Robert W. Malone

Cholesterol enhances cationic liposome-mediated DNA transfection of human respiratory epithelial cells.

Cationic liposome transfection reagents are useful for transferring polynucleotides into cells, and have been proposed for human pulmonary gene therapy. The effect of adding cholesterol to cationic lipid preparations has been tested by first formulating the cationic lipid N-[1-(2,3-dioleoyloxy)propyl-N-[1-(2-hydroxy)ethyl]-N,N-dimethyl ammonium iodide (DORI) with varying amounts of dioleoylphos-phatidylethanolamine (DOPE) and cholesterol. Cholesterol was found to enhance lipid-mediated transfection in both the respiratory epithelial cells and mouse fibroblasts. These findings will facilitate nucleic acid transfection of many cell types including differentiated epithelial cell monolayers, and therefore may be useful for examining gene regulation in various cell types and for developing pulmonary gene therapy.

Improved method for the removal of endotoxin from DNA

Contaminating endotoxin in solutions used in gene therapy and genetic immunization can result in various deleterious effects both in vitro and in vivo. In order to avoid such complications, attempts were made to characterize the extent of the problem of endotoxin contamination and develop a solution to this problem. After screening for endotoxin in plasmid DNA preparations using the Limulus Amoebocyte Lysate (LAL) assay, nearly half of all samples displayed high endotoxin levels. Therefore, a simple one-step procedure was developed for the removal of endotoxin using a polymyxin B resin.

THE COUNTERION INFLUENCE ON CATIONIC LIPID-MEDIATED TRANSFECTION OF PLASMID DNA

A panel of DOTAP analogs was prepared by altering the anionic counterion that accompanies the trimethylammonium polar domain. The transfection of plasmid DNA into NIH3T3 cells and mouse lung was examined using the counterion analogs. The in vitro transfection activity decreased as follows: DOTAP.bisulfate > trifluoromethanesulfonate approximately equal to iodide approximately equal to bromide > dihydrogenphosphate approximately equal to chloride approximately equal to acetate > sulfate. A similar activity trend was observed in vivo.

A flexible approach to synthetic lipid ammonium salts for polynucleotide transfection

Abstract The synthesis of novel 2,3-acyloxy-1-propylammonium salts is described. The route incorporates the use of glycidol and N-methyl-2-aminoethanol to prepare cationic lipids with control of absolute stereochemistry and regioselective introduction of fatty acid side chains.

Considerations for the Design of Improved Cationic Amphiphile-Based Transfection Reagents

AbstractCationic amphiphiles (cytofectins) are widely used for the transfection of cultured cells, and may become useful for the development of genetic medicines. Although fundamental research focused on clarification of physicochemical structure/biologic function correlations has been limited, general principles relating to optimization of cytofectin structure are beginning to emerge. We review these general conclusions and suggest considerations which may assist in the development of improved compounds. Data which relates to formulation is also discussed. The formulation studies address the tendency of high concentration cytofectin:polynucleotide complexes to precipitate. From these observations we postulate that a thermodynamically stable product can be formed by sonication with heating of cytofectin:polynucleotide complexes, and that this process reduces the kinetically driven aggregation and precipitation which currently complicates many in vivo studies.

Developing dendritic cell polynucleotide vaccination for prostate cancer immunotherapy.

Immunotherapy has been successfully used to treat some human malignancies, principally melanoma and renal cell carcinoma. Genetic-based cancer immunotherapies were proposed which prime T lymphocyte recognition of unique neo-antigens arising from specific mutations. Genetic immunization (polynucleotide vaccination, DNA vaccines) is a process whereby gene therapy methods are used to create vaccines and immunotherapies. Recent findings indicate that genetic immunization works indirectly via a bone marrow derived cell, probably a type of dendritic antigen presenting cell (APC). Direct targeting of genetic vaccines to these cells may provide an efficient method for stimulating cellular and humoral immune responses to infectious agents and tumor antigens. Initial studies have provided monocytic-derived dendritic cell (DC) isolation and culture techniques, simple methods for delivering genes into these cells, and have also uncovered potential obstacles to effective cancer immunotherapy which may restrict the utility of this paradigm to a subset of patients.

Hepatic gene expression after direct DNA injection

Abstract The liver is an attractive target tissue for gene therapy. Current techniques which may be useful for in vivo hepatic gene delivery include viral vectors such as retroviruses, adenoviruses and adeno-associated viruses, and non-viral methods including liposome/DNA complexes, peptide/DNA complexes, dendrimer delivery and direct injection of DNA. While, previous studies have suggested that genes cannot be delivered to liver tissue by direct DNA injection, this review focuses on the factors that lead to significant gene expression in liver, as compared to other tissues, after direct injection of plasmid DNA. Several factors are discussed, including injection technique, DNA dose, DNA diluent and enhancement of transfected gene expression by dexamethasone administration. The current limitations and potential uses of direct DNA injection into liver are reviewed. Direct DNA injection is currently a reliable, reproducible and simple alternative for hepatic gene transduction that provides a tool for molecular research and may have future potential for therapeutic gene delivery.