhanced by combination with in vivo CD25 depletion.

Cytofectin amine head group modification and degree of liposome pegylation: factors influencing gene transfer.

The effectiveness of liposome-mediated gene transfer methods hinges, in part, on the nature of the interaction between the DNA cargo and the liposomes. Here we have examined the effect of quaternization of the cytofectin cationic head group on this interaction and the effect of concentration of the biocompatible, protective polymer polyethylene glycol2000 (PEG2000) on transfection activity. Thus 3β[N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) and 3β[N-(N’,N’,N’-trimethylammonium propane)-carbamoyl] cholesterol iodide (Chol-Q), differing only in the degree of head group methylation, have been formulated into liposomes with polyethylene glycol2000-distearoylphosphatidyl ethanolamine (DSPE PEG2000) and the neutral co-lipid dioleoylphosphatidylethanolamine (DOPE). Their DNA-binding characteristics have been determined and the gene transfer capabilities of resulting lipoplexes have been examined in HEK 293 human embryonic kidney cells. Quaternary ammonium Chol-Q liposomes were found to bind DNA more avidly than their tertiary amine Chol-T counterparts. The inclusion of PEG2000 in liposome formulations resulted in an increase in the optimal liposome-DNA binding ratio. Chol-T liposomes promoted transgene activity levels 5 times greater than those obtained with Chol-Q lipoplexes. Furthermore, a drop in transfection activity of only 17% was noted on increase of liposome pegylation from 2 to 5 mole percent. The study's findings suggest that strong association between cationic liposomes and DNA may lead to reduced levels of transfection activity as a result of poor release of nucleic acid after cellular uptake.

Liposome‐based vascular endothelial growth factor‐165 transfection with skeletal myoblast for treatment of ischaemic limb disease

The study aims to use cholesterol (Chol) + DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (VEGF165) gene transfer into skeletal myoblasts (SkMs) for treatment of acute hind limb ischaemia in a rabbit model. The feasibility and efficacy of CD liposome mediated gene transfer with rabbit SkMs were characterized using plasmid carrying enhanced green fluorescent protein (pEGFP) and assessed by flow cytometry. After optimization, SkMs were transfected with CD lipoplexes carrying plasmid-VEGF165 (CD-pVEGF165) and transplanted into rabbit ischaemic limb. Animals were randomized to receive intramuscular injection of Medium199 (M199; group 1), non-transfected SkM (group 2) or CD-pVEGF165 transfected SkM (group 3). Flow cytometry revealed that up to 16% rabbit SkMs were successfully transfected with pEGFP. Based on the optimized transfection condition, transfected rabbit SkM expressed VEGF165 up to day 18 with peak at day 2. SkMs were observed in all cell-transplanted groups, as visualized with 6-diamidino-2-phenylindole and bromodeoxyuridine. Angiographic blood vessel score revealed increased collateral vessel development in group 3 (39.7 ± 2.0) compared with group 2 (21.6 ± 1.1%, P < 0.001) and group 1 (16.9 ± 1.1%, P < 0.001). Immunostaining for CD31 showed significantly increased capillary density in group 3 (14.88 ± 0.9) compared with group 2 (8.5 ± 0.49, P < 0.001) and group 1 (5.69 ± 0.3, P < 0.001). Improved blood flow (ml/min./g) was achieved in animal group 3 (0.173 ± 0.04) as compared with animal group 2 (0.122 ± 0.016; P= 0.047) and group 1 (0.062 ± 0.012; P < 0.001). In conclusion, CD liposome mediated VEGF165 gene transfer with SkMs effectively induced neovascularization in the ischaemic hind limb and may serve as a safe and new therapeutic modality for the repair of acute ischaemic limb disease.

Local Transient Myocardial Liposomal Gene Transfer of Inducible Nitric Oxide Synthase Does Not Aggravate Myocardial Function and Fibrosis and Leads to Moderate Neovascularization in Chronic Myocardial Ischemia in Pigs

This study was designed to explore the effect of transient inducible nitric oxide synthase (iNOS) overexpression via cationic liposome-mediated gene transfer on cardiac function, fibrosis, and microvascular perfusion in a porcine model of chronic ischemia. Chronic myocardial ischemia was induced using a minimally invasive model in 23 landrace pigs. Upon demonstration of heart failure, 10 animals were treated with liposome-mediated iNOS-gene-transfer by local intramyocardial injection and 13 animals received a sham procedure to serve as control. The efficacy of this iNOS-gene-transfer was demonstrated for up to 7 days by reverse transcriptase-polymerase chain reaction in preliminary studies. Four weeks after iNOS transfer, magnetic resonance imaging showed no effect of iNOS overexpression on cardiac contractility at rest and during dobutamine stress (resting ejection fraction: control 27%, iNOS 26%; P = ns). Late enhancement, infarct size, and the amount of fibrosis were similar between groups. Although perfusion and perfusion reserve in response to adenosine and dobutamine were not significantly modified by iNOS-transfer, both vessel number and diameter were significantly increased in the ischemic area in the iNOS-treated group versus control (point score: control 15.3, iNOS 34.7; P < 0.05). Our findings demonstrate that transient iNOS overexpression does not aggravate cardiac dysfunction or postischemic fibrosis, while potentially contributing to neovascularization in the chronically ischemic heart.

Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma

BackgroundEpigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a prelimilary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking. The aim of this study was to investigate the expression profile and methylation status of RASSF1A gene, and to explore its concrete mechanisms as a tumor suppressor gene in Nasopharyngeal Carcinoma.MethodsWe examined the expression profile and methylation status of RASSF1A in two NPC cell lines, 38 primary nasopharyngeal carcinoma and 14 normal nasopharyngeal epithelia using RT-PCR and methylated specific PCR(MSP) respectively. 5-aza-dC was then added to confirm the correlation between hypermethylation status and inactivation of RASSF1A. The NPC cell line CNE-2 was transfected with exogenous pcDNA3.1(+)/RASSF1A plasmid in the presence or absence of mutated K-Ras by liposome-mediated gene transfer method. Flow cytometry was used to examine the effect of RASSF1A on cell cycle modulation and apoptosis. Meanwhile, trypan blue dye exclusion assays was used to detect the effect of RASSF1A transfection alone and the co-transfection of RASSF1A and K-Ras on cell proliferation.ResultsPromoter methylation of RASSF1A could be detected in 71.05% (27/38) of NPC samples, but not in normal nasopharyngeal epithelia. RASSF1A expression in NPC primary tumors was lower than that in normal nasopharyngeal epithelial (p < 0.01). Expression of RASSF1A was down-regulated in two NPC cell lines. Loss of RASSF1A expression was greatly restored by the methyltransferase inhibitor 5-aza-dC in CNE-2. Ectopic expression of RASSF1A in CNE-2 could increase the percentage of G0/G1 phase cells (p < 0.01), inhibit cell proliferation and induce apoptosis (p < 0.001). Moreover, activated K-Ras could enhance the growth inhibition effect induced by RASSF1A in CNE-2 cells (p < 0.01).ConclusionExpression of RASSF1A is down-regulated in NPC due to the hypermethylation of promoter. Exogenous expression of RASSF1A is able to induce growth inhibition effect and apoptosis in tumor cell lines, and this effect could be enhanced by activated K-Ras.

Influence of liposomal transfection of apolipoprotein E3 gene on neurologic and cognitive deficits dynamics at experimental traumatic brain injury

The aim of study was to estimate gene therapy influence, that promotes synthesis induction of 3 apolipoprotein E (apoE3) in damaged brain tissue, on motor and memory dysfunction and learning disorders after experimental traumatic brain injury (TBI). Heavy TBI in rats was inflicted under overall anesthesia by free falling from1.5 mof load weighting450 g. The mixture of DOTAP liposome and 25 µg of plasmid vector pCMV•SPORT6 with cDNA of APOE3 gene was infused intraventricularly using ALZET osmotic pump. Posttraumatic neurologic deficits was estimated according to the scale of motor dysfunction at the first day of experiment before TBI infliction and from 1st to 7th day after trauma. Cognitive functions (spatial memory and learning) were tested in Morris water maze during 7–10 days after injury. The obtained results testified that cationic liposome-mediated APOE3 gene transfer caused quicker regress of neurologic and cognitive disorders after experimental TBI.

Liposome-mediated gene therapy in the kidney.

Gene therapy directed to the kidney has been attempted to improve renal disorders such as inherited kidney diseases and common renal diseases that cause interstitial fibrosis, tubular atrophy, and glomerulosclerosis. Viral and non-viral vectors have been tried and been modulated to obtain sufficient transgene expression. However, gene delivery to the kidney is usually difficult because of characteristics of renal cell biology. Among non-viral vectors, the liposome system is a promising procedure for kidney-targeted gene therapy. Using cationic liposome, tubular cells were effectively transduced by retrograde injection of liposome/cDNA complex. Although transgene expression was reportedly modest using cationic liposomes, this method improved renal disease models such as carbonic anhydrase II deficiency and unilateral ureteral obstruction. In contrast, HVJ-liposome system is an effective transfection method to glomerular cells using intra-renal arterial infusion and improved glomerular disease models such as glomerulonephritis and glomerulosclerosis. In addition, intra-renal pelvic injection of DNA by HVJ-liposome system showed transgene expression in interstitial fibroblasts. In kidney-targeted gene therapy, liposome-mediated gene transfer is an attractive method because of its simplicity and reduced toxicity. In spite of modest transgene expression, several renal disease models were successfully modulated by liposome system. Although one limitation of liposome-mediated gene delivery is the duration of transgene expression, the liposome/cDNA complex can be repeatedly administered due to the absence of an immune response.

Liposome‐mediated gene transfer of endothelial nitric oxide synthase to cirrhotic rat liver decreases intrahepatic vascular resistance

Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) in sinusoidal endothelial cells is reduced in the injured liver and leads to intrahepatic portal hypertension. The present study evaluates the effects of liposome-mediated gene transfer of eNOS on the intrahepatic vascular resistance and portal venous pressure (PVP) in cirrhotic rats. Hepatic cirrhosis was induced in male Sprague-Dawley rats by intraperitoneal injection of carbon tetrachloride (CCl(4)), whereas the control normal rats were given the same dose of peanut oil. Plasmid eukaryotic expression vector (liposome-pcDNA3/eNOS) was injected into the portal vein of CCl(4) cirrhotic rats, whereas cirrhotic controls received the same dose of naked plasmid (liposome-pcDNA3) or Tris buffer, and control normal rats received the same dose of Tris buffer. Five days after gene transfer, the levels of eNOS mRNA and protein, NO production, PVP and the changes of hepatic intrahepatic vascular resistance were investigated. Five days after eNOS gene transfer, the levels of eNOS mRNA, eNOS protein and NO production in cirrhotic rats increased remarkably, while hepatic vascular resistance and PVP decreased significantly in cirrhotic rats. Liposome-mediated eNOS gene transfer via intraportal injection is feasible and the increase of intrahepatic eNOS leads to a marked decrease in introhepatic vascular resistance and PVP. These data indicate that intrahepatic eNOS plays an important role in the pathogenesis of portal hypertension and gene transfer of eNOS is a potential and novel therapy for portal hypertension.

HVJ-AVE liposome-mediated Tissue Factor Pathway Inhibitor (TFPI) gene transfer with recombinant TFPI (rTFPI) irrigation attenuates restenosis in atherosclerotic arteries

In this study, we investigate whether the combination of HVJ-AVE liposome-mediated TFPI gene transfer and recombinant TFPI (rTFPI) irrigation would reduce restenosis safely and more effectively. The results indicated that at 4 weeks after angioplasty, the MLD, EELA, IELA and LA of TFPI group and rTFPI group were markedly greater than those of the control groups, and those in the combination group were even greater. The mean IA, I/M, and the percentage of stenosis in TFPI gene group and rTFPI group were significantly reduced compared with control groups, and those in the combination group were even further reduced. Thrombosis in the TFPI gene group, rTFPI group and combination group was significantly reduced compared with the other control groups. The systemic coagulation status of treated animals was not significantly changed and no toxicity was observed in each group. So combination of TFPI gene transfer using HVJ-AVE liposomes and rTFPI irrigation could inhibit thrombosis and neointimal hyperplasia, and attenuate vascular remodeling and luminal stenosis more effectively than using each method alone. The combination method may be a more effective and safe strategy for the prevention of restenosis after angioplasty in humans.

Antisense in vivo knockdown of synaptotagmin I by HVJ–liposome mediated gene transfer attenuates ischemic brain damage in neonatal rats

Synaptic release of the excitatory amino acid glutamate is considered as an important mechanism in the pathogenesis of ischemic brain damage in neonates. Synaptotagmin I is one of exocytosis-related proteins at nerve terminals and considered to accelerate the exocytosis of synaptic vesicles by promoting fusion between the vesicles and plasma membrane. To test the possibility that antisense in vivo knockdown of synaptotagmin I modulates the exocytotic release of glutamate, thus suppressing the excitotoxic intracellular processes leading to neuronal death following ischemia in the neonatal brain, we injected antisense oligodeoxynucleotides (ODNs) targeting synaptotagmin I (0.3 (AS), 0.15 (0.5 AS), or 0.03 μg (0.1 AS), or vehicle) into the lateral ventricles of 7-day-old rats by using a hemagglutinating virus of Japan (HVJ)–liposome mediated gene transfer technique. At 10 days of age, these rats were subjected to an electrical coagulation of the right external and internal carotid arteries, then the insertion of a solid nylon thread into the right common carotid artery toward the ascending aorta up to 10–12 mm from the upper edge of the sternocleidomastoid muscle. Cerebral ischemia was induced by clamping the left external and internal carotid arteries with a clip, and ended by removing the clip 2 h later. Twenty-four hours after the end of ischemia, the extent of ischemic brain damage was neuropathologically and quantitatively evaluated in the neocortex and striatum. While the relative volume of damage in the cerebral cortex and striatum of the vehicle group was extended to 40% and 13.7%, respectively, that in the AS group was significantly reduced to 4.8% and 0.6%. In the 0.5 AS group, the relative volume of ischemic damage in the cerebral cortex and striatum was reduced to 20.5% and 15.4%, respectively, and the difference between the 0.5 AS group and vehicle group was statistically significant in the neocortex, but not in the striatum. These results indicated that antisense in vivo knockdown of synaptotagmin I successfully attenuated ischemic brain damage in neonatal rats and that the effect was dose-dependent. It was also suggested that this treatment was more effective in the neocortex than in the striatum in neonatal rats.

Efficient Delivery of the A-Gamma Globin Gene in Human Hematopoietic and Non-Hematopoietic Cells by Cationic Phosphonolipid- and Lipophosphoramide-Mediated Transfection.

Liposome-mediated gene transfer is known to exhibit a number of desirable features for gene delivery, such as decreased immunogenicity, lack of mutagenesis, efficient transgene expression, internalization of episomal DNA irrespective of size, reproducibility, compositional variability and ease of use. As we and others recently demonstrated, cationic Phosphonolipids (CPs) have pronounced gene delivery potency into human cells. Based on the well-established dependency of gene transfer efficiency upon lipoplex properties, the superior-performing CP EG.308 and a novel cationic lipophosphoramide vechicle (CL) KLN-5, were evaluated for delivery of the human A-gamma globin gene in K562 human erythroleukemia and 5637 human epithelial carcinoma cells. The plasmid vector used for Aγ-globin gene was pAγLuc (3.3 kb HindIII human Aγ-globin gene). Two other vectors were used as controls:i.pGL3bLuc+, which encodes for luciferase; and,ii.pEGFP-CI, which encodes for the green fluorescent protein.Dynamic Light Scattering experiments underlined the cooperative effect of the liposome vesicle diameter, complex formation medium and charge ratio upon lipoplex size. At optimized lipoplex generation conditions, the initial lipid size was not found to be always predictive of lipofection potency, rather transfection efficiency is a function of the liposome vehicle to generate lipoplexes of enhanced size (300–3500nm). Dot-blot analysis and reporter gene-expression assays revealed that among a number of varying composition solvent-systems, optimal nuclear transgene incorporation and expression was attained in 300mM NaCl. Lipoplexes EG.308- and KLN-5/pAγLuc formed in 300mM NaCl mediated maximal transgene delivery and expression 1 and 3 days-post transfection, respectively (330pg luciferase/ng genomic DNA, 80000 RLU/μg protein, respectively). On day 14, sustained plasmid DNA quantity was still detected in the nucleus. Enhanced plasmid integrity was confirmed by Southern blotting analysis. Both linear and circular pAγLuc DNA delivery resulted in pronounced transgene entry (330pg/ng genomic DNA); however, by introducing the latter, transgene expression was almost 100% higher. As opposed to the effective KLN-5-mediated transfection of K562 cells (330pg luciferase/ng genomic DNA, 80000 RLU/μg protein), transgene targeting and expression in 5637 cells was low (73pg luciferase/ng genomic DNA, 13150 RLU/μg protein). Confocal fluorescence microscopy revealed similar lipoplex-cell membrane association events and overlapping endocytic activity in EG.308- and KLN-5- mediated lipofection of target cells. However, KLN-5/pDNA nuclear co-localization in a greater number of GFP+ transfected cells compared to EG.308/pDNA lipofected ones, support a lipid-specific complex behaviour. These results indicate that effective transgene nuclear targeting is feasible with the tested CP and CL. Human Aγ-globin transgene nuclear incorporation and expression in target cells by CPs lends credence to their use as a vehicle of therapeutic transgene delivery.