Title:  Oral delivery of nucleic acid vaccines by particulate complexes

United States Patent:  6,475,995

Issued:  November 5, 2002

Inventors:  Roy; Krishnendu (Baltimore, MD); Huang; Shau-Ku (Towson, MD); Sampson; Hugh (Larchmont, NY); Leong; Kam W. (Ellicot City, MD)

Assignee:  The Johns Hopkins University (Baltimore, MD)

Appl. No.:  232167

Filed:  January 15, 1999

Nanoparticle coacervates of nucleic acids and polycations serve as effective vaccines when administered orally. They can induce immunity to a variety of disease causing agents and raise a protective response to allergens.

DETAILED DESCRIPTION OF THE INVENTION

The disclosures of prior U.S. patent applications Ser. Nos. 08/265,966, and 08/657,913 are expressly incorporated herein.

Our novel approach of using DNA nanospheres for oral administration has demonstrated the general utility of oral DNA-based immunization. The induced response may be either or both humoral or cellular. It may be used for inducing any type of immune response desired. This may be against bacterial, viral, fungal, parastic, or tumor-associated antigens.

Any type of expression construction can be used to obtain expression in the vaccinated mammal of the antigen gene. Typically the gene will be operably linked to a promoter which is active in the recipient cells. Viral promoters, such as CMV are particularly useful for this purpose, although any known promoter can be used. The gene or oligonucleotide encoding the antigen can be in the form of RNA, DNA, cDNA.

The antigen can be any known in the art, including but not limited to an allergen, particularly a food allergen, or a bacterial, viral, fungal, parastic, or tumor-associated antigen.

The crucial aspect of the invention is the administration of the vaccines by the oral route. Thus the formulation may be any which is safe and healthy for oral ingestion. Preferably the formulation contains no toxic substances. If in a liquid form, sterility may be desired. The form of the vaccine may be as a tablet, capsule, liquid, elixir, powder, granules, etc. It may be admixed with food or drink. It may self-adminstered for added convenience rather than requiring the expense of a health professional for administration as is often required for injections and other inocculations.

It is preferred that the nanospheres be less than 5 microns. More preferred are nanospheres of less than 3 microns, and even more proffered are nanospheres which are less than 2, 1, 0.5, and 0.1 microns. While size can be effected by the conditions of coacervation and the size of the component polyanion and polycation, nanospheres of the desired size can also be size selected using a technique which separates the nanospheres on the basis of size. The particles can be size-fractionated, e.g., by sucrose gradient ultracentrifugation. Particles with size less than 150 nanometers can access the interstitial space by traversing through the fenestrations that line most blood vessels walls.

The polymeric polycation from which the coacervate is formed can be any which is biologically degradable, and safe for oral ingestion. This includes, but is not limited to gelatin and chitosan. Polyamino acids, synthetic or naturally occurring, can also be used, such as polylysine, poly-lysine-poly-arginine, polyarginine, protamine, spermine, spermidine, etc. Polysaccharides may also be used.

Targeting ligands, if desired, can be directly bound to the surface of the nanosphere or can be indirectly attached using a "bridge" or "spacer". Because of the amino groups provided by the lysine groups of the gelatin, the surface of the nanospheres can be easily derivatized for the direct coupling of targeting moieties. For example, carbo-diimides can be used as a derivatizing agent. Alternatively, spacers (linking molecules and derivatizing moieties on targeting ligands) such as avidin-biotin can be used to indirectly couple targeting ligands to the nanospheres. Biotinylated antibodies and/or other biotinylated ligands can be coupled to the avidin-coated nanosphere surface efficiently because of the high affinity of biotin (k_a.about. 10¹⁵ M^-1) for avidin (Hazuda, et al., 1990, Processing of precursor interleukin 1 beta and inflammatory disease, J. Biol. Chem., 265:6318-22; Wilchek, et al., 1990, Introduction to avidin-biotin technology, Methods In Enzymology, 184:5-13). Orientation-selective attachment of IgGs can be achieved by biotinylating the antibody at the oligosaccharide groups found on the F_c portion (O'Shannessy, et al., 1984, A novel procedure for labeling immunoglobulins by conjugation to oligosaccharides moieties, Immunol. Lett., 8:273-277). This design helps to preserve the total number of available binding sites and renders the attached antibodies less immunogenic to F_c receptor-bearing cells such as macrophages. Spacers other than the avidin-biotin bridge can also be used, as are known in the art. For example, Staphylococcal protein A can be coated on the nanospheres for binding the F_c portions of immunoglobulin molecules to the nanosphetes.

Cross-linking of linking molecules or targeting ligands to the nanosphere is used to promote the stability of the nanosphere as well as to covalently affix the linking molecule or targeting ligand to the nanosphere. The degree of cross-linking directly affects the rate of nucleic acids released from the nanospheres. Cross-linking can be accomplished using glutaraldehyde, carbodiimides such as EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, DCC (N,N'-dicyclohexylcarbodiimide), carboxyls (peptide bond) linkage, bis (sulfosuccinimidyl) suberate, dimethylsuberimidate, etc.

Targeting ligands according to the present invention are any molecules which bind to specific types of cells in the body. These may be any type of molecule for which a cellular receptor exists. Preferably the cellular receptors are expressed on specific cell types only. Examples of targeting ligands which may be used are hormones, antibodies, cell-adhesion molecules, saccharides, drugs, and neurotransmitters.

The nanospheres of the present invention have good loading properties. Typically, following the method of the present invention, nanospheres having at least 5% (w/w) nucleic acids can be achieved. Preferably the loading is greater than 10 or 15% nucleic acids. Often nanospheres of greater than 20 or 30%, but less than 40 or 50% nucleic acids can be achieved. Typically loading efficiencies of nucleic acids into nanospheres of greater than 95% can be achieved.

The method of the present invention involves the coacervation of polymeric cations and nucleic acids. Because this process depends on the interaction of the positively charged polymeric cations and the negatively charged nucleic acids it can be considered as a complex coacervation process. However, sodium sulfate (or ethanol) induces the coacervation reaction by inducing a phase transition, and therefore it could also be considered as a simple coacervation reaction. Nucleic acids are present in the coacervation mixture at a concentration of between 1 ng/ml to 500 .mu.g/ml. Desirably the nucleic acids are at least about 2-3 kb in length. Sodium sulfate is present at between 7 and 43 mM. Gelatin or other polymeric cation is present at between about 2 and 7% in the coacervation mixture.

Nanosphere delivery vehicles synthesized by the complex coacervation of DNA with either gelatin or chitosan have several potential attractive features: 1) ligands may be conjugated to the nanosphere for targeting or stimulating receptor-mediated endocytosis; 2) lysosomolytic agents can be incorporated to reduce degradation of the DNA in the endosomal and lysosomal compartments; 3) other bioactive agents or multiple plasmids can be co-encapsulated; 4) bioavailability of the DNA can be improved because of protection from serum nuclease degradation by the matrix; 5) the nanosphere can be lyophilized for storage.

An attractive nanosphere delivery system requires a delicate balance among factors such as the simplicity of preparation, cost effectiveness, nucleic acids loading level, controlled release ability, storage stability, and immunogenicity of the components. The gene delivery system described here may offer advantages compared to other particulate delivery systems, including the liposomal system. The problems of instability, low loading level, and controlled release ability are better resolved with the polymeric nanosphere systems. Gelatin has received increasing biologic use ranging from surgical tissue adhesive (Weinschelbaum, et al., 1992, Surgical treatment of acute type A dissecting aneurysm with preservation of the native aortic valve and use of biologic glue. Follow-up to 6 years, J. Thorac. Cardiovasc. Surg., 130:369-74) to quantitative immunohistochemical assays (Izumi, et al., 1990, Novel gelatin particle agglutination test for serodiagnosis of leprosy in the field, J. Clinical Microbiol., 28:525-9) and as drug delivery vehicle (Tabata, et al., 1991, Effects of recombinant alpha-interferon-gelatin conjugate on in vivo murine tumor cell growth, Cancer Res., 51:5532-8), due to its biocompatibility and enzymatic degradability in vivo. Compared to other synthetic polymeric systems, such as the extensively studied polylactic/polyglycolic copolymers, the mild conditions of nanosphere formulation are appealing. Unlike the solvent evaporation and hot-melt techniques used to formulate synthetic polymeric nanospheres, complex coacervation requires neither contact with organic solvents nor heat. It is also particularly suitable for encapsulating bio-macromolecules such as nucleic acids not only through passive solvent capturing but also by direct charge-charge interactions.

Unlike viral vectors, which cannot deliver genes larger than 10 kb, the nanosphere delivery system of the present invention does not have such size limitations. Nucleic acid molecules of greater than about 2 kb can be used, and nucleic acid molecules even greater than 10 kb may be used. Typically the nucleic acid will be greater than 300 bases, and typically greater than 0.5, 1, 2, 5, or 10 kb. Typically the nucleic acid molecule will be less than 200, 100, or 50 kb.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

We have evaluated the modulation of peanut allergen (as a model allergen)-induced hypersensitivity using an oral chitosan/DNA-based immunization approach. The model for this study is the hypersensitivity responses to peanut allergens in an inbred strain of mice (C3H), in which several quantitative parameters of hypersensitivity have been established, and a significant induction of the immune response has. been demonstrated following gene immunization. This system demonstrates that antigens can be delivered to the systemic circulation of the animal by oral administration of a DNA.

Claim 1 of 6 Claims

What is claimed is:

1. A method of eliciting an immune response in a mammal against an antigen, comprising:

orally administering an immunogenic formulation comprising a solid nanoparticle of less than 5 .mu.m comprising a coacervate of a polymeric polycation and a polyanion, wherein the polymeric polycation is selected from the group consisting of gelatin and chitosan, and wherein the polyanion consists of nucleic acids encoding an antigen, whereby the antigen is expressed and elicits an immune response in the mammal.
 

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