WO1999029839A1 - Grafted copolymers as gene carriers - Google Patents

Grafted copolymers as gene carriers Download PDF

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Publication number
WO1999029839A1
WO1999029839A1 PCT/US1998/026451 US9826451W WO9929839A1 WO 1999029839 A1 WO1999029839 A1 WO 1999029839A1 US 9826451 W US9826451 W US 9826451W WO 9929839 A1 WO9929839 A1 WO 9929839A1
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composition
glycol
poly
polymer
nucleic acid
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PCT/US1998/026451
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English (en)
French (fr)
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Young-Hun Choi
Jong Sang Park
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Expression Genetics, Inc.
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Application filed by Expression Genetics, Inc. filed Critical Expression Genetics, Inc.
Priority to JP2000524412A priority Critical patent/JP2001526181A/ja
Priority to AU18209/99A priority patent/AU1820999A/en
Priority to EP98963119A priority patent/EP1032659A1/en
Priority to KR1020007006209A priority patent/KR20010032879A/ko
Priority to IL13667998A priority patent/IL136679A0/xx
Priority to BR9814273-9A priority patent/BR9814273A/pt
Publication of WO1999029839A1 publication Critical patent/WO1999029839A1/en

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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
    • 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

Definitions

  • This invention relates to a gene carrier for use in connection with in vivo applications. More particularly, the invention relates to a non-toxic composition and a method for effectively delivering a selected nucleic acid into a host cell by forming an electrostatic complex of the nucleic acid with a cationic polymer. Graft copolymers of poly-L-lysine (PLL) and a polyoxyalkyl glycol are particularly effective. Genes are very attractive candidates for therapeutic use in a variety of disease states due to their ability to produce bioactive proteins using the bio synthetic machinery provided by host cells. M.S. Wadhwa et al., 6 Bioconjugate Chemistry 283 (1995). There .are many established protocols for transferring genes into cells, including calcium phosphate precipitation, electroporation, particle bombardment, liposomal delivery, viral- vector delivery, and receptor-mediated gene delivery. Id.
  • Retro viral vectors in particular, have been successfully used for introducing exogenous genes into the genomes of actively dividing cells such that stable transformants are obtained. D.G. Miller et al., 10 Mol. Cell Biol.4239 (1990).
  • the method of using retroviral vectors for inserting genes into the host cell's genome depends on the viral infection pathway. Applying the retroviral method in human gene therapy raises serious concerns about possible recombination with endogenous viruses, oncogenic effects, and immunologic reactions.
  • compositions for delivery of a selected nucleic acid into a target cell wherein the composition is configured for forming an electrostatic complex with the selected nucleic acid, comprising a biocompatible graft copolymer of a cationic first polymer and an arnphiphilic second polymer.
  • the first polymer is a member selected from the group consisting of poly(L-lysine), derivatives thereof and mixtures thereof, .and more preferably is poly(L-lysine).
  • the second polymer is a polyoxyalkyl glycol, such as those selected from the group consisting of polyethylene glycol homopolymers, polypropylene glycol homopolymers, alpha- substituted poly(oxyalkyl) glycols, poly(oxyalkyl) glycol copolymers and block copolymers, and activated derivatives thereof.
  • Polyethylene glycol is particularly preferred.
  • the graft copolymer comprises polyethylene glycol grafted to an e-amino group of poly(L-lysine).
  • the graft copolymer preferably comprises about 5 mole% to about 25 mole% of polyethylene glycol and, more preferably, about 10 mole% of polyethylene glycol.
  • a composition for delivery of a selected nucleic acid into a host cell comprises an electrostatic complex of a selected nucleic acid and a biocompatible graft copolymer comprising a cationic first polymer and an amphiphilic second polymer.
  • the nucleic acid and the graft copolymer are present in a weight ratio of about 0.3 to 10.
  • the composition further comprises an effective amount of an anti- endosome functional agent, such as chloroquine.
  • an anti- endosome functional agent such as chloroquine.
  • Such effective amount of chloroquine is preferably about 25-250 ⁇ M and more preferably about 75-150 ⁇ M.
  • a method of transforming a host cell with a selected nucleic acid comprises contacting the host cell with an effective amount of an electrostatic complex comprising the selected nucleic acid and a biocompatible graft copolymer, wherein the biocompatible graft compolymer comprises a cationic first polymer and an amphiphilic second polymer; such that the host cell internalizes the selected nucleic acid.
  • a method of using a composition for delivering a selected nucleic acid to an individual comprises administering an effective amount of an electrostatic complex comprising the selected nucleic acid and a biocompatible graft copolymer, comprising a cationic first polymer and an amphiphilic second polymer, such that the complex is systemically circulated and contacts a host cell such that the host cell internalizes the selected nucleic acid.
  • FIG. 1 is a schematic representation of complex formation between plasmid DNA and an illustrative gene delivery composition according to the present invention.
  • FIG.2 shows an illustrative synthesis scheme of a grafted copolymer, PEG-g-
  • FIG. 3 shows data from a fluorescence quenching assay: ( ⁇ ) PLL control; (0) 5 mole% PEG-g-PLL; (o) 10 mole% PEG-g-PLL; ( ⁇ ) 25 mole% PEG-g-PLL.
  • FIG.4 shows size (diameter) determinations by dynamic laser light scattering of pSV- ⁇ -gal (DNA), a complex of pS V- ⁇ -gal and PLL (PLL), a complex of pSV- ⁇ - gal and 5 mole% PEG-g-PLL (5 mol% PEG), a complex of pSV- ⁇ -gal and 10 mole% PEG-g-PLL (10 mol% PEG), and a complex of pS V- ⁇ -gal and 25 mole% PEG-g- PLL (25 mol% PEG).
  • DNA pSV- ⁇ -gal
  • PLL pS V- ⁇ -gal and PLL
  • FIG.5 shows transfection efficiency for transfection ofhiiman liver carcinoma (HepG2) cells with pSV- ⁇ -gal using LIPOFECTIN reagent (Lipofectin), a PLL complex (PLL), a 5 mole% PEG-g-PLL complex (5 mol% PEG), a 10 mole% PEG- g-PLL complex (10 mol% PEG), and a 25 mole% PEG-g-PLL complex (25 mol% PEG).
  • FIG. 6 shows the transfection efficiency of human liver carcinoma (HepG2) cells as a function of the weight ratio of DNA to PEG-g-PLL.
  • FIG. 7 shows cell viability of human liver carcinoma HepG2 cells after transfection with pSV- ⁇ -gal using a control (media), LIPOFECTIN reagent
  • FIG. 8 shows transfection efficiency of human liver carcinoma HepG2 cells with pSV- ⁇ -gal using a 10 mole% PEG-g-PLL complex as determined 24, 48, 72, and 96 hours after transfection.
  • FIG. 9 shows the effect of chloroquine concentration on transfection of human liver carcinoma HepG2 cells with pSV- ⁇ -gal using a 10 mole% PEG-g-PLL complex.
  • an anti-endosome function agent includes a mixture of two or more of such agents
  • reference to “an amphiphilic polymer” includes reference to one or more of such polymers
  • reference to “a cationic polymer” includes reference to a mixture of two or more of such cationic polymers.
  • PEG-g-PLL means a grafted copolymer wherein PEG or another poly(oxyalkyl)glycol is conjugated to an e-amino group of a lysine residue ofPLL.
  • x mol% PEG-g-PLL refers to PEG-g-PLL having x mole% of PEG.
  • x is a number between 1 and 100
  • PLL is PEG-g-PLL containing 5 mole% of PEG.
  • poly(oxyalkyl)glycol refers to polyether glycol polymers that when grafted to PLL render the resulting composition non-toxic and water soluble. Each monomer portion of the polymer contains a carbon chain having up to about 5 carbon atoms.
  • Preferred poly(oxyalkyl) glycols are selected from the group consisting of polyethylene glycol (PEG) homopolymers, polypropylene glycol homopolymers, alpha- substituted poly(oxyalkyl) glycols (such as methoxypolyethylene glycols or other suitable alkyl-substituted derivatives such as those containing C,-C 4 alkyl groups), poly(oxyalkyl) glycol copolymers and block copolymers, and activated derivatives thereof.
  • the poly(oxyalkyl) glycols used in the present invention preferably have a molecular weight of about 200 to about 50,000, and more preferably about 200 to about 20,000.
  • PEG polyethylene glycol
  • PLL poly(L-lysine), derivatives thereof, and mixtures thereof.
  • the PLL preferably has a molecular weight in the range of about 200 to 50,000 and more preferable in the range of about 500 to 30,000.
  • ⁇ ективное amount means .an .amount of a nucleic acid that is nontoxic but sufficient to provide the selected local or systemic effect and performance at a reasonable benefit/risk ratio attending any medical treatment.
  • administering means delivering the complex formed by admixing the nucleic acid to be delivered with a gene carrier composition according to the present invention to the individual being treated such that the complex is capable of being circulated systemically to the parts of the body where the complex can contact the target cells.
  • the composition is preferably administered to the individual by systemic administration, typically by subcutaneous, intramuscular, or intravenous administration, or intraperitoneal administration.
  • injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion.
  • Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances such as wetting or emulsifying agents, buffers, and the like can be added.
  • nucleic acid i.e. DNA and/or RNA
  • Delivery of a nucleic acid can be used to achieve expression of a polypeptide or to inhibit expression of a polypeptide through the use of an "antisense" nucleic acid, especially antisense RNA.
  • an "antisense" nucleic acid especially antisense RNA.
  • polypeptide means peptides of any length and includes proteins.
  • the term "polypeptide” is used herein without .any particular intended size limitation, unless a particular size is otherwise stated.
  • Typical of polypeptides that can be expressed are those selected from the group consisting of oxytocin, vasopressin, adrenocorticotrophic hormone, epidermal growth factor, prolactin, luteinizing hormone releasing hormone, growth hormone, growth hormone releasing factor, insulin-like growth factors, insulin, erythropoietin, obesity protein such as leptin, somatostatin, glucagon, glucagon-like insulinotropic factors, parathyroid hormone, interferon, gastrin, interleukin-2.and other interleukins and lymphokines, tetragastrin, pentagastrin, urogastrin, secretin, calcitonin, enkephalins, endorphins, angiotensins, renin, brad
  • the nucleic acid when it is DNA, it can be a DNA sequence that is itself non- replicating, but is inserted into a plasmid wherein the plasmid further comprises a replicator.
  • the DNA may also contain a transcriptional promoter, such as the CMV
  • the DNA can also encode a polymerase for transcribing the DNA.
  • Many expression vectors for expression of a cloned gene in a mammal are known in the art, and many such expression vectors are commercially available, for example, pEUK-Cl (Clontech, Palo Alto, Calif). A gene of interest can be inserted into such an expression vector according to recombinant
  • the method can be used for treating a disease associated with a deficiency or absence or mutation of a specific polypeptide.
  • the method provides for immunizing an individual , wherein such individual can be a human or an animal, comprising delivering a DNA and/or RNA to the individual wherein the DNA and/or RNA codes for an immunogenic translation product that elicits an immune response against the immunogen.
  • the method can be used to elicit a humoral immune response, a cellular immune response, or a mixture thereof.
  • An illustrative method of forming the gene carriers according to the present invention is accomplished by grafting polymer monomethoxy polyethylene glycol
  • PEG mPEG
  • PEG a straight-chain amphiphilic polymer has been used to modify several enzymes giving them longer half-lives in vivo. F.F. Davis et al., in 4 Enzyme Engineering 169 (1978); A. Abuchowski et al., 1 Cancer Biochem. Biophys.175 (1984). PEG has also been used to modify interleukin-2 to give it increased solubility and increased half-life in vivo. N.V. Katre et al, 84 Proc. Nafl. Acad. Sci. USA 1487 (1987).
  • the PEG-grafted PLL provides a solubility increase when it forms an electrostatic complex with genes to be delivered (FIG. 1) because of the solubilizing effect of the PEG chains.
  • FOG. 1 genes to be delivered
  • the solubility increase induced by the added PEG chains favorably affects the transfection efficiency as compared to a plasmid D ⁇ A/PLL complex without increasing the cytotoxicity of PLL. Rather, the presence of the PEG chains also acts to reduce the cytotoxicity of the PLL base and improve transfection duration.
  • the electrostatic complex is formed by the affinity of the positively-charged polymer (e.g. PLL) and the negatively- charged nucleic acid.
  • Graft copolymers of PEG-g-PLL having PEG contents of 5 mol %, 10 mol
  • % and 25 mol % were synthesized according to the procedure outlined in FIG. 2.
  • synthesis of 5 mol % PEG-g-PLL was performed as follows: Methoxy PEGOCH 2 CH 2 C0 2 H was synthesized by alkylation of mPEGOCH 2 CH 2 OH with ethyl bromoacetate. Thionyl chloride (1.5 ml) was added to a round bottom flask containing 6 mg of mPEGOCH 2 CH 2 C0 2 H and refluxed for 40 min, followed by evaporation of thionyl chloride under vacuum. The resulting product was dissolved in 100 ⁇ l of DMSO, and added to 1 ml of DMSO solution containing 25 mg of PLL-hydrobromide and 160 ⁇ l of TEA while stirring at room temperature.
  • the plasmid pSV- ⁇ -gal (Promega Corp., Madison, Wisconsin; EMBL accession No. X65335) is a positive control vector for monitoring transfection efficiencies of mammalian cells.
  • the pSV- ⁇ -gal plasmid contains a SV40 early promoter and enhancer sequence, transcription start sites, E. coli lacZ coding region encoding ⁇ -galactosidase, and SV40 small T antigen polyadenylation signals.
  • SV40 early promoter and enhancer drive the transcription of the lacZ gene.
  • gel retardation assays were carried out to further determine whether PEG-g-PLL formed complexes with DNA.
  • PEG-g-PLL prepared as in Example 2 were fractionated by electrophoresis in a 1% agarose gel. After fractionation, the gel was stained with ethidium bromide (0.5 ⁇ g/ml) and illuminated on a UV illuminator. The movement of free plasmid DNA
  • Example 4 the sizes of plasmid DNA/PEG-g-PLL complexes prepared according to the procedure of Example 2 were measured by dynamic laser light scattering (Brookhaven BI-DS) at a 90° angle using a 1:3 weight ratio of plasmid
  • FIG. 4 shows the size of the plasmid alone and the size of complexes with various compounds.
  • the complexes have an average diameter of about 300 nm.
  • compositions according to the present invention for mediating in vitro transfection of mammalian cells was demonstrated.
  • HepG2 cells human liver carcinoma cells
  • Transfection was performed using PEG-g-PLL in 96-well plates seeded at a cell density of 20 x
  • the plasmid pSV- ⁇ -gal DNA PEG-g-PLL complex was prepared by mixing 1 ⁇ g of plasmid DNA and 3 ⁇ g of PEG-g-PLL in 100 ⁇ l of serum-free MEM medium and incubating it for 30 min at room temperature, followed by the addition of 10% (v/v) fetal bovine serum (Hyclone Laboratories, Logan, Utah) and 100 ⁇ M chloroquine (Sigma). Chloroquine, a cell permeant base, was used to partially neutralize acidic compartments of the cells and prevent the fusion of endosomes with lysosomes. P.
  • X-gal solution (1 mg/ml X-gaL 2 mM MgCl 2 , 5 mM K 4 Fe(CN) 6 and 5 mM K 3 Fe(CN) 6 ) was added, followed by incubation for 16 hr in a tissue culture incubator at 37°C.
  • X-gal solution was the removed and cells were covered with IX PBS.
  • Cells expressing the ⁇ -galactosidase enzyme from transfection of pSV- ⁇ -gal plasmid were stained blue by X-gal and could be seen and counted under a microscope.
  • transfection efficiency increased by up to 30-fold as the degree of PEG-modification increased.
  • 10 mol % PEG-g-PLL showed the best transfection efficiency.
  • the 25 mol% PEG- g-PLL showed significantly lower transfection efficiency.
  • the optimal range of PEG-modification in PLL seems to be around 10 mol %.
  • a commercially available transfection agent, LIPOFECTIN reagent (GIBCO BRL) showed slightly higher transfection efficiency than 10 mol % PEG-g-PLL in HepG2 cells.
  • LIPOFECTIN reagent is a 1:1 (w/w) liposome formulation of the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-n,n,n-trimethyl.ammonium chloride (DOTMA), and dioleoyl phosphotidylethanolamine (DOPE) in membr.ane filtered water.
  • DOTMA cationic lipid N-[l-(2,3-dioleyloxy)propyl]-n,n,n-trimethyl.ammonium chloride
  • DOPE dioleoyl phosphotidylethanolamine
  • Example 7 the cytotoxicity of PEG-g-PLL was determined.
  • the percentage of living cells was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetr.azolium bromide) assay of T. Mosman, 65 J. Immunol. Methods 55 (1983), hereby incorporated by reference.
  • the transfection mixture was replaced by fresh growth medium containing 26 ⁇ l of 2 mg/ml MTT solution. Plates were incubated for an additional 4 hr at 37°C ina tissue culture incubator, then MTT-containing medium was removed by aspiration, and 150 ⁇ l of DMSO was added to dissolve the formazan crystals formed by living cells. Absorbance was measured at 570 nm using a microplate reader (Model EL311 , Bio- Tek instrument Co.), and the percentage of living cells was calculated from the following equation:
  • compositions of the present invention are improvements over known substances with respect to cytotoxicity.
  • Example 7 the procedure of Example 7 was followed except that the cell viability assay was performed at 24, 48, 72, or 96 hours after transfection. Generally, a transfection assay is performed 48 hrs after transfection, but almost the same transfection efficiency was obtained when the assay was performed after only a 24 hr incubation (FIG. 8). In the case of LIPOFECTIN reagent, ⁇ -galactosidase -activity after a 24 hr incubation was half the ⁇ -galactosidase activity after a 48 hr incubation (data not shown). Further, the cells transfected by plasmid pS V- ⁇ -gal DNA/PEG-g- PLL mixture maintained its gene expression level up to 96 hr (FIG. 8).
  • Example 9 In this example, the role of chloroquine in transfection efficiency was determined. The procedure of Example 5 was followed except that chloroquine concentration was varied from 0-100 ⁇ M. FIG. 9 shows that chloroquine was not essential for successfully transfecting cells, however, chloroquine played an important role in increasing transfection efficiency. As the concentration of chloroquine increased up to 100 ⁇ M, the transfection efficiency increased up to 30-fold.
  • a nucleic acid encoding the leptin obesity protein such as human leptin or a rat leptin cDNA, C. Guoxun et al, Disappearance of Body Fat in Normal Rats Induced by Adenovirus-mediated Leptin, 93 Proc. NatT Acad. Sci. USA 14795-99 (1996), or a mouse leptin cDNA, P. Muzzin et al., Correction of Obesity and
  • the mammalian expression vector pEUK-Cl (Clontech, Palo Alto, Calif.) is designed for transient expression of cloned genes.
  • This vector is a 4.9 kb plasmid comprising a pBR322 origin of replication and an ampicillin resistance marker for propagation in bacteria, and also comprising the SV40 origin of replication, SV40 late promoter, and SV40 late polyadenylation signal for replication and expression of a selected gene in a mammalian cell.
  • MCS multiple cloning site
  • DNA fragments cloned into the MCS are transcribed as RNA from the S V40 late promoter and are translated from the first ATG codon in the cloned fragments.
  • Transcripts of cloned DNA are spliced .and polyadenylated using the SV40 VPI processing signals.
  • the leptin gene is cloned into the MCS of pEUK-Cl using techniques well known in the art, e.g. J. Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., 1989).
  • the resulting plasmid is delivered to a human or animal after incorporation into a complex according to the present invention illustrated in Example 2.
  • An effective .amount of the resulting complex is systemically administered to an individual such that complex enters the bloodstream and contacts target cells.
  • the target cells that are contacted by the complex take up the complex, thus internalizing the leptin DNA.
  • the leptin DNA is then expressed in the recipient cell, resulting in a positive effect in treatment for obesity or diabetes.
  • compositions of the present invention offer improved transfection ability than that obtained with PLL.
  • the present compositions also demonstrate low cytotoxicity, early gene expression, and maintenance of the early gene expression level up to 96 hrs. These characteristics are advantages over prior art compounds such as LIPOFECTIN reagent.

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PCT/US1998/026451 1997-12-12 1998-12-11 Grafted copolymers as gene carriers WO1999029839A1 (en)

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Application Number Priority Date Filing Date Title
JP2000524412A JP2001526181A (ja) 1997-12-12 1998-12-11 遺伝子輸送体であるグラフト共重合体
AU18209/99A AU1820999A (en) 1997-12-12 1998-12-11 Grafted copolymers as gene carriers
EP98963119A EP1032659A1 (en) 1997-12-12 1998-12-11 Grafted copolymers as gene carriers
KR1020007006209A KR20010032879A (ko) 1997-12-12 1998-12-11 유전자 담체로서 융합된 공중합체
IL13667998A IL136679A0 (en) 1997-12-12 1998-12-11 Grafted copolymers as gene carriers
BR9814273-9A BR9814273A (pt) 1997-12-12 1998-12-11 Copolìmeros enxertados como carreadores de genes

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US60/069,351 1997-12-12

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WO2003020966A2 (de) * 2001-08-27 2003-03-13 Zeptosens Ag Oberfläche zur immobilisierung von nukleinsäuren
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WO2012118189A1 (ja) 2011-03-03 2012-09-07 中外製薬株式会社 アミノ－カルボン酸により修飾されたヒアルロン酸誘導体
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KR101486989B1 (ko) 2013-10-15 2015-02-03 부산대학교 산학협력단 공중합체 및 이를 포함하는 유전자 전달체
CN106554499A (zh) * 2015-09-25 2017-04-05 南京理工大学 一种含二硫键的聚（β-氨基酯）类聚合物基因载体及其合成方法和应用
CN106978444A (zh) * 2016-01-15 2017-07-25 江苏命码生物科技有限公司 一种向细胞中导入核酸的方法
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US10758623B2 (en) 2013-12-09 2020-09-01 Durect Corporation Pharmaceutically active agent complexes, polymer complexes, and compositions and methods involving the same

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IL136679A0 (en) 2001-06-14
JP2001526181A (ja) 2001-12-18
AU1820999A (en) 1999-06-28
ZA9811378B (en) 1999-08-05
AR013017A1 (es) 2000-11-22
EP1032659A1 (en) 2000-09-06

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