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WO1997034483A1 - Procedes pour augmenter ou diminuer l'efficacite d'une transfection - Google Patents

Procedes pour augmenter ou diminuer l'efficacite d'une transfection Download PDF

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Publication number
WO1997034483A1
WO1997034483A1 PCT/US1997/004217 US9704217W WO9734483A1 WO 1997034483 A1 WO1997034483 A1 WO 1997034483A1 US 9704217 W US9704217 W US 9704217W WO 9734483 A1 WO9734483 A1 WO 9734483A1
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cells
transfection
cationic
expression
genetic material
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PCT/US1997/004217
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English (en)
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Kimberly Ann Mislick
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California Institute Of Technology
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Priority claimed from US08/644,095 external-priority patent/US5783566A/en
Application filed by California Institute Of Technology filed Critical California Institute Of Technology
Priority to AU22145/97A priority Critical patent/AU2214597A/en
Publication of WO1997034483A1 publication Critical patent/WO1997034483A1/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
    • 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
    • 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

Definitions

  • This invention is in the field of transfection of cells with genetic material, and relates to methods for increasing or decreasing transfection efficiency.
  • the genetic material may be introduced into the cell to correct a functional cellular defect, to augment an immune response or to express a foreign protein.
  • the inhibition may be inhibition of viral infection or minimization of transfection at a non-desired site.
  • the transfection efficiency can be controlled in vivo, ex vivo and in vitro.
  • Gene transfer is a term that broadly encompasses methods for introducing exogenous gene sequences into a cell or group of cells. There are a variety of methods known to those of skill in the art for introducing exogenous genetic material into a host cell. These methods include, but are not limited to, calcium precipitation, viral vector-mediated delivery, electroporation, and complexing the DNA with a cationic substance.
  • Such cationic substances include, but are not limited to, cationic lipids such as lipofectin and DOTMA (N-[1-(2,3-dioleyloxy)propyl]- N,N,N-trimethylammonium), cationic liposomes prepared from cationic lipids, cationic polyamino acids such as poly-L-lysine and polyornithine, cationic amphiphiles, polyethyleneimine, dendrite polymers with cationic substituents, and DEAE-dextran.
  • cationic lipids such as lipofectin and DOTMA (N-[1-(2,3-dioleyloxy)propyl]- N,N,N-trimethylammonium)
  • cationic liposomes prepared from cationic lipids
  • cationic polyamino acids such as poly-L-lysine and polyornithine
  • cationic amphiphiles polyethyleneimine
  • Gene transfer /t7 vitro may be used to study the effect of a given gene and its resulting gene product on a given population of cells. Gene transfer is typically directed to the areas of gene replacement and augmentation. Gene augmentation introduces a correct copy of a mutated gene into defective cells or a copy of a foreign sequence for gene expression within that cell. Gene replacement corrects defective genetic sequences, permitting targeted homologous recombination for a known gene sequence. Both approaches generally require that the exogenous genetic material, for example, polynucleotide sequences, be stably expressed, rather than being transiently expressed.
  • Genes can also be introduced that make cells chemosensitive.
  • a disease is encoded by multiple, discontinuous genes. In this case, replacing a single gene is unlikely to lead to eradication of the disease.
  • a "suicide" gene may be transfected to induce the self-destruction of diseased cells and tissues. This approach is currently being developed to treat a number of cancers.
  • target tissues are transfected with the Herpes Simplex Virus-thymidine kinase (HSV-tk) gene and then treated with gancyclovir, a nucleotide analog. Phosphorylated gancyclovir produced by transfected cells is incorporated into genomic DNA and further elongation is prevented.
  • HSV-tk Herpes Simplex Virus-thymidine kinase
  • This mode of treatment is enhanced by the "by ⁇ stander effect" i.e., the passage of phosphorylated gancyclovir into neighboring non-transduced cells through gap junctions.
  • the growth of the tumor is inhibited by transfecting only a fraction of the tumor mass.
  • expression can be either stable or transient.
  • genetic material such as modified and un- modified DNA and RNA sequences, is not used for gene replacement or augmentation, but for binding to or interacting with various sites.
  • SELEX process The identification and use of such oligomers is referred to as the SELEX process, and the oligomers identified by the process are referred to as SELEX oligomers.
  • SELEX process and SELEX oligomers are described in U.S. Patent No. 5,270,163, the contents of which are hereby incorporated by reference in its entirety. Uses for the oligomer include in vitro diagnostic applications and in vivo or ex vivo therapeutic applications.
  • Ex vivo gene augmentation or gene replacement involves removing cells from a patient, optionally expanding these cells in culture, transfecting the cells and identifying stable transfectants. After stable transfectants are identified, the cells are optionally expanded and returned to the host. This procedure has been used most successfully to transfect cells of hematopoietic origin.
  • fibroblasts Other cell types that have been stably transfected in culture include fibroblasts, myoblasts and hepatocytes. Stable cell lines have been used experimentally for certain applications in place of autologous host cells. These procedures require that the cells are maintained in culture.
  • a major limitation of in vitro expansion before transfection and the selection of stably transfected cells following transfection is the length of time the cells spend in culture. The time required to obtain cells from a patient, treat the cells, select stable transfectants and return these to their host can be a matter of weeks to months. In the absence of the in vivo cellular milieu, protein expression may change. Over time, certain subpopulations of cells survive under selection conditions and replicate better than others.
  • the conditions used to select stable transfectants also selects for cells able to survive in culture.
  • Cell survival in vitro does not necessarily translate into improved cell survival in vivo.
  • Cells adapted to culture or cells expanded in culture may not be able to survive in vivo.
  • a method that minimizes cell time in culture, i.e., by increasing the transfection efficiency, may reduce the likelihood that cell changes occur in vitro.
  • DOTMA cationic lipids
  • DOTMA cationic lipids
  • DNA complexes that purportedly fuse with negatively charged lipids associated with cell membranes (Feigner, P.L. et al, Proc. Natl. Acad. Sci. (USA) 84:7413-7417 (1987)) and U.S. Patent No. 4,897,355 to Eppstein, D. et al).
  • Cationic liposomes have been used in vitro to introduce genetic sequences into tissue culture cells (Mannino, R.J., Fould-Fogerite, S., Biotechniques 6:682 - 690, 1988).
  • Liposome-mediated gene delivery can deliver mRNA, RNA, DNA, a modified polynucleotide or oligonucleotide, or a combination thereof directly into the cell cytoplasm (Malone et al., Proc. Natl. Acad. Sci. (USA) 86:6077-6081 , (1989)).
  • Variables thought to affect the efficiency of gene expression include the type of lipid in the cationic liposomes, the relative amounts of DNA and lipid, the day of assay following transfection, the media used for lipid:DNA complex formation, the promoter driving expression observed with the reporter gene, the physiological state of the cells (e.g., whether or not the cells were differentiated) and the type of cell. Maximum expression in transfected primary cells was one to two orders magnitude lower than with cell lines. Harrison et al., Biotechniques, 19(5):816-823 (November 1995).
  • Sulfated proteoglycans are among the most negatively charged components of the cell. They consist of a core protein covalently linked to one or more of four types of sulfated glycosaminoglycans. Heparin and heparan sulfate are the most anionic glycosaminoglycans, followed by dermatan sulfate, chondroitan sulfate, and keratan sulfate. Although most well-known for their structural contribution to basement membranes and the extracellular matrix, proteoglycans participate in a variety of cellular events as membrane-bound proteins.
  • Membrane-associated heparan sulfate proteoglycans mediate infection by the Herpes Simplex Virus the binding of low-density lipoprotein and epidermal growth factor. Chondroitan sulfate proteoglycans promote the attachment of human foreskin fibroblasts to cationic diamine derivatized glass. Moreover, cellular proteoglycans bind to gold-labeled polylysine, a technique used to localize them by electron microscopy However, the role of proteoglycans and glycosaminoglycans in cationic liposome-mediated transfection has not been extensively studied.
  • Non-differentiated cells are extremely difficult to transfect. However, because these cells are non-differentiated, it would be extremely advantageous to transfect such cells. For example, treating cells when non- differentiated can correct hereditary disorders such as beta-thalassemia and sickle cell anemia. It is known that the surface concentration of proteoglycans on non-differentiated cells is very low When these cells begin to differentiate, proteoglycan expression on the cell surface is increased. By understanding the reasons why the transfection efficiency of these cells is typically low, it may be possible to alter the transfection conditions to transfect these cells successfully It is therefore an object of the present invention to provide methods for increasing the efficiency of transfection, whether performed in vitro, in vivo, or ex vivo. It is a further object of the present invention to provide methods for decreasing the efficiency of transfection. It is still a further object of the present invention to provide a method for improving the transfection efficiency for non-differentiated cells.
  • the present invention provides methods for controlling transfection mediated by complexes of genetic material with a cationic species, and, in particular, cationic liposomes. Transfection mediated by these complexes is known in the art. This method of transfection is described with respect to complexes including cationic liposomes, for example, in PCT WO 93/14889 to Vical, Inc., the contents of which are hereby incorporated by reference.
  • the genetic material is preferably used herein as a complex with a cationic species.
  • the size of poly-L-lysine-DNA complexes and complexes of genetic material and cationic liposomes used for transfection is typically between approximately 10 and 300 nm.
  • complexes for use in the present methods have a size between approximately 10 and 300 nm, and, more preferably, between 20 and 150 nm.
  • Transfection efficiency can be lowered by lowering the proteoglycan expression on the cell surface, and can be increased by increasing the proteoglycan expression on the cell surface.
  • Various compounds are known to increase the amount of proteoglycans on the cell surface. These compounds include, but are not limited to, phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12-O-myristoyl-13- acetate (PMA), EGF, IGF-1 , IL-3, IL-5, FGF, Interferon, PDGF, and TGF beta.
  • Preferred compounds for increasing the amount of proteoglycans on the cell surface are phorbol esters. More preferably, the phorbol esters are TPA or PMA.
  • cells to be transfected are contacted with an effective amount of a complex of a cationic substance and genetic material to effectively transfect all or a portion of the cells, and an effective amount of a material known to increase the expression of proteoglycans on the cell surface.
  • Genetic material can also be delivered to a cell ex vivo.
  • live cells are first removed from the organism to be transfected. Then, the cells are contacted with a preparation that includes an effective amount of a complex of a desired genetic material and a cationic substance, preferably a cationic lipid, to deliver the genetic material into the cells, in combination with an effective amount of a substance known to increase proteoglycan expression on the cell surface. After transfection, the cells are returned to the organism.
  • the amount of proteoglycans on the cell surface is increased to increase the binding of the complex with the cell surface. In this fashion, the efficiency of transfection is increased.
  • ex vivo transfection can include a selection step to separate or expand the transfected cells.
  • the cells are substantially separated from the surrounding extracellular matrix.
  • the expression may be transient, or may persist for a substantial length of time.
  • the genetic material encodes a polypeptide that is adapted to treat a disease caused by a functional gene deficiency.
  • the polypeptide is an immunogenic polypeptide in the organism.
  • the organism which is preferably a mammal, and, more preferably a human, develops an immune response against the immunogen after the transfected cells are returned. This method may be used to immunize the organism.
  • the genetic material operatively codes for lymphokine.
  • Cells that may be transfected include, but are not limited to, white blood cells, myoblasts, and bone marrow cells.
  • Transfection efficiency can be increased by lowering the plasma concentration of glycosaminoglycans and, optionally, other polyanionic species, and can be decreased by increasing the plasma concentrations of glycosaminoglycans.
  • Various compounds are known to lower the plasma concentration of glycosaminoglycans. These compounds include, but are not limited to, protease inhibitors, plasma lipoproteins, growth factors, lipolytic enzymes, extracellular matrix proteins, and platelet factor 4.
  • Preferred compounds for minimizing the plasma concentration of glycosaminoglycans are protease inhibitors and plasma lipoproteins.
  • the compounds are protease inhibitors.
  • the complex is administered with an effective amount of a substance that increases the expression of proteoglycans on the cell surface, and optionally with an effective amount of a compound that decreases the plasma concentration of glycosaminoglycans.
  • the complex and the compounds can be administered at the same time or within a reasonable time of each other, so long as the overall effect is that the cell surface concentration of proteoglycans increases and, optionally, the plasma concentration of glycosaminoglycans decreases, when transfection occurs.
  • the mode of administration can be any mode that effectively targets the complex to the desired cells.
  • Suitable modes of administration for use in the present invention include, but are not limited to, oral, parenteral, intravenous, intramuscular, intrauteral, intraperitoneal, and intranasal.
  • lung tissue can be targeted by administering the complex intranasally via an aerosol.
  • the liposome can be prepared to specifically target a certain cell type.
  • certain antibodies are known to target liposomes to various cells.
  • Tumor cells or ischemic tissue can be targeted by using unilamellar vesicles less than 200 nm in diameter.
  • Non- selectively minimizing transfection efficiency can be important in preventing or minimizing viral infections, in cases where viruses bind and enter cells through polyanionic sites on the cell surface, rather than to other cell receptors.
  • the amount of proteoglycans on the cell surface can be reduced, and/or the concentration of glycosaminoglycans or other polyanionic substances in the plasma can be increased.
  • the amount of proteoglycans on the cell surface can be minimized by adding various compounds that are known to lower cell surface concentration of proteoglycans.
  • Examples of these compounds include, but are not limited to, xylosides and catabolic cytokines such as GM-CSF, IL-1 alpha and beta, and TNF-alpha.
  • Preferred compounds for minimizing the amount of proteoglycans on the cell surface are xylosides and catabolic cytokines such as IL-1 alpha and beta, and TNF-alpha.
  • the plasma concentration of glycosaminoglycans can be lowered by adding an effective amount of a compound that decreases the plasma concentration of glycosaminoglycans.
  • these compounds include, but are not limited to, protease inhibitors, plasma lipoproteins, growth factors, lipolytic enzymes, extracellular matrix proteins, and platelet factor 4.
  • Plasma concentration of glycosaminoglycans and other polyanions can be increased by either direct administration of glycosaminoglycans or other polyanions, or by administration of compounds known to increase the plasma concentration of these species.
  • Methods for selectively reducing transfection efficiency can be used to target cells in which transfection is not desired, by lowering the proteoglycan expression on the surface of those cells, and then introducing genetic material to cells in which transfection is desired.
  • an effective amount of a compound that minimizes proteoglycan expression on the cell surface is administered to the cells in which transfection is not desired.
  • the administration to these cells can be by any suitable mode that localizes these compounds in the desired cells. Suitable modes for administering these compounds include those described above for in vivo transfection.
  • Transfection efficiency can also be controlled by modifying proteoglycan expression such that the cells express the various proteoglycans on the cell surface in a different ratio than untreated cells.
  • higher concentrations of chondroitan-based proteoglycans on the surface of cells causes increased transfection efficiency, whereas in others, higher concentrations of heparan sulfate-based proteoglycans on the surface of cells causes increased transfection efficiency.
  • One of skill in the art can readily determine which proteoglycans are preferred. Methods for modulating the ratio of heparan sulfate to chondroitin sulfate using glycosaminoglycan biosynthesis inhibitors is known in the art, as described in Timar, et al., Int.
  • glycosaminoglycan biosynthesis inhibitors for use in the present invention include, but are not limited to, ⁇ -D-xyloside, 2-deoxy-D-glucose, ethane-1-hydroxy-1 ,1- diphosphonate and 5-hexyl-2-deoxyuridine.
  • Transfection efficiency can be increased by complexing genetic material with a cationic lipid that is covalently or ionically bound to an agent known to increase the amount of proteoglycans on the cell surface.
  • a cationic lipid that is covalently or ionically bound to an agent known to increase the amount of proteoglycans on the cell surface.
  • neutral lipids, lysolipids and neutral phospholipids can be covalently or ionically bound to an agent known to increase the amount of proteoglycans on the cell surface, and these modified lipids can be included in a cationic liposome formulation.
  • Cationic liposomes prepared from the resulting lipids also increase transfection efficiency by increasing the concentration of cell surface proteoglycans.
  • phorbol esters such as TPA and PMA can be reacted with a suitable lipid with one or more hydroxy or amine groups to form an ester or amide linkage.
  • Anabolic cytokines with reactive functional groups can be similarly coupled to suitable lipids using known chemistry.
  • lipids covalently or ionically linked with substances known to decrease the cell surface expression of proteoglycans can be administered to those cells in which transfection is not desired, before a complex of genetic material and a cationic species is administered to cells in which transfection is desired.
  • xylosides or catabolic cytokines with reactive functional groups can be covalently linked to suitably functionalized lipids to prepare modified lipids that reduce the cell surface expression of proteoglycans.
  • Cytokines whether anabolic, catabolic or modulatory, can be covalently linked to these lipids to form compounds that increase, decrease or otherwise modulate expression of proteoglycans on the cell surface.
  • the complex is co-administered with a therapeutic agent.
  • Suitable therapeutic agents for use in practicing the present invention include, but are not limited to, cytotoxic agents, antifungal agents, antibacterial agents, antiviral agents, immunomodulating agents, anti ⁇ inflammatory agents, vasoconstrictors, and vasodilators.
  • the efficiency of transfection of non- differentiated cells can be increased by causing the non-differentiated cells to express proteoglycans at or near the time the cells are transfected.
  • Figure 1 is a graph of the effect of net complex charge on expression of DNA, showing the percent of maximum expression versus the ratio by charge of lysine to nucleotide.
  • Figure 2A is a graph of the effect of the concentration of chlorate ion on luciferase expression, showing the percent of expression in untreated cells versus the millimolar concentration of chlorate ion.
  • Figure 2B is a bar graph of the effect of the chlorate ion treatment on the binding of PLL-DNA to HeLa cells. The percent binding of control is shown for chlorate ions, a control group without chlorate ions, and a combination of chlorate and sulfate ions.
  • Figure 3A is a bar graph of the effect of lyases on expression. The percent expression of untreated cells is shown for a group treated with chondroitinase ABC and for a group treated with heparinase.
  • Figure 3B is a bar graph of the effect of exogenous glycosaminoglycans on expression. The percent expression of untreated cells is shown for groups treated with heparan sulfate, heparin, hyaluronic acid, chondroitin sulfate A, chondroitin sulfate B, and chondroitin sulfate C.
  • Figure 3C is a bar graph of the effect of glycosaminoglycans on binding. The percent binding of untreated cells is shown for groups treated with heparan sulfate (HS), heparin (H), hyaluronic acid (HUA), chondroitin sulfate A (CSA), chondroitin sulfate B (CSB), and chondroitin sulfate C (CSC).
  • HS heparan sulfate
  • H heparin
  • HUA hyaluronic acid
  • CSA chondroitin sulfate A
  • CSB chondroitin sulfate B
  • CSC chondroitin sulfate C
  • Figure 4A is a bar graph showing luciferase expression in wild-type and mutant Chinese hamster ovary (CHO) cells.
  • Figure 4B is a bar graph of the effect of proteoglycan expression on the binding of wild-type and mutant CHO cells.
  • Figure 5A is a bar graph showing the luciferase expression in relative light units (RLU) for cationic lipid-mediated transfection in wild-type and mutant Chinese hamster ovary (CHO) cells.
  • the solid lines represent expression in mutant cells, and the dashed lines represent expression in wild- type cells.
  • Figure 5B is a bar graph showing the dependence of the correlation factor X (the ratio of the luciferase expression in wild-type and mutant Chinese hamster ovary (CHO) cells) on the cationic lipids used for transfection.
  • the correlation factor X the ratio of the luciferase expression in wild-type and mutant Chinese hamster ovary (CHO) cells
  • Transfection is defined herein as the intracellular delivery of genetic material, for example, DNA and mRNA, into the cells of an organism, preferably a mammal, and more preferably, a human.
  • the genetic material is expressible, and produces beneficial or interesting proteins after being introduced into the cell.
  • the genetic material is used to bind to or interact with a site within the cell, or encodes a material that binds to or interacts with a site within the cell.
  • a virus binds to and enters cells via polyanionic sites on the cell surface
  • the methods for increasing or decreasing transfection efficiency also have an effect on viral infection.
  • polypeptide and "protein” are used interchangeably.
  • Suitable cell types that can be transfected using the methods described herein include, but are not limited to, fibroblasts, myoblasts, hepatocytes, cells of hematopoetic origin such as white blood cells and bone marrow cells, cancer cells and ischemic tissue. Transfection can be performed in vitro, ex vivo, or in vivo.
  • the genetic material can be transiently expressed or stably expressed.
  • In vitro transfection involves transfecting cells outside of the living organism, for example, using cell cultures.
  • In vivo transfection involves transfecting cells within a living organism.
  • Ex vivo transfection involves removing cells from an organism, transfecting all or a portion of the cells, and returning the cells to the organism.
  • removing is used to describe any method known to those with skill in the art to obtain a sample of live cells from an organism.
  • Methods to remove a live cell sample include, but are not limited to, venipucture, cell scraping, and biopsy techniques that include punch biopsy, needle biopsy and surgical excision.
  • returning includes methods known to those with skill in the art to replace cells in the body. These include, but are not limited to, intravenous introduction, surgical implantation and injection.
  • Transient gene expression is generally defined as temporary gene expression that diminishes over time under selective conditions. Transient expression can more broadly be defined as gene expression occurring over periods of less than one year to periods as short as one week or one month.
  • the gene therapy application, the vector construct, whether or not chromosome integration has occurred, the cell type and the location of cell implantation following transfection will all influence the length of time that a particular gene is expressed.
  • Transient expression is often desirable in the practice of the present invention to permit use of the transiently transfected cells as a drug.
  • a gene can be administered periodically, the dosage can be adjusted, and the effect is ultimately transient.
  • Stable gene expression is generally defined as gene expression that does not significantly diminish over time, wherein the transfected cells manufacture a relatively constant level of gene product for relatively long periods of time.
  • Genetic material is defined herein as DNA, RNA, mRNA, ribozymes, antisense oligonucleotides, modified polynucleotides and oligonucleotides, including SELEX oligomers, protein nucleic acid (PNA) or a combination thereof.
  • Modified polynucleotides and oligonucleotides are defined as those polynucleotides and oligonucleotides that contain non-naturally occurring nucleotides.
  • Modified nucleosides can be incorporated into the genetic material to impart in vivo and in vitro stability of the oligonucleotides to endo and exonucleases, alter the charge, hydrophilicity or lipophilicity of the molecule, and/or provide differences in three dimensional structure. Nucleoside modifications that have been previously described include
  • the size of poly-L-lysine-DNA complexes and complexes of genetic material and cationic liposomes typically used for transfection is typically between approximately 10 and 300 nm.
  • complexes for use in the present methods have a size between approximately 10 and 300 nm, and, more preferably, between approximately 20 and 150 nm.
  • Cationic species suitable for use in the present invention include, but are not limited to, cationic lipids, cationic liposomes, cationic polyamino acids such as poly-L-lysine, polybrene, and polyornithine, lipopolyamine, polyethylene imine, DEAE-dextran, cationic amphiphiles, calcium ions, and dendrite polymers containing cationic functional groups.
  • the cationic species is a cationic lipid or a cationic liposome.
  • Cationic liposomes are liposomes that contain lipid components that have an overall positive charge at physiological pH.
  • Cationic amphiphiles are species that contain both a lipid component and a polycationic component. Examples of these include cationic polyamino acids, for example poly-L-lysine, that contain lipid sidechains.
  • Suitable cationic lipids for use in the present invention include, but are not limited to, DOTMA, Lipofectin (GIBCO/BRL, Gaithersburg, Maryland), 1 ,2-bis(oleoyloxy)3-(trimethylammonio)propane (DOTAP), N-(w, w-1-dialkoxy) -alkyl-N, N, N-trisubstituted ammonium surfactants, complex cationic lipids having similar structures and properties and mixtures of these.
  • Particularly preferred cationic lipids are those cationic lipids that are readily degradable in vivo.
  • DORI DL-1 ,2-dioleyl-3-dimethylaminopropyl- ⁇ - hydroxyethylammonium
  • DORIE DL-1 ,2-O-dioleyl-3- dimethylaminopropyl- ⁇ -hydroxyethylammonium
  • DORI ester/ether compounds DL-1 -O-oleyl-2-oleyl-3- dimethylaminopropyl- ⁇ -hydroxyethylammonium or DL-1-oleyl-2-O oleyl-3-dimethyl-aminopropyl- ⁇ -hydroxyethylammonium.
  • lipids may be added to the desired cationic lipid or mixture of lipids.
  • lipids include, but are not limited to, lyso lipids, such as lysophosphatidylcholine (l-oleoyllysophosphatidylcholine), cholesterol, or neutral phospholipids such as dioleyl phosphatidyl ethanolamine (DOPE), dioleoyl phosphatidyl choline (DOPC), dimyristoyl phosphatidyl choline (DMPC), and dipalmitoyl phosphatidyl choline (DPPC).
  • DOPE dioleyl phosphatidyl ethanolamine
  • DOPC dioleoyl phosphatidyl choline
  • DMPC dimyristoyl phosphatidyl choline
  • DPPC dipalmitoyl phosphatidyl choline
  • the ratios of lipids may vary to include a majority of cationic lipid in combination with cholesterol and/or mixtures of lyso or neutral lipids.
  • the ratio by charge of cationic lipid to genetic material is between approximately 1.5 and 6. The optimum ratio varies with the type of cationic species. However, optimization is routine in the art.
  • a glycosaminoglycan is a linear heteropolysaccharide possessing a characteristic disaccharide repeat sequence.
  • One monosaccharide of the disaccharide repeat is an amino sugar with D- glucosamine or galactosamine, and the other unit is typically, but not always, a uronic acid residue of either D-glucuronic acid or iduronic acid. Both units are variably N- and O-sulfated, which adds to the heterogeneity of these complex macromolecules.
  • the most common GAG structures are chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid, heparin and heparan sulfate.
  • glycosaminoglycans include, but are not limited to, protease inhibitors, plasma lipoproteins, growth factors, lipolytic enzymes, extracellular matrix proteins, and platelet factor 4.
  • Preferred compounds for minimizing the plasma concentration of glycosaminoglycans are protease inhibitors, and plasma lipoproteins. More preferably, the compounds are protease inhibitors.
  • Various compounds are also known to increase the plasma concentration of glycosaminoglycans.
  • a proteoglycan is formed when a glycosaminoglycan is covalently attached at the reducing end through an O-glycosidic linkage to a serine residue or N-linked to asparagine in a core protein.
  • a major function of cell surface proteoglycans is in cell adhesion and migration. These processes are generally thought to be mediated by interactions between the proteoglycans and extracellular matrix components such as laminin, collagen, and fibronectin.
  • the negative charge of the proteoglycans is similar to the negative charge of the glycosaminoglycans, and is largely due to the sulfation of the glycosaminoglycan moiety in the proteoglycan.
  • phorbol esters such as TPA and PMA
  • cytokines such as EGF, IGF-1 , IL-3 , IL-5, FGF, Interferon, PDGF, and TGF beta.
  • Preferred compounds for increasing the amount of proteoglycans on the cell surface are phorbol esters. More preferably, the phorbol esters are TPA or PMA.
  • Various compounds are known to decrease the amount of proteoglycans on the cell surface. These compounds include, but are not limited to, xylosides, GM-CSF, IL-1 alpha and beta, and TNF-alpha.
  • Preferred compounds for decreasing the amount of proteoglycans on the cell surface are xylosides, IL-1 alpha and beta, and TNF-alpha.
  • Cytokines are polypeptide mediators which can be produced by a variety of cells. Cytokines such as interleukin 3 mediate proteoglycan expression on the surface of cells (Nietfeld, Experientia 49:456-469 (1993).
  • Suitable cytokines for use in practicing the present invention include, but are not limited to, epidermal growth factor (EGF), fibroblast growth factor (FGF), granulocyte colony stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GMC-SF), interferon gamma (IG), insulin-like growth factor-1 (ILGF), interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-1 receptor antagonist (IL-1 RA), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), macrophage colony stimulating factor (MCSF), platelet derived growth factor (PDGF), transforming growth factor beta (TGF beta), and tumor necrosis factor alpha (TNF alpha).
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • GCSF granulocyte colony stimulating factor
  • GMC-SF granulocyte macrophage colony stimulating factor
  • certain cytokines are anabolic, certain are both anabolic and modulatory, certain are catabolic, and certain are both catabolic and modulatory.
  • Catabolic cytokines inhibit proteoglycan synthesis and/or promote proteoglycan degradation.
  • Catabolic cytokines include IL-1 alpha and beta, and TNF-alpha.
  • Anabolic cytokines stimulate proteoglycan synthesis and/or inhibit proteoglycan degradation.
  • Anabolic cytokines include EGF, IGF-1 , IL-3 , and IL-5.
  • Modulatory cytokines regulate the effects of the anabolic and catabolic cytokines.
  • Modulatory cytokines include G-CSF, IL-4, and M-CSF.
  • cytokines are multifunctional, and may elicit more than one effect in a target cell.
  • one of skill in the art can readily determine the effect elicited from administration of a particular cytokine on a particular cell type, by determining whether proteoglycan expression on the cell surface has been increased or decreased.
  • Cytokines that are both anabolic and modulatory include FGF, Interferon, PDGF, and TGF beta.
  • Cytokines that are both catabolic and modulatory include GM-CSF.
  • IL-6 has all three properties.
  • the present invention provides methods for controlling transfection that is mediated by complexes of genetic material with cationic species such as cationic liposomes.
  • Transfection mediated by these complexes is known in the art. This method of transfection is described with respect to complexes including cationic liposomes, for example, in PCT WO 93/14889 to Vical, Inc., the contents of which are hereby incorporated by reference. Briefly, transfection efficiency is increased by increasing the cell surface expression of proteoglycans, and is decreased by decreasing the cell surface expression of proteoglycans.
  • transfection efficiency is also increased by decreasing the plasma concentration of glycosaminoglycans, and is decreased by increasing the plasma concentration of glycosaminoglycans, to the extent that effecting the concentration of glycosaminoglycans in the plasma can occur without significant effect on the cell surface concentration of proteoglycans.
  • This invention has a number of applications, some of which will be discussed in detail below.
  • the methods can be used to express exogenous polynucleotide in a variety of tissues. For example, liver cells can be transfected with the LDL receptor to reduce serum cholesterol in vivo, and muscle can be removed to treat the cells with the defective gene product involved in Duchennes muscular dystrophy.
  • Progenitor cells from the hematopoietic system can be treated at a predifferentiated stage to correct hereditary disorders such as beta-thalassemia.
  • the method is contemplated to be particulariy useful for the treatment of diseases caused by a functional gene deficiency.
  • the polynucleotide encoding exogenous polypeptide codes for the polypeptide deficient or malfunctioning in that particular genetic disease.
  • Transient expression of a gene product can be advantageously used to promote an immune response.
  • viral diseases can be treated by interferon expression and cytokines can be used to stimulate the immune system to react against foreign antigens or cancers.
  • foreign proteins can be expressed transiently from target cells to generate an immune response.
  • the mechanism by which the cationic species interacts with the cell to be transfected has been the subject of debate.
  • the present inventors have determined that the cationic species interact with the polyanionic proteoglycans present on the surface of cells that are to be transfected. Applicant's data show a direct correlation between transfection efficiency and proteoglycan expression on the cell surface.
  • Transfection efficiency can be lowered by lowering the proteoglycan expression on the cell surface, and can be increased by increasing the proteoglycan expression on the cell surface.
  • Complexes of DNA and cationic species are often used to increase the efficiency of transfection relative to using naked DNA. Transfection using these complexes, for example, complexes of DNA with cationic liposomes, has been extensively described.
  • Polyanionic glycosaminoglycans are present in blood plasma, and also interact with the complex. This interaction is one of the factors responsible for the non-reproducibility of transfection conditions used in vitro or ex vivo when the same conditions were attempted in vivo.
  • PLL DNA
  • membrane-associated proteoglycans mediate the delivery of polylysine-DNA complexes into cells.
  • transfection efficiency was measured by determining lucerifase expression of the transfected gene. Since ionic interactions between proteoglycans and cationic substrates often depend on the sulfation of glycosaminoglycan chains, transfection was assayed in HeLa cells cultured in the presence of sodium chlorate, an inhibitor of glycosaminoglycan sulfation, as shown in Example 2. Transfection was also tested in cells pre-treated with glycosaminoglycan lyases and exogenous glycosaminoglycans, as shown in Example 3.
  • transfection efficiency of polylysine-DNA in wild ⁇ type Chinese Hamster Ovary (CHO) cells was compared to that of mutant CHO cells unable to synthesize proteoglycans, as shown in Example 5.
  • the ratio of gene delivery agent to DNA is an important variable affecting transfection efficiency. Many laboratories have reported that on the basis of charge, gene delivery agents must be added in excess over oligonucleotide to obtain maximum expression. To determine the optimum charge ratio in the polylysine system, complexes prepared with 1.5 mg DNA and varying amounts of polylysine were transfected into HeLa cells. As shown in Figure 1 , complexes composed of a small lysine/oligonucleotide ratio yielded very little expression.
  • proteoglycans The interactions of proteoglycans with biological molecules are largely ionic and depend on the sulfation of the glycosaminoglycan chains. In fact, the involvement of proteoglycans in cellular processes is often demonstrated by treating cells with sodium chlorate, a potent inhibitor of glycosaminoglycan sulfation. Chlorate competitively inhibits ATP-sulfuryltransferase, an enzyme essential in the synthesis of PAPS, a co-substrate of protein sulfation.
  • DNA was labeled with YOYO, a fluorescent intercalator, complexed with polylysine and incubated with chlorate treated (35 mM) and untreated HeLa cells. After 4 hours, cells were washed, fixed, and imaged by confocal microscopy to localize intracellular fluorescence. As shown in Figure 2B uptake of PLL- YYDNA was severely impaired in chlorate treated cells.
  • HeLa cells were transfected in the presence of free glycosaminoglycans (40 mg/ml) in the transfection media.
  • free glycosaminoglycans 40 mg/ml
  • heparin and heparan sulfate competitively inhibited luciferase expression while, at this concentration, chondroitans A and C and hyaluronic acid did not ( Figure 3B).
  • Chondroitan sulfate B the third most sulfated glycosaminoglycan, reduced expression by 40%.
  • CHO-pgs745 cells are highly proliferative and are morphologically indistinct from their wild-type counterparts.
  • Both CHO-K1 and CHO p-pgs745 cells were transfected as described below in Example 4.
  • luciferase expression was nearly 60 times lower in the proteoglycan deficient cells.
  • the uptake of PLL-YYDNA into CHO-pgs 45 cells was dramatically lower than in the wild-type cultures.
  • Figure 4B cellular binding of radiolabeled complexes at 4°C was similarly impaired. Therefore, cells unable to express proteoglycans are transfection incompetent.
  • PerFect Transfection Kit (Invitrogen). The exact structures of the lipids in the kit are proprietary information. Luceriferase expression in wild-type cultures relative to proteoglycan deficient cells varied depending on the lipid.
  • lipid formulations consisting of a 1 :1 mixture of a cationic lipid and DOPE or a single cationic lipid.
  • Wild ⁇ type and mutant cells (approximately 2 X 10 ⁇ cells/well) were transfected with approximately 0.2 ⁇ g DNA and the optimum amount of lipid (as determined by the manufacturer). Cells were exposed to lipid:DNA complexes for four hours in serum free Ham's F-12 media, rinsed twice with PBS, and incubated in regular growth media.
  • transfection efficiency is higher when transfection is performed on wild-type cells that express proteoglycans relative to mutant cells that do not express significant levels of proteoglycans on the cell surface.
  • Transfection was measured in relative light units (RLU), which correlates with luciferase expression.
  • RLU relative light units
  • the ratio of luceriferase expression in wild-type cells versus mutant cells deficient in xylosyltransferase was measured as the ratio of relative light units observed following transfection of wild-type and mutant cells. Depending on the lipid formulation, the ratios ranged from 1.69 to 81 , as shown in Figure 5B. Summary
  • Chlorate diminished transfection from cell surface binding, to uptake and expression of DNA. Pre-treating cells with chlorate increases transfection due to increased proteoglycan expression. Thus, the transfection efficiency of a given cell type is largely determined by membrane proteoglycan expression.
  • K562 cells incapable of transfection by unmodified polylypine, yield high levels of expression by transferrin-polylysine mediated gene delivery. This implies that transfection efficiency may depend more on the mechanism of entry, and less on an intrinsic, intracellular deficiency.
  • Cell lines deficient in proteoglycan synthesis are unable to efficiently transfect polylysine: DNA, although wild-type cells yield measurable luciferase expression.
  • the invention disclosed herein can employ several types of genetic material, including DNA, mRNA, RNA, PNA, modified oligonucleotides, modified polynucleotides, ribozymes and antisense oligonucleotides.
  • the genetic material is delivered to bind to or interact with a site within the cell.
  • modified oligonucleotides or polynucleotides are preferred.
  • DNA or mRNA delivery is preferred.
  • RNA or DNA gene delivery can be used for transient gene expression applications. Without selection, it is likely that the majority of the cells will be transiently transfected. Transient expression permits gene expression for periods ranging from a few hours to several months. While repeated therapies may be required for some applications, the patient nevertheless benefits from the effect of gene expression. Like a drug, the dosage of polynucleotide and the number of delivery sites to the tissue can be monitored and adjusted accordingly.
  • DNA encoding a therapeutic protein may be circular or linear and should contain regulatory elements that facilitate expression in the target cells.
  • Current progress in molecular biology is directed to identifying and isolating tissue specific promoters. As these tissue specific promoters are identified, they may be incorporated into the gene expression vector and used for ex vivo gene therapy strategies in a particular tissue. Similarly, more promiscuous promoters may be selected for a given application based on their promotional strength. For example, the actin promoter, the Rous Sarcoma virus (RSV) promoter or the Myo D promoter may be used for myoblast and muscle transfection.
  • RSV Rous Sarcoma virus
  • Myo D promoter may be used for myoblast and muscle transfection.
  • CMV cytomegalovirus
  • IE immediate early gene/enhancer
  • RSV promoter enhancer Wolff, J.A. et al., Science 247: 1465-1468 (1990), hereby incorporated by reference
  • p-actin gene/promoter enhancer Kawamoto et al, Mol. Cell. Biol. 8:26z (1988), hereby incorporated by reference
  • CMV IE gene/enhancer promoter with the first intern, Chapman, B.S.
  • the transfected DNA additionally contains regions to mediate ribosome binding, polyadenylation signals and includes enhancer regions to facilitate in vivo gene expression.
  • RNA encoding the polypeptide of interest similarly contains appropriate promoter elements and ribosome binding sites.
  • the RNA is capped or stabilized to promote translation and to minimize exonuclease activity.
  • RNA in cationic-mediated gene transfer is described in a publication by Malone et al. (Proc. Natl. Acad. Sci. USA 86:6077-6081 (1989), hereby incorporated by reference).
  • Liposomes are microscopic delivery vesicles formed when amphiphilic lipids are mixed with water (i.e., hydrated), and include one or more spherical lipid bilayers which surround an internal aqueous phase. Amphiphilic lipids dispersed in aqueous solution spontaneously form bilayers with the hydrocarbon tails directed inward and the polar headgroups outward to interact with water. Simple agitation of the mixture generally produces multilamellar liposomes (MLVs), structures with many bilayers in an onion-like form. MLVs typically have a mean diameter of between 1 ,000 to 10,000 nm. MLVs, mainly because they are relatively large, are usually taken up by the reticuloendothelial system (RES).
  • RES reticuloendothelial system
  • Unilamellar liposomes can be formed, for example, by sonicating a dispersion of MLVs. Typical sizes of UVs range from approximately 30- 1000 nm. Preferably, the liposomes used in the present invention have a mean diameter less than 200 nm.
  • Hydrophilic drugs can be encapsulated in the aqueous core of the vesicles, and lipophilic drugs can be dissolved in the vesicle membrane.
  • Cationic lipids can be incorporated in liposomes, and the liposomes can be complexed with polyanionic genetic material. When an excess of cationic groups to anionic groups is present, the excess cationic groups can bind to the polyanionic species on the surface of cells to which genetic material is to be added.
  • Cationic lipids are amphipathic molecules, containing hydrophobic moieties such as cholesterol or alkyl side chains and a cationic group, such as an amine.
  • Phospholipids are amphipathic molecules containing a phosphate group and fatty acid side chains. Phospholipids can have an overall negative charge, positive charge, or neutral charge, depending on various substituents present on the side chains.
  • Typical phospholipid hydrophilic groups include phosphatidyl choline, phosphatidylglycerol, and phosphatidyl ethanolamine moieties.
  • Typical hydrophobic groups include a variety of saturated and unsaturated fatty acid moieties.
  • the liposomes used in the present invention include cationic lipids that form a complex with the polyanionic genetic material and also bind to polyanionic proteoglycans present on the surface of cells.
  • the cationic lipids can be phospholipids or lipids without phosphate groups.
  • Particularly preferred cationic lipids are esters of the Rosenthal Inhibitor (Rl) (DL-2,3-distearoyloxypropyl(dimethyl)- ⁇ - hydroxyethylammoniumbromide), as described in U.S. Patent No. 5,264,618, the contents of which are hereby incorporated by reference. These derivatives can be prepared, for example, by acyl and alkyl substitution of 3- dimethylaminopropane diol, followed by quaternization of the amino group. Analogous phospholipids can be similarly prepared.
  • Rl Rosenthal Inhibitor
  • Transfection efficiency can be increased by incorporating a lysophosphatide into the liposome formulation.
  • the lysophosphatides can be present in amounts up to approximately a third of the total lipid concentration.
  • Preferred lysophosphatides include lysophosphatidylcholines such as l-oleoyllysophosphatidylcholine and lysophosphatidylethanolamines.
  • Particularly preferred lysophosphatides are DOTMA, 1 ,2-bis(oleoyloxy)3- (trimethylammonio)propane (DOTAP), Lipofectin (GIBCO/BRL, Gaithersburg, Maryland) and mixtures of these.
  • cationic lipids are used that are readily degradable in vivo. These include analogs of DORI (DL-1 ,2-dioleyl-3 ⁇ dimethylaminopropyl- ⁇ - hydroxyethylammonium) and DORIE (DL- 1 ,2-O-dioleyl-3-dimethylaminopropyl- ⁇ -hydroxyethylammonium) as well as DORI ester/ether compounds (DL-1-O-oleyl-2-oleyl-3- dimethylaminopropyl- ⁇ -hydroxyethylammonium or DL-1-oleyl-2-O oleyl-3-dimethyl-aminopropyl- ⁇ -hydroxyethylammonium).
  • DORI DL-1 ,2-dioleyl-3 ⁇ dimethylaminopropyl- ⁇ - hydroxyethylammonium
  • DORIE DL- 1 ,2-O-dioleyl-3-dimethylaminopropyl- ⁇
  • Liposomes can be stabilized by incorporating a neutral lipid, such as cholesterol, into the liposome formulation.
  • a mole ratio of between approximately 4:1 and 55:45 lipid to cholesterol, and, preferably, approximately 2:1 lipid to cholesterol will provide liposomes that are stable.
  • Neutral phospholipids such as DOPE, DOPC, DMPC, and DPPC can also be added.
  • the ratios of lipids may vary to include a majority of cationic lipid in combination with cholesterol and/or mixtures of lyso or other neutral lipids.
  • the liposomes of the present invention can be targeted through various means.
  • the size of the liposome provides one means for targeting the liposomes.
  • relatively small UVs efficiently target ischemic tissue and tumor tissue, as described in U.S.S.N. 08/083,123 (allowed), and U.S. Patent Nos. 5,019,369, 5,435,989 and 5,441 ,745 to Presant et al., the contents of which are hereby incorporated by reference.
  • the liposomes can be targeted according to the mode of administration.
  • lung tissue can be targeted by intranasal administration
  • cervical cells can be targeted by intravaginal administration
  • prostate tumors can be targeted by intrarectal administration.
  • Skin cancer can be targeted by topical administration. Depending on location, tumors can be targeted by injection into the tumor mass.
  • liposomes can be targeted by incorporating a ligand such as an antibody, a receptor, or other compound known to target liposomes to various sites, into the liposomal formulation.
  • the ligands can be attached to cationic lipids used to form the liposomes, or to a neutral lipid such as cholesterol used to stabilize the liposome.
  • Ligands that are specific for one or more specific cellular receptor sites are attached to a vesicle to form a delivery vehicle that can be targeted with a high degree of specificity to a target cell population of interest.
  • Suitable ligands for use in the present invention include, but are not limited to, sugars, proteins such as antibodies, hormones, lectins, major histocompatibility complex (MHC), and oligonucleotides that bind to or interact with a specific site.
  • An important criteria for selecting an appropriate ligand is that the ligand is specific and is suitably bound to the surface of the vesicles in a manner which preserves the specificity.
  • the ligand can be covalently linked to the lipids used to prepare the liposomes. Altematively, the ligand can be covalently bound to cholesterol or another neutral lipid, where the ligand-modified cholesterol is used to stabilize the lipid bilayer. Cytokines are preferred ligands.
  • IL-2 is a preferred cytokine. This ligand is particularly useful for targeting specific activated T- and B-cell populations in view of the particular specificity for the high affinity IL-2 receptors on such cells.
  • the IL-2 ligand can be stabilized for in vivo use by certain amino acid substitutions, as described in Wang and Mark, Science 224:1431 (1984).
  • lipids already include ligands that are suitable for targeting various cell types. These include glycolipids, lipoproteins, glycoproteins, and hydrophobic proteins. Examples described in the literature include gangliosides (Jonah, et al., Biochem. Biophys. Acta 541 :321 (1978)), lactosyl ceramide (Spanjer and Scherphof, Biochem. Biophys. Acta, 734:40 (1983)), and sialoglycoprotein (Takada et al., Biochem. Biophys. Acta, 802:237 (1984).
  • Synthetic cholesterol derivatives covalently bound to sugars such as aminomannose have been described, for example, in Mauk, et al., Science 207:309 (1980). Vesicles including aminomannose derived cholesterol have been demonstrated to target EmT6 tumor cells. These compounds can be incorporated into the lipid bilayer when the liposomes are prepared. Dinitrophenyl caproylphosphatidylethanolamine and other phosphatidylethanolamine derivatives linking small peptides have also been directly incorporated into lipid bilayers. Proteins have been covalently linked to liposomes through thiol, hydroxy and/or amine groups on the protein and the lipid, using known coupling techniques, for example, carbodiimide or glutaraldehyde chemistry.
  • the ligand be covalently bound to the surface of a pre-formed lipid vesicle, to ensure that the ligand is present on the outside of the vesicle.
  • the binding can occur directly or through a suitable linker molecule.
  • transfection efficiency can be measured by using the techniques disclosed below in Example 4.
  • Ex vivo transfection requires first removing tissue or a cell sample from an organism.
  • the organism is preferably a mammal, and more preferably, a human.
  • the cells are then contacted with a preparation including an effective amount of a complex of the desired genetic material and a cationic species, together with an effective amount of a compound that increases expression of proteoglycans on the cell surface.
  • the cells are returned to the organism.
  • the cells are returned within less than 40 hours after removing the sample from the organism.
  • the procedure delivers a genetic material, preferably material that operatively codes for a polypeptide to the interior of the transfected cell.
  • the cells are substantially separated from the surrounding extracellular tissue before they are contacted with the preparation.
  • a skin biopsy is enzymatically digested to dissociate the cells from their extracellular matrix.
  • the cells are isolated, exposed to the gene of interest, and returned directly to the host with minimal time in culture to reduce the potential for cell change in vitro.
  • Some of the cells express the desired gene transiently, others stably; however, with a minimum turn around time, the cells are more likely to survive in vivo. Whether stable or transient expression is achieved, the result is beneficial. If needed, the therapy can be repeated to maintain the desired level of exogenous gene expression.
  • the methods discussed herein teach one of skill in the art to introduce gene sequences such that a variety of cells from a variety of tissues can be treated ex vivo and rapidly returned to a host for gene expression without selection for stable transfectants.
  • a biopsy or cell sample is preferably obtained from a patient in need of gene therapy.
  • the treated cells can be derived from a secondary source such as a cell line or a cell donor.
  • the cell sample is preferably obtained from a tissue where gene expression would be most advantageous.
  • the cell sample may be derived from a variety of tissues, including, but not limited to, skin, liver, pancreas, spleen, muscle, bone marrow, nervous system cells, blood cells, and tumor cells.
  • Biopsy of formed tissue may be obtained by needle, catheter, punch biopsy or surgical excision.
  • a skin biopsy may be best obtained by a skin punch, while a liver biopsy may be best obtained by a needle biopsy.
  • a simple blood draw into a heparinized, EDTA, or citrate tube can be processed by centrifuging at 1000 rpm for 10 min.
  • the cells can then be washed to remove serum, red blood cells, and debris, and then used directly.
  • Biopsies removed from a patient are preferably placed in sterile saline and washed to remove blood and debris.
  • the tissue is then preferably minced with a scalpel or single edge razor blade until the tissue resembles a thick paste.
  • the mincing is preferably performed in a small amount of Hanks Balanced Salt Solution (GIBCO, Grand Island, New York) or other suitable solution that permits the relative pH of the tissue sample to be monitored during processing, because maintaining physiologic pH is important to cell survival.
  • GEBCO Hanks Balanced Salt Solution
  • the paste can then be treated with an enzyme preparation such as trypsin and collagenase B (Boehringer Mannheim, Indianapolis, Indiana), and also treated to remove connective tissue fragments.
  • Other enzymes used in tissue dissociation include Versene, pancreatin and other collagenases.
  • the enzymatic solution is then preferably washed away from the cell suspension, and the cells are washed and then used for transfection. For some gene therapy applications, a uniform cell population is preferred. In these applications, the cells are first dissociated and then separated from other cell types. There are a variety of methods known to those with skill in the art for removing a particular cell type from a mixed suspension of cells. Cell types can be differentiated from one another by their density or size.
  • mesh screens or sucrose gradient centrifugation can be used to separate one cell population from another.
  • Cells can additionally be separated on the basis of their adherent properties. For example, fibroblasts will adhere and settle onto plastic more quickly than epithelial cells.
  • Populations of cells can also be separated from one another by virtue of surface protein.
  • Lectins or antibodies can be used to selectively separate one population of cells from another either using panning (passing cells over a surface with bound lectin or antibody), column chromatography or fluorescent activated cell sorting.
  • cells from a blood sample are separated in a device (The Collector, Applied Immune Sciences, Menlo Park, California) which includes multiple polystyrene plates to which monoclonal antibodies, which bind selectively to specific cells, have been permanently attached.
  • a device The Collector, Applied Immune Sciences, Menlo Park, California
  • targeted cells remain attached to the polystyrene surface, while other cells pass through the device.
  • the captured cells can then be released by mechanical or chemical means.
  • the washed and dissociated cells can be stored for up to 24 hours or longer in culture. However, the cells are preferably transfected immediately following tissue dissociation and cell purification.
  • the cells can be stored in tissue culture medium with a composition compatible to the particular cell type. Typically Eagles (EMEM) or Dulbecco's minimum essential medium (DMEM) and an antibiotic solution (Fungi Bact Solution, Irvine Scientific, Irvine, California; that contains penicillin, streptomycin and fungizone) are used to maintain the cells ex vivo.
  • ex vivo transfection can include a selection step to separate or expand the transfected cells.
  • Transfected cells can be frozen for storage prior to re-insertion into the organism.
  • the genetic material is added to bind to or interact with a site within the cell.
  • the genetic material encodes a peptide that is adapted to treat a disease caused by a functional gene deficiency.
  • the genetic material expresses the polypeptide in the live cells.
  • the expression of the polynucleotide by the cells may be transient, or may persist for a substantial length of time.
  • the polypeptide is an immunogenic polypeptide in the organism.
  • the organism preferably a mammal, more preferably a human
  • the operatively linked codes for a genetic material codes for lymphokine.
  • Cells that may be used include, but are not limited to, white blood cells, myoblasts, and bone marrow cells.
  • Biopsies and cells may be frozen before or after tissue dissociation. Dissociation and purification procedures should be performed as rapidly and expediently as possible after biopsy to maximize cell viability.
  • the cells are transfected. The transfected cells are preferably returned immediately to the patient without expansion or selection in culture.
  • the biopsied tissue can be dissociated, treated with the desired genetic material, and frozen without expansion or selection in culture. Samples can be thawed as necessary for future treatments. Similarly, a portion of the frozen cells can be thawed and assayed for transfection efficiency or for gene expression.
  • methods known to those with skill in the art for freezing cells There are a variety of methods known to those with skill in the art for freezing cells.
  • a muscle biopsy can be used to isolate muscle myoblasts.
  • Patient myoblasts can be isolated following the techniques of Blau et al. (Proc. Natl. Acad. Sci. USA 78:5623-5627, 1981 , the contents of which are hereby incorporated by reference).
  • Myoblasts are readily obtained from muscle without substantial inconvenience to the patient.
  • a small amount of tissue, processed according to the techniques of Blau et al. yields sufficient quantities of myoblasts for transfection.
  • the muscle myoblasts are then transfected ex vivo following the methods disclosed herein and returned to the muscle or to other tissues of the patient.
  • Myoblasts can additionally be stored or frozen for a time without a significant loss in cell viability or a significant change in cell phenotype.
  • myoblasts When myoblasts are reintroduced back into muscle, they are capable of fusing with the available mature myofibers. Accordingly, myoblast delivery is a particularly efficient method for gene delivery to muscle cells. Since the myoblasts are readily isolated and conveniently obtained from muscle tissue of the patient, there is no risk of tissue rejection. Transient gene expression is more likely to be sustained over time from repeated therapies in autologous cells. Another important advantage to myoblast transfection is that muscle tissue is accessible to injection and underlies most surfaces of the body, thus the transfected cells can be injected in multiple locations as often as needed. Using muscle specific promoters like MCK, gene product expression can be restricted to mature muscle fibers. The muscle cells thus express the desired polynucleotide sequence. Recent work indicates that stable myoblast transfectants injected into muscle tissue express their gene product into the blood stream (Hoffman, M. Science 254: 1455-1456, 1991).
  • the cells are washed, concentrated and returned to the patient.
  • the cells may be returned directly to their tissue of origin, or for some applications, the cells can be returned to a second location either within the tissue of origin or at a location distant from the tissue of origin.
  • a large bore needle (18 gauge or larger) is preferably used to return cells to the body. Any bore size may be used that does not destroy the cells or restrict cell passage into the tissue. It may be beneficial to introduce the treated cells into multiple locations within a given tissue. For example, in treating Duchenne's muscular dystrophy, multiple injections of treated cells will advantageously disperse the gene product throughout the tissue. If transfection occurs without tissue dissociation, the intact biopsy can be replaced by surgical implantation.
  • Other cells can be administered to their organ of origin by injecting them into a vein that leads to the organ. Methods to return treated cancer cells to a patient will depend on the cancer type.
  • Other transfected cancer cells of hematopoietic origin may be introduced directly into the blood stream. Cells from hard tissue tumors may be replaced directly into a tumor mass to elicit tumor regression through external injection or internal injection using catheters or the like.
  • Transfection efficiency can be increased by lowering the plasma concentration of glycosaminoglycans and, optionally, other polyanionic species, and can be lowered by increasing the plasma concentrations of glycosaminoglycans.
  • Transfection can be performed in vivo by administering an effective amount of the complex to cause transfection of a significant amount of the cells and an effective amount of compounds that increase proteoglycan expression on the cell surface, and, optionally, compounds that decrease the plasma concentration of glycosaminoglycans.
  • the complex and the compounds can be administered at the same time or within a reasonable time of each other, so long as the overall effect is that the cell surface concentration of proteoglycans increases and/or the plasma concentration of glycosaminoglycans decreases when transfection occurs.
  • the cationic species used to prepare the complex is a cationic liposome. More preferably, the liposome is prepared to specifically target a certain cell type. For example, certain antibodies are known to target liposomes to various cells. By using unilamellar vesicles less than 200 nm in diameter, tumor cells can be targeted for transfection. By administering the liposomes intranasally via an aerosol, lung tissue can be targeted.
  • proteoglycans in transfection also allows minimization of transfection efficiency. Minimizing transfection efficiency can be important in preventing or minimizing viral infections, in embodiments in which viral DNA infects cells by first binding to polyanionic sites on the cell surface, rather than to other cell receptors.
  • the amount of proteoglycans on the cell surface can be reduced, and/or the concentration of proteoglycans or other polyanionic substances in the plasma can be increased.
  • the amount of proteoglycans on the cell surface can be minimized by adding various compounds that are known to lower the concentration of proteoglycans.
  • the interaction of the viral DNA with the cell surface can also be minimized by increasing the plasma concentration of proteoglycans and/or other polyanionic species.
  • concentration of these species can be increased by directly adding the polyanionic species to the plasma, and/or by adding an effective amount of one or more substances known to increase the plasma concentration of these species.
  • Methods for minimizing transfection efficiency can also be used to target cells in which transfection is not desired, by lowering the proteoglycan expression on the surface of those cells, and then introducing genetic material to cells in which transfection is desired.
  • targeted administration of sodium chlorate to specific cells can effectively diminish the cell surface concentration of proteoglycans, and thus minimize transfection efficiency in the targeted cells.
  • General administration of chlorate ions will generally reduce the efficiency of transfection.
  • Transfection efficiency can also be controlled by modifying proteoglycan expression such that the cells express a different ratio of different types of proteoglycans on the cell surface.
  • higher concentrations of chondroitan-based proteoglycans on the surface of cells causes increased transfection efficiency, whereas in others, higher concentrations of heparan sulfate-based proteoglycans on the surface of cells causes increased transfection efficiency.
  • Suitable glycosaminoglycan biosynthesis inhibitors for use in the present invention include, but are not limited to, ⁇ -D-xyloside, 2- deoxy-D-glucose, ethane-1-hydroxy-1 ,1-diphosphonate and 5-hexyl-2- deoxyuridine.
  • Improved Cationic Liposomes include, but are not limited to, ⁇ -D-xyloside, 2- deoxy-D-glucose, ethane-1-hydroxy-1 ,1-diphosphonate and 5-hexyl-2- deoxyuridine.
  • Transfection efficiency can be increased by complexing genetic material with a cationic lipid that is covalently or ionically bound to an agent known to increase the amount of proteoglycans on the cell surface.
  • a cationic lipid that is covalently or ionically bound to an agent known to increase the amount of proteoglycans on the cell surface.
  • neutral lipids, lysolipids and neutral phospholipids can be covalently or ionically bound to an agent known to increase the amount of proteoglycans on the cell surface, and these modified lipids can be included in a cationic liposome formulation.
  • Cationic liposomes prepared from the resulting lipids also increase transfection efficiency by increasing the concentration of cell surface proteoglycans.
  • phorbol esters such as TPA and PMA can be reacted with a suitable lipid with one or more hydroxy or amine groups to form an ester or amide linkage.
  • Anabolic cytokines with reactive functional groups can be similarly coupled to suitable lipids using known chemistry.
  • lipids covalently or ionically linked with substances known to decrease the cell surface expression of proteoglycans can be administered to those cells in which transfection is not desired, before a complex of genetic material and a cationic species is administered to cells in which transfection is desired.
  • xylosides or catabolic cytokines with reactive functional groups can be covalently linked to suitably functionalized lipids to prepare modified lipids that reduce the cell surface expression of proteoglycans.
  • Cytokines whether anabolic, catabolic or modulatory, can be covalently linked to these lipids to form compounds that increase, decrease or otherwise modulate expression of proteoglycans on the cell surface.
  • the efficiency of transfection of non- differentiated cells can be increased by causing the non-differentiated cells to express proteoglycans at or near the time the cells are transfected.
  • Transfection of non-differentiated cells can be important where a genetic defect is present in the cells, which defect can be corrected before the cells become differentiated. After a cell differentiates, it is often difficult to correct the genetic defect.
  • the genetic material is preferably administered directly to the developing fetus.
  • Several genetic defects have been characterized, and an appropriate genetic "splice" has been identified. It is expected that, over time, additional genetic defects will be characterized. However, due to the low concentration of proteoglycans on the cell surface, non-differentiated cells have been difficult to transfect. The present method increases the transfection efficiency, allowing more efficient gene replacement for correcting genetic defects.
  • Cancer cells also exhibit low cell surface concentrations of proteoglycans. Although these cells can be transfected, the transfection efficiency can be increased by increasing the cell surface concentration of proteoglycans.
  • HeLa cells were obtained from the American Type Culture Collection and grown in Dulbecco's Modified Eagles Media (DMEM, Gibco) containing Basal Medium Eagle Amino Acids (Gibco), Non-essential Amino Acids (Gibco), 10% fetal bovine serum (Hyclone), and 40 mg/ml gentamicin (Gibco).
  • DMEM Dulbecco's Modified Eagles Media
  • Mutant (CHO-pgs 745) and wild-type (CHO-K1) cells were generously donated by Dr. J.D. Esko (University of Alabama, Birmingham, School of Medicine).
  • the mutant cell line lacks xylosyltransferase, an initiator of glycosaminoglycan synthesis, and makes little if any glycosaminoglycan. Both cell lines were proliferated in Ham's F-12 media, 7.5% FBS and subcultured every four days.
  • transfection media consisted of regular growth media supplemented with 100 ⁇ M chloroquine (Sigma).
  • the PGL2 plasmid (Promega) encoding the firefly luciferase reporter gene was amplified in competent JM109 Escherichia Coli (Promega) and purified by chromatographic methods (Qiagen).
  • DNA (1.5 mg) was labeled with a- 32 PdCTP (3000 Ci/mmol) using a Nick Translation Kit (Boehringer Mannhiem) and purified by repeated chloroform: phenol extractions and ethanol precipitation.
  • plasmid (100mg/ml) was incubated with a fifty-fold molar excess of YOYO-1 (Molecular Probes), a highly fluorescent DNA intercalator, and incubated for two hours at 4°C.
  • the solution was loaded onto a Centricon-30 desalting unit and spun for 3 hours at 6000 rpm and 4°C to remove unbound YOYO.
  • the retentate was diluted to its original concentration in sterile water.
  • the concentration of YOYO per mole plasmid was determined by UV-Vis analysis.
  • Example 2 Desulfation of Cells HeLa cells (20 K cells/ml) were seeded into 12 well plates (Falcon).
  • Example 3 Treatment of cells with glycosaminoglycan Lyases and Purified Glycosaminoglycans HeLa cells were seeded at a density of 50 K/ml into 12 well plates
  • DNA complexes and glycosaminoglycans (40 mg/ml, either heparan sulfate, heparan, chondroitan sulfate A, chondroitan sulfate B, chondroitan sulfate C, or hyaluronic acid) were added together at the time of transfection.
  • Transfected cells were treated as per the normal protocol.
  • Example 4 Preparation of Complexes and Transfections An aliquot of poly-L-lysine (100 mg/ml, Sigma) was added to DNA (1.5 mg, 100 mg/ml) diluted in 150 ml HBS (Hepes Buffered Saline, 150 mM, pH 7.4). Polylysine-DNA (PLL-DNA) samples were mixed gently, incubated for 30 minutes at room temperature, and added to cells in the presence of transfection media. Four hours later, cells were rinsed twice in 1 ml phosphate buffered saline (PBS, 150 mM NaCl, 150 mM NaHPO3, pH 7.4) and placed into fresh culture media at 37°C.
  • PBS phosphate buffered saline
  • Luciferase expression in cellular lysates was determined using an Enhanced Luciferase Assay Kit (Analytical Luminescence Laboratories) according to the manufacturer's instructions. Expression was quantitated in terms of Relative Light Units (RLU) on an Analytical Luminescence Laboratories Model 2010 Luminometer.
  • RLU Relative Light Unit
  • HeLa, CHO-pgs 745, and CHO-K1 cells were incubated with PLL- YYDNA for four hours and then rinsed three times in PBS.
  • cells were treated with DNAse (Sigma, 1 mg/ml) for 15 minutes and then detached by trypsin.
  • Cell suspensions were pelleted, washed twice in PBS, and fixed in 4% paraformaldehyde/PBS for 10 minutes at room temperature. After a final rinse in PBS, cell pellets were resuspended in Biomeda/PBS solution (90:10) and mounted between a slide and coverslip to dry. Fluorescence images were obtained using a BioRad Confocal Microscope.
  • the invention has been described with respect to its preferred embodiments. It will be readily apparent to those skilled in the art that further changes and modifications in the actual implementation of the concepts described herein can easily be made without departing from the spirit and scope of the invention as defined by the following claims.

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Abstract

Cette invention se rapporte à des procédés pour réguler l'efficacité d'une transfection transmise par des complexes d'espèces cationiques et d'un matériau génétique, en ajustant la quantité de protéoglycanes à association membranaire et éventuellement en ajustant la concentration en plasma de glycosaminoglycanes. L'efficacité de la transfection est régulée par la quantité de protéoglycanes à association membranaire se trouvant dans la cellule à transfecter et également par la concentration en plasma de glycosaminoglycanes. En augmentant la quantité de protéoglycanes à association membranaire présents dans la cellule et éventuellement en abaissant la concentration en plasma de glyclosaminoglycanes, on augmente l'efficacité de la transfection. En abaissant la quantité des protéoglycanes à association membranaire présents dans la cellule et éventuellement en abaissant la concentration en plasma des glyclosaminoglycanes, l'efficacité de la transfection peut être diminuée. L'efficacité de la transfection peut être régulée, qu'elle soit effectuée in vivo, ex vivo, ou in vitro.
PCT/US1997/004217 1996-03-18 1997-03-12 Procedes pour augmenter ou diminuer l'efficacite d'une transfection WO1997034483A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005092101A2 (fr) 2004-03-26 2005-10-06 Syngenta Participations Ag Combinaison herbicide
WO2010100781A1 (fr) * 2009-03-06 2010-09-10 国立大学法人東京大学 Composition pour l'administration d'acide nucléique
WO2011107258A2 (fr) 2010-03-03 2011-09-09 Syngenta Participations Ag Procédé de lutte contre les mauvaises herbes
WO2021001265A1 (fr) 2019-07-03 2021-01-07 Syngenta Crop Protection Ag Lutte sélective contre les mauvaises herbes
CN117070570A (zh) * 2023-08-28 2023-11-17 南通大学 提高脂质体转染试剂的转染效率的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5459127A (en) * 1990-04-19 1995-10-17 Vical, Inc. Cationic lipids for intracellular delivery of biologically active molecules

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US5459127A (en) * 1990-04-19 1995-10-17 Vical, Inc. Cationic lipids for intracellular delivery of biologically active molecules

Non-Patent Citations (3)

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Title
PROC. NATL. ACAD. SCI. U.S.A., November 1987, Vol. 84, WANG et al., "pH-Sensitive Immunoliposomes Mediate Target-Cell-Specific Delivery and Controlled Expression of a Foreign Gene in Mouse", pages 7851-7855. *
SCIENCE, 25 August 1995, Vol. 269, MARSHALL E., "Gene Therapy's Growing Pains", pages 1050-1055. *
THE FASEB JOURNAL, February 1995, Vol. 9, MILLER et al., "Targeted Vectors for Gene Therapy", pages 190-199. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005092101A2 (fr) 2004-03-26 2005-10-06 Syngenta Participations Ag Combinaison herbicide
WO2010100781A1 (fr) * 2009-03-06 2010-09-10 国立大学法人東京大学 Composition pour l'administration d'acide nucléique
WO2011107258A2 (fr) 2010-03-03 2011-09-09 Syngenta Participations Ag Procédé de lutte contre les mauvaises herbes
WO2021001265A1 (fr) 2019-07-03 2021-01-07 Syngenta Crop Protection Ag Lutte sélective contre les mauvaises herbes
CN117070570A (zh) * 2023-08-28 2023-11-17 南通大学 提高脂质体转染试剂的转染效率的方法

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