Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-05-26
2004-02-24
Nguyen, Dave T. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S450000, C564S152000, C564S157000, C564S160000
Reexamination Certificate
active
06696424
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to cytofectin and adjuvant compositions. More particularly, the present invention provides compositions useful as cytofectins and as adjuvants, as well as methods for facilitating the transfection of nucleic acids into cells and for enhancing the humoral immune response of vertebrates to polynucleotide-based vaccines.
BACKGROUND OF THE INVENTION
Cytofectins are used to enhance the delivery of biologically active agents, particularly polynucleotides, proteins, peptides, and drug molecules, by facilitating transmembrane transport or by encouraging adhesion to biological surfaces. Some bioactive substances do not need to enter cells to exert their biological effect, because they operate either by acting on cell surfaces through cell surface receptors or to cell surfaces by interacting with extracellular components. However, many natural biological molecules and their analogues, including proteins and polynucleotides, or foreign substances, such as drugs, which are capable of influencing cell function at the subcellular or molecular level are preferably incorporated within the cell in order to produce their effect. For these agents, the cell membrane presents an impermeable selective barrier.
Successful intracellular delivery of agents not naturally taken up by cells has been achieved by exploiting the natural process of intracellular membrane fusion, or by direct access of the cell's natural transport mechanisms, which include endocytosis and pinocytosis (Duzgunes, N.,
Subcellular Biochemistry
11:195-286 (1985)). In addition, the cell membrane barrier can be overcome by complexing the agent to be delivered or transfected with lipid formulations closely resembling the lipid composition of natural cell membranes. These lipids are able to fuse with the cell membranes on contact, and in the process, the agents associated with the lipid complexes or aggregates are delivered intracellularly. Lipid aggregates comprising charged lipids can not only facilitate intracellular transfers by fusing with cell membranes but also by overcoming charge repulsions between the cell membrane and the agent to be delivered.
Cellular delivery of beneficial or interesting proteins can be achieved by introducing expressible DNA or mRNA into cells, a technique known as transfection. Nucleotide sequences introduced in this way can produce the corresponding protein encoded by the nucleotide sequence. The therapy of many diseases could be enhanced by the induced intracellular production of peptides which could remain inside the target cell, be secreted into the local environment of the target cell, or be secreted into the systemic circulation to produce their effect. Various techniques for introducing the DNA or mRNA precursors of bioactive peptides into cells include the use of viral vectors, including recombinant vectors and retroviruses, which have the inherent ability to penetrate cell membranes. However, the use of such viral agents to integrate exogenous DNA into the chromosomal material of the cell carries a risk of damage to the genome and the possibility of inducing malignant transformation. Another aspect of this approach which restricts its use in vivo is that the integration of DNA into the genome accomplished by these methods implies a loss of control over the expression of the peptide it codes for, so that transitory therapy is difficult to achieve and potential unwanted side effects of the treatment could be difficult or impossible to reverse or halt.
A major advance in the area of DNA transfection was the discovery that certain synthetic cationic lipids, such as DOTMA, in the form of liposomes or small vesicles, could interact spontaneously with DNA to form lipid-DNA complexes that are capable of fusing with the negatively charged lipids of the cell membranes, resulting in both uptake and expression of the DNA (see, e.g., Feigner, P. L. et al.,
Proc Natl Acad Sci USA
84:7413-7417 (1987) and U.S. Pat. No. 4,897,355, the disclosures of which are incorporated herein by reference). The well-known Lipofectin™ reagent (Bethesda Research Laboratories, Gaithersburg, Md.), an effective agent for the delivery of highly anionic polynucleotides into living tissue culture cells, comprises positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. In part, the effectiveness of cationic lipids as cytofectins is thought to result from their enhanced affinity for cells, many of which bear regions of high negative charge on their membrane surfaces. Also in part, the presence of positive charges on a lipid aggregate comprising a cationic lipid enables the aggregate to bind polyanions, especially nucleic acids. Lipid aggregates prepared in this way can spontaneously attach to negative charges on cell surfaces, can fuse with the plasma membrane, and can efficiently deliver functional polynucleotides into cells. More recently, other cationic lipids, including diesters and diethers of modified Rosenthal Inhibitor (RI) compounds, have been found to be effective cytofectin compounds (see, e.g., U.S. Pat. Nos. 5,459,127 and 5,264,618, the disclosures of which are incorporated herein by reference).
In the late 1980s, it was discovered that direct intramuscular (i.m.) injection of lipid-DNA complexes results in measurable protein expression, and also that “naked” plasmid DNA (pDNA) can be taken up and expressed in muscle to a greater extent than lipid-DNA complexes (Felgner, 1997)). One of the first applications of pDNA injection technology was the induction of an immune response. In 1991, it was first reported that mice could be immunized against HIV gp120 by i.m. vaccination with gp120 plasmid DNA (Felgner et al., 1991), and that mice could be protected from a lethal challenge of influenza virus after DNA immunization with influenza nucleoprotein (NP) antigen. Protection obtained after immunization with the highly conserved NP antigen extended across two different viral strains (Ulmer et al., 1996)). Numerous publications in the field of polynucleotide-based vaccination followed thereafter (Boyer et al., 1996; Boyer et al., 1997; Davis et al., 1997; Wang et al., 1997; Agadjanyan et al., 1998; Heppell et al., 1998; Lodmell et al., 1998; Vanderzanden et al., 1998)).
A problem often encountered in the course of polynucleotide-based vaccination is insufficient or suboptimal humoral response. To obtain a stronger humoral and/or cellular response, it is common to administer such vaccines in an immunogenic composition containing an adjuvant, a material which enhances the immune response of the patient to the vaccine. Adjuvants are useful generally for improving the immune response of an organism to a particular immunogen and are commonly included in vaccine compositions to increase the amount of antibodies produced and/or to reduce the quantity of immunogen and the frequency of administration.
A variety of adjuvants have been reported to effect differing levels of immune response enhancement to polynucleotide-based vaccination. Examples of such adjuvant materials include semi-synthetic bacterial cell wall-derived mono-phosphoryl lipid A (Sasaki, S. et al.,
Infection and Immunity
65(9):3250-3258 (1997)), small molecule immunostimulators (Sasaki, S. et al.,
Clin Exp Immunol
111:30-35 (1998)), and saponins (Sasaki, S. et al.,
J Virol
72(6):4391-4939 (1998)). The immune response from i.m. pDNA vaccination has also been enhanced through the use of cationic lipids (e.g., Ishii, N. et al.,
Aids Res Hum Retroviruses
13(16):1421-1428 (1997)), Okada, E. et al.,
J Immunology
159:3638-3647 (1997); Yokoyama, M. et al.,
FEMS Immunol Med Microbiol
14:221-230 (1996); Gregoriadis, G. et al.,
FEBS Letters
402:107-110 (1997); Gramzinski, R. A. et al.,
Molecular Medicine
4:109-118 (1998); Klavinskis, L. S. et al.,
Vaccine
15(8):818-820 (1997); Klavinskis, L. S. et al.,
J Immunology
162:254-262 (1999); Etchart, N. et al,
J Gen Virology
78:1577-1580 (1997); Norman, J. et al., in
Methods in M
Nguyen Dave T.
Schnizer Richard
Sterne Kessler Goldstein & Fox P.L.L.C.
Vical Incorporated
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