Cationic Lipid-mediated Gene Transfer Research Articles

Evaluation of novel cationic gene based liposomes with cyclodextrin prepared by thin film hydration and microfluidic systems

In gene delivery, non-viral vectors have become the preferred carrier system for DNA delivery. They can overcome major viral issues such as immunogenicity and mutagenicity. Cationic lipid-mediated gene transfer is one of the most commonly used non-viral vectors, which have been shown to be a safe and effective carrier. However, their use in gene delivery often exhibits low transfection efficiency and stability. The aim of this study was to examine the effectiveness of novel non-viral gene delivery systems. This study has investigated the encapsulation and transfection efficiency of cationic liposomes prepared from DOTAP and carboxymethyl-β-cyclodextrin (CD). The encapsulation efficiency of the CD-lipoplex complexes were also studied with and without the addition of Pluronic-F127, using both microfluidic and thin film hydration methods. In vitro transfection efficiencies of these complexes were determined in COS7 and SH-SY5Y cell lines. Formulation stability was evaluated using liposomes size, zeta potential and polydispersity index. In addition, the external morphology was studied using transmission electron microcopy (TEM). Results revealed that formulations produced by microfluidic method had smaller, more uniform and homogenious size and zeta-potential as well as higher encapsulation efficiency when compared with liposomes manufactured by thin film hydration method. Overall, the results of this study show that carboxymethyl-β-cyclodextrin increased lipoplexes’ encapsulation efficiency using both NanoAssemblr and rotary evaporator manufacturing processes. However, this increase was reduced slightly following the addition of Pluronic-F127. The addition of carboxymethyl-β-cyclodextrin to cationic liposomes resulted in an increase in transfection efficiency in mammalian cell lines. However, this increase appeared to be cell line specific, COS7 showed higher transfection efficiency compared to SH-SY5Y.

Preparation, characterisation and cell transfection of cationic liposomes in gene therapy

Cationic lipid-mediated gene transfer is one of the most commonly used non-viralvectors. It has been shown to be a safe and effective carrier. However, its use ingene delivery was hampered by its low transfection efficiency and stability.DOTAP, DOPE, cholesterol (CHO) and carboxymethyl-β-cyclodextrin (CD) wereused to prepare cationic liposomes. Cationic liposomes were prepared using both,thin film hydration and a microfluidic method. Formulation stability wasevaluated using liposome size, zeta potential and polydispersity index (PDI).Promega QuantiFluor® ONE dsDNA System was used to investigate theencapsulation efficiency. COS7 and SH-SY5Y cell lines were used to determinetransfection efficiency. Results show that carboxymethyl-β-cyclodextrin increasedencapsulation efficiency by 15.5% and 8% using NanoAssemblr® and rotaryevaporator, respectively compared to liposomes without CD. The addition ofcarboxymethyl-β-cyclodextrin to cationic liposomes resulted in an increase intransfection efficiency in both cell lines.B

Enhanced gene delivery to the lung using biodegradable polyunsaturated cationic phosphatidylcholine-detergent conjugates

Lung diseases are among the more representative causes of mortality and morbidity worldwide and gene therapy is considered as a promising therapeutic approach for their treatment. However the design of efficient nucleic acid carriers for airway administration still is a challenge and there is a pressing need for new developments in this field. Herein, new synthetic DNA carriers based on the conjugation of a phospholipid and C12E4, a nonionic detergent, are developed. DNA complexes with phosphatidylcholine-detergent conjugates are administered in mouse airways, and transgene expression and inflammatory activity as an index of toxicity are investigated as a function of time, DNA dose, and presence of helper and stealth lipids. Introduction of a biodegradable linker between the phosphatidylcholine and detergent moieties significantly attenuates the severity of inflammatory response that characterizes cationic lipid-mediated gene transfer. Concurrent introduction of polyunsaturated fatty acid chains in the carrier scaffold improves transgene expression and further reduces airway inflammation. Finally, the biodegradable phosphatidylcholine-detergent conjugates favorably compare to GL67A, the gold standard for DNA delivery to the airway that is currently under clinical evaluation. Our findings indicate that the lipid formulations described herein may have great potential as nucleic acid carriers for gene therapy.

Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro

BackgroundThe porcine oocyte maturation in vivo occurs within the ovarian follicle and is regulated by the interactions between oocytes and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid. Therefore, the antral follicle is an essential microenvironment for efficient oocyte maturation and its developmental competence. Quantitative bioluminescence imaging of firefly luciferase reporter genes in an intact antral follicle would allow investigation of changes in cellular and molecular events and in the context of the whole follicles. In this study, we investigate factors influencing bioluminescence measurements as a first step towards developing a new bioluminescence imaging system for intact antral follicles.MethodsWe analyzed the time course of bioluminescence emitted from transfected living intact follicles using a cationic lipid mediated gene transfer method with increasing doses (1-3 μg) of firefly luciferase reporter gene (pGL4). In addition, a standard luciferase assay was used to confirm the luciferase expression in granulosa cells in the transfected intact antral follicles. Finally, the dose effects of substrate, D-luciferin, were determined for optimal quantitative bioluminescence imaging of intact porcine antral follicles in vitro.ResultsThe level of luciferase activity of follicles with 3 μg pGL4 was significantly (P < 0.05) greater than the 1 μg and 2 μg groups at 1 min after D-luciferin injection. The bioluminescence intensity of transfected follicles reached a peak at 1 min, and then it was significantly (P < 0.05) reduced within 2 min after injection of D-luciferin; with the level of bioluminescence emission remained constant from 2.5 to 10 min. The bioluminescence emission was maximal with 300 μg of D-luciferin.ConclusionsThe results of this study suggested that the investigation of factors influencing bioluminescence measurements is a critical step toward developing a new bioluminescence imaging model. This study is the first to demonstrate that reporter genes can be transferred to intact granulosa cells with a lipid-mediated gene transfer method within intact follicles in vitro, and the level of transgene expression can be assessed by bioluminescence imaging in living intact antral follicles.

Cationic Lipid Formulations Alter the In Vivo Tropism of AAV2/9 Vector in Lung

Physicochemical properties of gene transfer vectors play an important role in both transduction efficiency and biodistribution following airway delivery. Adeno-associated virus (AAV) vectors are currently used in many gene transfer applications; however, the respiratory epithelium remains a challenging target. We synthesized two cationic sterol-based lipids, dexamethasone-spermine (DS) and disubstituted spermine (D(2)S) for pulmonary gene targeting. Scanning and transmission electron micrographs (TEM) confirmed that AAV/lipid formulations produced submicron-sized clusters. When AAV2/9 or AAV2/6.2 were formulated with these cationic lipids, the complexes had positive zeta potential (zeta) and the transduction efficiency in cultured A549 cells increased by sevenfold and sixfold, respectively. Transduction of cultured human airway epithelium with AAV2/6.2-lipid formulations also showed approximately twofold increase in green fluorescence protein (GFP) positive cells as quantified by flow cytometry. Intranasal administration of 10(11) genome copies (GC) of AAV2/9 and AAV2/6.2 coformulated with lipid formulations resulted in an average fourfold increase in transgene expression for both vectors. Formulation of AAV2/9 with DS changed the tropism of this vector for the alveolar epithelium, resulting in successful transduction of conducting airway epithelium. Our results suggest that formulating AAV2/9 and AAV2/6.2 with DS and D(2)S can lead to improved physicochemical characteristics for in vivo gene delivery to lung.

Small Poly-L-Lysines Improve Cationic Lipid-Mediated Gene Transfer in Vascular Cells in vitro and in vivo

The potential of two small poly-L-lysines (sPLLs), low molecular weight sPLL (LMW-L) containing 7–30 lysine residues and L18 with 18 lysine repeats, to enhance the efficiency of liposome-mediated gene transfer (GT) with cationic lipid DOCSPER [1,3-dioleoyloxy-2-(N⁵-carbamoyl-spermine)-propane] in vascular smooth muscle cells (SMCs) was investigated. Dynamic light scattering was used for determination of particle size. Confocal microscopy was applied for colocalization studies of sPLLs and plasmid DNA inside cells. GT was performed in proliferating and quiescent primary porcine SMCs in vitro and in vivo in porcine femoral arteries. At low ionic strength, sPLLs formed small complexes with DNA (50–100 nm). At high ionic strength, large complexes (>1 µm) were observed without any significant differences in particle size between lipoplexes (DOCSPER/DNA) and lipopolyplexes (DOCSPER/sPLL/DNA). Both sPLLs were colocalized with DNA inside cells 24 h after transfection, protecting DNA against degradation. DOCSPER/sPLL/DNA formulations enhanced GT in vitro up to 5-fold, in a porcine model using local periadventitial application up to 1.5-fold. Both sPLLs significantly increased liposome-mediated GT. Poly-L-lysine L18 was superior to LMW-L since it enabled maximal GT at a 10-fold lower concentration. Thus, sPLLs may serve as enhancers for GT applications in SMCs in vitro and in vivo using local delivery.

Electroporation-mediated transfer of plasmids to the lung results in reduced TLR9 signaling and inflammation

Electroporation can deliver DNA efficiently and safely to tissues in live animals, including the lung where it causes little inflammation or lung injury. In contrast, cationic lipid-mediated gene transfer has been shown to induce an inflammatory response caused by unmethylated plasmid CpG residues, which activate the toll-like receptor (TLR9) signaling pathway. As TLR9 is located in the endosomal/lysosomal compartment, we hypothesized that plasmids do not activate TLR9 during electroporation because they enter the cytoplasm directly through transient pores in the plasma membrane. To test this, plasmids were transfected into kidney epithelial cells overexpressing TLR9 (HEK293-TLR9+) and cells lacking TLR9 (HEK293-TLR9-null). Interleukin (IL)-8 expression, an indicator of TLR9 activation, increased more than 10-fold at 24 h post-liposome transfection in HEK293-TLR9+ cells, but showed no significant increase in electroporated cells, compared with untransfected cells. In vivo liposome-mediated gene transfer caused increases in IL-6, IL-12, tumor necrosis factor alpha and interferon gamma in mouse bronchial alveolar lavage fluid, whereas the levels of these cytokines were more than 10-fold lower by comparison following electroporation. Depletion of alveolar macrophages suggested that this inflammatory response is mediated by resident pulmonary epithelial cells. These results suggest that electroporation-mediated gene transfer bypasses the TLR-9 pathway, thus accounting for the low levels of inflammation seen with this approach.

Optimizing Aerosol Gene Delivery and Expression in the Ovine Lung

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.

Intracellular Delivery of Poly(ethylene glycol) Conjugated Antisense Oligonucleotide Using Cationic Lipids by Formation of Self-Assembled Polyelectrolyte Complex Micelles

A polyelectrolyte complex (PEC) micelle-based antisense oligodeoxynucleotide (ODN) delivery system was designed to overcome intrinsic limitations of cationic lipid-mediated gene transfer. Cationic lipid (Lipofectamine, LF) and ODN conjugated poly(ethylene glycol) (PEG) were ionically complexed to form self-assembled spherical PEC micelles. They have a distinctive structural feature: a charge-neutralized core surrounded by highly flexible PEG corona. The PEC micelles could be visualized as a nano-sized sphere by atomic force microscopy (AFM). The DNA/LF PEC micelles exhibited far improved transfection efficiency compared to those of conventional lipoplex formulations (ODN/LF) in the presence of serum. They showed enhanced cellular uptake followed by rapid nuclear localization of ODN in human epithelial carcinoma (KB) cells. In addition, anti-proliferative activity of c-raf gene-directed antisense ODN was almost fully maintained in KB cells in the presence of serum.

551. The Use of Electroporation To Transfer Plasmids to the Lung Results in Reduced TLR9 Signaling and Inflammation

Electroporation is an efficient and safe technique to deliver naked DNA to both cells in culture and in living animals. Application of brief square wave pulses to cells induces transient pores to open in the plasma membrane, allowing DNA to enter the cytoplasm directly. We have used this approach to deliver plasmid DNA to the murine lung and found high level gene expression with almost undetectable inflammation or lung injury. By contrast, cationic lipid mediated gene transfer routinely induces inflammatory responses characterized by increased levels of cytokines. This inflammatory response is caused by unmethylated CpG residues in plasmid DNA, which activate toll-like receptor 9 (TLR9). Since TLR9 has been reported to be located in endosomes, we hypothesized that plasmid DNA does not activate the TLR9 pathway when delivered by electroporation, because electroporation bypasses endocytosis. To test this, plasmids were transfected into HEK293-TLR9 cells and HEK293-Null cells, using electroporation or liposomes. Twenty- four hours later, expression of IL-8 was measured by qPCR. IL-8 mRNA increased more than 10-fold in Lipofectin transfected HEK293-TLR9 cells, but no significant increase was seen in electroporated cells. Neither method caused increases in IL-8 expression in HEK293-Null cells. Western blotting using an antibody against phosph-IRAK1 shows the activation of TLR signaling pathway after cells were transfected by lipofectin, but activation is greatly reduced following electroporation of the same plasmids. To follow this signaling in living animals, 20 μg plasmid DNA was transferred to the lungs of mice by tracheally delivered lipoplex or electroporation and activation of cytokine expression was measured 24 hours later by either qPCR or by Cytometric Bead Array to measure cytokine proteins in BALF. The levels of IL-6, IL-12, and IFN-γ were only slightly higher with electroporation gene transfer compared with DNA only and liposome only. By contrast, liposome/DNA complex caused more than 10-fold increase in these three cytokines compared to electroporation gene transfer. Taken together, these results suggest that electroporation-mediated transfer of DNA appears to bypass the TLR-9 pathway, accounting for the very low levels of inflammation seen in animals.

556. Proteasome Inhibitors Increase Cationic-Lipid Mediated Gene Transfer in A549 Cells In Vitro

It has previously been described that proteasome inhibitors improve transduction efficiency of adeno-associated virus in vitro. More recently it was shown that proteasome-inhibitors increase peptide-based non-viral gene transfer in certain cell lines. We have assessed if proteasome inhibitors doxorubicin and LLnL increase Lipofectamine 2000-mediated gene expression in A549 (type II pneumocyte-like) and CFTE cells (cystic fibrosis tracheal epithelium). Cells were grown in 96-well plates and transfected (0.05 mg pLux/well complexed to 0.25 mg Lipofectamine) over-night. During transfection, and for 24 hrs after, cells were exposed to doxorubicin (0.5 mM to 20 mM and 0.5 mM to 8 mM for A549 and CFTE cells, respectively, n = 8 wells/condition, at least 2 independent experiments). 48 hrs after transfection luciferase expression was measured. The effect of doxorubicin on transfection was dose-dependent and peaked at 15 mM (no dox: 6.8|[times]|104|[plusmn]|1.2|[times]|104, dox: 1.3|[times]|10|[plusmn]|1.9|[times]|10 RLU/mg, p<0.05) for A549 cells. In contrast exposure of CFTE cells to 1-4 mM doxorubicin decreased gene expression (p<0.05) compared to no drug control. In parallel experiments, cells were exposed to LLnL (10 mM to 100 mM and 10 mM to 60 mM for A549 and CFTE cells, respectively, n=8 wells/condition, at least 2 independent experiments). Transfection efficiency was dose-dependent in A549 cells and all concentrations of LLnL lead to a significant increase in gene expression (6-18 fold, p<0.05). In CFTE cells LLnL did not significantly increase gene expression. Although, the intracellular processes that lead to gene expression after transfection are not well understood and the mode of action of proteasome inhibitors in the context of lipid-mediated gene transfer is unclear, the data presented here suggest follow up in in vivo studies is warranted.

Inhibition by TNF-alpha and IL-4 of cationic lipid mediated gene transfer in cystic fibrosis tracheal gland cells

In vivo, tracheal gland serous cells highly express the cystic fibrosis transmembrane conductance regulator (cftr) gene. This gene is mutated in the lethal monogenic disease cystic fibrosis (CF). Clinical trials in which the human CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in weak and transient gene expression. As CF is characterized by mucus inspissation, airway infection, and severe inflammation, we tested the hypothesis that inflammation and especially two cytokines involved in the Th1/Th2 inflammatory response, interleukin 4 (IL-4) and TNFalpha, could inhibit gene transfer efficiency using a model of human CF tracheal gland cells (CF-KM4) and Lipofectamine reagent as a transfection reagent. The specific secretory defects of CF-KM4 cells were corrected by Lipofectamine-mediated human CFTR gene transfer. However, this was altered when cells were pre-treated with IL-4 and TNFalpha. Inhibition of luciferase reporter gene expression by IL-4 and TNFalpha pre-treated CF-KM4 cells was measured by activity and real-time RT-PCR. Both cytokines induced similar and synergistic inhibition of transgene expression and activity. This cytokine-mediated inhibition could be prevented by anti-inflammatory agents such as glucocorticoids but not by non-steroidal (NSAI) agents. This data suggests that an inflammatory context generated by IL-4 and TNFalpha can inhibit human CFTR gene transfer in CF tracheal gland cells and that glucocorticoids may have a protecting action.

53 Inhibition by TNF-alpha and IL-4 of cationic lipid mediated gene transfer in cystic fibrosis tracheal gland cells

53 Inhibition by TNF-alpha and IL-4 of cationic lipid mediated gene transfer in cystic fibrosis tracheal gland cells

Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool.

Effective gene therapy depends on the efficient transfer of therapeutic genes to target cells. None of the current technologies, however, satisfy all of the requirements necessary for gene therapy, because the plasma and nuclear membranes of mammalian cells are tight barriers against gene transfer using synthetic delivery systems. The protein transduction domain (PTD) of human immunodeficiency virus type 1 (HIV-1) Tat protein greatly facilitates protein transfer via membrane destabilisation. We synthesised polylysine peptides containing Tat PTD (TAT-pK), or other sequences, and investigated their potential as agents for gene transfer. The synthesised polypeptide TAT-pK retains DNA binding function and mediates delivery of a reporter gene to cultured cells. RGD motif binds with low affinity to alpha integrins which induce cell activation. Two control polypeptides, GGG-pK and RGD-pK, were synthesised and tested, but their gene transfer abilities were weaker than those of TAT-pK. TAT-pK-mediated gene transfer was enhanced in the presence of chloroquine or ammonium chloride, to a greater extent than that of cationic lipid-mediated gene transfer in most cancer cell lines tested. These data suggest that TAT-pK may be a potent candidate delivery vehicle that promotes gene transfer, dependent on the endocytic pathway. We conclude that the TAT-pK/DNA complex is useful as a minimal unit to package therapeutic genes and to transduce them into mammalian cells.

Calorimetry of Cationic Liposome–DNA Complex and Intracellular Visualization of the Complexes

This chapter describes cationic liposome–mediated gene transfer that has become a widely used tool for in vitro transfection of eukaryotic cells and as a promising and safe alternative for in vivo gene therapy applications. It involves the formation of a cationic lipid–DNA complex that interacts efficiently with the cell surface, leading to the entry and expression of the exogenous DNA. The use of cationic lipids as DNA delivery systems for gene therapy is limited by the relatively low efficiency of the gene transfer process compared with viral vectors. Improvement of the in vitro and in vivo cationic lipid–mediated gene transfection efficiency can be attempted by use of trial and error approaches consisting of synthesizing and testing a large number of cationic lipid derivatives. Alternatively, a deeper understanding of the intermediate steps and mechanisms involved in the gene transfer process mediated by cationic lipids could allow the design of new rational strategies to improve the transfection efficiency. Two key steps are involved in the cationic lipid–mediated gene transfer process: (1) interaction between the cationic liposomes and DNA leading to the formation of a complex and (2) interaction between the complex and the target cells leading to DNA entry and expression of the transgene.

Cationic lipid–DNA complexes—lipoplexes—for gene transfer and therapy

Cationic lipid-mediated gene transfer and delivery still attract great attention of many gene therapy laboratories. From the point of view of the most important characteristics of lipoplex particles, e.g. its charge and size, we reviewed recent studies available. In general, the paper deals with non-viral systems of gene transfer into eukaryotic cell based on various lipids. Having usually less efficiency in gene transfer, lipid-based gene transfer vehicles (lipoplexes/genosomes) are characterized with certain advantages even over viral ones: they are less toxic and immunogenic, could be targetable and are easy for large-scale production, a size of transferred DNA being quite high. Conditions of DNA condensation during interactions with lipids are described. Results of the studies of mechanism of DNA–lipid complex interactions with the cell membrane and their transport into the nucleus are summarized. Dependence of efficiency of gene transfer on lipoplex structure and physical–chemical properties is reviewed. Advantages and disadvantages of different macromolecule complexes from the point of view of transfection efficiency, possibility of use in vivo, cytotoxicity and targeted gene transfer in certain organs and tissues are also discussed. Results of transfection of different cells using neutral, anion and cation liposomes are reviewed. The conclusion reached was that efficiency and specificity of gene transfer may grow considerably when mixed macromolecule lipid systems including polycations and glycolipids are used.

The role of non-ionic surfactants on cationic lipid mediated gene transfer

Cationic lipid carriers were made of 1,2-dioleoyl- sn-glycero-3-trimethylammoniumpropane (DOTAP), squalene and different amounts of non-ionic surfactants. Various non-ionic surfactants were selected to elucidate the role of Tween 80 in the cationic lipid mediated gene delivery. They had a similar structure to Tween 80 such as various poly(ethyleneglycol) (PEG) chain lengths and acyl chain with different headgroups. For comparison, lipid carriers were also prepared with 1,2-dioleoyl- sn-glycero-3-trimethylammoniumpropane (DOTAP) and 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE). Addition of non-ionic surfactants decreased the emulsion–DNA interaction and affected the transfection activity depending on the chain length and the content of PEG in the surfactant. Among the surfactants, Tween 80 yielded the best transgene expression without showing toxicity in COS-1 cells. The delivery mechanism of the complex was investigated by measuring the effects of endocytosis inhibitors (chloroquine and wortmannin). The emulsion–DNA complex seems to be taken up by the cells via endocytosis.

Role of intracellular cationic liposome-DNA complex dissociation in transfection mediated by cationic lipids.

The cationic lipid-mediated gene transfer process involves sequential steps: internalization of the cationic lipid-DNA complexes inside the cells via an endocytosis-like mechanism, escape from endosomes, dissociation of the complex, and finally entry of free DNA into the nucleus. However, cationic lipid-DNA complex dissociation in the cytoplasm and the ability of the subsequently released DNA to enter the nucleus have not yet been demonstrated. In this report we showed, using confocal laser scanning analysis, that microinjection of a double fluorescent-labeled cationic lipid-pCMV-LacZ plasmid complex into the cytoplasm of HeLa cells results in efficient complex dissociation. However, the released DNA did not enter the nucleus, and no significant transfection could be detected. In contrast, nuclear microinjection of the cationic lipid-pCMV-LacZ plasmid complex resulted in efficient complex dissociation and transfection of all the cells. Taken together, the data suggest that intracellular dissociation of the cationic lipid-DNA complex is not a limiting step for transfection as previously thought.

Analysis of differential lipofection efficiency in primary and established myoblasts.

In this study we have compared the process of lipid-mediated transfection in primary and established myoblasts, in an attempt to elucidate the mechanisms responsible for the scarce transfectability of the former. We determined the metabolic stability of cytoplasmically injected and lipofected DNA in primary and established myoblasts and carried out a comparative time course analysis of luciferase reporter-gene expression and DNA stability. The efficiency of the transcription-translation machinery of the two cell types was compared by intranuclear injection of naked plasmid DNA encoding luciferase. Subcellular colocalization of fluorescein-labeled lipopolyplexes with specific endosomal and lysosomal markers was performed by confocal microscopy to monitor the intracellular trafficking of plasmid DNA during transfection. The metabolic stability of plasmid DNA was similar in primary and established myoblasts after both lipofection and cytoplasmic injection. In both cell types, lipofection had no detectable effect on the rate of cell proliferation. Confocal analysis showed that nuclear translocation of transfected DNA coincided with localization in a compartment devoid of endosome- or lysosome-specific marker proteins. The residency time of plasmid DNA in this compartment differed for primary and established myoblasts. Our findings suggest that the lower transfectability of primary myoblasts is mostly due to a difference in the intracellular delivery pathway that correlates with more rapid delivery of internalized complex to the lysosomal compartment.

OPTIMIZATION OF CATIONIC LIPID MEDIATED GENE TRANSFER: STRUCTURE-FUNCTION, PHYSICO-CHEMICAL, AND CELLULAR STUDIES

ABSTRACTThe rationale design aimed at the enhancement of cationic lipid mediated gene transfer is discussed. These improvements are based on the straight evaluation of the structure-activity relationship and on the introduction of new structures. Much attention have been given to the supramolecular structures of the lipid/DNA complexes, to the effect of serum on gene transfer and to the intracellular trafficking of the lipoplexes. Finally new avenue using reducible cationic lipids has been discussed.