Transfection reagents

Abstract

The present invention provides optimized transfection reagents comprising mixtures of cationiclipoids. In particular, the present invention provides DNA delivery vehicles based on identifying the optimal hydrophobicity of novel cationic phospholipid derivatives that, alone or in combination, form complexes with DNA (lipoplexes) and exhibit enhanced transfection activity.

Description

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 60 / 508,544, filed Oct. 3, 2003, which is incorporated herein by reference in its entirety.[0002] The present invention was made, in part, under funds from the National Institutes of Health Grant No. GM 52329. The government may have certain rights in the invention.FIELD OF THE INVENTION [0003] The present invention provides optimized transfection reagents comprising mixtures of cationic lipoids. In particular, the present invention provides DNA delivery vehicles based on identifying optimal hydrophobicity of novel cationic phospholipid derivatives that, alone or in combination, form complexes with DNA (lipoplexes) and exhibit enhanced transfection activity. BACKGROUND [0004] There are approximately four thousand different genetic diseases, many highly debilitating and frequently resulting in death at an early age. Because almost all of these diseases involve a defective protein, conventional trea...

AI Technical Summary

Benefits of technology

[0019] In some embodiments, the mixture comprises first and second cationic lipoids. The present invention provides optimized transfection reagents comprising mixtures of cationic lipoids. In particular, the present invention provides DNA delivery vehicles based on identifying the optimal hydrophobicity of novel cationic phospholipid derivatives that, alone or in combination, form complexes with DNA (lipoplexes) and exhibit enhanced transfection activity.

[0021] In some embodiments, the second cationic lipoid is a lipoid with a hydrophobic structure that is significantly different from that of the first lipoid. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, the mechanism of action of such a hydrophobic structure may disrupt the packing of the bilayer of a liposome or other structure so that the lipid organization of the array may be different in the mixture, thereby enhancing transfection. Differences in physical organization of lipids of transfection agents may translate into effects on transfection, as measured by detecting transfection efficiency with and without the second cationic lipoid or by comparing the transfection reagents having the second cationic lipoid to other transfection reagents (see e.g., Example 1 for such a method). Optimization to determine the optimal composition for liposomes or other lipoid arrays can be achieved by a variety of methods. For example, in some embodiments, the second cationic lipoid has a smaller hydrophobic mass (e.g., shorter hydrophobic tail or net shorter hydrophobic tails in a molecule with multiple tails). In some embodiments, the second cationic lipoid is functionalized to add hydrophilicity (e.g., canceling some of the hydrophobic mass). For example, in some embodiments, a lipid head group is made larger or more hydrophilic. In some embodiments, a lipid tail is functionalized to reduce hydrophobicity. For example, in some embodiments, the functionalization comprises addition of one or more polar groups. In some embodiments, a polar fluorophore is added (e.g., NBD), providing the added feature of fluorescent detectability. In some embodiments, the second cationic lipoid comprises a head group and a second component with sufficient hydrophobicity to allow it to form a bilayer with the first cationic lipoid, but otherwise with low hydrophobicity.

[0023] In some embodiments, the present invention provides a composition comprising lipoid transfection reagents, wherein the reagents comprise a first cationic lipoid (e.g., having a head group and a lipid tail), and a second cationic lipoid (e.g., having the same or a different head group and a different, second lipoid tail), wherein the second cationic lipoid, when combined with said first cationic lipoid in said reagents, decreases the hydrophobicity of the reagents compared to said reagents in the absence of the second cationic lipoid, and wherein said decrease increases the ability of the reagents to transfect cells. In some preferred embodiments, the second cationic lipoid comprises either a short, medium, or long chain fatty acid and the first cationic lipoid comprises either a short, medium or long chain fatty acid. A short chain fatty acid is a fatty acid chain having 7 or less carbons. A medium chain fatty acid is a fatty acid chain having between 8 and 15 carbons (e.g., laurate, myristate, etc.). A long chain fatty acid is a fatty acid chain having 16 or more carbons (e.g., palmitate, stearate, oleate, etc.). The lipid tails may be saturated or unsaturated.

[0024] In other embodiments, the second lipoid has lipid tails that may be the same length as those of the first lipoid, but are of a different shape. Such different shapes arise by incorporating different carbon chain branches along the lipid tails (e.g., as when methyl branches are incorporated along the lipid tail, the result of which is to increase the cross-sectional area (in the plane of the bilayer) of the lipid). Generally, the chains may be either shorter and / or fatter than the chains of the first lipoid.

Problems solved by technology

There are approximately four thousand different genetic diseases, many highly debilitating and frequently resulting in death at an early age.

Because almost all of these diseases involve a defective protein, conventional treatment is difficult.

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