WO1999058134A1

WO1999058134A1 - Methods and compositions for nucleic acid delivery

Info

Publication number: WO1999058134A1
Application number: PCT/US1999/010235
Authority: WO
Inventors: Suresh K. Mittal; Neeraj Aggarwal; Harm Hogenesch; Peixuan Guo
Original assignee: Purdue Research Foundation
Priority date: 1998-05-11
Filing date: 1999-05-11
Publication date: 1999-11-18
Also published as: AU3895499A

Abstract

Nucleic acid delivery compositions, methods for delivery of nucleic acid in vivo to an animal or in vitro to target cells and cellular populations, such as lymphoid cells, having internalized therein the inventive nucleic acid delivery compositions are provided. In one form of the invention, a nucleic acid delivery composition includes polymeric microparticle matrices, especially polysaccharide microparticles such as alginate microparticles, and a nucleic acid embedded in the microparticles. In other embodiments, the nucleic acid delivery composition further includes a virus composition, such as an adenovirus composition. The polymeric microparticles may be coated with a cationic polymer and are, in one inventive form, microspherical. Methods of in vivo delivery of a nucleic acid to a target cell are also described. The methods include administering to an animal an effective amount of a nucleic acid delivery composition as described above. Methods of in vitro delivery of nucleic acid to target cells that include incubating the cells with the nucleic acid delivery compositions of the present invention are also provided.

Description

METHODS AND COMPOSITIONS FOR NUCLEIC ACID DELIVERY

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the benefit of U.S. Provisional Application Serial Number 60/085,050, filed on May 11, 1998, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Many procedures for introducing foreign genes into various cells of animals typically reguire removing the target cells from the host, in vi tro propagation and transformation of the cells, and reintroduction of the cells into the animal. These procedures are technically demanding and are not applicable to all cell types, especially terminally differentiated, non-dividing cells. In attempts to overcome these drawbacks, various methods of in vivo introduction of plasmid deoxyribonucleic acid (DNA) encoding a protein of interest for vaccination and gene therapy purposes have been evaluated for their transfection efficiency in cultured cells and animals, as such procedures are believed to be less technically demanding and may be applied to a wider variety of cell types. For example, some of the methods include injecting naked DNA into muscle tissue, bombarding tissue with gold microparticles coated with DNA, and incorporating DNA into liposomes and other polycationic lipids. However, many of these alternate strategies also suffer from drawbacks. For example, the cellular uptake of naked DNA when injected into muscle tissue is significantly less and, following endocytosis, a large portion of the DNA is degraded after fusion of endosomes (carrying the DNA) with lysosomes. Moreover, bombarding tissue with gold microparticles coated with DNA is not useful for a wide variety of cell types.

There is therefore a need for improved compositions and methods for delivering nucleic acid into cells. The present invention addresses this need.

SUMMARY OF THE INVENTION

Compositions and methods for delivery of nucleic acid, either in vivo to an animal or in vi tro to a target cell, are provided. In one aspect of the invention, a nucleic acid delivery composition includes polymeric microparticles having nucleic acid embedded therein. In one inventive form, the polymeric microparticles are polysaccharide microparticles, especially alginate microparticles. The microparticles advantageously are microspherical wherein at least about 50% of the microspheres have a diameter of 10 microns or less. The microparticles, in certain embodiments, are formed by non-covalent, ionic cross- linking with divalent cations, such as zinc cations, and may further be coated with a cationic polymer.

Moreover, it has surprisingly been discovered that inclusion of a virus composition, such as an adenovirus composition, into the nucleic acid delivery compositions of the present invention augments the transfer of nucleic acid, such as nucleic acid that produces a desired product, into a target cell and thereby increases production of the desired product. For example, a nucleotide sequence may express a desired protein and the virus may cause an increase in the expression of the protein. Accordingly, another preferred embodiment of the present invention includes a nucleic acid delivery composition that includes polymeric microparticles, nucleic acid and a virus composition, such as an adenovirus composition, wherein the virus composition and the nucleic acid are embedded in the polymeric microparticles. In another aspect of the invention, methods of in vivo delivery of nucleic acid are provided. In one embodiment, a method of in vivo delivery of nucleic acid includes administering to an animal an effective amount of one of the inventive nucleic acid delivery compositions above-described. The desired product, such as a protein, may be expressed in a wide variety of animal tissues, including, for example, intestinal epithelial tissue, spleen tissue and liver tissue. In alternate embodiments, the methods may be applied to delivery of nucleic acid to target cells in vi tro by incubating target cells, such as lymphoid cells that include macrophages, with the inventive nucleic acid delivery compositions. In another aspect of the invention, cellular populations comprising lymphoid cells, such as dendritic cells, macrophages, B and T lymphocytes or a combination thereof, or other cells including muscle cells, liver cells, and epithelial cells, having internalized therein the inventive nucleic acid delivery compositions are provided.

It is therefore an object of the invention to provide nucleic acid delivery compositions that can efficiently deliver nucleic acid to an animal or target cells.

It is a further object of the invention to provide a method of in vivo delivery of nucleic acid to target cells in an animal.

It is a further object of the invention to provide a method of in vitro delivery of nucleic acid to target cells . It is yet another object of the present invention to provide a cellular population of target cells, such as lymphoid cells, having internalized therein the inventive nucleic acid delivery compositions.

These and other objects and advantages of the present invention will be apparent from the descriptions herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows expression of LacZ in cells initially transfected with plasmid DNA carrying the LacZ gene and subsequently infected with bovine adenovirus type 3 (BAd3) in order to test the efficiency of various promoters. Each bar represents the mean of two independent samples. FIG. 1A: PK-15 cells transfected with pTK21CMVgalSV40; FIG. IB: PK-15 cells transfected with pREP9gal; FIG. 1C: 3T3 cells transfected with pTK21CMVgalSV40. 1+5, 1 μg DNA and 5 μg liposomes; 5+5, 5 μg DNA and 5 μg liposomes; 1+5+V, 1 μg DNA + 5 μg liposomes + BAd3; 5+5+V, 5 μg DNA + 5 μg liposomes + BAd3.

FIG. 2 depicts expression of LacZ in tissues of mice inoculated with microspheres containing plasmid DNA and BAd3. Each bar represents the mean value from three mice. pTK-21, pTK21CMVgalSV40; pREP, pREP9gal; pTK-21+V, pTK21CMVgalSV40 + BAd3; pREP+V, pREP9gal + BAd3.

FIG. 3 shows a histochemical analysis for LacZ in tissues of mice inoculated with microspheres containing plasmid DNA and BAd3. FIGS. 3A-C show sections from the indicated organs from animals inoculated with BAd3 and FIGS. 3D-F show sections from the indicated organs from animals inoculated with pTK21CMVgalSV40 + BAd3. FIGS. 3A and 3D: liver; FIGS. 3B and 3E: intestine; FIGS. 3C and 3F: spleen. FIG. 4 depicts expression of LacZ in cells initially transfected with plasmid DNA carrying the LacZ gene and subsequently infected with BAd3 as discussed in Example 5. Each bar represents the mean of two independent samples. 1+10, 1 μg DNA and 10 μg liposomes; 5+10, 5 μg DNA and 10 μg liposomes; 1+10+V, 1 μg DNA + 10 μg liposomes + BAd3; 5+10+V, 5 μg DNA + 10 μg liposomes + BAd3.

FIG. 5 depicts a LacZ-specific IgG response in sera of mice inoculated with microspheres containing DNA as discussed in Example 5. Mice were immunized with microspheres containing either PBS, DNA or DNA + BAd3 at days 0, 14 and 28 by either the A) oral (FIG. 5A) , B) intranasal (FIG. 5B) , C) intramuscular (FIG. 5C) , D) subcutaneous (FIG. 5D) or E) intraperitoneal (FIG. 5E) route. Each point represents the mean value for 3-4 animals + SD. i.n., intranasal; i.m., intramuscular; s.c, subcutaneous; i.p., intraperitoneal.

FIG. 6 shows a BAd3-specific IgG response in sera of mice inoculated with microspheres containing BAd3 as discussed in Example 5. Mice were immunized with microspheres containing either PBS, BAd3 or DNA + BAd3 at days 0, 14 and 28 by either the A) oral (FIG. 6A) , B) intranasal (FIG. 6B) , C) intramuscular (FIG. 6C) , D) subcutaneous (FIG. 6D) or E) intraperitoneal (FIG. 6E) route. Each point represents the mean value for 3-4 animals + SD. i.n., intranasal; i.m., intramuscular; s.c, subcutaneous; i.p., intraperitoneal. FIG. 7 depicts a LacZ-specific IgA response in sera of mice inoculated with microspheres containing DNA as discussed in Example 5. Mice were immunized with microspheres containing either PBS, DNA or DNA + BAd3 at days 0, 14 and 28 by either the A) oral (FIG. 7A) , B) intranasal (FIG. 7B) , C) intramuscular (FIG. 7C) , D) subcutaneous (FIG. 7D) or E) intraperitoneal (FIG. 7E) route as described in Example 5. Each point represents the mean value for 3-4 animals + SD. i.n., intranasal; i.m., intramuscular; s.c, subcutaneous; i.p._. intraperitoneal .

FIG. 8 depicts a BAd3-specific IgA response in sera of mice inoculated with microspheres containing BAd3 as discussed in Example 5. Mice were immunized with microspheres containing either PBS, BAd3 or DNA + BAd3 at days 0, 14 and 28 by either the A) oral (FIG. 8A) , B) intranasal (FIG. 8B) , C) intramuscular (FIG. 8C) , D) subcutaneous (FIG. 8D) or E) intraperitoneal (FIG. 8E) route. Each point represents the mean value for 3-4 animals + SD. i.n., intranasal; i.m., intramuscular; s.c, subcutaneous; i.p., intraperitoneal.

FIGS. 9 depicts a LacZ-specific IgA response in lung lavages and fecal samples of mice inoculated with microspheres containing DNA as discussed in Example 6. Mice were immunized with microspheres containing either PBS, DNA or DNA + BAd3 at days 0, 14 and 28 by either the A) oral (FIG. 9A) , B) intranasal (FIG. 9B) , C) intramuscular (FIG. 9C) , D) subcutaneous (FIG. 9D) or E) intraperitoneal (FIG. 9E) route. Each point represents the mean value for 3-4 animals + SD. i.n., intranasal; i.m., intramuscular; s.c, subcutaneous; i.p., intraperitoneal .

FIG. 10 depicts a BAd3-specific IgA response in lung lavages and fecal samples of mice inoculated with microspheres containing BAd3 as discussed in Example 6. Mice were immunized with microspheres containing either PBS, BAd3 or BAd3 + DNA at days 0, 14 and 28 by either the A) oral (FIG. 10A) , B) intranasal (FIG. 10B) , C) intramuscular (FIG. IOC), D) subcutaneous (FIG. 10D) or E) intraperitoneal (FIG. 10E) route. Each point represents the mean value for 3-4 animals + SD. i.n., intranasal; i.m., intramuscular; s.c, subcutaneous; i.p., intraperitoneal.

FIG. 11 shows lymphocyte proliferation in response to LacZ in freshly isolated spleen cells from immunized mice as discussed in Example 7. Mice were immunized with microspheres containing either PBS, DNA, BAd3 or DNA + BAd3 at days 0, 14 and 28 by either the oral, intranasal, intramuscular, subcutaneous or intraperitoneal route. Each point represents the mean stimulation indices for 3-4 animals + SD. DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications of the invention, and such further applications of the principles of the invention as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the invention relates.

The present invention provides compositions and methods for delivery of nucleic acids into target cells. In one aspect, a nucleic acid delivery composition is provided that includes solid polymeric microparticles having nucleic acid dispersed therein. The solid (i.e., non-hollow) microparticles, especially alginate microparticles, are preferably microspheres of specified diameter. The composition is advantageous for delivering nucleic acid constructs, including plasmid vectors carrying nucleotide sequences for producing a desired product, to target cells in vivo or in vi tro . The products produced by the nucleotide sequences are preferably proteins. The proteins may be antigenic, and are thus able to produce an immune response for vaccination procedures. The nucleotide sequence may, alternatively, produce proteins for other purposes, such as those that provide other benefits to the animal. The compositions may also include a virus composition that includes a virus, such as an adenovirus, to aid in delivery of the nucleotide sequences and increase production of the desired product. In other aspects of the invention, methods for in vivo and in vi tro delivery of nucleic acid are also provided. Cellular populations that include target cells having the inventive nucleic acid delivery compositions internalized therein are also provided.

As discussed above, in one aspect of the invention, a nucleic acid delivery composition is provided. The composition includes polymeric microparticles, or matrices, and nucleic acid. The nucleic acid is advantageously dispersed in the polymeric microparticles. A wide variety of polymers may be used to form the inventive polymeric microparticles in which nucleic acid may be embedded. For example, such polymers include polysaccharides, such as alginate, collagen, gelatin, polyacrylamide, polymethacrylamide, polyvinyl acetate, poly-N- vinylpyrrolidone, polyvinyl alcohol, polyacrylic acids, and other similar polymers.

In one form of the invention, the polymeric microparticles are advantageously polysaccharide microparticles and are especially alginate microparticles formed from an alginate gel. Alginate is a hydrophilic, colloidal polysaccharide that can be extracted from seaweed and is composed of a copolymer of 1,4-linked β-D-mannuronic and α-L-guluronic acid. Alginate may be obtained by methods known in the art or may be purchased commercially. Alginate forms hydrogels when exposed to multivalent cations, including divalent cations such as Ca²⁺. Other multivalent cations such as Ba²⁺, Sr²⁺, Zn²⁺, and Mn²⁺ may also be used. However, it has been discovered that hydrogels of improved stability may be formed by ionically cross-linking the polysaccharide with a combination of Ca²⁺ and Zn²⁺. More specifically, it has been determined that the amount of Zn²⁺ used in forming the hydrogel can be regulated to control particle characteristics, such as hardness and integrity. An appropriate amount of Zn²⁺ will provide effective release of the encapsulated nucleic acid.

In forming alginate microparticles, it is preferred that divalent cations (preferably Ca²⁺ and Zn²⁺) are used and the weight (g) of alginate per mole of divalent cations is about 26:1 to about 31:1. It is further preferred that the Ca²⁺:Zn²⁺ divalent cation mole ratio be about 12:1.

In one form of the invention, charged polymeric microparticles, including microparticles which are negatively charged such as alginate microparticles, may be coated with a cationic polymer. Although not being limited by theory, it is believed the ionic interaction between the cationic polymer and the negatively-charged microparticle may serve to neutralize negative charges on the microparticle and thus make the microparticle more hydrophobic. A hydrophobic microparticle is preferred as the hydrophobicity allows for increased cellular uptake of the microparticle, thus leading to increased uptake of the desired nucleic acid. The negatively-charged microparticles, or matrices, may be coated with a wide variety of cationic polymers. Cationic polymers that reduce the negative charge on the microparticle, without affecting the structural integrity of the microparticle or the embedded nucleic acid and allow for increased uptake of polymeric microparticles are preferred. Examples of cationic polymers include proteins, such as polyamino acids and preferably include poly-L-lysine, poly-D- lysine and poly-ornithine . Poly-L-lysine is preferred.

The microparticles may be variously shaped, but are advantageously microspherical, wherein at least about 50% of the microspheres have a diameter of no more than about 10 microns. If the microspheres are larger, entry into the target cell will be diminished or prevented. It is preferred that at least about 60%, more preferably at least about 70%, further preferably at least about 80% and most preferably at least about 90% of the microspheres have a diameter no greater than about 10 microns. Although such small-sized hydrogel microspheres may be formed without use of an emulsion, it is preferred that an emulsion is used in order to form a larger amount of such small-sized microspheres. A wide variety of emulsions may be used, including mineral oil or vegetable oils, with Span 85 or Tween 20 as emulsifiers. It is preferred to use a vegetable oil, especially canola oil, to form the microspheres.

In forming the nucleic acid delivery compositions, the desired nucleic acid is mixed with the polymer, such as alginate, selected to form the micoparticle. A wide variety of nucleic acids, including deoxyribonucleic acid (DNA) , ribonucleic acid (RNA) and modified versions thereof, may be dispersed within the microparticles. It is preferred that the nucleic acid is a DNA that may act as a template for a desired product, such as RNA or protein. The DNA preferably includes a nucleotide sequence, such as a gene sequence, that encodes a protein product. The nucleotide sequence is preferably incorporated into a vector . The term "nucleotide sequence," as used herein, is intended to refer to a natural or synthetic linear and sequential array of nucleotides and/or nucleosides, and derivatives thereof. The terms "encoding" and "coding" refer to the process by which a nucleotide sequence, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce a polypeptide.

A wide variety of vectors may be employed, including plasmids, and phage vectors such as cosmids. However, plasmid vectors are preferred. Many such plasmid vectors are known to the skilled artisan, including high and low copy number plasmids. Such vectors may include a promoter, a selectable marker, and transcriptional enhancer sequences, all as known in the art.

The nucleotide sequence may be advantageously operably linked to a promoter sequence as known in the art. As defined herein, a nucleotide sequence is "operably linked" to another nucleotide sequence when it is placed into a functional relationship with another nucleotide sequence. For example, if a nucleotide sequence is operably linked to a promoter sequence, this generally means that the nucleotide sequence is contiguous with the promoter and the promoter may promote transcription of the gene. A wide variety of promoters are known in the art, including cell-specific promoters, inducible promoters and constitutive promoters. The promoters may be selected so that the desired product produced from the nucleotide sequence template is produced constitutively in the target cells. Alternately, promoters may be selected that require activation by activating elements known in the art, so that production of the desired product may be regulated as desired. An effective amount of the nucleic acid is embedded in the polymeric microparticles. The amount is typically effective for its desired purpose, such as producing a sufficient quantity of protein for an antigenic response, or other purpose. Although this quantity may vary depending on the application, the amount of nucleic acid typically included within a polymer when forming the microparticles is about 0.5 μg to about 0.8 μg of nucleic acid per 100 μg polymer. This typically results in production of microparticles with about 0.2 μg to about 0.5 μg nucleic acid per 100 μg polymer.

In a further embodiment, it has unexpectedly been discovered that inclusion of a virus composition, preferably a composition that includes a DNA virus, such as an adenovirus, in the nucleic acid delivery composition leads to increased transfer of nucleic acid to target cells, as well as increased expression of protein-encoding nucleic acids that are delivered.

Accordingly, in a preferred embodiment, the nucleic acid delivery composition further includes a virus composition, preferably a composition that includes a non-replicative virus (i.e., a virus that does not have the ability to replicate viral proteins and reproduce in a host cell) . Replicative viruses may also advantageously be used. Although adenovirus may be used, it is also believed that other non-enveloped viruses may be used, including picornaviruses, as well as enveloped virus including paramyxovirus, rhabdovirus, poxvirus and togavirus . In one form of the invention, the virus is embedded in the polymeric microparticles along with the desired nucleic acid. In alternate embodiments, a recombinant viral genome may be constructed, by methods known in the art, that includes a desired nucleic acid that includes a nucleotide sequence template used to form a desired product, such as a protein product. In such an embodiment, only the recombinant virus is dispersed within the microparticle for nucleic acid delivery.

The amount of the virus composition that is included in the microparticle is an amount effective in increasing transfer of intact, unmodified (i.e., not intentionally modified, or altered in any way by cellular enzymes during the delivery process) nucleic acid into the target cell and/or increasing production of the desired product, such as increasing expression of a nucleotide sequence to produce a desired protein. Although this amount may vary due to factors including the size of the plasmid, the microparticles are formed advantageously with about 5,000 to about 10,000 plaque forming units of virus composition per μg of polymer. This typically results in production of microparticles with about 2,000 to about 5,000 plaque forming units of virus per μg polymer.

Nucleic acid may be delivered to a wide variety of target cells. The target cells to which the nucleic acid is preferably delivered are typically located in mammalian or avian lymphoid tissue, preferably human lymphoid tissue. For example, the dome region of gut- associated lymphoid tissue contains specialized intestinal epithelial cells, follicle-associated epithelial cells, also known as microfold or membranous cells (M cells) . These cells are specialized cells that are involved in antigen transport. The M cells have the capacity to internalize the microparticles of the present invention. Without being limited by theory, it is believed that the M cells transfer the microparticles to lymphoid cells, such as B or T lymphocytes, macrophages or dendritic cells. These lymphoid cells may migrate to draining lymph nodes. Moreover, the cells may migrate through the blood stream to various organs, such as the liver and spleen. Additionally, lymphoid cells in the intestine, or other locations, may also internalize the microparticles directly. It is also believed that such microparticles may be internalized directly by other target cells, including other epithelial cells, muscle cells, liver cells and lung cells. Alternatively, the target cells may be present as a cell culture. Accordingly, in yet another aspect of the invention, a cellular population which has internalized therein the inventive nucleic acid delivery compositions is also provided. A wide variety of cells may be cultured, including lymphoid cells, epithelial cells, muscle cells, liver cells, and lung cells. Cells from a wide variety of animals, preferably mammalian, and further preferably human, as described above, may be cultured. In one form, lymphoid cells may be cultured and incubated with the nucleic acid delivery compositions under conditions which allow the compositions to be internalized within the cells. A wide variety of lymphoid cells may be cultured, including B and T lymphocytes, dendritic cells and macrophages. After internalization, such cells, if the nucleotide sequence is a template for a desired product, such as a protein product, have the capacity to produce the product . In a further aspect of the invention, a method of in vivo delivery of a nucleic acid to target cells in an animal is provided. The method includes providing the nucleic acid delivery compositions described above and administering an effective amount of the composition to an animal. A wide variety of routes of administration may be utilized, depending on the specific need. For example, the inventive compositions may be delivered orally, intranasally, intramuscularly, subcutaneously, intraperitonealy, intravaginally and any combination thereof. When the nucleic acid includes a gene sequence and the oral route of delivery is desired, expression of the desired gene may be observed in various tissues. For example, expression may be observed in intestinal tissue, spleen tissue and liver tissue. Expression of the gene may also be observed in various target cells, especially when the microparticle with nucleic acid embedded therein is delivered intranasally, intravaginally, subcutaneously, or intraperitoneally.

Although the method of nucleic acid delivery is preferably performed to obtain in vivo delivery of the nucleic acid, the method may be performed to deliver nucleic acid to target cells in culture ( in vi tro) , such as by incubating target cells with the composition. In this alternate embodiment, when the target cells having the nucleic acid delivery composition internalized therein are lymphoid cells, they may be administered to an animal, if desired, by methods known to those skilled in the art. For example, the cells could be introduced into the blood stream of the animal, by, for example, injection, and may then migrate to a target organ. Furthermore, the cells may be introduced intraperitoneally. Alternately, the target cells, including lymphoid cells, liver cells, muscle cells, epithelial cells, lung cells, and a variety of other cells may be introduced directly into a target organ, such as by injection.

In yet other embodiments, the method of nucleic acid delivery described above may be performed to vaccinate an animal. In these embodiments, a gene sequence may encode the desired antigenic protein. Vaccination may be accomplished against a wide variety of pathogens that cause diseases in animals, including humans, swine, cattle, canine or avian species. Desired antigenic proteins that may be used to stimulate an immune response include antigenic portions of a virus or bacteria, including surface glycoproteins, other structural or non-structural proteins, nucleoproteins, or antigenic portions thereof. For example, antigenic proteins in humans include hemagglutinin matrix protein, nucleoprotein from influenza A virus, glycoproteins 120 and 160 from the human immunodeficiency virus, and human hepatitis B surface antigen and are more particularly described in Donnelly, et al., DNA Vaccines, Life Sci. 60, pp. 163- 172 (1997). Antigenic proteins in non-human animals that may be used for vaccination protocols include spike glycoprotein of transmissible gastroenteritis virus, and envelope glycoproteins of viruses, such as bovine herpes type I virus, pseudorabies virus, and bovine respiratory synctia virus. In yet further embodiments, the method of nucleic acid delivery may be performed so that a protein will be expressed within an animal for some other desired purpose such as providing some other beneficial effect to the animal. In such embodiments, a gene preferably encodes a protein that is needed by an animal, either because the protein is no longer produced, is produced in insufficient quantities to be effective in performing its function, or is mutated such that it either no longer functions or is only partially active for its intended function. The nucleic acid delivery compositions may be administered to a wide variety of animals. Preferred vertebrates include mammals, such as mice, rabbit, dogs, cats, birds, and preferably humans. Farm animals are also preferred, including cattle, pigs, goats, and horses .

Reference will now be made to specific examples illustrating the compositions and methods above. It is to be understood that the examples are provided to illustrate preferred embodiments and that no limitation to the scope of the invention is intended thereby.

EXAMPLE 1 Microencapsulation of Plasmid DNA and BAd3 In order to determine whether alginate microspheres could be used for encapsulating plasmid DNA for gene delivery and the effect of BAd3 on the level of foreign gene expression by plasmid DNA in vivo, microspheres were generated that contained either plasmid DNA, virus or both.

The protocol for preparing alginate microspheres was modified from a procedure described in Bowersock, T.L., et al., J. Control . Release 39:209-220 (1996). To obtain a total of 10 mL suspension of microspheres for each preparation, 5 x 10⁹ p.f.u. of purified preparation of BAd3, 1.5 mg of pTK21CMVgalSV40, 1.5 mg of pREPgal, 1.5 mg of pTK21CMVgal SV40 + 5 x 10⁹ p.f.u. of BAd3 or 1.5 mg of pREPgal + 5 x 10⁹ p.f.u. of BAd3 in a volume of 2 ml of phosphate-buffered saline, pH 7.2 (PBS), were mixed with 8 ml of a 1.5% sodium alginate solution, emulsified with 40 ml canola oil and 0.9 ml of Span-85, and stabilized by addition of ten ml of a solution containing 0.5% CaCl₂ and 0.05% ZnCl₂. Thus, 1 mL suspension of microspheres may contain a maximum of either 5 x 10⁸ p.f.u. of BAd3, 150 μg of plasmid DNA or 5 x 10⁸ p.f.u. of BAd3 and 150 μg of plasmid DNA. The majority of microspheres were within 5-10 μm (microns) in diameter as measured by a Microtrak Particle Analyzer. All washings generated during the process of microencapsulation were collected and divided into two portions. One portion was extracted with phenol and chloroform, DNA was precipitated with ethanol and resuspended in a minimum volume of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Uncut DNA was run on an agarose gel by electrophoresis, stained with ethidium bromide and visualized under UV light. The second portion of all washings was centrifuged at 27,000 r.p.m. for 2 hrs at 4°C in Beckman Ti50.1 rotor. The pellets were resuspended in minimum volumes of PBS and titrated for infectious virus particles by plaque assay on MDBK cells.

Analysis

None of the washings showed DNA in detectable amount (data not shown) indicating that the efficiency of microencapsulation of plasmid DNA was high. Infectious virus titers from various washings were insignificant (data not shown) . As the process of microencapsulation did not involve any treatment that obviously could decrease virus infectivity, the microencapsulation of BAd3 was also most likely efficient. EXAMPLE 2

Efficiency of Various Promoters in Transient Expression of the LacZ Gene

This example illustrates that the cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus (RSV) early promoter are efficient in driving transient expression of the LacZ gene in various cell types .

Cell culture and Virus

MDBK, PK-15, and 3T3 cells of bovine, porcine and murine origin, respectively were obtained from American Type Culture Collection (ATCC) , and grown as monolayer cultures using Eagle's minimum essential medium (MEM) [Life Technologies, Inc.] supplemented with 10% fetalClone III (HyClone Laboratories, Inc.) and 50 μg/mL gentamicin. BAd3, obtained from ATCC, was grown in MDBK cells and purified by cesium chloride density- gradient centrifugation as described in Graham, F.L., Manipulation of Adenovirus Vectors, In: Murray, E.J. ed., Methods in Molecular Biology: Gene Transfer and Expression Protocols, v. 7 Clifton: The Humana Press, 109-128, (1991) . The titer of the purified virus preparation was determined by plaque assay on MDBK cells .

Plasmids

Plasmids pTK21CMVgalSV40 (provided by Dr. Guo, Department of Veterinary Pathobiology, Purdue

University, West Lafayette, Indiana) and pREP9gal (as described in Scholz E. et al., J. Virol . Method. 45:291-303 (1993)) contain the bacterial β- galactosidase (LacZ) gene under the control of the cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus (RSV) early promoter, respectively. The LacZ gene in both of these plasmids was under the control of the simian virus 40 (SV40) polyadenylation signal. Plasmid DNA was purified by isopycnic centrifugation in cesium chloride-ethidium bromide gradients as described by Sambrook, J. et al . , Molecular Cloning: A Labora tory Manual, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989.

Transfection of cells

One day before transfection, semiconfluent monolayers of PK-15 and 3T3 cells were harvested with trypsin, counted, and 1-2 x 10⁵ cells were added into each well of 6-well plates and incubated at 37°C in 5% C0₂. The cell monolayers were washed twice with OPTI- MEM I (Life Technologies, Inc.). Plasmid DNA (1 or 5 μg) was mixed with 5 μg of Lipofectin (Life Technologies, Inc.) and incubated at room temperature following the manufacturer's protocol. The DNA- liposome mixture was added dropwise to the cell monolayers covered with OPTI-MEM I and incubated for 0.5 hour at 37°C. At this time point, a set of monolayer transfected with either pTK21CMVgalSV40 or pREP9gal were infected with BAd3 at a multiplicity of infection (m.o.i.) of 500 plaque forming unit (p.f.u.) per cell. After 24 hours post-transfection, the medium was replaced with MEM containing 5% fetalClone III. The cells were harvested by scraping at 48 and 72 hours post-transfection and the cell pellet was assayed for LacZ activity.

β-galactosidase assay The protocol to measure LacZ activity was adapted from a previously described procedure found in Sambrook, J. et al . , Molecular Cloning: A Labora tory Manual , Cold Spring Harbor: Cold Spring Harbor

Laboratory Press, 1989. The cell pellets were resuspended in the cell extraction buffer [250 mM Tris- HC1 (pH 7.8), 0.5% NP40, 1 mM phenylmethylsulfonylfluoride (PMSF)], vortexed and supernatants were saved for LacZ assay. The mouse tissues were homogenized in the cell extraction buffer using a tissumizer and supernatants were used to assay for LacZ activity. A 40 μL sample of various dilutions of cell or tissue extracts were mixed with 350 μL of the sodium phosphate solution [100 mM sodium phosphate (pH 7.5), 10 mM KC1, 1 mM MgS0₄, and 50 mM 2- mercaptoethanol] . After incubation at 37°C for 5 minutes, 132 μL of the ONGP solution [0.4% o- nitrophenol β-D-galactopyranoside (ONGP) in 100 mM sodium phosphate, pH 7.5] was added and the incubation was continued for 1 hour. The enzyme reaction was stopped with 172 μL of 1 M Na₂C0₃ and the intensity of the yellow color developed was measured spectrophotometrically at 420 nm. Various dilutions of purified bacterial LacZ (Sigma, Inc.) were used as a standard for LacZ assay. Analysis

We tested the efficiency of two heterologous promoters, the cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus (RSV) early promoter, to drive transient expression of the LacZ gene when cells initially transfected with plasmid DNA are subsequently infected with BAd3. Since BAd3 does not replicate in either PK-15 cells or 3T3 cells (data not shown) , virus infection of these cells will not cause cell lysis. PK-15, and 3T3 cells were transfected with either pTK21CMVgalSV40 (containing the CMV promoter) or pREP9gal (containing the RSV promoter) and subsequently infected with BAd3 at a multiplicity of infection (m.o.i.) of 500 plaque forming units (p.f.u.) per cell. At 48 and 72 hrs post-transfection the cells were harvested, cell extracts were prepared and used to assay for LacZ activity. Mock and virus-infected cell extracts were used as negative controls. Purified bacterial LacZ was used as a standard. Expression of LacZ in PK-15 cells transfected with pTK21CMVgalSV40 or pREPgal and subsequently infected with BAd3 was approximately 1.7 to 6.4- or 0.7 to 2.3- fold higher, respectively compared to transfected cells without virus infection (Fig. 1A; IB) . Similarly, expression of LacZ in 3T3 cells transfected with pTK21CMVgalSV40 and subsequently infected with BAd3 was approximately 4.3 to 8.6-fold higher compared to transfected cells without virus infection (Fig. IC) . We did not get detectable levels of LacZ expression with pREPgal in 3T3 cells under the conditions used for pTK21CMVgalSV40. The maximum level of LacZ expression with pREPgal + BAd3 even in PK-15 cells was approximately half compared to that of pTK21CMVgalSV40 + BAd3. Expression of LacZ in 3T3 cells with pTK21CMVgalSV40 + BAd3 was approximately half of that obtained in PK-15 cells. The choice of both promoter and cell line seems to be responsible for the levels of reporter gene expression in a transient expression assay. We did not find reporter gene expression proportionate to the amount of plasmid DNA used. This was mainly due to limiting amounts of liposomes when higher amounts of plasmid DNA were used since, by increasing quantities of liposomes with increasing amounts of DNA, the levels of foreign gene expression were proportionate to the amount of DNA used (data not shown) . However, this does not have a significant effect on the interpretation of the results.

EXAMPLE 3

Expression of β-galactosidase in Tissue of Mice Transfected In Vivo with Plasmid DNA Alone or with

Virus

This example illustrates that β-galactosidase is expressed in liver tissue, spleen tissue and intestinal tissue of mice when LacZ is included in the inventive nucleic acid delivery compositions of the present invention. This example further illustrates that expression of LacZ is generally increased when BAd3 is included in the composition. Animal inoculation

A total of 18, 6- to 8-week-old BALB/c mice were randomly grouped into six groups (three animals/group) and inoculated orally using a gavage needle and a syringe at day 1, day 2 and day 3 with 1 mL suspension of alginate microspheres containing either PBS, BAd3, pTK21CMVgalSV40, pREP9gal, pTK21CMVgalSV40 + BAd3 or pREP9gal + BAd3. Animals were sacrificed at day 5 by an overdose of sodium barbiturate and the small intestine, spleen and liver were collected and divided into two portions. One portion of various tissues was weighed and used to assay for LacZ activity. Purified bacterial LacZ was used as a standard. The other portion was embedded in Tissue-Tek Optimal Cutting Temperature (O.C.T.) compound (Miles Scientific, Inc.) and stored at -70°C until use. This portion was used for immunohistochemical and histochemical analyses.

Histochemical Staining Frozen tissue sections from 6-8 week old BALB/c mice inoculated with alginate microspheres containing either BAd3 or pTK21CMVgalSV40 + BAd3 were prepared using a cryomicrotome (Leica CM1800) , fixed with acetone, and stored at -70°C. The histochemical assay to detect LacZ activity in si tu was modified from a previously described protocol found in Engelhardt, J.F., Proc . Na t . Acad. Sci . USA 91:6196-6200 (1994). Briefly, sections were cut from frozen tissues and fixed with 0.5% glutaraldehyde in PBS. After rinsing twice with PBS containing 1 mM MgCl₂, sections were overlayed with the X-gal solution [1 mg/mL 5-bromo-4- chloro-3-indolyl-β-D-galactoside (X-gal), 5.0 mM K₃Fe(CN)₆, 5.0 mM K₄Fe(CN)₆ and 1.0 mM MgCl₂ in PBS] and incubated overnight at room temperature. The slides were washed with PBS and counterstained with eosin. The blue deposits indicate LacZ activity.

Expression of LacZ in Various Organs of Mice Determined by Enzymatically Assaying for β-Galactosidase Mice were orally inoculated with microspheres containing either plasmid DNA, virus or both and the intestine, spleen and liver were collected and analyzed for LacZ expression. In mice inoculated with microspheres containing only pTK21CMVgalSV40, reporter gene expression was observed in intestine, whereas in mice receiving microspheres containing pTK21CMVgalSV40 and BAd3, LacZ expression was observed in intestine, spleen and liver (FIG. 2). In mice inoculated with microspheres containing either pREPgal or pREPgal + BAd3, LacZ expression was observed in intestine, spleen and liver (FIG. 2). Expression of reporter gene was comparatively less in liver with either plasmid. In mice inoculated with plasmid DNA and virus, expression of LacZ in intestine, spleen and liver was approximately 2-fold higher than the expression obtained with the plasmid DNA only.

Expression of LacZ Determined by Histochemical Staining

The intestine, spleen and liver sections from mice inoculated with microspheres containing plasmid DNA and

BAd3 were analyzed for LacZ expression by histochemical staining. The reporter gene expression was evident from blue color development in some cells in all three tissues tested (FIGS. 3 D, E and F) . None of the control tissues had color development above background (FIGS. 3 A, B and C) . Similarly, the intestine, spleen and liver sections from mice inoculated with microspheres containing plasmid DNA and virus were analyzed for expression of LacZ by immunohistochemical staining using a monoclonal antibody against LacZ. The reporter gene expression was evident from brown color deposits in some cells in all three tissues tested (data not shown) . As expected, control tissues did not develop color above background. Since cytochemical and immunohistochemical examinations were not quantitative, mouse tissues were not analyzed from all groups.

EXAMPLE 4

Expression of β-galactosidase in Cells Transfected with

Plasmid DNA

To determine the effect of BAd3 infection on transient LacZ expression in cells transfected with plasmid DNA, 3T3 cells initially transfected with pMNe- gal-SV40 were subsequently infected with BAd3. It was found that expression of LacZ in 3T3 cells transfected with plasmid DNA and subsequently infected with BAd3 was approximately 2.4 to 7.4-fold higher compared to transfected cells without virus infection

Cell Culture and Virus

The cell cultures and virus were the same as described in Example 2. Plasmid

A 3.8 kb Xbal-Bglll fragment containing the bacterial β-galactosidase (LacZ) gene under the control of the murine cytomegalovirus (MCMV) immediate-early promoter and the simian virus 40 (SV40) polyadenylation signal was excised from pCA36 (kindly provided by Dr. F. L. Graham, Departments of Biology and Pathology, McMaster University, Hamilton, Ontario, Canada) and inserted into the Xbal-BamHI site of pUClδ to yield pMNe-gal-SV40. Plasmid DNA was purified by isopycnic centrifugation in cesium chloride-ethidium bromide gradients (Sambrook, J. , et al . , Molecular Cloning: A Labora tory Manual . Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989) .

Transfection and β-galactosidase assays

The transfection protocol was similar to that described previously [Aggarwal, N. et al., Can . J. Vet . Res . 63:148-152 (1999)]. 3T3 cells in 6-well plates (Nalge Nunc International) were transfected with pMNe- gal-SV40 (1 or 5 μg) mixed with 10 μg of Lipofectin (Life Technologies, Inc.). Following a 0.5 hour incubation at 37°C, cells were infected with BAd3 at a multiplicity of infection (m.o.i.) of 500 plaque forming unit (p.f.u.) per cell. The cells were harvested by scraping at 48 and 72 hrs post- transfection and the cell pellet was assayed for LacZ activity. Mock and virus-infected cell extracts were used as negative controls. Purified bacterial LacZ was used as a standard. The protocol to measure LacZ activity was essentially the same as previously described [Aggarwal , N . , et al . , Can . J. Vet . Res . 63 : 148-152 ( 1999 ) ] .

Analysis

Expression of LacZ in 3T3 cells transfected with plasmid DNA and subsequently infected with BAd3 was approximately 2.4 to 7.4-fold higher compared to transfected cells without virus infection (FIG. 4). These results suggest that there is adenovirus-mediated augmentation of the transgene gene expression in cells transfected with plasmid DNA.

EXAMPLE 5 Systemic Immune Response in Mice Inoculated with Microspheres Containing Plasmid DNA and BAd3

This example shows the levels of LacZ-specific and

BAd3-specific IgG and IgA antibody titers in serum samples from mice immunized with microspheres containing plasmid DNA and/or BAd3 when the microspheres are administered by various routes.

Microencapsulation of plasmid DNA and BAd3 The protocol for preparing alginate microspheres was modified from a previously described procedure

[Aggarwal, N., et al., Can . J. Vet . Res . 63:148-152

(1999)]. To obtain a total of 10 mL suspension of microspheres for each preparation, phosphate-buffered saline, pH 7.2 (PBS), 1.8 x 10¹⁰ p.f.u. of purified preparation of BAd3, 3.8 mg of pMNe-gal-SV40 or 3.8 mg pMNe-gal-SV40 + 1.8 x 10¹⁰ p.f.u. of BAd3 were mixed with sodium alginate solution and emulsified with oil to form microspheres which were stabilized by calcium chloride and zinc chloride. One mL suspension of microspheres was expected to contain a maximum of either 1.8 x 10⁹ p.f.u. of BAd3, 380 μg of plasmid DNA or 1.8 x 10⁹ p.f.u. of BAd3 and 380 μg of plasmid DNA. The majority of microspheres were of 5-10 μm in diameter as measured by a Microtrak Particle Analyzer.

Animal inoculation

Eighty 6- to 8-week-old BALB/c mice were randomly grouped into 20 groups (four animals/group) and inoculated by either the oral, intranasal, intramuscular, subcutaneous or intraperitoneal route at days 0, 14, and 28 with alginate microspheres containing either PBS, BAd3, pMNe-gal-SV40 or pMNe-gal- SV40 + BAd3. For oral, intramuscular, subcutaneous, and intraperitoneal inoculations 250 μl of the microsphere suspension was used, whereas for intranasal inoculation only 100 μl of the microsphere suspension was used. The blood samples were collected at days 0, 28, and 40 to monitor the development of LacZ-specific and BAd3-specific IgG and IgA antibodies by enzyme- linked immunosorbent assays (ELISA) . Animals were sacrificed at day 40 by an overdose of sodium barbiturate and spleens were collected for lymphocyte proliferation assay. At day 40, 1 ml of PBS was infused into the lungs through trachea and then recovered to collect lung lavages to evaluate the development of LacZ-specific and BAd3-specific mucosal immune responses by ELISA. The fecal samples were collected from intestines at day 40 and homogenized in PBS (1 g/mL) and supernatants were used to evaluate the development of LacZ-specific and BAd3-sρecific mucosal immune responses by ELISA.

Enzyme-Linked Immunosorbent Assay (ELISA)

The serum samples were used to detect LacZ-specific and BAd3-specific IgG and IgA antibodies by ELISA following the protocol as previously described [Mittal, S. K., et al., Virology 213:131-139 (1995)]. The intestinal fecal samples and lung lavages were used to detect LacZ-specific and BAd3-specific IgA antibody by ELISA. Ninety six-well microtiter plates (Becton Dickinson & Co.) were coated either with purified LacZ (Boehringer Mannheim Corp.) or purified BAd3 and incubated with different dilutions of each sample to detect the development of LacZ-specific or BAd3-specific antibody response, respectively. A horse radish peroxidase (HRP) -conjugated goat anti-mouse IgG (BioRad Laboratories) , or HRP-conjugated goat anti-mouse IgA (Southern Biotechnology Assoc, Inc.) were used as a secondary antibody.

Analysis In order to determine whether alginate microspheres containing plasmid DNA could be used for immunization, mice were immunized with microspheres containing either PBS, DNA or DNA + BAd3 at days 0, 14 and 28 by either the oral, intranasal, intramuscular, subcutaneous or intraperitoneal route. The serum samples were collected at days 0, 28 and 40, and LacZ- specific and BAd3-specific IgG and IgA antibody titers were determined by ELISA. LacZ-specific IgG antibody titers obtained from the serum samples collected at day 28 (second bleed) from mice immunized with microspheres containing only plasmid DNA by an oral, intranasal, intramuscular, subcutaneous or intraperitoneal route were 1,000 ± 400, 1,200 ± 461, 2,000 + 800, 4,800 ± 1,847 and 8,533 ± 3,695, respectively (FIG. 5). Another inoculation at day 28 resulted in a further increase in LacZ-specific IgG antibody titers. Animals immunized with microspheres containing plasmid DNA and BAd3 yielded higher LacZ-specific IgG antibody titers compared to titers obtained with microspheres containing only plasmid DNA. BAd3-specific IgG antibody titers obtained from serum samples collected at day 28 (second bleed) from mice immunized with microspheres containing BAd3 by an oral, intranasal, intramuscular, subcutaneous or intraperitoneal route were 4,800 ± 1,847, 30,000 ± 11,547, 480,000 ± 184,752, 560,000 ± 160,000 and 1120,000 ± 320,000, respectively (FIG. 6). Another inoculation at day 28 resulted in a further increase in BAd3-specific IgG antibody titers. Animals immunized with microspheres containing BAd3 and plasmid DNA yielded similar or slightly higher BAd3-specific IgG antibody titers compared to titers obtained with microspheres containing only BAd3.

LacZ-specific IgA antibody titers obtained from the serum samples collected at day 28 (second bleed) from mice immunized with microspheres containing only plasmid DNA by an oral, intranasal, intramuscular, subcutaneous or intraperitoneal route were 100 + 40, 480 ± 184, 63 ± 23, 70 + 20 and 280 ± 80, respectively (FIG. 7) . Another inoculation at day 28 resulted in a further increase in LacZ-specific IgA antibody titers. Animals immunized with microspheres containing plasmid DNA and BAd3 yielded higher LacZ-specific IgA antibody titers compared to titers obtained with microspheres containing only plasmid DNA.

BAd3-specific IgA antibody titers obtained from the serum samples collected at day 28 (second bleed) from mice immunized with microspheres containing BAd3 by an oral, intranasal, intramuscular, subcutaneous or intraperitoneal route were 500 ± 200, 1,333 ± 460, 666 ± 230, 1,200 ± 461 and 1,800 ± 1,000, respectively (FIG. 8) . Another inoculation at day 28 resulted in a further increase in BAd3-specific IgG antibody titers. Animals immunized with microspheres containing BAd3 and plasmid DNA yielded similar or higher BAd3-specific IgG antibody titers compared to titers obtained with microspheres containing only BAd3. LacZ-specific and BAd3-specific IgG and IgA antibody titers in animals immunized with microspheres containing PBS were close to background (FIGS. 5-8) .

These results suggest that there was BAd3-mediated enhancement of a LacZ-specific systemic immune response irrespective of the route of inoculation. The maximum LacZ-specific systemic IgG antibody titers were observed in mice inoculated intraperitoneally followed by subcutaneously, intramuscularly, intranasally, and orally inoculated animal groups. Whereas, the maximum LacZ-specific systemic IgA antibody titers were observed in animals inoculated intranasally followed by intraperitoneally, subcutaneously, orally and intramuscularly inoculated mouse groups.

EXAMPLE 6

Mucosal Immune Response in Mice Incolutated with Microspheres Containing Plasmid DNA and BAd3

To determine the effect of the route of inoculation on the mucosal immune response, mice were immunized, as in Example 5, with microspheres containing either PBS, DNA or DNA + BAd3, prepared as in Example 5, at days 0, 14 and 28 by either the oral, intranasal, intramuscular, subcutaneous or intraperitoneal route. The lung lavages and fecal samples were collected at day 40, and LacZ-specific and BAd3-specific IgA antibody titers were determined by ELISA as described in Example 5. LacZ-specific IgA antibody titers in the lung lavages of mice immunized with microspheres containing only plasmid DNA by an oral, intranasal, intramuscular, subcutaneous or intraperitoneal route were 4.5 + 2.5, 10 ± 4, 4.5 ± 2.5, 6 ± 2.3 and 10 ± 4, respectively (FIG. 9).

LacZ-specific IgA antibody titers in the fecal samples of mice immunized with microspheres containing only plasmid DNA by an oral, intranasal, intramuscular, subcutaneously or intraperitoneal route were 20 ± 8, 12

± 4.6, 6 ± 2.3, 12 ± 4.6 and 13.3 ± 4.6, respectively

(FIG. 9) . Animals immunized with microspheres containing plasmid DNA and BAd3 yielded higher LacZ-specific IgA antibody titers compared to titers obtained with microspheres containing only plasmid DNA.

BAd3-specific IgA antibody titers in the lung lavages of mice immunized with microspheres containing only BAd3 administered by an oral, intranasal, intramuscular, subcutaneous or intraperitoneal route were 31 ± 12.5, 56 ± 31, 33 ± 14.4, 31 ± 12.5 and 62.5 ± 25, respectively (FIG. 10) .

BAd3-specific IgA antibody titers in the fecal samples of mice immunized with microspheres containing only BAd3 administered by an oral, intranasal, intramuscular, subcutaneous or intraperitoneal route were 260 ± 120, 70 ± 20, 53 ± 23, 120 ± 46 and 213 ± 92, respectively (FIG. 10) . Animals immunized with microspheres containing BAd3 and plasmid DNA yielded similar or slightly higher BAd3-specific IgA antibody titers compared to titers obtained with microspheres containing only BAd3. LacZ-specific and BAd3-specific IgA antibody titers in animals immunized with microspheres containing PBS were close to background (FIGS. 9-10).

These results suggest that there was BAd3-mediated enhancement of a LacZ-specific mucosal immune response irrespective of the route of inoculation. The maximum LacZ-specific IgA antibody titers in the lung lavages were observed in mice inoculated intranasally followed by intraperitoneally, subcutaneously, orally, and intramuscularly inoculated animal groups. The maximum LacZ-specific IgA antibody titers in the fecal samples were observed in animals inoculated orally followed by intraperitoneally, intranasally, subcutaneously, and intramuscularly inoculated mouse groups.

EXAMPLE 7 Cellular Immune Response in Mice Inoculated with Microspheres Containing Plasmid DNA and BAd3

This example shows that the route of inoculation influences the type of immune response elicited in animals inoculated with microspheres containing plasmid DNA. Microspheres were prepared and mice were inoculated as described in Example 5.

Lymphocyte Proliferation Assay Spleens were removed aseptically from sacrificed mice and homogenized individually in sterilized tissuemizers to obtain single-cell suspensions. Viable spleen cells were counted by Trypan blue dye exclusion. The spleen cells resuspended in RPMI 1640 supplemented with 10% Fetal Clone III, 100 units penicillin/ml and 100 μg streptomycin/ml and the concentration of spleen cells were adjusted to 2X10⁶ cells/ml. 200 μl of the cell suspension (2X10⁵ cells) were added in each well of 96-well flat-bottom plates (Corning, Inc.). 10 μl of RPMI 1640 containing various amounts of purified LacZ (0.1, 4, and 8 μg/well) was added in each set of wells (3 wells/antigen concentration) to induce an antigen- specific proliferative response. For a negative control, 10 μg of RPMI 1640 was added in each set of wells. For a positive control, concanavalin (ConA) (2 μg/well) was used for a proliferative response. In order to measure proliferation of spleen cells, the cells in the presence of, or absence of, antigen were then incubated at 37°C for 4 days and then labeled with 0.5 μCi ³H-thymidine (6.7 Ci/mmol, ICN Pharmaceutical, Inc.) per well for 18 h. Cells were harvested and incorporation of ³H-thymidine was determined by a Packard scintillation counter. Mean value of counts per minute

(cpm) of triplicate wells was calculated and used to determine stimulation index (mean cpm of wells with antigen/mean cpm of wells without antigen) .

Analysis

To determine the effect of the route of inoculation on the cellular immune response, mice were immunized with microspheres containing either PBS, DNA or DNA + BAd3 at days 0, 14 and 28 by either the oral, intranasal, intramuscular, subcutaneous or intraperitoneal route. Spleens were collected at day 40, and spleen cells were analyzed for LacZ-specific stimulation by a lymphocyte proliferation assay. Lymphocyte proliferation, as indicated by a stimulation index, yields slightly better results with animals inoculated by orally, intranasally or intraperitoneally than animals immunized intramuscularly or subcutaneously, as shown in FIG. 11.

These results suggest that the route of inoculation influences the type of immune response elicited in animals inoculated with microspheres containing plasmid DNA. EXAMPLE 8

Delivery of Micr©encapsulated Nucleic Acid to Cells in

Culture

Macrophages are obtained from American Type Culture Collection. The cells are cultured on tissue culture plates in a RPMI 1640/10% FCS culture medium. Alginate microparticles are prepared as described in Example 1, with plasmid pMNe-gal-SV40 embedded therein. The cells are incubated with the alginate matrices with plasmid DNA embedded therein for a period of about 1 hour at 37°C so that the matrices are internalized within the cells.

Macrophages are also incubated as described above with alginate microparticles that also include adenovirus embedded therein, along with plasmid pMNe- gal-SV40, as prepared in Example 1.

While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. In addition, all references cited herein are indicative of the level of skill in the art and are hereby incorporated by reference in their entirety.

Claims

CLAIMSWhat is claimed is:

1. A method of in vivo delivery of nucleic acid, comprising administering to an animal an effective amount of a nucleic acid delivery composition, said composition comprising alginate microspheres having nucleic acid embedded therein, wherein at least about 50% of said microspheres have diameters no greater than about 10 microns, said microspheres internalizable by lymphoid cells, said nucleic acid having a nucleotide sequence for producing a desired product.

2. The method of claim 1, wherein said nucleic acid is deoxyribonucleic acid.

3. The method of claim 1, wherein said nucleic acid is in a plasmid vector.

4. The method of claim 2, wherein said nucleotide sequence is operably linked to a promoter.

5. The method of claim 1, wherein said desired product is produced in lymphoid cells.

6. The method of claim 1, wherein said desired product is produced in intestinal epithelial tissue.

7. The method of claim 1, wherein said desired product is produced in liver tissue.

8. The method of claim 1, wherein said desired product is produced in spleen tissue.

9. The method of claim 1, wherein said administering said composition is performed by oral administration.

10. The method of claim 1, wherein administering said composition is performed through a route selected from the group consisting of oral, intranasal, intramuscular, subcutaneous, intravaginal intraperitoneal, and combinations thereof.

11. The method of claim 1, wherein at least about 70% of said microspheres have diameters no greater than about 10 microns.

12. The method of claim 1, wherein said product is a protein.

13. A method of in vivo delivery of nucleic acid, comprising administering to an animal an effective amount of a nucleic acid delivery composition, said composition comprising: (a) alginate microparticles having nucleic acid embedded therein, said microparticles formed by ionic cross- linking with zinc cations; and (b) a cationic polymer, said microparticles coated with said cationic polymer.

14. The method of claim 13, wherein said composition further comprises a virus composition embedded in said microparticles.

15. The method of claim 14, wherein said virus composition comprises a virus selected from the group consisting of replicative and non-replicative virus.

16. The method of claim 14, wherein said virus composition comprises an adenovirus.

17. The method of claim 13, wherein said cationic polymer is a protein.

18. The method of claim 17, wherein said protein is poly-L-lysine.

19. The method of claim 13, wherein said nucleic acid is deoxyribonucleic acid.

20. The method of claim 19, wherein said deoxyribonucleic acid is in a plasmid vector.

21. The method of claim 19, wherein said nucleotide sequence is operably linked to a promoter.

22. The method of claim 13, wherein said alginate microparticles are alginate microspheres.

23. The method of claim 22, wherein at least about 50% of said alginate microspheres have diameters no greater than about 10 microns.

24. The method of claim 22, wherein at least about 70% of said alginate microspheres have diameters no greater than about 10 microns.

25. The method of claim 22, wherein at least about 80% of said alginate microspheres have diameters no greater than about 10 microns.

26. A method of in vivo delivery of nucleic acid, comprising administering to an animal an effective amount of a nucleic acid delivery composition, said composition comprising alginate microspheres having nucleic acid and a virus composition embedded therein, wherein at least about 50% of said microspheres have diameters no greater than about 10 microns, said microspheres internalizable by lymphoid cells, said nucleic acid having a nucleotide sequence for producing a desired product.

27. The method of claim 26, wherein said virus composition comprises a non-replicative virus.

28. The method of claim 27, wherein said non- replicative virus is a deoxyribonucleic acid virus.

29. The method of claim 27, wherein said non- replicative virus is an adenovirus.

30. The method of claim 26, wherein said nucleic acid is deoxyribonucleic acid.

31. A method of in vivo delivery of nucleic acid, comprising administering to an animal an effective amount of a nucleic acid delivery composition, comprising polymeric microparticles having nucleic acid and a virus composition embedded therein.

32. A method of in vi tro delivery of nucleic acid to target cells, said method comprising incubating said target cells with an effective amount of a nucleic acid delivery composition, said composition comprising alginate microspheres having nucleic acid embedded therein, wherein at least about 50% of said microspheres have diameters no greater than about 10 microns, said microspheres internalizable by lymphoid cells, said nucleic acid having a nucleotide sequence for producing a desired product.

33. The method of claim 32, wherein said composition includes a virus composition, said virus composition embedded in said alginate microspheres.

34. A method of in vivo delivery of nucleic acid, said method comprising administering to said animal an effective amount of a nucleic acid delivery composition comprising alginate microspheres having nucleic acid embedded therein, wherein at least about 50% of said matrices have diameters no greater than about 10 microns, said microspheres internalizable by lymphoid cells, said nucleic acid having a nucleotide sequence encoding a desired protein, said protein expressible in tissues of said animal, said tissues selected from the group consisting of liver tissue, intestinal epithelial tissue, spleen tissue, and combinations thereof.

35. The method of claim 34, said composition further including a virus composition embedded in said alginate microspheres.

36. The method of claim 35, wherein said virus composition comprises an adenovirus.

37. The method of claim 34, wherein said alginate microspheres are coated with a cationic polymer.

38. A nucleic acid delivery composition, comprising alginate microspheres having nucleic acid embedded therein, wherein at least about 50% of said matrices have diameters no greater than about 10 microns, said microspheres internalizable by lymphoid cells, said nucleic acid having a nucleotide sequence for producing a desired product.

39. The composition of claim 38, wherein said nucleic acid is deoxyribonucleic acid.

40. The composition of claim 38, wherein said nucleic acid is in a plasmid vector.

41. The composition of claim 38, wherein said alginate microspheres are formed by ionic cross- linking.

42. The composition of claim 41, wherein said ionic cross-linking comprises ionic cross-linking with divalent cations, said divalent cations including zinc

43. The composition of claim 38, wherein said composition further comprises a virus composition, said virus composition embedded in said alginate microspheres .

44. The composition of claim 43, wherein said virus composition comprises a deoxyribonucleic acid virus.

45. The composition of claim 44, wherein said deoxyribonucleic acid virus is an adenovirus.

46. The composition of claim 38, wherein at least about 70% of said microspheres have diameters no greater than about 10 microns.

47. The composition of claim 38, wherein said alginate microspheres are coated with a cationic polymer.

48. The composition of claim 47, wherein said cationic polymer is a protein.

49. The composition of claim 48, wherein said protein is poly-L-lysine.

50. A nucleic acid delivery composition, comprising:

(a) alginate microparticles having nucleic acid embedded therein, said microparticles formed by ionic cross- linking with zinc cations; and

(b) a cationic polymer, said microparticles coated with said cationic polymer.

51. The composition of claim 50, wherein said nucleic acid is deoxyribonucleic acid.

52. The composition of claim 51, wherein said deoxyribonucleic acid is in a plasmid vector.

53. The composition of claim 51, wherein said deoxyribonucleic acid is operably linked to a promoter.

54. The composition of claim 50, where said alginate microparticles are internalizable by lymphoid cells.

55. The composition of claim 50, wherein said alginate microparticles are microspherical.

56. The composition of claim 55, wherein at least about 50% of said microspherical alginate microparticles have diameters no greater than about 10 microns .

57. The composition of claim 56, wherein at least about 70% of said microspherical alginate microparticles have diameters no greater than about 10 microns .

58. A nucleic acid delivery composition, comprising alginate microspheres having nucleic acid and a virus composition embedded therein, wherein at least about 50% of said microspheres have diameters no greater than about 10 microns, said microspheres internalizable by lymphoid cells, said nucleic acid having a nucleotide sequence for producing a desired product.

59. A nucleic acid delivery composition, comprising polymeric microparticles having nucleic acid and a virus composition embedded therein, wherein said microparticles are internalizable by lymphoid cells.

60. The composition of claim 59, wherein said polymeric microparticles are polysaccharide microparticles .

61. The composition of claim 60, wherein said polysaccharide microparticles are alginate microparticles .

62. A cellular population comprising lymphoid cells, said cells having internalized therein a nucleic acid delivery composition, said composition comprising alginate microspheres having nucleic acid embedded therein, wherein at least about 50% of said microspheres have diameters no greater than about 10 microns, said nucleic acid having a nucleotide sequence for producing a desired product.

63. The cellular population of claim 62, wherein said lymphoid cells are macrophages.

64. The cellular population of claim 62, wherein said composition further includes a virus composition, said virus composition embedded in said alginate microspheres.

65. A cellular population comprising lymphoid cells, said cells having internalized therein a nucleic acid delivery composition, said composition comprising:

(b) a cationic polymer, said microparticles coated with said cationic polymer.

66. The cellular population of claim 65, wherein said composition further includes a virus composition, said virus composition embedded in said alginate microparticles .

67. The cellular population of claim 65, wherein said lymphoid cells are macrophages.