Title:  Materials and methods for the intracellular delivery of substances

United States Patent:  6,169,078

Assignee:   University of Florida (Gainesville, FL)

Filed:  May 12, 1998

The subject invention concerns novel materials and methods for the delivery of substances into cells. In a specific embodiment substances are delivered into cells using a novel class of lipid compounds. These compounds, cationic lipid compounds having a disulfide bond, can be complexed with DNA to be inserted into a cell in gene therapy. Once inside the cell, enzymes present within the cell cleave the disulfide bond and the DNA is released.

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 cationic lipid was mixed with an equal molar ration of DOPE. Lipid/DNA complexes were treated either with media containing 10 mM DTT or DTT free-media for 12 hours at 37^oC. to evaluate the dissociation of DNA from complexes in reductive media. Only DNA complexed by DOGSDSO/DOPE liposomes and treated with DTT was released. DNA complexed by DOGSHDO/DOPE liposome was not released in either environment. Without treatment of DTT, no DNA was released from the DOGSDSO/DOPE/DNA complex after incubation for 12 hours at 37^oC. This indicates that the dissociation of DNA from lipid complexes is difficult and may act as a barrier for gene transfection. When exposed to high concentrations of intracellular reductive substances, the disulfide bond of DOGSDSO is reduced and DNA released resulting in higher plasmid DNA concentrations within the cytoplasm and greater production of the transgene.

Thus, in one embodiment, the materials and methods of the subject invention exploit differences between intracellular and extracellular environments with regard to the presence of glutathione. High levels of glutathione in intracellular spaces results in the reduction in disulfide bonds which have been introduced between the desired molecule and the second molecule or moiety. The reduction of the disulfide bond inside a target cell frees the desired molecule from the carrier molecule or moiety.

Similar transport and release of desired molecules can be achieved according to the subject invention using systems which exploit other differences between intracellular and extracellular environments. Thus, in another embodiment of the subject invention, the desired molecule is attached to a second molecule such that the attachment is stable in the extracellular high sodium environment but the attachment is broken and the desired molecule is released in the low sodium and/or high potassium intracellular environment.

In a further embodiment of the subject invention the association between the desired molecule and the second molecule is stable in the low viscosity extracellular spaces but, upon exposure to the higher viscosity intracellular environment, the desired molecule is released from the second molecule.

Claim 1 of 3 Claims

What is claimed is:

1. A method of delivering a desired molecule into a cell wherein said method comprises attaching said desired molecule to a transport molecule thereby forming a transport complex wherein said transport molecule remains associated with said desired molecule in extracellular conditions and releases said desired molecule in intracellular conditions; said method further comprising contacting said transport complex with said cell, wherein said transport molecule is DOGSDSO.

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