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US20090258932A1 - Lyophilized DNA Formulations for Enhanced Expression of Plasmid DNA - Google Patents

Lyophilized DNA Formulations for Enhanced Expression of Plasmid DNA Download PDF

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Publication number
US20090258932A1
US20090258932A1 US12/421,425 US42142509A US2009258932A1 US 20090258932 A1 US20090258932 A1 US 20090258932A1 US 42142509 A US42142509 A US 42142509A US 2009258932 A1 US2009258932 A1 US 2009258932A1
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Prior art keywords
hgf
dna
formulation
lyophilized
dna formulation
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US12/421,425
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English (en)
Inventor
Jong-mook Kim
Sujeong Kim
Woong Hahn
Wonsun Yoo
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Helixmith Co Ltd
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Viromed Co Ltd
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Priority to US12/421,425 priority Critical patent/US20090258932A1/en
Assigned to VIROMED CO., LTD. reassignment VIROMED CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KIM, JONG-MOOK, HAHN, WOONG, KIM, SUJEONG, YOO, WONSUN
Publication of US20090258932A1 publication Critical patent/US20090258932A1/en
Priority to US13/045,460 priority patent/US8389492B2/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • Lyophilization is often a preferred formulation for therapeutic materials because the long-term stability of many materials increases in the lyophilized state.
  • lyophilized formulations are not the formulations of choice.
  • the preferred formulation has been a liquid formulation.
  • lyophilized plasmid DNA may be a preferred form of storage
  • lyophilized formulations for plasmid DNA have been considered to cause a reduction in gene expression efficiency. Lyophilization causes the removal of the hydration sphere around a molecule. For DNA, it appears that there are approximately 20 water molecules per nucleotide pair bound most tightly to DNA that do not form an ice-like structure upon low-temperature cooling. Upon DNA dehydration over hygroscopic salts at 0% relative humidity, only five or six water molecules remain. Thus, lyophilization may increase the stability of DNA under long-term storage, but may also cause some damage upon the initial lyophilization process, potentially through changes in the DNA secondary structure or the concentration of reactive elements such as contaminating metals. Therefore, a potential mechanism for loss of gene expression efficiency of lyophilized plasmid DNA may be through a gross structural change to the plasmid.
  • Poxon et al used carbohydrates to ameliorate the in vitro decreased transfection activity of a non-therapeutic plasmid, pRL-CMV expressing Renilla luciferase, stored in EDTA buffer, Poxon et al did not address the use of lyophilized naked DNA formulations in vivo for disease treatment or prevention.
  • the present invention provides for a lyophilized formulation for plasmid DNA that not only preserves the biological activity of the expressed gene but, in certain instances, is able to enhance biological activity.
  • a DNA formulation prior to lyophilization, comprises a plasmid DNA, salt and a carbohydrate; and where the plasmid DNA comprises an HGF gene, or variant thereof.
  • the DNA formulation is lyophilized.
  • the lyophilized DNA formulation is reconstituted.
  • the carbohydrate of the DNA formulation of the present invention is a mono-, oligo-, or polysaccharide such as sucrose, glucose, lactose, trehalose, arabinose, pentose, ribose, xylose, galactose, hexose, idose, mannose, talose, heptose, fructose, gluconic acid, sorbitol, mannitol, methyl ⁇ -glucopyranoside, maltose, isoascorbic acid, ascorbic acid, lactone, sorbose, glucaric acid, erythrose, threose, allose, altrose, gulose, erythrulose, ribulose, xylulose, psicose, tagatose, glucuronic acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, neuram
  • the carbohydrate is sucrose or mannitol.
  • the carbohydrate of the DNA formulation of the present invention is in an amount selected from the group consisting of between about 0.05% to about 30%, between about 0.1% to about 15%, between about 0.2% to about 10%, between about 0.5% and 5%, between about 0.75% and 3%, between about 0.8% and 2%, and between about 0.8% and 1.5%.
  • the carbohydrate is sucrose or mannitol.
  • the carbohydrate of the DNA formulation is in an amount of about 1.1%.
  • the salt of the DNA formulation is selected from the group consisting of NaCl or KCl. In further embodiments, the salt of the DNA formulation is in an amount selected from the group consisting of between about 0.01% and 10%, between about 0.1% and 5%, between about 0.1% and 4%, between about 0.5% and 2%, between about 0.8% and 1.5%, between about 0.8% and 1.2% w/v. In certain embodiments, the salt of the DNA formulation is in an amount of about 0.9% w/v.
  • the plasmid DNA of the invention comprises an HGF gene, or variant thereof.
  • the HGF gene is a mammalian HGF gene or variant thereof.
  • the HGF gene is a human HGF gene or variant thereof.
  • the HGF gene is a hybrid HGF gene, e.g., a hybrid HGF gene comprising HGF cDNA and an inherent or foreign intron or fragment thereof, e.g., an inherent intron 4 or fragment thereof of the human HGF gene.
  • the hybrid HGF gene comprises HGF-X2 (SEQ ID NO: 13), HGF-X3 (SEQ ID NO: 14), HGF-X6 (SEQ ID NO: 8), HGF-X7 (SEQ ID NO: 9) or HGF-X8 (SEQ ID NO: 10).
  • the plasmid DNA comprising a hybrid HGF gene is selected from the group consisting of: pCK-HGF-X2, pCK-HGF-X3, pCK-HGF-X6, pCK-HGF-X7, pCK-HGF-X8, pCP-HGF-X2, pCP-HGF-X3, pCP-HGF-X6, pCP-HGF-X7 and pCP-HGF-X8, where the HGF-X2, HGF-X3, HGF-X6, HGF-X7 and HGF-X8 correspond to SEQ ID NOs: 13-14 and 8-10, respectively.
  • the lyophilized DNA formulations maintain or enhance the expression of the plasmid DNA.
  • the lyophilized DNA formulation provides enhanced biological activity of the expressed protein.
  • the enhanced expression of the plasmid DNA or the enhanced biological activity of the expressed protein is due to the presence of the carbohydrate in the formulation. In certain embodiments, this carbohydrate is sucrose or mannitol.
  • the invention also provides for a reconstituted lyophilized plasmid DNA formulation.
  • the lyophilized DNA is reconstituted in a pharmaceutically acceptable solution.
  • the pharmaceutically acceptable solution is selected from the group consisting of water, PBS, TE, Tris buffer and normal saline.
  • the plasmid DNA of the reconstituted lyophilized formulation is at a final concentration of about 1 ng/mL, about 5 ng/mL, about 10 ng/mL, about 50 ng/mL, about 100 ng/mL, about 250 ng/mL, about 500 ng/mL, about 1 ⁇ g/mL, about 5 ⁇ g/mL, about 10 ⁇ g/mL, about 50 ⁇ g/mL, about 100 ⁇ g/mL, about 200 ⁇ g/mL, about 300 ⁇ g/mL, about 400 ⁇ g/mL, about 500 ⁇ g/mL, about 600 ⁇ g/mL, about 700 ⁇ g/mL, about 800 ⁇ g/mL, about 900 ⁇ g/mL, about 1 mg/mL, about 2 mg/mL, about 2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5 mg/mL, about 5 mg
  • the final concentration of the plasmid DNA of the reconstituted lyophilized formulation is from about 1 ng/mL to about 30 mg/mL. In certain aspects, the final concentration of the plasmid DNA of the reconstituted lyophilized formulation is from about 100 ⁇ g/mL to about 2.5 mg/mL. In further aspects, the final concentration of the plasmid DNA of the reconstituted lyophilized formulation is from about 500 ⁇ g/mL to about 1 mg/mL.
  • the present invention is also directed to a method of treating or preventing ischemic or liver disease in a subject, comprising administering a composition reconstituted from a lyophilized hepatocyte growth factor (HGF) DNA formulation, where the DNA formulation comprises a plasmid DNA, salt and a carbohydrate; and where the plasmid DNA comprises an HGF gene, or variant thereof.
  • HGF hepatocyte growth factor
  • the composition reconstituted from a lyophilized HGF DNA formulation is administered by direct injection.
  • the present invention is further directed to a method of making a lyophilized HGF DNA formulation comprising: (a) preparing a DNA formulation comprising a plasmid DNA, a salt and a carbohydrate, where the plasmid DNA comprises an HGF gene, or variant thereof; and (b) lyophilizing the DNA formulation.
  • the steps for lyophilization may include subjecting a DNA formulation of the invention to the process of being frozen at subzero temperatures (e.g, ⁇ 10° C. to ⁇ 500° C.), and then subjected to one or more drying cycles which comprises gradually heating the DNA formulation to a temperature of about 20° C. to less than or equal to about 30° C., wherein the lyophilization occurs over a period of about 50 to about 100 hours.
  • subzero temperatures e.g, ⁇ 10° C. to ⁇ 500° C.
  • the method for lyophilization comprises: (a) forming an aqueous DNA formulation comprising a plasmid DNA, a salt and a carbohydrate, where the plasmid DNA comprises an HGF gene, or variant thereof; (b) cooling the DNA formulation solution to a temperature of about ⁇ 10° C. to about ⁇ 50° C., until frozen; (c) drying the DNA formulation by heating to a temperature of about 20° C. to about 30° C.; and (d) recovering a lyophilized DNA formulation composition having a water content of from about 0.1 weight percent to about 5 weight percent based on the total weight of the recovered DNA formulation.
  • the DNA formulation is lyophilized under conditions comprising (a) about 30 hours to about 50 hours at a temperature greater than or equal to about ⁇ 50° C. and less than about 0° C., and (b) about 20 hours to about 50 hours at a temperature greater than or equal to about 0° C. to less than or equal to about 30° C., progressively, wherein the lowest (a) temperature is about ⁇ 50° C. to about ⁇ 30° C. and the highest (b) temperature is between about 20° C. to about 30° C.
  • the DNA formulation is lyophilized under conditions of ⁇ 50° C. for 4 hours, ⁇ 40° C. for 12 hours, ⁇ 30° C. for 6 hours, ⁇ 20° C.
  • the DNA formulation is lyophilized under conditions of 5° C. for 1 minute, ⁇ 50° C. for 2 hours, ⁇ 40° C. for 6 hours, ⁇ 35° C. for 3 hours, ⁇ 30° C. for 6 hours, ⁇ 25° C. for 3 hours, ⁇ 20° C. for 3 hours, ⁇ 15° C. for 3 hours, ⁇ 10° C. for 6 hours, ⁇ 5° C. for 3 hours, 0° C. for 6 hours, and 30° C. for 17 hours, progressively.
  • the DNA formulation is lyophilized under conditions of 5° C. for 1 minute, ⁇ 10° C. for 1 minute, ⁇ 20° C. for 1 minute, ⁇ 30° C. for 1 minute, ⁇ 50° C. for 1 minute, ⁇ 50° C. for 2 hours, ⁇ 45° C. for 6 hours, ⁇ 40° C. for 3 hours, ⁇ 35° C. for 6 hours, ⁇ 30° C. for 3 hours, ⁇ 25° C. for 6 hours, ⁇ 20° C. for 3 hours, ⁇ 15° C. for 6 hours, ⁇ 10° C. for 3 hours, ⁇ 5° C. for 6 hours, 0° C. for 12 hours, 10° C. for 3 hours, 20° C. for 6 hours, and 30° C. for 29 hours, progressively.
  • the invention is further directed to a lyophilized nucleic acid formulation or a reconstituted lyophilized nucleic acid formulation, as set forth above, where the nucleic acid is an RNA that encodes for HGF, or variant thereof.
  • FIG. 1 depicts a bar graph comparing in vitro HGF expression among various formulations.
  • HGF expression levels were measured using ELISA in culture supernatants isolated from 293T cells transfected with a lyophilized plasmid DNA pCK-HGF-X7 formulated in 0.9% NaCl at a final DNA concentration of 0.5 mg/mL, with sucrose at 0.25% (lane 3), 1.1% (lane 4), 5% (lane 5), 10% (lane 6) or 20% (lane 7) or with mannitol at 1.2% (lane 8), 4.85% (lane 9) or 10% (lane 10). Control reactions with a negative control (lane 1) and non-lyophilized DNA (lane 2) were used as comparison.
  • FIG. 2 depicts a bar graph comparing in vivo HGF expression between non-lyophilized and lyophilized pCK-HGF-X7.
  • Mice were injected with 100 ⁇ g of non-lyophilized pCK-HGF-X7 containing 0.9% NaCl (NL-HGF-X7) or pCK-HGF-X7 lyophilized with 1.1% Sucrose and 0.9% NaCl (L-HGF-X7) into the tibialis cranialis.
  • HGF expression levels were measured using ELISA in muscle tissue lysates after sacrificing the mice at day 7.
  • HGF expression levels are shown for negative control (lane 1), non-lyophilized pCK-HGF-X7 containing 0.9% NaCl (NL-HGF-X7; lane 2), and pCK-HGF-X7 lyophilized with 1.1% sucrose and 0.9% NaCl (L-HGF-X7; lane 3).
  • FIG. 3 shows a schematic diagram of the experimental procedure using the porcine ischemic heart disease model.
  • NL-HGF-X7 corresponds to non-lyophilized pCK-HGF-X7 containing 0.9% NaCl.
  • L-HGF-X7 corresponds to pCK-HGF-X7 lyophilized with 1.1% sucrose and 0.9% NaCl.
  • FIG. 4 depicts a bar graph showing the effect of non-lyophilized and lyophilized pCK-HGF-X7 on myocardial perfusion. The percent improvement of myocardial perfusion as compared to baseline is shown when the porcine ischemic heart disease model is utilized. Results are shown for pigs injected with plasmid alone (pCK; lane 1), non-lyophilized pCK-HGF-X7 containing 0.9% NaCl (NL-HGF-X7; lane 2), and pCK-HGF-X7 lyophilized with 1.1% sucrose and 0.9% NaCl (L-HGF-X7; lane 3).
  • FIG. 5 depicts a bar graph showing the effect of non-lyophilized and lyophilized pCK-HGF-X7 on wall thickening.
  • the percent improvement on wall thickening in the injected ischemic border area of the left ventricle as compared to baseline is shown when the porcine ischemic heart disease model is utilized.
  • Results are shown for pigs injected with plasmid alone (pCK; lane 1), non-lyophilized pCK-HGF-X7 containing 0.9% NaCl (NL-HGF-X7; lane 2), and pCK-HGF-X7 lyophilized with 1.1% sucrose and 0.9% NaCl (L-HGF-X7; lane 3).
  • DNA or “nucleic acid” or “nucleic acid fragment” refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide or construct.
  • a nucleic acid or fragment thereof may be provided in linear (e.g., mRNA) or circular (e.g., plasmid) form as well as double-stranded or single-stranded forms.
  • isolated nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide contained in a vector is considered isolated for the purposes of the present invention.
  • an isolated polynucleotide examples include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present invention.
  • Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically.
  • a “coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, and the like, are not part of a coding region.
  • Two or more nucleic acids or nucleic acid fragments of the present invention can be present in a single polynucleotide construct, e.g., on a single plasmid, or in separate polynucleotide constructs, e.g., on separate (different) plasmids.
  • any nucleic acid or nucleic acid fragment may encode a single HGF polypeptide or fragment, derivative, or variant thereof, e.g., or may encode more than one polypeptide, e.g., a nucleic acid may encode two or more polypeptides.
  • a nucleic acid may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator, or may encode heterologous coding regions fused to the HGF coding region, e.g., specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
  • a polynucleotide comprising a nucleic acid which encodes a polypeptide normally also comprises a promoter and/or other transcription or translation control elements operably associated with the polypeptide-encoding nucleic acid fragment.
  • An operable association is when a nucleic acid fragment encoding a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).
  • a DNA polynucleotide of the present invention may be a circular or linearized plasmid or vector, or other linear DNA which may also be non-infectious and nonintegrating (i.e., does not integrate into the genome of vertebrate cells).
  • a linearized plasmid is a plasmid that was previously circular but has been linearized, for example, by digestion with a restriction endonuclease. As used herein, the terms plasmid and vector can be used interchangeably.
  • lyophilized DNA refers to any DNA that is prepared in dry form by rapid freezing and dehydration, in the frozen state under high vacuum. “Lyophilizing” or “lyophilization” refers to a process of freezing and drying a solution. Lyophilized DNA is often made ready for use by addition of sterile distilled water.
  • a “vector” refers to any vehicle for the cloning of and/or transfer of a nucleic acid into a host cell.
  • a vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.
  • the term “vector” includes vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.
  • a large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc.
  • Possible vectors include, for example, plasmids such as pBR322 or pUC plasmid derivatives, or the Bluescript vector.
  • the insertion of the DNA fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate DNA fragments into a chosen vector that has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) into the DNA termini.
  • Such vectors may be engineered to contain selectable marker genes that provide for the selection of cells. Such markers allow identification and/or selection of host cells that express the proteins encoded by the marker.
  • Additional vectors include lipoplexes (cationic liposome-DNA complex), polyplexes (cationic polymer-DNA complex), and protein-DNA complexes.
  • a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).
  • Plasmid refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.
  • plasmid refers to a construct made up of genetic material (i.e., nucleic acids). Typically a plasmid contains an origin of replication which is functional in bacterial host cells, e.g., Escherichia coli , and selectable markers for detecting bacterial host cells comprising the plasmid.
  • Plasmids of the present invention may include genetic elements as described herein arranged such that an inserted coding sequence can be transcribed and translated in eukaryotic cells.
  • a plasmid is a closed circular DNA molecule.
  • RNA product refers to the biological production of a product encoded by a coding sequence.
  • a DNA sequence including the coding sequence, is transcribed to form a messenger-RNA (mRNA).
  • mRNA messenger-RNA
  • the messenger-RNA is then translated to form a polypeptide product which has a relevant biological activity.
  • the process of expression may involve further processing steps to the RNA product of transcription, such as splicing to remove introns, and/or post-translational processing of a polypeptide product.
  • expression vector refers to a vector, plasmid or vehicle designed to enable the expression of an inserted nucleic acid sequence following transformation into the host.
  • the cloned gene i.e., the inserted nucleic acid sequence, e.g., a HGF gene or variant thereof, is usually placed under the control of control elements such as a promoter, a minimal promoter, an enhancer, or the like.
  • control elements such as a promoter, a minimal promoter, an enhancer, or the like.
  • Initiation control regions or promoters which are useful to drive expression of a nucleic acid in the desired host cell are numerous and familiar to those skilled in the art.
  • any promoter capable of driving expression of these genes can be used in an expression vector, including but not limited to, viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue specific promoters, pathogenesis or disease related promoters, developmental specific promoters, inducible promoters, light regulated promoters; including, but are not limited to, the SV40 early (SV40) promoter region, the promoter contained in the 3′ long terminal repeat (LTR) of Rous sarcoma virus (RSV), the E1A or major late promoter (MLP) of adenoviruses (Ad), the human cytomegalovirus (HCMV) immediate early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, the baculovirus IE1 promoter, the elongation factor 1 alpha (EF1) promoter, the glyceraldehyde-3-phosphat
  • expression sequences may be modified by addition of enhancer or regulatory sequences and the like.
  • Non-limiting examples of expression vectors of the invention include pCK (Lee et al., Biochem. Biophys. Res. Commun. 272:230 (2000); WO 2000/040737) and pCP (pcDNA3.1, Invitrogen, USA).
  • a “construct” as used herein generally denotes a composition that does not occur in nature.
  • a construct can be produced by synthetic technologies, e.g., recombinant DNA preparation and expression or chemical synthetic techniques for nucleic or amino acids.
  • a construct can also be produced by the addition or affiliation of one material with another such that the result is not found in nature in that form.
  • a “gene” refers to a polynucleotide comprising nucleotides that encode a functional molecule, including functional molecules produced by transcription only (e.g., a bioactive RNA species) or by transcription and translation (e.g., a polypeptide).
  • the term “gene” encompasses cDNA and genomic DNA nucleic acids.
  • “Gene” also refers to a nucleic acid fragment that expresses a specific RNA, protein or polypeptide, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence.
  • “Native gene” refers to a gene as found in nature with its own regulatory sequences.
  • a chimeric gene refers to any gene that is not a native gene, comprising regulatory and/or coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A chimeric gene may comprise coding sequences derived from different sources and/or regulatory sequences derived from different sources. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene or “heterologous” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the cell by a gene transfer procedure.
  • Heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
  • the heterologous DNA may include a gene foreign to the cell.
  • isolated or “biologically pure” refer to material that is substantially or essentially free from components which normally accompany the material as it is found in its native state.
  • isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.
  • the DNA formulation of the invention prior to lyophilization, is formulated with certain excipients, including a carbohydrate and a salt.
  • the stability of a lyophilized formulation of DNA to be utilized as a diagnostic or therapeutic agent can be increased by formulating the DNA prior to lyophilization with an aqueous solution comprising a stabilizing amount of carbohydrate.
  • a carbohydrate of the DNA formulation of the invention is a mono-, oligo-, or polysaccharide, such as sucrose, glucose, lactose, trehalose, arabinose, pentose, ribose, xylose, galactose, hexose, idose, mannose, talose, heptose, fructose, gluconic acid, sorbitol, mannitol, methyl ⁇ -glucopyranoside, maltose, isoascorbic acid, ascorbic acid, lactone, sorbose, glucaric acid, erythrose, threose, allose, altrose, gulose, erythrulose, ribulose, xylulose, psicose, tagatose, glucuronic acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, neuraminic acid
  • the carbohydrate is mannitol or sucrose.
  • the carbohydrate solution prior to lyophilization can correspond to carbohydrate in water alone, or a buffer can be included.
  • buffers include PBS, HEPES, TRIS or TRIS/EDTA.
  • the carbohydrate solution is combined with the DNA to a final concentration of about 0.05% to about 30% sucrose, typically 0.1% to about 15% sucrose, such as 0.2% to about 5%, 10% or 15% sucrose, preferably between about 0.5% to 10% sucrose, 1% to 5% sucrose, 1% to 3% sucrose, and most preferably about 1.1% sucrose.
  • a salt of the DNA formulation of the invention is NaCl or KCl.
  • the salt is NaCl.
  • the salt of the DNA formulation is in an amount selected from the group consisting of between about 0.001% to about 10%, between about 0.1% and 5%, between about 0.1% and 4%, between about 0.5% and 2%, between about 0.8% and 1.5%, between about 0.8% and 1.2% w/v. In certain embodiments, the salt of the DNA formulation is in an amount of about 0.9% w/v.
  • the final concentration of DNA is from about 1 ng/mL to about 30 mg/mL of plasmid.
  • a formulation of the present invention may have a final concentration of about 1 ng/mL, about 5 ng/mL, about 10 ng/mL, about 50 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, about 1 ⁇ g/mL, about 5 ⁇ g/mL, about 10 ⁇ g/mL, about 50 ⁇ g/mL, about 100 ⁇ g/mL, about 200 ⁇ g/mL, about 400 ⁇ g/mL, about 500 ⁇ g/mL, about 600 ⁇ g/mL, about 800 ⁇ g/mL, about 1 mg/mL, about 2 mg/mL, about 2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5 mg/mL, about 5 mg/mL
  • the DNA formulation of the invention is lyophilized under standard conditions known in the art.
  • a method for lyophilization of the DNA formulation of the invention may comprise (a) loading a container, e.g., a vial, with a DNA formulation, e.g., a DNA formulation comprising a plasmid DNA, a salt and a carbohydrate, where the plasmid DNA comprises an HGF gene, or variant thereof, into a lyophilizer, wherein the lyophilizer has a starting temperature of about 5° C. to about ⁇ 50° C.; (b) cooling the DNA formulation to subzero temperatures (e.g., ⁇ 10° C. to ⁇ 50° C.); and (c) substantially drying the DNA formulation.
  • a DNA formulation e.g., a DNA formulation comprising a plasmid DNA, a salt and a carbohydrate, where the plasmid DNA comprises an HGF gene, or variant thereof
  • the lyophilizer has a starting temperature of about 5°
  • the conditions for lyophilization, e.g., temperature and duration, of the DNA formulation of the invention can be adjusted by a person of ordinary skill in the art taking into consideration factors that effect lyophilization parameters, e.g., the type of lyophilization machine used, the amount of DNA used, and the size of the container used.
  • the container holding the lyophilized DNA formulation may then be sealed and stored for an extended period of time at various temperatures (e.g., room temperature to about ⁇ 180° C., preferably about 2-8° C. to about ⁇ 80° C., more preferably about ⁇ 20° C. to about ⁇ 80° C., and most preferably about ⁇ 20° C.).
  • the lyophilized DNA formulations are preferably stable within a range of from about 2-8° C. to about ⁇ 80° C. for a period of at least 6 months without losing significant activity.
  • Stable storage plasmid DNA formulation can also correspond to storage of plasmid DNA in a stable form for long periods of time before use as such for research or plasmid-based therapy. Storage time may be as long as several months, 1 year, 5 years, 10 years, 15 years, or up to 20 years. Preferably the preparation is stable for a period of at least about 3 years.
  • the present invention provides for a lyophilized DNA formulation, where the DNA formulation, prior to lyophilization, comprises a plasmid DNA, and the plasmid DNA comprises an HGF gene, or variant thereof.
  • Hepatocyte growth factor is a heparin binding glycoprotein also known as scatter factor or hepatopoietin-A.
  • An endogenous gene encoding human HGF is located at chromosome 7q21.1 and comprises 18 exons and 17 introns, having the nucleotide sequence of SEQ ID NO: 1 (Seki T., et al., Gene 102:213-219 (1991)).
  • a transcript of about 6 kb is transcribed from the HGF gene, and then, a polypeptide HGF precursor consisting of 728 amino acids (SEQ ID NO: 2) is synthesized therefrom.
  • a polypeptide of dHGF precursor consisting of 723 amino acids is also synthesized by an alternative splicing of the HGF gene.
  • the biologically inactive precursors may be converted into active forms of disulfide-linked heterodimer by protease in serum.
  • the alpha chain having a high molecular weight forms four kringle domains and an N-terminal hairpin loop like a preactivated peptide region of plasminogen.
  • the kringle domains of a triple disulfide-bonded loop structure consisting of about 80 amino acids may play an important role in protein-protein interaction.
  • the low molecular weight beta chain forms an inactive serine protease-like domain.
  • dHGF consisting 723 amino acids is a polypeptide with deletion of five amino acids in the 1st kringle domain of the alpha chain, i.e., F, L, P, S and S.
  • HGF secreted from mesoderm-derived cells has various biological functions, e.g., 1) inducing epithelial cells into a tubular structure; 2) stimulating vascularization from endothelial cells in vitro and in vivo; 3) regeneration of liver and kidney, owing to its anti-apoptosis activity; 4) organogenesis of kidney, ovary and testis; 5) controlling osteogenesis; 6) stimulating the growth and differentiation of erythroid hematopoietic precursor cells; and 7) axon sprouting of neurons (Stella, M. C. and Comoglio, P. M., The International Journal of Biochemistry & Cell Biology 31:1357-1362 (1999)).
  • HGF or a gene encoding HGF or a variant thereof may be developed as a therapeutic agent for treating ischemic or liver diseases.
  • the HGF may exist as either HGF or dHGF, and therefore, the coexpression of HGF and dHGF is important for maximizing the therapeutic effect.
  • a hybrid HGF gene which can simultaneously express HGF and dHGF with a high efficiency for gene therapy is an HGF variant that would be advantageous to utilize in the plasmid DNA formulation of the present invention.
  • the hybrid HGF gene has been previously described in Intl. Appl. No. WO 03/078568 and U.S. Publ. No. 2005/0079581 A1, the contents of each which are herein incorporated by reference.
  • the hybrid HGF gene is prepared by inserting an inherent or foreign intron between exons 4 and 5 in HGF cDNA.
  • the hybrid HGF gene has a higher expression efficiency than HGF cDNA and simultaneously expresses two heterotypes of HGF and dHGF (deleted variant HGF).
  • isoform of HGF refers to any HGF polypeptide having an amino acid sequence that is at least 80% identical (e.g., at least 90% or 95% identical) to a HGF amino acid sequence that is naturally produced in an animal, including all allelic variants. In one embodiment, the term refers to isoforms that are known to have cell proliferation activity. Isoforms of HGF include, without limitation, flHGF, dHGF, NK1, NK2, and NK4, e.g., corresponding to SEQ ID NOs: 2-6, and variants thereof (e.g., NK2 variants, SEQ ID NOs: 11-12).
  • flHGF refers to the full length HGF protein of an animal, e.g., a mammal, e.g., amino acids 1-728 (SEQ ID NO: 2) of human HGF.
  • HGF refers to the deleted variant of HGF protein produced by alternative splicing of the HGF gene in an animal, e.g., a mammal, e.g., human HGF consisting of 723 amino acids (SEQ ID NO: 3) with deletion of five amino acids in the 1st kringle domain of the alpha chain (F, L, P, S and S) from the full length HGF sequence.
  • NK1 refers to an isoform of HGF from an animal, e.g., a mammal, e.g., a human, consisting of the N-terminal hairpin loop and kringle1 domains.
  • NK2 refers to an isoform of HGF from an animal, e.g., a mammal, e.g., a human, consisting of the N-terminal hairpin loop, kringle1, and kringle2 domains.
  • NK4 refers to an isoform of HGF from an animal, e.g., a mammal, e.g., a human, consisting of the N-terminal hairpin loop, kringle1, kringle2, kringle3, and kringle4 domains.
  • HGF HGF-like protein sequence
  • the structure and function of HGF has been extensively studied and one of skill in the art is aware of the amino acids in the HGF sequence that are important for retaining substantially all of the biological activity of the protein and that are preferably not changed or only conservatively changed in any sequence variant of HGF. See, e.g., Hartmann et al., Proc. Natl. Acad. Sci. USA 89:11574 (1992); Lokker et al., EMBO J. 11:2503 (1992), Zhou et al., Structure 6:109 (1998), Ultsch et al., Structure 6:1383 (1998), Shimizu et al., Biochem. Biophys. Res. Commun.
  • An embodiment of the hybrid HGF gene of the present invention comprising the inherent intron is 7113 bp long and has the nucleotide sequence of SEQ ID NO: 7.
  • a hybrid HGF gene may comprise a fragment of inherent intron optionally having a small recombinant sequence inserted thereinto between exons 4 and 5 of HGF cDNA.
  • HGF-X such a hybrid HGF gene comprising a fragment of inherent intron.
  • Examples of hybrid HGF genes include HGF-X2 (SEQ ID NO: 13), HGF-X3 (SEQ ID NO: 14), HGF-X6 (SEQ ID NO: 8), HGF-X7 (SEQ ID NO: 9) and HGF-X8 (SEQ ID NO: 10).
  • HGF has various biological functions, and based on these various functions, HGF, a gene encoding HGF, or a variant thereof, may be developed as a therapeutic agent for treating ischemic or liver diseases.
  • an HGF DNA formulation is administered after reconstitution of the lyophilized DNA formulation.
  • reconstituted refers to the restoration to the original form, e.g., by rehydration, of a substance previously altered for preservation and storage, e.g., the restoration to a liquid state of a DNA plasmid formulation that has been previously dried and stored.
  • the lyophilized composition of the present invention may be reconstituted in any aqueous solution which produces a stable, mono-dispersed solution suitable for administration.
  • aqueous solutions include, but are not limited to: sterile water, TE, PBS, Tris buffer or normal saline.
  • the concentration of reconstituted lyophilized DNA in the methods of the current invention is adjusted depending on many factors, including the amount of a formulation to be delivered, the age and weight of the subject, the delivery method and route and the immunogenicity of the antigen being delivered.
  • the reconstituted lyophilized DNA formulation of the invention may be administered orally or via parenteral routes such as intravenous, intramuscular, intraendocardial, intramyocardial, intrapericardial, intraventricular, intraarticular, intradermal, intracerebral, intrarenal, intrahepatic, intrasplenic, intralymphatic, subcutaneous, intraabdominal, intratesticular, intraovarian, intrauterine, sternal, intratracheal, intraplueral, intrathoracic, intradural, intraspinal, intramedullary, intramural, intrascorionic and arterial injection or infusion, or topically through rectal, intranasal, inhalational or intraocular administration.
  • the method of delivery is intramuscular, intramyocardial, intravenous, intracerebral, or intrarenal.
  • the typical daily dose of the reconstituted lyophilized DNA formulation of the present invention ought to be determined in light of various relevant factors including the conditions to be treated, the chosen route of administration, the age, sex and body weight of the individual patient, and the severity of the patient's symptom, and can be administrated in a single dose or in divided dose. Therefore, the daily dose should not be construed as a limitation to the scope of the invention in any way.
  • treat refers to the administration to a subject of a factor, e.g. a HGF, e.g., a hybrid HGF, or variant thereof, in an amount sufficient to result in amelioration of one or more symptoms of the ischemic or liver disease, or prevent advancement of the ischemic or liver disease.
  • a factor e.g. a HGF, e.g., a hybrid HGF, or variant thereof.
  • ischemic disease refers to a disease associated with a deficient supply of blood to a body part (as the heart or brain) that is due to obstruction of the inflow of arterial blood (as by the narrowing of arteries by spasm or disease).
  • ischemic diseases include coronary artery disease (CAD) and peripheral artery disease (PAD).
  • liver disease applies to many diseases and disorders that cause the liver to function improperly or cease functioning.
  • HGF is a major agent promoting hepatocyte proliferation, and acts in concert with transforming growth factor-alpha and heparin-binding epidermal growth factor during liver regeneration. Additionally, HGF ameliorates hepatic injury via anti-apoptotic effects in animal models of fulminant hepatic failure, and attenuates hepatic fibrosis in animals with liver cirrhosis. Consequently, HGF is considered to not only induce liver regeneration, but also to inhibit disease progression and ameliorate hepatic fibrosis in patients suffering from intractable liver diseases.
  • the reconstituted lyophilized DNA formulation of the invention may be administered according to the delivery methods as set forth above.
  • the method of delivery in the treatment of liver disease will be intravenous, intraarterial, or intrahepatic.
  • the reconstituted HGF DNA formulation can comprise two or more isoforms of HGF.
  • the HGF isoforms may be previously lyophilized separately, or in the same DNA formulation. Both of these lyophilized isoforms, after reconstitution, can be administered separately or at the same time, i.e., co-administered; separate reconstituted plasmid DNA formulations for the two or more isoforms of HGF may be administered or co-administered or a single expression plasmid containing genes for two or more isoforms of HGF and capable of expressing the genes for the two or more isoforms of HGF may be administered.
  • the two isoforms flHGF and dHGF may be administered using two separate plasmids.
  • the two separate plasmids containing genes for flHGF and dHGF may be used for co-administration.
  • a single expression plasmid containing genes for both flHGF and dHGF may be administered.
  • the flHGF and dHGF on a single expression plasmid is encoded by the same polynucleotide or by separate polynucleotides.
  • HGF HGF-like growth factor
  • approaches to include more than one polynucleotide capable of expressing an HGF isoform on a single plasmid include, for example, the use of Internal Ribosome Entry Site (IRES) sequences, dual promoters/expression cassettes, and fusion proteins.
  • IRS Internal Ribosome Entry Site
  • the two or more isoforms expressed from the same plasmid or on two separate plasmids, as discussed above, are selected from the group consisting of flHGF, dHGF, NK1, NK2, and NK4 or selected from the group consisting of SEQ ID NOs: 2 to 6.
  • the two or more isoforms can also include additional HGF isoforms known to one of ordinary skill in the art.
  • the plasmid DNA is administered through direct intracellular injection and, more preferably, by the use of a syringe or a catheter.
  • Catheters have been used to introduce recombinant genes in vivo (see, e.g., E. G. Nabel, et al., Proc. Natl. Acad. Sci. USA 89, 5157 (1992); E. G. Nabel, et al., Science 249, 1285 (1990); E. G. Nabel, et al., Science 244, 1342 (1989); E. G. Nabel, et al., J. Clin. Invest. 91, 1822 (1993); G. Plautz, et al., Circ.
  • Utilization of a catheter provides the ability to deliver the plasmid DNA into the cells which are difficult to access by the use of a syringe.
  • the plasmid DNA can be administered through intraarterial or intravenous injection and, more preferably, by the use of a syringe or a catheter.
  • Administration of the plasmid DNA of the invention can also be accomplished by gene transfer into target cells, in situ, to optimize the subsequent delivery of genes in vivo.
  • the plasmid pCK-HGF-X7 (WO 03/078568) which is designed to express hepatocyte growth factor (HGF) protein was used in the experiment.
  • E. coli (TOP10, Invitrogen, USA) were transformed with pCK-HGF-X7, and a single colony was isolated. The isolated colony was then cultured in LB media containing 30 ⁇ g/mL kanamycin. Plasmid DNA was purified using an EndoFree plasmid Giga kit (Qiagen, USA), and re-suspended in saline containing 0.9% NaCl at a final DNA concentration of 1.0 to 2.0 mg/mL.
  • Formulations of pCK-HGF-X7 were prepared in saline containing 0.9% NaCl at a final DNA concentration of 0.5 mg/mL or 1 mg/mL, with sucrose (0.25, 1.1, 5, 10 or 20% w/v) or mannitol (1.2, 4.85 or 10% w/v).
  • Table 1A and 1B show the percentage sucrose and mannitol, respectively, and the corresponding carbohydrate/DNA (w/w) ratios for the tested pCK-HGF-X7 formulations.
  • the suspended plasmid DNA was then lyophilized with Production-Master Freeze Dryer (C&H Cooling & Heating Systems, Korea). The temperature was lowered to ⁇ 50° C. for 4 hours at 100 mTorr. Then, the temperature was raised to ⁇ 40° C. for 12 hours, ⁇ 30° C. for 6 hours, ⁇ 20° C. for 6 hours, ⁇ 10° C. for 6 hours, 0° C. for 6 hours, 10° C. for 6 hours and 30° C. for 24 hours, progressively, at 28 ⁇ 29 mTorr. The lyophilized plasmid DNA was kept at ⁇ 20° C. until analyzed.
  • the suspended plasmid DNA was also lyophilized with Production-Master Freeze Dryer (C&H Cooling & Heating Systems, Korea). The temperature was lowered to 5° C. for 1 minute, and ⁇ 50° C. for 2 hours at 100 mTorr. Then, the temperature was raised to ⁇ 40° C. for 6 hours, ⁇ 35° C. for 3 hours, ⁇ 30° C. for 6 hours, ⁇ 25° C. for 3 hours, ⁇ 20° C. for 3 hours, ⁇ 15° C. for 3 hours, ⁇ 10° C. for 6 hours, ⁇ 5° C. for 3 hours, 0° C. for 6 hours, and 30° C. for 17 hours, progressively, at 28 ⁇ 29 mTorr. The lyophilized plasmid DNA was kept at ⁇ 20° C. until analyzed.
  • the suspended plasmid DNA was also lyophilized with Production-Master Freeze Dryer (C&H Cooling & Heating Systems, Korea). The temperature was lowered to 5° C. for 1 minute, ⁇ 10° C. for 1 minute, ⁇ 20° C. for 1 minute, ⁇ 30° C. for 1 minute, and ⁇ 50° C. for 1 minute at 150 mTorr. The temperature was maintained at ⁇ 50° C. for another 2 hours at 150 mTorr. Then, the temperature was raised to ⁇ 45° C. for 6 hours, ⁇ 40° C. for 3 hours, ⁇ 35° C. for 6 hours, ⁇ 30° C. for 3 hours, ⁇ 25° C. for 6 hours, ⁇ 20° C.
  • the lyophilized plasmid DNA was kept at ⁇ 20° C. until analyzed.
  • the lyophilized formulations prepared above were analyzed for in vitro gene expression efficiency according to the methods described in Example 3. The in vitro results for these preparations were the same.
  • the lyophilized plasmid DNA was transfected into 293T cells, and the level of HGF expression was measured. As a control, non-lyophilized plasmid DNA was also transfected.
  • mice Thirteen 5-week old BALB/c mice (males, Charles River) were obtained for each group, and provided with food and water ad libitum. The mice were allowed 7 days of rest before being subjected to the experiment.
  • mice were injected with 100 ⁇ g of non-lyophilized pCK-HGF-X7 containing 0.9% NaCl (NL-HGF-X7) or pCK-HGF-X7 lyophilized with 1.1% sucrose and 0.9% NaCl (L-HGF-X7) into the tibialis cranialis, and were sacrificed at day 7 after treatment.
  • the lyophilized plasmid DNA was reconstituted with water to the final concentration of 0.5 mg/mL before injection.
  • HGF protein expression To measure the level of HGF protein expression, the injected muscles were collected, and the muscle tissue was lysed with 500 ⁇ L of cell lysis buffer (50 mM NaCl, 0.2% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 2% IGEPAL CA-630, 25 mM Tris-HCl, pH7.4, 1 mM phenylmethylsulfonyl fluoride) for 16 hours at 4° C. The lysates were centrifuged at 12,000 rpm for 5 minutes, and the supernatants were harvested and analyzed for HGF expression using a human HGF ELISA kit (R&D Systems).
  • cell lysis buffer 50 mM NaCl, 0.2% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 2% IGEPAL CA-630, 25 mM Tris-HCl, pH7.4, 1 mM phenylmethylsulfonyl fluoride
  • the ELISA results were statistically assessed by one way ANOVA and subsequent Tukey's Test using SPSS program (version 13.0).
  • HGF protein An average of 246 ng/mL of HGF protein was produced from the animals administered with pCK-HGF-X7 lyophilized with 1.1% sucrose and 0.9% NaCl (L-HGF-X7), while the animals administered with non-lyophilized pCK-HGF-X7 expressed 76 ng/mL of HGF ( FIG. 2 ). This result indicates that pCK-HGF-X7 lyophilized with 1.1% sucrose and 0.9% NaCl can express HGF protein more efficiently than non-lyophilized pCK-HGF-X7 (p ⁇ 0.001).
  • Xylazine (2 mg/kg), ketamine (20 mg/kg), and atropine (0.05 mg/kg) were injected intramuscularly into each pig. Twenty minutes later, a 22-gauge Medicut sheath was inserted into the superficial femoral artery for continuous monitoring of the blood pressure. Thiopental sodium (10 mg/kg) was injected intravenously, and endotracheal intubation was performed via the orotracheal route. Anesthesia was maintained by inhalation of enflurane. During the operation, positive pressure ventilation and an oxygen fraction of 30% ⁇ 40% were maintained. Electrocardiograms, oxygen saturation and arterial blood pressure were monitored continuously.
  • lidocaine (1 mg/kg) was injected intravenously 15 minutes after the ligation, and the pericardium and thoracotomy wounds were closed. A single 28 Fr chest tube connected to wall suction was removed immediately after enough spontaneous respiration returned, followed by the removal of the endotracheal tube.
  • the lyophilized plasmid DNA was reconstituted with water to the final concentration of 1 mg/mL before injection.
  • the injection points were marked with suture tags using metal rings.
  • SPECT single photon emission computed tomography
  • a 20-segment model was chosen for a segmental analysis. Six segments corresponding to the cardiac base were excluded from the analysis because this region could be easily influenced by the diaphragmatic attenuation or some artifacts around the heart; also because the heart base was far away from the sites of the distal coronary ligation and plasmid injection.
  • the SPECT images constructed by electrocardiography gating were analyzed by an auto-quantitation program (AutoQUANT, ADAC Labs, CA., USA), which is believed to eliminate the possible bias by any associated technician's manipulation.
  • segmental perfusion was quantified by measuring the uptake of 99m Tc-MIBI and calculated as a percentage of the maximum uptake.
  • segmental perfusion thus estimated was less than 70%, it was defined as an underperfused segment and used as the target of plasmid delivery. Segments remaining well perfused even after the coronary ligation were also excluded, as they would probably get no benefit from the therapeutic angiogenesis.
  • Wall thickening in the systolic phase was indicated as a percentage of the end diastolic wall thickness on the gated images.
  • segmental perfusion and wall-thickening were significantly increased in the lyophilized pCK-HGF-X7 treated group as compared to those of the non-lyophilized pCK and pCK-HGF-X7 treated groups.

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