Lipid Vectors Research Articles

Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection.

Intravenous gene delivery via cationic lipidic vectors gives systemic gene expression particularly in the lung. In order to understand the mechanism of intravenous lipofection, a systematic study was performed to investigate the interactions of lipidic vectors with mouse serum emphasizing how serum affects the biophysical and biological properties of vectors of different lipid compositions. Results from this study showed that lipidic vectors underwent dynamic changes in their characteristics after exposure to serum. Addition of lipidic vectors into serum resulted in an immediate aggregation of vectors. Prolonged incubation of lipidic vectors with serum led to vector disintegration as shown in turbidity study, sucrose-gradient centrifugation analysis and fluorescence resonance energy transfer (FRET) study. Vector disintegration was associated with DNA release and degradation as shown in EtBr intercalation assay and DNA digestion study. Serum-induced disintegration of vectors is a general phenomenon for all cationic lipidic vectors tested in this study. Yet, vectors of different lipid compositions vary greatly in the rate of disintegration. There is an inverse correlation between the disintegration rate of lipidic vectors and their in vivo transfection efficiency. Vectors with a rapid rate of disintegration such as those containing dioleoyl-phosphatidylethanolamine (DOPE) poorly stayed in the lung and were barely active in transfecting cells. In contrast, cholesterol-containing vectors that had a rapid aggregation and a slow disintegration were highly efficient in transfecting cells in vivo. The results of this study explain why cationic lipidic vectors of different lipid compositions have a dramatic difference in their in vivo transfection efficiency. These results also suggest that the study of the interactions of lipidic vectors with serum may serve as a predictive model for the in vivo efficiency of a lipidic vector. Further study of the numerous interactions of lipidic vectors with serum might lead to the development of a vector which can deliver a gene to target cells in a tissue-specific manner.

Safety study and characterization of E1A-liposome complex gene-delivery protocol in an ovarian cancer model.

A phase I clinical trial of E1A-liposome complex is currently ongoing in patients with HER-2/neu-overexpressing breast or ovarian cancers. To optimize the E1A-liposome complex for a further stage of clinical trial, several aspects of the current protocol have been examined in an animal model. In the orthotopic ovarian cancer model, different doses of lipid in the the E1A-liposome complex, which is currently used in clinical trials, were tested for the in vivo gene-transfer efficacy and tumor-suppression function. A lowered lipid dose--1/13 of the previous amount--produced gene expression level and E1A tumor-suppression efficacy similar to that of the original protocol. Mini-E1A, an E1A construct without its immortalization domain and yet capable of repressing HER-2/neu, was proved to be as potent as E1A in suppressing tumor development in vivo. These changes in the E1A-liposome complex will significantly reduce any potential adverse effects caused by lipid vector and E1A DNA. To examine further whether residual E1A DNA may still exist in normal organs after the E1A-liposome treatment, PCR was used to detect E1A DNA in mice that survived for 1 1/2 years after the last treatment. E1A DNA was detected only in the lungs and kidneys, but not in livers, hearts, spleens, brains, uterus or the ovaries. Furthermore, resistance of the E1A DNA extracted from tissues to the digestion of Dpnl restriction enzyme, which can cleave the methylated E1A plasmid DNA generated by methylation-competent bacteria, suggested integration of E1A DNA into the chromosome of the lungs and kidneys. Experimental results presented here provide important information for safety concerns and for the design of future phase II and phase III trials.

New Biocompatible Cationic Amphiphiles Derivative from Glycine Betaine: A Novel Family of Efficient Nonviral Gene Transfer Agents

With the aim of developing new efficient agents for transfecting of eukaryotic cells we have designed and synthesized a novel family of cationic lipid vectors derived from glycine betaine. In this study we present three novel molecules differing by the length of their aliphatic chains (R=12,R=14,R=16). The lyotropic properties of these cationic lipids have been determined, and their transfection efficiency on different cell lines evaluated, using a luminescent assay. Two of these compounds, GB14 and GB12 are efficientin vitroexperiments. Cytoxicity evaluation of these new molecules showed promising results with a low cytotoxicity, especially when co-lipids were included in the formulation. These compounds represent a new family of gene transfer vectors which display good transfection efficiency and low toxicity, possibly due to the natural properties of glycine betaine. These compounds have great potential for the future development ofin vivogene transfer protocols.

Targeted Delivery of Antisense Oligodeoxynucleotides Formulated in a Novel Lipidic Vector

A novel lipidic system has been developed to selectively deliver oligo-deoxynucleotides (ODN) to target cells. It involves the complexation of ODN with polylysine, followed by the addition of pH-sensitive liposomes that contain a targeting ligand, folate. The resulting particles, named LPDII, efficiently and selectively deliver ODN to human KB cells which overexpress folate-binding proteins (FBP). When an ODN against epidermal growth factor receptors (EGFR) was formulated in LPDII, delivery of these particles to KB cells resulted in down-regulation of EGFR and also cell growth inhibition. Free ODN exhibited no inhibitory effect on the growth of KB cells in the same concentration range. An ODN of scrambled sequence, free or formulated in LPDII, also failed to inhibit the growth of KB cells. Finally, the antisense effect of ODN on KB cells was inhibited by an excess amount of free folate, suggesting that the sequence-dependent effect of the ODN was mediated by folate binding protein.

Lipidic Vector Systems for Gene Transfer

Clinical application of gene therapy depends on the development of suitable gene transfer vehicles (vectors). Although generally not as efficient as viral vectors, nonviral systems such as lipidic vectors have the potential advantages of being less toxic, nonrestrictive in cargo DNA size, potentially targetable, and easy to produce in relatively large amounts. More important, lipidic vectors generally lack immunogenicity, allowing repeated in vivo transfection using the same vector. In this paper, we will attempt to summarize some of the recent advances in lipidic gene delivery vectors. Three types of lipidic gene transfer vectors are described: 1) DNA/cationic liposome complexes, 2) DNA encapsulated in neutral or anionic liposomes, and 3) liposome-entrapped, polycation-condensed DNA (LPDI and LPDII). We review the various factors affecting vector structure and gene delivery efficiency, and we discuss the possible mechanisms of gene transfer and their implications in vector design.

Folding and aggregation of DNA chains induced by complexation with lipospermine: formation of a nucleosome-like structure and network assembly

Dioctadecylamidoglycylspermine (DOGS) is a cationic lipid vector capable of efficiently introducing DNA into various eukaryotic cells. We investigated the higher-order structure of the DNA/DOGS complex using fluorescence and electron microscopy. Our results show that the DNA/DOGS complex exhibits a nucleosome-like structure in which DNA wraps around an aggregate of DOGS molecules. In addition, DNA/DOGS complexes tend to associate with each other to form network structures. The resulting network assembly may play a role in effective gene transfection.

Inhibition of Neurogenic Precursor Proliferation by Antisense α Thyroid Hormone Receptor Oligonucleotides

Thyroid hormone 3,5,3'-triiodo-L-thyronine (T3) is required for normal brain development in vertebrates. T3 acts through two classes of nuclear receptors (TR alpha and TR beta) that have distinct developmental spatial and temporal distributions suggesting different functions during neuronal development. One possibility is that TR alpha, which is expressed early in embryogenesis, is involved in neuroblast proliferation. To test this hypothesis we used the embryonic chick optic lobe, as we found that T3 stimulates [3H]thymidine incorporation in this tissue both in vivo and in vitro during embryonic days 6-9. We applied oligonucleotides (ODNs) against TR alpha and TR beta to primary cultures of chick optic lobes. By employing a cationic lipid vector we could use very low ODN concentrations (< 150 nM). Antisense ODNs against TR alpha significantly inhibited [3H]thymidine incorporation, whereas antisense TR beta had no significant effect. However, both ODNs inhibited expression of TRs, as they blocked transcription from a T3-activated reporter gene. Random ODNs used as controls had no significant effect on [3H]thymidine incorporation or on T3-dependent transcription. These observations suggest that TR alpha is implicated in neuroblast proliferation and add credence to the hypothesis that the multiplicity of nuclear receptors allows for specific actions of T3 during development.