Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use of gene transfer

Abstract

Novel lipid-nucleic acid particulate complexes which are useful for in vitro or in vivo gene transfer are described. The particles can be formed using either detergent dialysis methods or methods which utilize organic solvents. Upon removal of a solubilizing component (i.e., detergent or an organic solvent) the lipid-nucleic acid complexes form particles wherein the nucleic acid is serum-stable and is protected from degradation. The particles thus formed have access to extravascular sites and target cell populations and are suitable for the therapeutic delivery of nucleic acids.

Description

[0001] This application is a continuation-in-part of U.S. application Ser. No. 08 / 485,458 and of U.S. application Ser. No. 08 / 484,282, both filed on Jun. 7, 1995.FIELD OF THE INVENTION [0002] This invention relates to lipid-nucleic acid particles which are useful for the introduction of nucleic acids into cells, and methods of making and using them. The invention provides a circulation-stable, characterizable delivery vehicle for the introduction of plasmids or antisense compounds into cells. These vehicles are safe, stable, and practical for clinical use. BACKGROUND OF THE INVENTION [0003] Gene transfer into genetically impaired host cells in order to correct the genetic defects has vast potential for succesfully treating a variety of thus far hitherto untreatable medical conditions. There are currently six major non-viral methods by which genes are introduced into host cells: (i) direct microinjection, (ii) calcium phosphate precipitation, (ii) DEAE-dextran complexes, (iv) electro...

AI Technical Summary

Benefits of technology

The present invention is about novel, lipid-nucleic acid particles and methods of making and using them. These particles can be used for the therapeutic delivery of nucleic acids. The particles are stable in serum and can be administered to patients through intravenous nucleic acid transfer or cell removal and reinjection. The particles are made through a hydrophobic lipid-nucleic acid intermediate and are protected from degradation. The technical effect of this invention is to provide a stable and effective way to deliver nucleic acids for therapeutic purposes.

Problems solved by technology

In vivo gene transfer is complicated by serum interactions, immune clearance, enzymatic degradation of the genes, toxicity and biodistribution.

In in vivo administration, selection is not possible, and a reasonably high frequency of transformation is necessary to achieve sufficient expression to compensate for a defective endogenous gene.

Although viral vectors have the inherent ability to transport nucleic acids across cell membranes and some can integrate exogenous DNA into the chromosomes, they can carry only limited amounts of DNA.

In addition, their use poses significant risks.

One such risk is that the viral vector may revert to a pathogenic genotype either through mutation or genetic exchange with a wild type virus.

However, most plasmids do not possess the attributes required for intracellular delivery and therefore sophisticated delivery systems are required.

However, both DOTMA and DOSPA based formulations, despite their efficiency in effecting gene transfer, are prohibitively expensive.

DDAB on the other hand is inexpensive and readily available from chemical suppliers but is less effective than DOTMA in most cell lines.

Another disadvantage of the current lipid systems is that they are not appropriate for intravenous injection.

The complexes thus formed have undefined and complicated structures and the lipofection efficiency is severely reduced by the presence of serum.

These complexes are often prepared in the presence of ethanol and are not stable in water.

Additionally, degradation either outside or inside the target cell remains a problem (see, Duzghines, Subcellular Biochemistry 11:195-286 (1985)).

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