WO1999058134A1 - Methodes et compostions d'apport d'acide nucleique - Google Patents

Methodes et compostions d'apport d'acide nucleique Download PDF

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WO1999058134A1
WO1999058134A1 PCT/US1999/010235 US9910235W WO9958134A1 WO 1999058134 A1 WO1999058134 A1 WO 1999058134A1 US 9910235 W US9910235 W US 9910235W WO 9958134 A1 WO9958134 A1 WO 9958134A1
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composition
nucleic acid
microspheres
virus
microparticles
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PCT/US1999/010235
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English (en)
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Suresh K. Mittal
Neeraj Aggarwal
Harm Hogenesch
Peixuan Guo
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Purdue Research Foundation
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Priority to AU38954/99A priority Critical patent/AU3895499A/en
Publication of WO1999058134A1 publication Critical patent/WO1999058134A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • 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.
  • many of these alternate strategies also suffer from drawbacks.
  • 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.
  • bombarding tissue with gold microparticles coated with DNA is not useful for a wide variety of cell types.
  • a nucleic acid delivery composition includes polymeric microparticles having nucleic acid embedded therein.
  • 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.
  • a virus composition such as an adenovirus composition
  • 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.
  • a nucleotide sequence may express a desired protein and the virus may cause an increase in the expression of the protein.
  • 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.
  • 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
  • the desired product may be expressed in a wide variety of animal tissues, including, for example, intestinal epithelial tissue, spleen tissue and liver tissue.
  • 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.
  • 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.
  • nucleic acid delivery compositions that can efficiently deliver nucleic acid to an animal or target cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • a nucleic acid delivery composition 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.
  • viruses such as an adenovirus
  • 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.
  • a nucleic acid delivery 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.
  • such polymers include polysaccharides, such as alginate, collagen, gelatin, polyacrylamide, polymethacrylamide, polyvinyl acetate, poly-N- vinylpyrrolidone, polyvinyl alcohol, polyacrylic acids, and other similar polymers.
  • 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 ⁇ -D-mannuronic and ⁇ -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 Ca 2+ . Other multivalent cations such as Ba 2+ , Sr 2+ , Zn 2+ , and Mn 2+ may also be used.
  • hydrogels of improved stability may be formed by ionically cross-linking the polysaccharide with a combination of Ca 2+ and Zn 2+ . More specifically, it has been determined that the amount of Zn 2+ used in forming the hydrogel can be regulated to control particle characteristics, such as hardness and integrity. An appropriate amount of Zn 2+ will provide effective release of the encapsulated nucleic acid.
  • alginate microparticles it is preferred that divalent cations (preferably Ca 2+ and Zn 2+ ) 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 Ca 2+ :Zn 2+ divalent cation mole ratio be about 12:1.
  • divalent cations preferably Ca 2+ and Zn 2+
  • the weight (g) of alginate per mole of divalent cations is about 26:1 to about 31:1. It is further preferred that the Ca 2+ :Zn 2+ divalent cation mole ratio be about 12:1.
  • charged polymeric microparticles including microparticles which are negatively charged such as alginate microparticles, may be coated with a cationic polymer.
  • a cationic polymer 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.
  • 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.
  • the desired nucleic acid is mixed with the polymer, such as alginate, selected to form the micoparticle.
  • the polymer such as alginate
  • 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 .
  • nucleotide sequence is intended to refer to a natural or synthetic linear and sequential array of nucleotides and/or nucleosides, and derivatives thereof.
  • 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.
  • 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.
  • a nucleotide sequence is "operably linked" to another nucleotide sequence when it is placed into a functional relationship with another nucleotide sequence.
  • this generally means that the nucleotide sequence is contiguous with the promoter and the promoter may promote transcription of the gene.
  • 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.
  • 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 ⁇ g to about 0.8 ⁇ g of nucleic acid per 100 ⁇ g polymer. This typically results in production of microparticles with about 0.2 ⁇ g to about 0.5 ⁇ g nucleic acid per 100 ⁇ g polymer.
  • a virus composition preferably a composition that includes a DNA virus, such as an adenovirus
  • inclusion of a virus composition leads to increased transfer of nucleic acid to target cells, as well as increased expression of protein-encoding nucleic acids that are delivered.
  • 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) .
  • 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.
  • 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 .
  • the virus is embedded in the polymeric microparticles along with the desired nucleic acid.
  • 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.
  • a desired product such as a protein product.
  • 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.
  • 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 ⁇ g of polymer. This typically results in production of microparticles with about 2,000 to about 5,000 plaque forming units of virus per ⁇ g 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • routes of administration may be utilized, depending on the specific need.
  • the inventive compositions may be delivered orally, intranasally, intramuscularly, subcutaneously, intraperitonealy, intravaginally and any combination thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cells could be introduced into the blood stream of the animal, by, for example, injection, and may then migrate to a target organ.
  • the cells may be introduced intraperitoneally.
  • 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.
  • the method of nucleic acid delivery described above may be performed to vaccinate an animal.
  • 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.
  • 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.
  • 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.
  • 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 .
  • EXAMPLE 1 Microencapsulation of Plasmid DNA and BAd3
  • alginate 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).
  • 5 x 10 9 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 10 9 p.f.u. of BAd3 or 1.5 mg of pREPgal + 5 x 10 9 p.f.u.
  • 1 mL suspension of microspheres may contain a maximum of either 5 x 10 8 p.f.u. of BAd3, 150 ⁇ g of plasmid DNA or 5 x 10 8 p.f.u. of BAd3 and 150 ⁇ g of plasmid DNA.
  • microspheres were within 5-10 ⁇ m (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.
  • CMV cytomegalovirus
  • RSV Rous sarcoma 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 ⁇ g/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 pTK21CMVgalSV40 (provided by Dr. Guo, Department of Veterinary Pathobiology, Purdue
  • pREP9gal (as described in Scholz E. et al., J. Virol . Method. 45:291-303 (1993)) contain the bacterial ⁇ - galactosidase (LacZ) gene under the control of the cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus (RSV) early promoter, respectively.
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • 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.
  • 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.
  • m.o.i. 500 plaque forming unit
  • 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.
  • 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 ⁇ L sample of various dilutions of cell or tissue extracts were mixed with 350 ⁇ L of the sodium phosphate solution [100 mM sodium phosphate (pH 7.5), 10 mM KC1, 1 mM MgS0 4 , and 50 mM 2- mercaptoethanol] . After incubation at 37°C for 5 minutes, 132 ⁇ L of the ONGP solution [0.4% o- nitrophenol ⁇ -D-galactopyranoside (ONGP) in 100 mM sodium phosphate, pH 7.5] was added and the incubation was continued for 1 hour.
  • the sodium phosphate solution 100 mM sodium phosphate (pH 7.5), 10 mM KC1, 1 mM MgS0 4 , and 50 mM 2- mercaptoethanol
  • the enzyme reaction was stopped with 172 ⁇ L of 1 M Na 2 C0 3 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
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • 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.
  • m.o.i. 500 plaque forming units
  • p.f.u. plaque forming units
  • 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) .
  • 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) .
  • 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 histo
  • 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.
  • 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).
  • 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.
  • 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.
  • mice inoculated with microspheres containing plasmid DNA
  • 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) .
  • 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.
  • a 3.8 kb Xbal-Bglll fragment containing the bacterial ⁇ -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 pUCl ⁇ 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) .
  • 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 ⁇ g) mixed with 10 ⁇ g 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.
  • m.o.i. multiplicity of infection
  • p.f.u. plaque forming unit
  • phosphate-buffered saline, pH 7.2 (PBS), 1.8 x 10 10 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 10 10 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 10 9 p.f.u.
  • mice 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.
  • alginate microspheres containing either PBS, BAd3, pMNe-gal-SV40 or pMNe-gal- SV40 + BAd3.
  • 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.
  • ELISA enzyme- linked immunosorbent assays
  • 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-s ⁇ ecific mucosal immune responses by 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.
  • HRP horse radish peroxidase
  • 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.
  • mice 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).
  • 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.
  • 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.
  • 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
  • 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).
  • 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 ⁇ g streptomycin/ml and the concentration of spleen cells were adjusted to 2X10 6 cells/ml. 200 ⁇ l of the cell suspension (2X10 5 cells) were added in each well of 96-well flat-bottom plates (Corning, Inc.).
  • 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.
  • 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.

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Abstract

L'invention concerne des compositions d'apport d'acide nucléique, des méthodes d'apport in vivo d'acide nucléique à un animal ou in vitro à des cellules cibles et à des populations cellulaires, telles que des cellules lymphoïdes, en y introduisant lesdites compositions d'apport d'acide nucléique. Dans un mode de réalisation, une composition d'apport d'acide nucléique contient des matrices de microparticules polymères, notamment des microparticules de polysaccharide, telles que des microparticules d'alginate, et un acide nucléique enchâssé dans ces microparticules. Dans d'autres modes de réalisation, la composition d'apport d'acide nucléique contient également une composition virale, telle qu'une composition adénovirale. Les microparticules polymères peuvent être enduites d'un polymère cationique et, dans un mode de réalisation, sont microsphériques. L'invention concerne également des méthodes d'apport in vivo d'un acide nucléique à une cellule cible. Ces méthodes consistent à administrer à un animal une quantité efficace d'une composition d'apport d'acide nucléique selon l'invention. L'invention concerne également des méthodes d'apport in vitro d'acide nucléique à des cellules cibles, lesquelles méthodes consistent à incuber ces cellules avec les compositions d'apport d'acide nucléique selon l'invention.
PCT/US1999/010235 1998-05-11 1999-05-11 Methodes et compostions d'apport d'acide nucleique WO1999058134A1 (fr)

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KR20020028419A (ko) * 2000-10-10 2002-04-17 서만석 유전자 또는 유전자 백신의 전달방법
KR100514092B1 (ko) * 2002-11-23 2005-09-13 한국생명공학연구원 양이온성 고분자 물질과 음이온성 고분자 물질을 이용한 새로운 다중 유전자 전달 복합체

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