Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Utility Patent
1998-05-12
2001-01-02
Priebe, Scott D. (Department: 1633)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S002600, C424S450000, C435S455000, C435S458000
Utility Patent
active
06169078
ABSTRACT:
BACKGROUND OF THE INVENTION
Although gene therapy technology is becoming more advanced and sophisticated there are still a number of technical hurdles which limit the usefulness of this technology. One of the important technical hurdles pertains to the difficulty of delivering DNA into a cell and having that DNA reach its intended target so that genetic transformation can occur. There are several steps in this process including finding an appropriate vehicle for delivering the DNA, increasing the efficiency by which DNA enters the cell, and increasing the likelihood that the DNA will be released by the delivery vehicle so that it reaches its intended location within the cell.
The use of cationic organic molecules to deliver heterologous genes in gene therapy procedures has been reported in the literature. Not all cationic compounds will complex with DNA and facilitate gene transfer. Currently, a primary strategy is routine screening of cationic molecules looking for good candidates. The types of compounds which have been used in the past include cationic polymers such as polyethyleneamine, ethylene diamine cascade polymers, and polybrene. Proteins, such as polylysine with a net positive charge have also been used. The largest group of compounds, cationic lipids; includes DOTMA, DOTAP, DMRIE, DC-chol, and DOSPA. All of these agents have proven effective but suffer from potential problems such as toxicity and expense in the production of the agents.
Cationic liposomes are currently the most popular system for gene transfection studies. Cationic liposomes serve two functions: protect DNA from degradation and increase the amount of DNA entering the cell. While the mechanisms describing how cationic liposomes function have not been fully delineated, such liposomes have proven useful in both in vitro and in vivo studies. However, these liposomes suffer from several important limitations. Such limitations include low transfection efficiencies, expense in production of the lipids, low suspenibility when complexed to DNA, and toxicity.
Compounds containing disulfide bonds are able to participate in disulfide exchange reactions over a broad range of conditions from acid to basic pH and in a wide variety of buffer constitutes and physiological conditions (Hermanson, G. T. (1996) Bioconjugate Techniques, pp. 150-152, Academic Press, San Diego). Because of their special chemical properties, disulfide conjugate techniques have been widely used in drug delivery to achieve high delivery efficiencies (Trail, P. A., D. Willner, S. J. Lasch, A. J. Henderson, S. J. Hofstead, A. M. Casazza, R. A. Firestone, I. Hellstrom, K. E. Hellstrom (1993)
Science
261:212-215; Legendre, J. Y., A. Trzeciak, B. Bohrmann, U. Deushle, E. Kitas, A. Supersaxo (1997)
Bioconjugate Chem.
8:57-63; Kostina, E. V., A. S. Boutorine (1993)
Biochimie
75:35-41; Trail, P. A., D. Willner, J. Knipe, A. J. Henderson, S. J. Lasch, M. E. Zoeckler, G. R. Braslawsky, J. Brown, S. J. Hofstead, R. S. Greenfield, R. A. Firestone, K. Mosure, K. F. Kadow, M. B. Yang, K. E. Hellstron, I. Hellstrom (1997)
Cancer Research
57:100-105). The most common method used in bioconjugates involves cross-linking or modification reactions using disulfide exchange processes to form disulfide linkage with sulfhydryl-containing molecules. However, this method is not suitable for the syntheses of most cationic lipids due to their specific chemical structures. The use of cationic compounds containing disulfide bonds to deliver DNA in gene therapy procedures has not previously been described.
BRIEF SUMMARY OF THE INVENTION
The subject invention provides novel materials and methods for efficiently delivering polynucleotides or other molecules across cell membranes into intracellular regions. Advantageously, this transport of molecules from extracellular to intracellular locations is accomplished in a manner whereby the transported molecule is released in the intracellular regions and does not remain complexed or otherwise associated with a carrier molecule upon exposure to the intracellular environment. The introduction into a cell of molecules which are not associated with carrier molecules is highly advantageous because the transported molecule, free from association with a carrier molecule, is able to provide a desired biological activity without any inhibition which could be caused by a carrier molecule.
To accomplish efficient molecular transport followed by intracellular release, the materials and methods of the subject invention exploit differences which exist between intracellular and extracellular environments. A specific embodiment of the subject invention involves attaching or complexing a desired molecule with a second molecule (or other moiety) wherein said second molecule facilitates the efficient transfer of the desired molecule across a cell membrane. The association between the desired molecule and the second molecule remains intact in the extracellular environment and as the desired molecule crosses the cell membrane. However, upon exposure to the intracellular environment, the association between the desired molecule and the second molecule is broken thereby freeing the desired molecule in the intracellular space.
The desired molecules which can be transported into cells according to the subject invention include, for example, polynucleotides such as DNA or RNA. The transport of polynucleotides into target cells is necessary in a variety of procedures where it is desired, for example, to transform a target cell with heterologous polynucleotides. The efficiency of such transformation procedures can be improved by the materials and methods of the subject invention which make it possible to delivery uncomplexed polynucleotides to the intracellular environment. Such uncomplexed polynucleotides (or other desired molecules) are not hindered from performing an intended function within the cell by the presence of a carrier molecule.
In a specific embodiment, the subject invention provides a new class of lipid molecules for use in non-viral gene therapy. Advantageously, these novel compounds effectively complex with DNA and facilitate the transfer of DNA through a cell membrane into the intracellular space of a cell to be transformed with heterologous DNA. Furthermore, these lipid molecules facilitate the release of heterologous DNA in the cell cytoplasm thereby increasing gene transfection during gene therapy in a human or animal.
The novel compounds of the subject invention provide a disulfide linker between a polar head group and a lipophilic tail group of the lipid. Upon entering a cell, these lipids are exposed to high intracellular concentrations of glutathione or other reducing substances which reduce the disulfide bond thereby releasing the heterologous DNA in the cytoplasm. This process increases the efficiency of gene transfection. The lipid molecules of the subject invention are particularly advantageous because they can be selectively destabilized within the cytosol of the cell.
A further aspect of the subject invention pertains to convenient methods of synthesis for disulfide-containing cationic lipids. In a specific embodiment, the lipid, 1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide ornithine conjugate (DOGSDSO), can be synthesized and used to prepare liposomes in combination with L-dioleoyl phosphatidylethanolamine (DOPE). The disulfide bond of DOGSDSO is cleaved by reductive media leading to destabilization of the liposome/DNA complex, thus increasing the release of DNA compared to a non-disulfide-containing analog.
To demonstrate the importance of the disulfide bond a comparison was made of the gene transfection activities and the transfection efficiency of the disulfide bond containing-cationic lipid DOGSDSO, its analog 1′,2′-dioleyl-sn-glycero-3′-succinyl-1,6-hexanediol ornithine conjugate (DOGSHDO) which has a similar structure to DOGSDSO but does not contain a disulfide bond, and commercially available 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In all liposome preparations the cati
Hughes Jeffrey Allen
Tang Fuxng
Nguyen Dave Trong
Priebe Scott D.
Saliwanchik Lloyd & Saliwanchik
University of Florida
LandOfFree
Materials and methods for the intracellular delivery of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Materials and methods for the intracellular delivery of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Materials and methods for the intracellular delivery of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2550804