Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-06
2001-06-26
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S023740, C536S023400, C536S023500, C514S04400A
Reexamination Certificate
active
06251599
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to compositions that are useful in biological applications involving nucleic acids. In one embodiment, the present invention relates to stabilized nucleic acid-polycation condensates for delivery of genes to cells for the purpose of altering the function, gene expression, or viability of the cells.
BACKGROUND
The ability to introduce nucleic acid into cells is the cornerstone of many molecular biology techniques and their pharmaceutical applications. Many different delivery systems have been developed to introduce exogenous DNA into cells. These include viruses, liposomes, electroporation, cell fusion, microinjection and salt precipitation. (See, e.g., Sambrook et al.,
Molecular Cloning, a Laboratory Manual
, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.) Although virus proteins and particles can provide efficient means of introducing exogenous DNA into target cells, problems associated with immunogenicity and potential infection have led to the production of synthetic delivery vehicles, such as cationic liposomes. Although many synthetic delivery systems may involve nonspecific cellular uptake, a variety of cell-specific delivery systems are also available.
Synthetic delivery vehicles for introduction of heterologous DNA into specific cells are architecturally complicated and often unstable under various preparatory and/or storage conditions. Not only must the DNA be maintained in a condition that ensures its structural integrity and functionality, the delivery vehicle itself, if attached to a ligand, should maintain the capability of being recognized and internalized by the target cell. It is often the case that a mechanism capable of achieving one of these goals has a negative effect on the other. For example, polycation-nucleic acid condensates in the form of compact particles show great promise as gene delivery vehicles. However, the stability of such condensates in the liquid and frozen state is limited due to their propensity to aggregate and fall out of solution.
Therefore, there exists a need in the art for nucleic acid molecule compositions which maintain their stability under a variety of different conditions that have been shown to destabilize various prior compositions. The present invention fulfills this need, while further providing other related advantages.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a composition comprising a nucleic acid condensed with a polycation in a liquid medium, thereby forming a particle, wherein the particle increases in size by less than one-fold during storage in the liquid medium for one week at about 2° C. to about 8° C. In certain embodiments the composition further comprises at least one excipient selected from a zwitterion, an amorphous cryoprotectant, a crystalline bulking agent, and mixtures thereof. In other embodiments the composition further comprises a ligand. In yet another embodiment the ligand is a polypeptide reactive with a cell growth factor receptor.
In another aspect, the invention provides a condensate comprising a nucleic acid, a polycation, and at least one excipient selected from a zwitterion, an amorphous cryoprotectant, a crystalline bulking agent, and mixtures thereof. In one embodiment, the excipient comprises a zwitterion, an amorphous cryoprotectant, and/or a crystalline bulking agent. In other embodiments the excipient comprises a cryoprotectant and a crystalline bulking agent. In yet other embodiments the condensate comprises a mixture of a first zwitterion and a second zwitterion.
In a further embodiment, the condensate has a nucleic acid concentration of less than about 20 mg/ml. In yet another embodiment the condensate has a polycation concentration of less than about 40 mg/ml. In additional embodiments the condensate comprises a nucleic acid:polycation charge ratio of less than 1:1. In other embodiments the condensate is a particle, and the particle increases in size less than one-fold during storage in a liquid medium for one week at about 2° C. to about 8° C. In other various embodiments the composition further comprises a ligand.
In yet another aspect, the invention provides a lyophile prepared by the process comprising the steps: (a) combining water, nucleic acid, polycation, and an excipient, thereby forming a plurality of particles; and (b) removing water from (a). In one embodiment the excipient is selected from a zwitterion, an amorphous cryoprotectant, a crystalline bulking agent, and mixtures thereof. In another embodiment substantially all of the water is removed from the lyophile. In the various embodiments the excipient may comprise multiple components including multiple zwitterions. In preferred embodiments, the polycation is selected from a polyamino acid, protamine, histone, and a polymer. In yet another embodiment, the lyophile is reconstituted in a liquid medium to provide particles, wherein the particles increase in size by less than one-fold as compared to particles before lyophilization. In a further embodiment, the lyophile comprises a ligand.
In a further aspect, the present invention provides a composition comprising a nucleic acid, a polycation, and an excipient, and further comprising a ligand covalently attached to at least one of these components. In one embodiment the ligand is covalently conjugated to the polycation forming a polycation-ligand conjugate. In another embodiment the ratio of the polycation-ligand conjugate to nucleic acid is less than about 5:1 (w:w). In yet a further embodiment, the ligand is a polypeptide reactive with a cell growth factor receptor. In a preferred embodiment the ligand is a polypeptide reactive with a fibroblast growth factor (FGF) receptor. In yet another embodiment the excipient is selected from a zwitterion, an amorphous cryoprotectant, a crystalline bulking agent, and mixtures thereof. In another embodiment the composition forms a plurality of particles wherein the particles increase in size by less than one-fold during storage in a liquid medium for one week at about 2° C. to about 8° C.
In yet another aspect, the present invention provides a method of preparing condensed nucleic acid comprising the steps of: (a) admixing a nucleic acid and a polycation in a liquid medium to form an admixture; (b) incubating the admixture under conditions in which the nucleic acid and the polycation condense to form a plurality of particles; (c) lyophilizing the admixture to remove the liquid medium thereby producing a lyophile comprising particles; and (d) reconstituting the lyophile with a predetermined volume of a reconstituting medium to form a reconstituted composition comprising a plurality of particles that increase in size less than one-fold during storage in the reconstituting liquid for one week at about 2° C. to about 8° C.
In one embodiment, the average size of the particles in the reconstituted composition of step (d) is less than twice the average particle size of the particles in step (b). In a further embodiment, the concentration of particles in the reconstituted composition of step (d) is greater than the concentration of particles in the composition prepared in step (b). In yet another embodiment, the method further comprises the additional step of admixing an amorphous cryoprotectant into the liquid medium before step (c). In further embodiments, various excipients selected from a zwitterion, an amorphous cryoprotectant, crystalline bulking agent, and mixtures thereof are added before step (c). In additional embodiments the polycation has a ligand attached thereto. In yet another embodiment, the particles in the reconstituted composition have a hydrodynamic diameter of less than about 200 nm. In still another embodiment, the average size of the particles in the reconstituted composition of step (d) increases less than one fold during storage at about 2° C. to about 8° C. for one week.
In still another aspect, the present invention provides a composition for the delivery of a nucleic acid to a mammalian cell prepared by the aforeme
Chen Xian
D'Andrea Mark J.
Ma Chenglie
Houtteman Scott W.
Seed Intellectual Property Law Group PLLC
Selective Genetics Inc.
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