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07/02/09 - USPTO Class 536 |  1 views | #20090171077 | Prev - Next | About this Page  536 rss/xml feed  monitor keywords

Delivery of nucleic acid-like compounds

USPTO Application #: 20090171077
Title: Delivery of nucleic acid-like compounds
Abstract: A process for preparing a microparticulate complex is provided. The process comprises (a) combining a particle-forming component (“PFC”) and a nucleic acid-like component (“NAC”) in a monophasic composition comprising water and a water-miscible, organic solvent to form a mixture wherein the PFC and the NAC are independently molecularly or micellarly soluble in the aqueous/organic solvent system, and (b) reducing the amount of the organic solvent in the mixture. This effects formation of the microparticulate complex of the NAC and the PFC. Also provided is a microparticulate complex that comprises a particle-forming component complexed to a nucleic acid-like component forming an approximately spherical particle, wherein the particle-forming component encloses an interior of the particle containing the nucleic acid-like component and the so-enclosed interior volume has less than about 50% (preferably less than 20%) of the volume containing free water. Also disclosed composition comprising water and particles of the microparticulate complex. According to the invention a nucleic acid-like component is delivered to a cell by (a) contacting the cell with a composition comprising water and the microparticulate complex, and (b) maintaining the contact for a time sufficient to allow the nucleic acid-based moiety to enter the cell. A therapeutic nucleic acid-like component is delivered into a patient in need thereof by administering a composition comprising water and particles of the microparticulate complex. Also disclosed is a charge-changing composition represented by the formula A-X-B, wherein X represents a chemical bond capable of irreversible dissociation in reaction to a factor in a physiological or bioprocess environment; A represents a molecular moiety that upon dissociation of the bond X produces a ionically charged product; and B represents a molecular moiety, which upon the dissociation of bond X, separates from the composition leaving the remaining ionically charged product more positive than that of A-X-B itself. (end of abstract)



Agent: Foley & Lardner LLP - Palo Alto, CA, US
Inventors: Keelung Hong, Wei-Wen Zheng, Daryl C. Drummond, Dmitri B. Kirpotin, Mark Eamon Hayes
USPTO Applicaton #: 20090171077 - Class: 536 253 (USPTO)

Delivery of nucleic acid-like compounds description/claims


The Patent Description & Claims data below is from USPTO Patent Application 20090171077, Delivery of nucleic acid-like compounds.

Brief Patent Description - Full Patent Description - Patent Application Claims
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This application claims priority to U.S. provisional patent application 60/381,471 filed 15 May 2002 and hereby converts the provisional to a full utility application.

FIELD OF THE INVENTION

The present invention relates to biotechnology, human and veterinary medicine, particularly to the methods and compositions for delivery of nucleic acid-like components to living cells.

BACKGROUND OF THE INVENTION

Introducing nucleic acids into living cells is an important process in modern biological research, industry, and medicine. Efficient delivery of a functional nucleic acid into a living cell is an indispensable component of genetic engineering, recombinant protein production, and medical technologies known as gene therapy.

For example, gene therapy involves the transfer of normal, functional genetic material into specific cells to correct an abnormality due to a deficient or defective gene product. A variety of methods have been developed to facilitate both in vivo, in vitro, or ex vivo gene transfer. One of the most frequently used delivery systems for achieving gene therapy involves viral vectors, most commonly adenoviral and retroviral vectors. However, the viral vectors have inherent problems including immunogenic and inflammatory responses, limited size of expression cassettes, possibility of viral infection or permanent viral gene integration. Non-viral delivery systems, for example, cationic liposomes and polycations, provide alternative methods which generally do not possess the disadvantages of viral vectors.

Alternatively, gene therapy involves the transfer of natural or synthetic oligonucleotides and polynucleotides into normal and/or pathological cells with the purpose of correcting or eliminating the diseased cells. For example, antisense oligonucleotides are used to block undesirable pathways of protein expression in the cells. Polynucleotide inductors of immunity, such as poly(I, C) or oligo- and polynucleotides having methylated GC pairs are used to increase the patients\' defense against pathogens such as viruses or cancer cells. Ribozymes are nucleic acids that catalyze selective degradation of other polynucleotides in the diseased cells, for example, in cancer or virus-infected cells. Because oligo- and polynucleotides generally have low permeability through cell membranes, and are quickly eliminated from the body, there is the need for oligo/polynucleotide delivery vehicles that would allow enhanced intracellular delivery and protection from degradation and/or elimination from the body.

In theory, the positively charged liposomes can complex to the negatively charged nucleic acids, for example, plasmids, via electrostatic interactions. To date many publications demonstrate that liposome-plasmid DNA complexes can mediate efficient transient expression of a gene in cultured cells but poor in vivo transfection efficiencies. Unlike viral vector preparations, liposome-DNA complexes are insufficiently stable in regard to their size or activity, and thus unsuitable for systemic injection. A large excess of cationic lipids is frequently used in these formulations, and contributes considerable toxicity to target cells.

In the past, methods based on detergent dialysis and extrusion have produced small lipid-DNA particles. Other methods of preparing lipid-DNA particles are based on solvent extraction of cationic lipid-neutralized DNA in lipid-soluble solvent from an immiscible two-phase system, with subsequent hydration and either extrusion or sonication of the solvent-free complexes to reduce the size. Although these preparations can be prepared by including neutral lipids and/or hydrophilic polymer derivatized lipid for prolonging such particle in circulation, in vivo transfection activities of such preparations are low.

It would be desirable to have methods and materials that can be scaled up easily for manufacture and that can produce nucleic acid-carrying particles that are small, active, and biocompatible.

Nucleic acid complexes for gene delivery are generally known in the art.

Wheeler et al., U.S. Pat. Nos. 5,976,567 and 5,981,501 disclose preparation of serum-stable plasmid-lipid particles by contacting an aqueous solution of a plasmid with an organic solution containing cationic and non-cationic lipids to provide a clear single phase. The clear single phase of Wheeler et al. encompasses organic phases in which aqueous component is present in a microemulsion form (“reverse phase” methods).

Thierry et al., U.S. Pat. No. 6,096,335 disclose preparing of a complex comprising a globally anionic biologically active substance, a cationic constituent, and an anionic constituent, by mixing anionic and cationic constituents, of which one is preferably a lipid, in a non-aqueous hydrophilic polar solvent, adding to said mixture an excess of an aqueous solution, and adding to the above mixture a globally anionic biologically active substance, such as nucleic acid, whereby a stable particular complex is formed having lamellar, rolled, and condensed structure.

Allen and Stuart, PCT/US98/12937 (WO 98/58630) disclose forming polynucleotide-cationic lipid particles in a lipid solvent suitable for solubilization of the cationic lipid, adding neutral vesicle-forming lipid to the solvent containing said particles, and evaporating the lipid solvent to form liposomes having the polynucleotide entrapped within.

Allen and Stuart, U.S. Pat. No. 6,120,798, disclose forming polynucleotide-lipid microparticles comprising dissolving a polynucleotide in a first, e.g. aqueous, solvent, dissolving a lipid in a second, e.g. organic, solvent immiscible with said first solvent, adding a third solvent to effect formation of a single phase, and further adding an amount of the first and second solvents to effect formation of two liquid phases.

Bally et al., U.S. Pat. No. 5,705,385, and Zhang et al., U.S. Pat. No. 6,110,745 disclose a method for preparing a lipid-nucleic acid particle by contacting a nucleic acid with a solution comprising a non-cationic lipid and a cationic lipid to form a lipid-nucleic acid mixture wherein the solution comprises 15-35% water and 65-85% of organic solvent; removing the aqueous portion of said mixture to form a non-aqueous lipid-nucleic acid mixture; removing the organic solvent, leaving behind a lipid-nucleic acid complex in the form of a film; and hydrating the film to form the particle.

Maurer et al., PCT/CA00/00843 (WO 01/06574) disclose a method for preparing fully lipid-encapsulated therapeutic agent particles of a charged therapeutic agent including combining preformed lipid vesicles, a charged therapeutic agent, and a destabilizing agent to form a mixture thereof in a destabilizing solvent that destabilizes, but does not disrupt, the vesicles, and subsequently removing the destabilizing agent, wherein the vesicles comprise a charged lipid having the charge opposite to that of the therapeutic agent, and wherein the vesicles contain a modified lipid having a steric barrier moiety in the amount to retard aggregation of the vesicles. The charged therapeutic agent may be anionic, for example, a nucleic acid, in which case the vesicles comprise a cationic lipid.

The above methods generally teach forming nucleic acid-lipid complexes in single phase solutions comprising organic solvents and water (aqueous-organic solutions). They do not teach the nucleic acid and lipid to be independently molecularly or micellarly soluble in said aqueous-organic solutions or the plasmid within said organic-aqueous solution to be in a condensed state.

SUMMARY OF THE INVENTION

One aspect of this invention is a process for preparing a microparticulate complex, which process comprises combining a particle-forming component and a nucleic acid-like component in a monophasic composition comprising water and a water-miscible, organic solvent to form a mixture wherein the particle forming component and the nucleic acid moiety are independently molecularly or micellarly soluble in the water/organic solvent system, and reducing the amount the organic solvent in the mixture. This effects formation of the microparticulate complex comprising the nucleic acid-like component and the particle-forming component. It is preferred that during such reducing step, the system remains monophasic, which means that its liquid components do not undergo phase separation resulting in the presence of a liquid-liquid interface, such as emulsion or liquid phase separation, but rather maintain a single liquid phase.



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Patent Applications in related categories:

20090299046 - Self-assembling oligonucleotides and methods - A composition comprising individual single-stranded oligonucleotides capable of self assembling to form a pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament, and a method for forming such filaments are disclosed. ...


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