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WO1998040499A1 - Apport de genes dans l'epithelium muqueux a des fins therapeutiques ou d'immunisation - Google Patents

Apport de genes dans l'epithelium muqueux a des fins therapeutiques ou d'immunisation Download PDF

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WO1998040499A1
WO1998040499A1 PCT/US1997/003421 US9703421W WO9840499A1 WO 1998040499 A1 WO1998040499 A1 WO 1998040499A1 US 9703421 W US9703421 W US 9703421W WO 9840499 A1 WO9840499 A1 WO 9840499A1
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mixture
lipid
polynucleotide
ratio
complex
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PCT/US1997/003421
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English (en)
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Heather Lynn Davis
Joel Jessee
Gulilat Gebeyehu
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Heather Lynn Davis
Joel Jessee
Gulilat Gebeyehu
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Application filed by Heather Lynn Davis, Joel Jessee, Gulilat Gebeyehu filed Critical Heather Lynn Davis
Priority to PCT/US1997/003421 priority Critical patent/WO1998040499A1/fr
Priority to AU19871/97A priority patent/AU1987197A/en
Publication of WO1998040499A1 publication Critical patent/WO1998040499A1/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

Definitions

  • the present invention is in the fields of gene therapy and immunology.
  • this invention is directed to methods of immunization and gene therapy using compositions comprising cationic lipids and polynucleotide molecules which code for immunogens or therapeutic genes, respectively.
  • This invention is also directed to methods for producing polyclonal and monoclonal antibodies from genetically immunized animals.
  • vaccines In order to be effective, vaccination must generate humoral and/or cell- mediated immunity which will prevent the development of disease upon subsequent exposure to the corresponding pathogen.
  • the pertinent antigenic determinants must be presented to the immune system in a manner that mimics a natural infection.
  • Conventional vaccines may consist of inactivated virulent strains, or live-attenuated strains (Old et al. , Principles of Gene Manipulation: An Introduction to Genetic Engineering, Blackwell Scientific Publications, 4th edition, 1989).
  • a general problem with using a vaccine consisting of whole virus is that many viruses (such as hepatitis B virus) have not been adapted to grow to high titre in tissue culture and thus, cannot be produced in sufficient quantity (Id.).
  • inactivated viruses present a potential danger of vaccine-related disease resulting from replication-competent virus may remain in the inoculum. Outbreaks of foot-and-mouth disease in Europe have been attributed to this cause (Id.).
  • attenuated virus strains have the potential to revert to virulent phenotype upon replication in the vaccinee. This problem has been reported to occur about once or twice in every million people who receive live polio vaccine (Id.).
  • encephalitis can occur following measles immunization with attenuated virus (Roit, I.M. Essential Immunology, Blackwell Scientific Publications, Sixth Ed., 1988).
  • a major disadvantage associated with the use of live virus vaccines is that persons with congenital or acquired immunodeficiency risk severe infections. Such persons include children in developing countries who are often immunodeficient because of malnutrition and/or infection with viruses or parasites (Id., Old et al., supra).
  • purified viral proteins or synthetic peptides for use in immunoprophylaxis (Murphy et al. , "Immunization Against Viruses," in Virology, Fields et al. , Eds., Raven Press, New York, pp.
  • Purified antigens may be produced by synthesizing peptides which represent immunologically important domains of surface antigens of the pathogen.
  • the synthetic peptide approach has been successfully used with an antigenic determinant of the foot and mouth disease virus (Id.).
  • One problem with this approach is that the poor antigenicity of synthetic peptides has required the use of Freund's adjuvant to enhance the immune response in experimental animals (Id. ) . Since Freund ' s adjuvant cannot be used in humans , an effective adjuvant for human use such as the saponins has been developed.
  • a single antigenic site may not be sufficient to induce resistance since large surface antigens usually contain several distinct immunological domains that elicit a protective humoral and/or cell-mediated response (Braciale et al, J. Exp. Med. 153:910-923 (1981); Wiley et al.,
  • An alternative to producing the recombinant antigen in vitro is to introduce nucleic acid sequences coding for the antigen into the cells of the vaccinee.
  • the antigen is produced in vivo by the vaccinee 's cells and provokes the immune response.
  • Tang et al. (Nature 356:152-154 (1992)) have shown that it is possible produce an immune response to human growth hormone protein in mice by propelling gold microprojectiles coated with plasmids containing human growth hormone genomic sequences. The resultant variability in the production of antibody production was hypothesized to arise from the operation of the microprojectile device, or the coating of the DNA onto the microprojectiles.
  • Liposomes have been used as carriers of genetic information in the transfection of tissue culture cells.
  • a fundamental problem of liposome- mediated transfection with liposomes comprising neutral or anionic lipids is that such liposomes do not generally ruse with the target cell surface. Instead, the liposomes are taken up phagocytically, and the polynucleotides are subsequently subjected to the degradative enzymes of the lysosomal compartment (Straubinger et al. , Methods Enzymol. 101:512-521 (1983); Mannino et al., Biotechniques 6:682-690 (1988)).
  • aqueous space of typical liposomes may be too small to accommodate large macromolecules such as DNA or RNA.
  • typical liposomes have a low capturing efficiency (Feigner, "Cationic Liposome- Mediated Transfection with LipofectinTM Reagent," in Gene Transfer and Expression Protocols Vol. 7, Murray, E.J., Ed., Humana Press, New Jersey, pp. 81-89 (1991)).
  • Liposomes comprising cationic lipids interact spontaneously and rapidly with polyanions such as DNA and RNA, resulting in liposome/nucleic acid complexes that capture 100% of the polynucleotide (Feigner et al. , Proc.
  • the polycationic complexes are taken up by the anionic surface of tissue culture cells with an efficiency that is about ten to one hundred times greater than negatively charged or neutral liposomes (Feigner, "Cationic Liposome-Mediated Transfection with LipofectinTM Reagent," in Gene Transfer and Expression Protocols Vol. 7, Murray, E.J., Ed., Humana Press, New Jersey, pp. 81-89 (1991)).
  • polycationic complexes fuse with cell membranes, resulting in an intracellular delivery of polynucleotide that bypasses the degradative enzymes of the lysosomal compartment (D ⁇ zg ⁇ nes et al, Biochemistry 25:9179-9184 (1989); Feigner et al , Nature 337:381-388 (1989)).
  • Various formulations of cationic lipids have been used to transfect cells in vitro (WO 91/17424; WO 91/16024; U.S. Patent No. 4,897,355; U.S. Patent No. 4,946,787; U.S. Patent No. 5,049,386; and U.S. Patent No. 5,208,036).
  • Cationic lipids have also been used to introduce foreign polynucleotides into frog and rat cells in vivo (Holt et al, Neuron 4:203-214 (1990); Hazinski et al, Am. J. Respr. Cell Mol Biol 4:206-209 (1991)).
  • cationic lipids may be used, generally, as pharmaceutical carriers to provide biologically active substances (for example, see WO 91/17424; WO 91/16024; and WO 93/03709).
  • CF cystic fibrosis
  • Delivery of the cystic fibrosis (CF) transmembrane conductance regulator gene may be used to treat CF (Alton, E.W.F.W., et al., Nature Gen. 5:135-142 (1993); Caplen, N.J., et al, Nature Med. 7:39-46 (1995)); Yoshimura, K., et al, Nucleic Acids Res.
  • CF cystic fibrosis
  • the present invention is directed to a method for transfecting mucosal epithelial cells of a mammal with a polynucleotide molecule, comprising: (a) mixing at least one cationic lipid with a polynucleotide molecule, wherein the amount of lipid is sufficient to complex substantially said DNA molecule, thereby forming a cationic lipid-polynucleotide complex; and
  • the polynucleotide molecule may be effective for gene therapy or code for an antigen capable of raising an immune response in the mammal.
  • the present invention is also directed to a method for generating an immune response against an infectious disease in an animal, comprising the steps of:
  • Complexes ofthe invention delivered to the epithelium are not only easy to administer, but also induce specific mucosal immunity, which is desirable to prevent entry of pathogens via the respiratory system or any ofthe other extensive mucosal surfaces ofthe body.
  • the present invention is further directed to a method for producing polyclonal antibodies comprising the use of the method of inducing an immune response described above. After production of the anitbodies, the invention may further comprise isolating the polyclonal antibodies from the immunized animal.
  • the present invention is also directed to a method for producing monoclonal antibodies, comprising:
  • the invention also relates to a cationic lipid mixture selected from the group consisting of: (a) TMTPS:DOPE (preferably at a ratio of 1 : 1.5);
  • TMTPS:C-16dl-PE preferably at a ratio of 1 : 1.5
  • TMTOS DOPE (preferably at a ratio of 1 : 1.5) ; and
  • TMTOS:C-16dl-PE preferably at a ratio of 1:1.5).
  • the invention also relates to a complex between a polynucleotide molecule and a cationic lipid mixture selected from the group consisting of:
  • TMTPS:DOPE preferably at a ratio of 1 : 1.5
  • TMTPS:C-16dl-PE preferably at a ratio of 1 : 1.5
  • DMRIExholesterol preferably at a ratio of 1:1
  • TMTOS:DOPE preferably at a ratio of 1 : 1.5
  • TMTOS:C-16dl-PE preferably at a ratio of 1 : 1.5.
  • the invention also relates to a method of obtaining a polynucleotide/cationic lipid complex, comprising admixing a polynucleotide with at least one cationic lipid in an amount sufficient to complex substantially said polynucleotide molecule, thereby forming a cationic lipid-polynucleotide complex.
  • the invention also relates to a polynucleotide/cationic lipid complex obtained by the method of the invention.
  • FIG. 3. depicts a bar graph showing luciferase reporter gene activity in mouse lung tissue at various times after IN inhalation of 100 ⁇ g of pCMV-luc
  • FIG.4. depicts a graph showing luciferase reporter gene activity in mouse lung tissue at 24 (triangles), 48 (circles) or 72 (squares) hours after administration of 100 ⁇ g of pCMV-luc DNA in saline (open symbols) or associated with lipid 301 at a 1:1 DNA:lipid (w/w) ratio (closed symbols).
  • DNA solutions were administered by intranasal (IN) instillation or inhalation, or by intratracheal (IT) injection or cannulation. Each point represents the value obtained from a single animal.
  • FIG. 5. depicts a bar graph showing luciferase reporter gene activity in mouse lung tissue 48 hr after IN inhalation of 100 ⁇ g of pCMV-luc DNA in saline or associated with one of 14 different lipids at 1 : 1 DNA:lipid (w/w) ratio.
  • FIGs. 6A-6C depict the structures of preferred cationic lipids for use in the present invention.
  • FIG.7 depicts the general procedure for the synthesis of C12, C14, C16 and C18 tetramethyl tetraalkyl spermine analogs. Detailed Description ofthe Preferred Embodiments
  • Cloning vector A plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of restriction endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological fiinction of the vector, and into which a DNA fragment may be spliced in order to bring about its replication and cloning.
  • the cloning vector may further contain a marker suitable for use in the identification of cells transformed with the cloning vector. Markers, for example, provide tetracycline resistance or ampicillin resistance.
  • Expression vector A vector similar to a cloning vector but which is capable of enhancing the expression of a gene which has been cloned into it, after transformation into a host.
  • the cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.
  • Promoter sequences may be either constitutive or inducible.
  • a recombinant host may be any prokaryotic or eukaryotic microorganism or cell which contains the desired cloned genes on an expression vector or cloning vector. This term is also meant to include those microorganisms that have been genetically engineered to contain the desired gene(s) in the chromosome or genome of that organism.
  • Recombinant vector Any cloning vector or expression vector which contains the desired cloned gene(s).
  • Host Any prokaryotic or eukaryotic microorganism or cell that is the recipient of a replicable expression vector or cloning vector.
  • a "host,” as the term is used herein, also includes prokaryotic or eukaryotic microorganisms or cells that can be genetically engineered by well known techniques to contain desired gene(s) on its chromosome or genome. For examples of such hosts, see
  • Promoter A DNA sequence generally described as the 5' region of a gene, located proximal to the start codon. The transcription of an adjacent gene(s) is initiated at the promoter region. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. Examples of promotors that may be used to drive gene expression in epithelial mucosal cells in vivo include, but are not limited to the CMV promoter (InVitrogen, San Diego, CA), the
  • Gene A DNA sequence that contains information needed for expressing a polypeptide or protein.
  • Structural gene A DNA sequence that is transcribed into messenger
  • RNA that is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • Therapeutic Gene A gene that when transfected and expressed in vivo into mammalian host cells results in the cure, amelioration or prevention of a mammalian disease.
  • Antisense oligonucleotide A DNA or RNA molecule or a derivative of a DNA or RNA molecule containing a nucleotide sequence which is complementary to that of a specific mRNA.
  • An antisense oligonucleotide binds to the complementary sequence in a specific mRNA and inhibits translation ofthe mRNA.
  • S-oligos are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom.
  • the S-oligos of the present invention may be prepared by treatment of the corresponding O-oligos with 3H-l,2-benzodithiol- 3-one- 1,1 -dioxide which is a sulfur transfer reagent. See Iyer et al, J. Org. Chem. 55:4693-4698 (1990); and Iyer et al, J. Am. Chem. Soc. 712:1253-1254 (1990).
  • Antisense Therapy A method of treatment wherein antisense oligonucleotides are administered to a patient in order to inhibit the expression ofthe corresponding protein.
  • Expression is the process by which a polypeptide is produced from a structural gene. The process involves transcription ofthe gene into mRNA and the translation of such mRNA into polypeptide(s).
  • Transfection refers to the stable of transient transformation of a host cell, e.g. a mucosal epithelial cell, with a nucleic acid molecule, e.g. a DNA molecule.
  • the recombinant host cell expresses protein which is encoded by the transfected DNA.
  • Antigenic Determinant A protein or peptide which contains one or more epitopes.
  • Immunogen A protein or peptide which is capable of eliciting an immune response due to the presence of one or more epitopes.
  • the present invention is directed to a method for transfecting mucosal epithelial cells of a mammal with a polynucleotide molecule, comprising:
  • this method may be used to generate an immune response in a mammal or to practice gene therapy, e.g. with therapeutical effective genes, antisense oligonucleotides, ribozymes, external guide sequences and the like.
  • any of the cationic lipids known in the prior art may be employed in the practice of the claimed invention. See, for example, Feigner et al. (Proc. Natl Acad. Sci. U.S.A. 84:1413-1411 (1987)); Feigner et al. (Focus 77:21-25
  • Example structures of cationic lipids useful in this invention are provided in Table 1.
  • any cationic lipid either monovalent or polyvalent, can be used in the compositions and methods of this invention.
  • Polyvalent cationic lipids are generally preferred.
  • Cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides or derivatives thereof.
  • Straight-chain and branched alkyl and alkene groups of cationic lipids can contain from 1 to about 25 carbon atoms.
  • Preferred straight-chain or branched alkyl or alkene groups have six or more carbon atoms.
  • Alicyclic groups can contain from about 6 to 30 carbon atoms.
  • Preferred alicyclic groups include cholesterol and other steroid groups.
  • Cationic lipids can be prepared with a variety of counter ions (anions) including among others: Cl “ , Br, I " , F ⁇ , acetate, trifluoroacetate, sulfate, nitrite, and nitrate.
  • a well-known cationic lipid is N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA). See Feigner, P.L. et al. Proc. Natl. Acad. Sci. USA 84:1413-1411 (1987).
  • DOTMA and the analogous diester DOTAP (l,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane), see Table 1 for structures, are commercially available. Additional cationic lipids structurally related to DOTMA are described in U.S. Patent No. 4,897,355.
  • DORI-ethers Another useful group of cationic lipids related to DOTMA and DOTAP are commonly called DORI-ethers or DORI-esters.
  • DORI lipids differ from DOTMA and DOTAP in that one of the methyl groups of the trimethylammonium group is replaced with a hydroxyethyl group, see structure in Table 1.
  • the DORI lipids are similar to the Rosenthal Inhibitor (Rl) of phospholipase A (Rosenthal, A.F. and Geyer, R.P., J Biol. Chem. 235:2202-2206 (1960).
  • the oleoyl groups of DORI lipids can be replaced with other alkyl or alkene groups such as palmitoyl or stearoyl groups.
  • the hydroxyl group ofthe DORI-type lipids can be used as a site for further functionalization, for example, for esterification to amines, like carboxyspermine.
  • Additional cationic lipids which can be employed in the compositions and methods of this invention include those described as useful for transfection of cells in PCT application WO 91/15501 published Oct. 17, 1991, Pinnaduwage, P. et al, Biochem. Biophys. Acta. 985:33-31 (1989); Rose, J.K., et al, BioTechniques 70:520-525 (1991), Ho, A. et al, Biochem. Intern. 22:235-241
  • DC-Choi Cationic sterol derivatives, like 3 ⁇ [N-(N',N'-dimethyl- aminoethane)carbamoyl] cholesterol (DC-Choi) in which cholesterol is linked to a trialkylammonium group, see Table 1, can also be employed in the present invention.
  • DC-Choi is reported to provide more efficient transfection and lower toxicity than DOTMA-containing liposomes for some cell lines. (Goa, X. and Huang, L., Biochem. Biophys. Res. Comm. 779:280-285 (1991).
  • the polycationic lipid formed by conjugating polylysine to DOPE (Zhou, X. et al, Biochem. Biophys. Acta 1065:8-14 (1991)), as well as other lipopolylysines, can also be employed in this invention.
  • Polycationic lipids containing carboxyspermine are also useful in the practice of this invention.
  • Behr, J-P. etal, Proc. Natl. Acad Sci. (USA) 52:6982- 6986 (1989), and EPO published application 304 111 (1990) describe carboxyspermine-containing cationic lipids including 5-carboxyspermylglycine dioctadecyl-amide (DOGS) and dipalmitoyl-phosphatidylethanolamide 5- carboxyspermylamide (DPPES). Additional cationic lipids can be obtained by replacing the octadecyl and palmitoyl groups of DOGS and DPPES, respectively, with other alkyl or alkene groups.
  • DOGS 5-carboxyspermylglycine dioctadecyl-amide
  • DPPES dipalmitoyl-phosphatidylethanolamide 5- carboxyspermylamide
  • Additional cationic lipids can be obtained by replacing the
  • R, and R 2 separately or together are Cj. 23 alkyl or alkenyl or (-CO-C,. ⁇ ) alkyl or alkenyl, q is 1 to 6, Z x and Z 2 , separately or together, are H or an unbranched alkyl group having one to six carbon atoms and where X can be a variety of groups including haloalkyl, alkylamines, a-l-kyldiamines, alkyltriamines, a-lkyltetraamines, carboxyspermine and related amines, or polyamines including polylysine or polyarginine.
  • Y is H or a group attached by an amide or alkyl amino group (X7) are particularly useful for complexation to nucleic acids.
  • Polycationic lipids such as those of Formula la where X is a spermine (e.g. X5) are preferred.
  • the compounds of Formula lb may also be employed:
  • cationic lipids can optionally be combined with non-cationic lipids, preferably neutral lipids, to form lipid aggregates that complex with nucleic acids.
  • Neutral lipids useful in this invention include, among many others: lecithins; phosphotidylethanolamme; phosphatidyllethanolamines, such as DOPE (dioleoylphosphatidylethanolamine), POPE
  • phosphatidylcholines such as DOPC (dioleoylphosphotidylcholine), DPPC (dipalmitoylphosphatidylcholine), POPC(palmitoyloleoylphosphatidylcholine) and distearoylphosphatidylcholine; phosphatidylglycerol; phosphatidylglycerols, such as DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), and distearoylphosphatidylglycerol; dipalmitoyl phosphatidyl ethanolamine (C-16-PE, FIG.6A); dipalmitoleoyl phosphatidyl ethanolamine (C-16-PE, FIG.6A); dipalmitoleoyl phosphatidyl ethanolamine (C-16-PE, FIG.6A); dipalmitoleoyl
  • phosphatidylserine phosphatidylserines, such as dioleoyl- or dipalmitoylphosphatidylserine; diphosphatidylglycerols; fatty acid esters; glycerol esters; sphingolipids; cardolipin; cerebrosides; and ceramides; and mixtures thereof.
  • Neutral lipids also include cholesterol and other 3 ⁇ OH-sterols. Table 1: Examples of Cationic Lipids
  • R A and R B are selected from the group consisting of H, or an alkyl, hydroxyalkyl or thiol substituted alkyl group having from 1 to about 6 carbon atoms;
  • R] and R 2 are selected from the group consisting of alkyl groups having from 1 to about 6 carbon atoms, where r is either 1 or 0, such that r is 0 or 1 when X is N, r is 0 when X is S or SO, and r is 1 when X is P;
  • A,-A 2 independently of one another, are selected from the group consisting ofthe following groups Z r Z 6 :
  • Zj is a straight-chain alkyl, alkenyl, or alkynyl group having from 2 to about 22 carbon atoms wherein one or more non-neighboring — CH — groups can be replaced with an 0 or S atom;
  • Zj is a branched alkyl, alkenyl, or alkynyl group having from 2 to about 22 carbon atoms wherein one or more non-neighboring -CH 2 - groups can be replaced with an 0 or S atom;
  • Z 3 is a straight-chain or branched alkyl group substituted with one or two
  • Z 3 and X; Z 4 is a substituted straight-chain or branched alkyl, alkenyl or alkynyl group having from 2 to about 22 carbon atoms wherein the substituent is an aromatic, alicyclic, heterocyclic or polycyclic ring and wherein one or more of the non-neighboring -CH 2 - groups of said alkyl, alkenyl or alkynyl group can be substituted with an 0 or S atom.
  • Z 5 is a -B-L group wherein B is selected from the group -CO-, -CO 2 -, -OCO-, -CO-N-, -O-CO-N-, -O-CH 2 -, -CH 2 -O-, -S-, CH 2 - -CH 2 -S- or
  • -CH 2 - and L is selected from the group consisting of: ⁇ j ⁇ 2 > ⁇ -i or an aromatic, alicyclic, heterocyclic or polycyclic ring moiety;
  • Z 6 is a -CH(D-L) 2 or a -C(D-L) 3 group wherein D is selected from the group consisting of -CO-, -CO 2 - -OCO-, -CO-N-, -O-CO-N-, -O-, or -S- and L is selected from the group consisting of: an aromatic, alicyclic, heterocyclic or polycyclic ring moiety.
  • the chain length can vary from 1 to 6.
  • x is 3 where j is 1 to
  • n beaut n 2 and n 3 can all have the same value, any two can have the same value or all three can have different values.
  • the chain length n j vary in the repeating pattern 3, 4, 3, 3, 4, 3, other particular embodiments of this invention include those in which differ from each other by +/- 1.
  • a groups, A,-A t include those in which two substituents on different X's, preferably neighboring X groups, are covalently linked with each other to form a cyclic moiety.
  • the oxygen or sulfur atoms introduced into Z x , and Z 2 groups are preferably introduced within about 3 carbon atoms from the bond to the X group.
  • the cationic lipids of Formula II are useful, either alone or in combination with other neutral lipid aggregate-forming components (e.g., DOPE or cholesterol) for formulation into liposomes or other lipid aggregates.
  • Such aggregates are polycationic, able to form stable complexes with anionic macromolecules, such as nucleic acids.
  • anionic macromolecules such as nucleic acids.
  • the polyanion-lipid complex interacts with cells making the polyanionic macromolecule available for absorption and uptake by the cell.
  • N-alkylated polyamines and their quaternary ammonium salts are particularly useful for intracellular delivery of negatively charged macromolecules.
  • R ⁇ Rj,, IL, and R ⁇ are C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 or C22 straight chain alkyl or alkenyl groups. See, for example, FIG. 6C (compound XXVII) and FIG. 7.
  • the longer chain lipids (C18-C22) lipids are employed.
  • Other preferred lipids include those in the following Table 2:
  • Direct gene transfer to mammalian cells in vivo may be carried out with the use of polynucleotides, e.g. plasmid DNA, or by using a viral or bacterial vector delivery system.
  • polynucleotides e.g. plasmid DNA
  • viral or bacterial vector delivery system e.g. plasmid DNA
  • the use of plasmid DNA vectors is highly preferable to live attenuated vectors owing to the ease of production, non-immunogenic nature of the vector itself, and absence of risk of inadvertent infection.
  • circular DNA is preferable to linear DNA.
  • Plasmid DNA may be used either in a pure form ("naked") or formulated with cationic lipids, however there are conflicting reports in the literature as to their relative efficiencies for directly transferring foreign genes into lung tissue.
  • the present invention relates to studies undertaken to examine the efficiency of gene transfer with different techniques of DNA delivery to the lungs and compare the use of pure plasmid DNA and DNA formulated with 14 different lipids.
  • CF cystic fibrosis
  • CF cystic fibrosis
  • IFN- ⁇ gamma interferon
  • Therapeutic application may also be ex vivo, in which the treated cells, e.g. epithelial cells, are returned to the body. (See, Ponder et al. , Proc. Natl. Acad. Sci. USA 55:1217-1221 (1991).)
  • Antisense oligonucleotides have been described as naturally occurring biological inhibitors of gene expression in both prokaryotes (Mizuno et al. ,
  • Antisense oligonucleotides are short synthetic DNA or RNA nucleotide molecules formulated to be complementary to a specific gene or RNA message. Through the binding of these oligomers to a target DNA or mRNA sequence, transcription or translation of the gene can be selectively blocked and the disease process generated by that gene can be halted (see, for example, Jack
  • Antisense therapy is the administration of exogenous oligonucleotides which bind to a target polynucleotide located within the cells.
  • antisense oligonucleotides may be ad ⁇ iinistered for anticancer therapy (U.S.
  • Ribozymes provide an alternative method to inhibit mRNA function. Ribozymes may be RNA enzymes, self-splicing RNAs, and self-cleaving RNAs (Cech et al. , Journal of Biological Chemistry 267: 17479- 17482 (1992)). It is possible to construct de novo ribozymes which have an endonuclease activity directed in trans to a certain target sequence. Since these ribozymes can act on various sequences, ribozymes can be designed for virtually any RNA substrate. Thus, ribozymes are very flexible tools for inhibiting the expression of specific genes and provide an alternative to antisense constructs.
  • RNA enzymes constructed according to this model have already proved suitable in vitro for the specific cleaving of RNA sequences (Haseloff et al. , supra) .
  • hairpin ribozymes may be used in which the active site is derived from the minus strand of the satellite RNA of tobacco ring spot virus (Hampel et al, Biochemistry 25:4929 ⁇ 933 (1989)). Recently, a hairpin ribozyme was designed which cleaves human immunodeficiency virus type 1 RNA (Ojwang et al, Proc. Natl Acad. Sci. USA 59:10802-10806 (1992)).
  • RNA activities are associated with hepatitis delta virus (Kuo etal, J. Virol 62:4429-4444 (1988)). See also U.S. 5,574,143 for methods of preparing and using ribozymes.
  • ribozyme molecules are designed as described by Eckstein et al (International Publication No. WO 92/07065) who disclose catalytically active ribozyme constructions which have increased stability against chemical and enzymatic degradation, and thus are useful as therapeutic agents.
  • an external guide sequence can be constructed for directing the endogenous ribozyme, RNase P, to intracellular mRNA, which is subsequently cleaved by the cellular ribozyme (Altman et al. , U.S. Patent No. 5,168,053).
  • the EGS comprises a ten to fifteen nucleotide sequence complementary to the target mRNA (corresponding to the miss-sequenced regions) and a 3'-NCCA nucleotide sequence, wherein N is preferably a purine (Id.).
  • the molecules bind to the targeted mRNA species by forming base pairs between the mRNA and the complementary NTP EGS sequences, thus promoting cleavage of mRNA by RNase P at the nucleotide at the 5 'side of the base-paired region (Id.).
  • genes coding for antigenic proteins capable of inducing an immune response when transfected into mucosal epithelial cells include outer membrane proteins from N meningitidis (U.S. 5,439,808), outer membrane proteins from H. influenzae (U.S. 5,196,338, EP 320,289, EP 378,929, WO95/03069, Munson and Tolan, Infect. Immun. 57:88 (1989)), pneumococcal proteins (U.S. 5,476,929); pseudorabies virus proteins (U.S. 4,753,884, U.S. 4,609,548), measles virus proteins (U.S. 5,503,834), herpesvirus proteins (U.S.
  • liposomes comprising the lipids are first prepared by the reverse evaporation method.
  • Cationic lipid-polynucleotide complexes are formed by mixing a cationic lipid solution, e.g. containing liposomes, with a polynucleotide solution.
  • the complexes are mixed by vortexing.
  • the cationic lipid and polynucleotides can be dissolved in any sterile physiologically-compatible aqueous carrier.
  • cationic lipid and polynucleotides are dissolved in sterile saline (150 mM NaCl).
  • the solutions are mixed at ambient temperatures.
  • the solutions are mixed at 25 °C.
  • the cationic lipid-polynucleotide complexes are incubated at room temperature, preferably for 15 to 45 minutes. It has been discovered that the ratio of lipid to DNA is important to the transfection efficiency of the complex. In general, one must combine an amount of lipid that is sufficient to complex substantially the polynucleotide molecule. This amount of lipid can be determined empirically titrating the polynucleotide molecule with the lipid and separating the reaction mixture on an agarose gel. The presence of uncomplexed polynucleotide molecule indicates that more lipid should be added.
  • the ratio of polynucleotide to lipid may range from about 5:1 to 1:50 (w/w), more preferably, about 2:1 ot 1:5 or 1:10, even more preferably about 1:1 to 1:3, and even more preferably about 1:2.
  • the most prefered ratio of polynucleotide to lipid is where the DNA is fully complexed without substantial amounts of excess lipid as determined on a gel. An excess of lipid is to be avoided due to possible toxicity of the lipid.
  • plasmid DNA may be formulated with cationic liposomes, which are themselves composed of a mixture of a cationic lipid and a neutral co-lipid.
  • cationic liposomes which are themselves composed of a mixture of a cationic lipid and a neutral co-lipid.
  • the electrostatic interactions between the net-positively charged lipid and a negatively charged DNA result in the spontaneous formation of lipid-DNA complexes.
  • the neutral lipid acts to increase the stability ofthe liposome, reduce cytotoxity and improve transfection efficiency.
  • the lipid/polynucleotide complex is used to carry out an in vivo transfection of mammalian mucosal epithelial cells.
  • mucosal epithelial cells that may be transfected include those cells in the nasal passageways, the lung, inside the cheek, under the tongue, the rectum, intestines and vagina.
  • Administration of lipid/polynucleotide complexes of the present invention may be by any suitable means that will result in administration to target mucosal epithelium. Such means include intranasal administration, instillation, inhalation, injection and cannulation.
  • the lipid complexes are administered as part of a nasal spray, or inhaled via nebulization into the mouth and/or nose, or via an endotracheal tube.
  • the methods of the invention may be practiced on any mammal which may experience the beneficial effects of the invention.
  • mammals are humans, although the invention is not intended to be so limiting.
  • Transfected cells express a foreign protein encoded by the polynucleotide, and may present the foreign protein on the cell surface.
  • the host animal mounts an immune response to the foreign protein, or immunogen.
  • the lipid/polynucleotide complex can be used as a vaccine to induce an immune response in a mammal.
  • the vaccines comprising the cationic lipid and polynucleotide may be administered in a wide range of dosages. Effective dosages and formulations will depend upon a variety of factors (such as the species of the recipient), and can be determined by one of ordinary skill in the art. Illustrative dosages range from about 1 ⁇ g to about 500 ⁇ g, depending on the animal. For example, the dose for a mouse may be only 10-50% of the human dose. A human baby will require a dose of about 50% of the adult dose. Such doses may be optimized by the clinician with no more than routine experimentation.
  • Illustrative dosages range from about 100 ⁇ g to about 5 mg.
  • the specific dosage administered may be dependent upon the age, weight, kind of current treatment, if any, and nature of the gene which will be expressed.
  • the initial dose may be followed by a booster dosage after a period of about four weeks to enhance the immunogenic response.
  • DNA is administered can require as much as 500 ⁇ g or more of a DNA construct per inoculation.
  • use of particular cationic lipids as a carrier for DNA constructs according to the claimed invention permits genetic immunization with much lower amounts of a DNA construct. Use of lower amounts of DNA constructs is important when the construct is not available in large quantities.
  • the lipid/polynucleotide complexes may be administered to the epithelium of the mammal as part of a pharmaceutical composition comprising a pharmaceutically acceptable carrier such as buffered physiologic saline solution.
  • a pharmaceutically acceptable carrier such as buffered physiologic saline solution.
  • the present invention is also directed to methods of producing immunogen-specific antibodies.
  • Polyclonal antibodies may be isolated and purified from vaccinated animals using procedures well-known in the art (for example, see Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988).
  • This invention is also directed to the use of immunization to produce monoclonal antibodies.
  • a mammal e.g. a mouse
  • B-lymphocytes are isolated from the mammal.
  • the immune response may be monitored by checking the antibody titer of the sera.
  • Monoclonal antibodies are produced following the procedure of K ⁇ hler and Milstein (Nature 256:495-497 (1975) (for example, see Harlow et al., supra). Briefly, monoclonal antibodies can be produced by immunizing mammals with a cationic lipid-polynucleotide complex, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce anti-immunogen antibody, culturing the anti-immunogen antibody-producing clones, and isolating anti-immunogen antibodies from the hybridoma cultures.
  • Tetra alkyl spermine (2.5 mmole) was dissolved in 60 mL of methyl iodide. The mixture was kept in the dark without stirring for 4.5 hours. After removal ofthe methyl iodide on a rotary evaporator, the residue was dissolved in 300 mL of methylene chloride, and extracted twice with 150 mL of 10% NaHCO 3 solution. After concentration, the desired tetramethyl tetraalkyl spermine was obtained as a light brown solid. Characterization was done by IR, NMR and FAB-MS. Example 2 In Vivo Transfection Studies
  • DNA concentration was calculated based on absorbance of ultraviolet light (OD 260) with final concentrations usually being 5-10 mg/ml. DNA solutions were stored at -20°C until required for administration. DNA was administered either as pure plasmid DNA in saline ("naked" DNA) or formulated with the cationic lipids described below.
  • the lipids were formulated into a liposome using the reverse evaporation method.
  • a known amount of cationic lipid was mixed with the neutral co-lipid in a 2L round bottom flask to obtain the desired molar ratio of cationic:neutral co- lipid.
  • Methylene chloride or 10% methanol in methylene chloride is added to make a 10-30 mg/mL solution.
  • GIBCO water is added to give approximately a
  • the lipid-formulated DNA was administered in a total volume of 150 ⁇ l that contained 1-100 ⁇ g DNA and an amount of lipid that was from 0.2 to 5 times by weight the amount of DNA. Prior to formulation with DNA, the solutions of lipids were thoroughly mixed by vortexing.
  • DNA was added and the mixture was briefly vortexed again. The final mixture was left, undisturbed on ice for 30 minutes to allow DNA/liposome complex formation. Immediately before administration, the DNA/lipid mixture was mixed gently by tapping on the side of Eppendorf tube. To determine if all the DNA was complexed with the lipid, mixtures were run on a 0.5% agarose gel and stained with 0.04 ⁇ g/ml ethidium bromide. The presence of free plasmid was detected as a band at the expected distance whereas
  • DNA-lipid complexes failed to enter the gel.
  • mice (Charles River, Montreal, QC) aged 6-8 weeks with 10 mice per experimental or control group. Each animal received naked or lipid-formulated DNA in a total volume of 150 ⁇ l, although this contained different doses of DNA and different relative proportions of DNA and lipid.
  • the DNA was administered to the respiratory system ofthe mice via intranasal (IN) or intratracheal (IT) routes.
  • D ⁇ A solution was then instilled bilaterally into the nasal cavity using a gel-loading tip and a Gilson pipette. The tip was inserted a few mm into the nasal cavity and each nostril was instilled 3 times at 15 minute intervals with 25 ⁇ l of D ⁇ A solution (for a total volume of 150 ⁇ l/mouse).
  • the D ⁇ A was delivered by a pipette tip, but this was not placed inside the nasal cavity. Rather, the D ⁇ A solution was deposited as droplets applied bilaterally directly over the external nares of mice under Halothane anesthesia. A volume of 25 ⁇ l was placed over each nostril 3 separate times (for a total volume of 150 ⁇ l) with 15 minute intervals between administrations.
  • mice fully anaesthetized with Somnotol® (75 mg/kg IP; MTC Pharmaceuticals, Cambridge, ON) had their trachea exposed through an anterior midline incision.
  • the DNA solution 150 ⁇ l was then injected through the anterior wall of the trachea using a 0.3 cc insulin syringe with a 29-gauge needle attached (Becton Dickenson, Franklin Lakes, New Jersey, USA). This was given either as a single injection or as three injections of 50 ⁇ l each, with 15 minute intervals between administrations.
  • the incision was sutured and the mouse was placed in an incubator until fully recovered from the anesthetic (approximately 45 min).
  • mice under Somnotol® anesthesia had their trachea exposed via an anterior midline incision, and this was used to visualize the insertion ofthe cannula into the trachea.
  • a 20-gauge olive tip steel feeding tube
  • mice were killed by cervical dislocation under Halothane anesthesia at various times from 6 hr to 19 days after gene transfer.
  • the lungs were dissected free, homogenized and assayed for luciferase reporter gene activity using the Promega Luciferase Assay System (Madison, WI, USA) according to a method previously described for muscle (Davis, H.L., et al. , Human Gene Ther. 4:151-
  • mice received a total of 1, 10, 25, 50 or 100 ⁇ g of pCMV-luc DNA, formulated with lipid 301 at a 1:1 DNA:lipid ratio (w/w). This was given by IN inhalation in a total volume of 150 ⁇ l. Lungs were assayed for luciferase activity at 48 hr. Longevity of reporter gene expression. A total of 100 ⁇ g pCMV-luc DNA formulated with lipid 301 at a 1 :1 DNA:lipid (w/w) ratio was administered by IN inhalation to each mouse. Lungs were removed from mice killed at 6 hr, 1, 2, 3,
  • Lipid composition A total of 100 ⁇ g pCMV-luc DNA was formulated with each of the 14 different lipids (see Table 2) at a 1 : 1 DNA:lipid (w/w) ratio.
  • the lipid-formulated DNA is superior to pure plasmid DNA, giving approximately 10-fold higher levels of luciferase reporter gene activity.
  • the two IN routes are basically equivalent to each other, which is not unexpected since both methods deliver the DNA into the nasal cavity, from where a large portion of it reaches the lungs.
  • the IT cannulation results in about 10-fold higher reporter gene activity than IT injection. This difference is likely due to leakage from the perforated trachea with the injection method.
  • the cannulated IT route has somewhat higher efficiency of transfection that the two IN routes and the IT injection, however the latter methods result in less variability (coefficient of variation >150% and ⁇ 100% respectively).
  • the IN inhalation method was chosen for assay of different lipids since it is easy and non-invasive and has less variability.
  • lipids that worked poorly (304, 305 [lipofectamine], 201, 202, 203, 204, 205, 206 and lipofectin), all resulted in mean luciferase activities less than that obtained with naked DNA (FIG. 5). All lipids had been given the same 1:1 DNA:lipid (w/w) ratio since it had been verified by agarose gel electrophoresis that they each bound similar quantities of DNA.
  • the present study demonstrates that it is possible for epithelial mucosal cells in the lung to take up and express foreign genes after administration of plasmid DNA in naked form or formulated with cationic lipids.
  • the efficiency of gene transfer with cationic lipid-formulated DNA can be higher then, the same as, or less than that with pure plasmid DNA.
  • DNA solutions may be administered intranasally (inhaled or instilled) or directly into the lungs via the trachea (injected or cannulated), with all four methods resulting in roughly equivalent efficiencies of transfection.
  • Pure plasmid DNA results in an extremely low efficiency of transient transfection of cells in vitro, and thus this procedure is usually carried out by formulating the DNA with cationic lipids or by some other method such as calcium phosphate precipitation or electroporation.
  • the situation is somewhat different in vivo in that some types of cells, most notably mature muscle fibers, actually take up and express genes significantly better if given in the form of pure plasmid DNA (Wolff, J.A., et al, Hum. Mol. Genet. 7:363-369 (1992)).
  • Cationic lipids form complexes with plasmid DNA via ionic interactions and are thought to bring about gene transfer by attaching to cell surfaces where they can either fuse directly or be endocytosed with subsequent intracellular fusion with endosomal membranes. As the lipid diffuses into the membrane, the lipid-DNA ionic interactions are disrupted, thereby releasing the DNA into the cytoplasm (Feigner et al., Ann. N.Y. Acad. Sci. 772:126-139 (1995)). Liposomes may also help improve the efficiency of transfection owing to increased retention time and slower degradation ofthe complexed DNA. There has been conflicting reports as to the relative efficiencies of pure plasmid DNA and liposome-formulated DNA for the direct gene transfer in the lung. Some investigators have reported that liposome-formulated but not naked
  • the DNA can be delivered to the lung by a wide variety of techniques including IN inhalation or instillation, or IT injection or cannulation.
  • IT cannulation which delivers all ofthe DNA directly to the lungs, and the lowest with IT injection, which appears to result in losses of DNA through the punctured trachea.
  • the two IN methods were equivalent to each other and although the DNA must first pass through the nose and throat, where some is most certainly swallowed, the efficiency of lung transfection was only slightly lower and much less variable than that with IT cannulation. This, in addition to the fact that IN administration is easy and quick to perform and non-invasive, makes IN the preferred method for animal studies.
  • lipid 305 Half ofthe lipids tested, including the two commercially available lipids lipofectin and lipofectamine (lipid 305), resulted in luciferase activity lower than that obtained with pure plasmid DNA. It was also found that different lipids have different capacities for complexing with DNA and the best results are obtained when the lipid is mixed with the maximum amount of DNA it can complex, but not more. Thus, it is important to determine the optimal DNA:lipid ratio by checking for non-associated DNA with gel electrophoresis. As well, it is necessary to be aware of the kinetics of gene expression as there is a rather brief period of peak expression.
  • DNA vaccines need to transfect only relatively few cells in order to induce potent immune responses. As such, the level of reporter gene activity in lungs of mice after direct introduction of either pure plasmid DNA or liposome-formulated DNA will be sufficient to induce immune responses to an antigenic protein.
  • most previous studies on DNA vaccines have involved transfection of cells in muscle tissue, the epidermis or dermis (Davis, H.L., and Brazolot Millan, C.L., Blood
  • DNA-based immunization via the mucosal epithelium, e.g. the respiratory system is an easy, non-invasive method to immunize individuals without the use of trained medical personnel.
  • the possibility to carry out mucosal immunization is highly desirable since it triggers both a mucosal and a systemic response, in contrast to systemic immunization which induces solely systemic immunity.
  • immunization at one mucosal surface has been shown to induce immunity at distant mucosal sites. Mucosal immunity is important to prevent pathogen entry at mucosal surfaces, whereas systemic immunity can only deal with pathogens once they have entered the body.
  • Luciferase reporter gene expression in the lung peaked at about 4 days and then fell off rapidly between 5 and 9 days, similar to as has been reported by other investigators (Meyer, K.B., etal, Gene Ther. 2:450-460 (1995); Wheeler, C.J., et ⁇ /., Proc. Natl. Acad. Sci. USA 93: 1454-11459 (1996)).

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Abstract

La présente invention concerne des compositions et procédés de transfection des épithéliums muqueux de mammifères avec des complexes acides nucléiques - lipides cationiques. Ces complexes acides nucléiques - lipides cationiques peuvent s'administrer dans les poumons, soit directement, soit par voie intranasale. On obtient les meilleurs résultats lorsque les lipides sont mélangés à la quantité maximale d'ADN avec laquelle il peuvent constituer un complexe.
PCT/US1997/003421 1997-03-10 1997-03-10 Apport de genes dans l'epithelium muqueux a des fins therapeutiques ou d'immunisation WO1998040499A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000027795A1 (fr) * 1998-11-12 2000-05-18 Invitrogen Corporation Reactifs de transfection
US6110898A (en) * 1996-05-24 2000-08-29 University Of Maryland, Baltimore DNA vaccines for eliciting a mucosal immune response
FR2794648A1 (fr) * 1999-06-10 2000-12-15 Merial Sas Vaccins adn pour animaux de compagnie et de sport
WO2000077043A3 (fr) * 1999-06-10 2001-07-19 Merial Sas Vaccins adn pour animaux de compagnie et de sport
FR2804028A1 (fr) * 2000-01-21 2001-07-27 Merial Sas Vaccins adn ameliores pour animaux de rente
WO2001015755A3 (fr) * 1999-09-01 2002-02-28 Genecure Pte Ltd Procedes et compositions d'administration d'agents pharmaceutiques
US6852705B2 (en) 2000-01-21 2005-02-08 Merial DNA vaccines for farm animals, in particular bovines and porcines
US6943152B1 (en) 1999-06-10 2005-09-13 Merial DNA vaccine-PCV
US7078388B2 (en) 2000-01-21 2006-07-18 Merial DNA vaccines for farm animals, in particular bovines and porcines
US7390494B2 (en) 1997-12-05 2008-06-24 Wyeth Circovirus sequences associated with piglet weight loss disease (PWD)
WO2013086243A3 (fr) * 2011-12-07 2013-09-19 Omega Protein Corporation Compositions de phospholipides enrichies pour les acides palmitoléique, myristoléique ou lauroléique, leur préparation et leur utilisation dans le traitement de maladie métabolique et cardiovasculaire
US9879243B2 (en) 2001-03-27 2018-01-30 Lifetechnologies Corporation Culture medium for cell growth and transfection
US9987348B2 (en) 2013-09-25 2018-06-05 Zoetis Services Llc PCV2B divergent vaccine composition and methods of use
US10195280B2 (en) 2014-07-15 2019-02-05 Life Technologies Corporation Compositions and methods for efficient delivery of molecules to cells
WO2025021083A1 (fr) * 2023-07-24 2025-01-30 National Institute Of Biological Sciences, Beijing Compositions de nanoparticules lipidiques pour le traitement localisé de maladies de la peau

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5589466A (en) * 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5589466A (en) * 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALBRECHT et al., "Cationic Lipid Mediated Transfer for c-abl and bcr Antisense Oligonucleotides to Immature Normal Myeloid Cells: Uptake, Biological Efects and Modulation of Gene Expression", ANNALS OF HEMATOLOGY, February 1996, Vol. 72, No. 2, pages 73-79. *
HARRISON et al., "Optimization of Gene Transfer Using Cationic Lipids in Cell Lines and Primary Human CD4+ and CD34+ Hematopoietic Cells", BIOTECHNIQUES, 1995, Vol. 19, No. 5, pages 816-820 and 822-823. *
NABEL et al., "Direct Gene Transfer for Treatment of Human Cancer", ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, November 1995, Vol. 772, pages 227-231. *
NABEL et al., "Immune Response in Human Melanoma After Transfer of an Allogeneic Class I Major Histocompatibility Complex Gene with DNA-Liposome Complexes", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, USA., December 1996, Vol. 93, pages 15388-15393. *
STOPECK et al., "Phase I Study of Direct Gene Transfer of an Allogeneic Histocompatibility Antigen, HLA-B7, in Patients with Metastatic Melanoma", JOURNAL OF CLINICAL ONCOLOGY, January 1997, Vol. 5, No. 1, pages 341-349. *

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US7145039B2 (en) 1998-11-12 2006-12-05 Invitrogen Corp. Transfection reagents
US7166745B1 (en) 1998-11-12 2007-01-23 Invitrogen Corporation Transfection reagents
US7173154B2 (en) 1998-11-12 2007-02-06 Invitrogen Corp. Transfection reagents
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US7470817B2 (en) 1998-11-12 2008-12-30 Invitrogen Corporation Transfection reagents
WO2000077043A3 (fr) * 1999-06-10 2001-07-19 Merial Sas Vaccins adn pour animaux de compagnie et de sport
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US6943152B1 (en) 1999-06-10 2005-09-13 Merial DNA vaccine-PCV
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US6852705B2 (en) 2000-01-21 2005-02-08 Merial DNA vaccines for farm animals, in particular bovines and porcines
FR2804028A1 (fr) * 2000-01-21 2001-07-27 Merial Sas Vaccins adn ameliores pour animaux de rente
AU783865B2 (en) * 2000-01-21 2005-12-15 Boehringer Ingelheim Animal Health USA Inc. Improved DNA vaccines for production-type animals
US9879243B2 (en) 2001-03-27 2018-01-30 Lifetechnologies Corporation Culture medium for cell growth and transfection
US9758536B2 (en) 2011-12-07 2017-09-12 Omega Protein Corporation Phospholipid compositions enriched for palmitoleic, myristoleic or lauroleic acid, their preparation and their use in treating metabolic and cardiovascular disease
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