WO2000006759A9 - Systemes d'administration et plasmides anti-angiogenese et leurs procedes de preparation et d'utilisation - Google Patents

Systemes d'administration et plasmides anti-angiogenese et leurs procedes de preparation et d'utilisation

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Publication number
WO2000006759A9
WO2000006759A9 PCT/US1999/016388 US9916388W WO0006759A9 WO 2000006759 A9 WO2000006759 A9 WO 2000006759A9 US 9916388 W US9916388 W US 9916388W WO 0006759 A9 WO0006759 A9 WO 0006759A9
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plasmid
sequence
composition
coding sequence
disease
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PCT/US1999/016388
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English (en)
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WO2000006759A3 (fr
WO2000006759A2 (fr
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Priority to CA002337496A priority Critical patent/CA2337496A1/fr
Priority to EP99938769A priority patent/EP1100941A2/fr
Priority to JP2000562541A priority patent/JP2002524036A/ja
Priority to AU53182/99A priority patent/AU5318299A/en
Publication of WO2000006759A2 publication Critical patent/WO2000006759A2/fr
Publication of WO2000006759A3 publication Critical patent/WO2000006759A3/fr
Publication of WO2000006759A9 publication Critical patent/WO2000006759A9/fr

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  • the present invention relates to gene delivery and gene therapy, and provides novel nucleic acid constructs for expression of anti-angiogenic gene products in a mammal, formulations for delivery that incorporate a nucleic acid construct for expression, and methods for preparing and using such constructs and formulations.
  • Immunotherapy is to strengthen the tumor-host interaction by introduction of tumor-specific antigens or to boost immune function by local cytokine expression.
  • cytokine gene medicines such as IL-2, IFN- ⁇ and IL-12 have been developed.
  • tumor-bearing mice develop an immune response, which leads to inhibition of tumor growth in murine syngeneic tumor model.
  • An alternative approach, anti-angiogenic gene medicine has been recognized as therapeutic strategy for both immunogenic and non- immunogenic tumors.
  • Tumors get oxygen and nutrients through the blood vessels.
  • the formation of blood vessels, or angiogenesis is required for tumor growth and metastases.
  • microvessel density in the tumor show a positive relationship with tumor growth, the risk of metastases, tumor recurrence or death (Folkman, J. , J. Natl . Cancer Inst . , 82:4-6 (1990); Folk an, J. Nat . Med. , 1:27-31 (1995)).
  • angiogenic factors such as VEGF and bFGF
  • anti-angiogenic factors such as endostatin, angiostatin and thromospondin-1
  • reconstitution of angiogenic inhibitors would provide a plausible strategy for cancer therapy (Hori, A., Cancer Res. , 51:6180-4 (1991); Kim, K.J., Na ture, 362:841-4 (1993); O'Reilly, M.S. et al., Cell, 79:315- 28 (1994); O'Reilly, M. S.
  • Angiostatin and Endostatin are two members of an expanding family of proteins that are angiogenesis inhibitors.
  • Angiostatin is an internal proteolytic fragment of mature plasminogen. It contains 4 triple loop disulfide-linked structures, known as kringle domains (Residues 98-440) . It has been shown that a form of 3 kringle domain (residues 98- 333) is more potent in vitro (Cao et al., The Journal of Biological Chemistry, 271:29461-29467 (1996)) and in vivo (Griscelli et al., Proc. Natl . Acad. Sci . USA, 95:6367-6372 (1998)).
  • Endostatin is the C-terminal proteolytic fragment of collagen 18a. Its structure resembles that of E-selectin, an adhesion molecule on endothelial cell (EC) surface (Hohenester, E. et al., EMBO J. , 17:1656-64 (1998)).
  • angiostatin and endostatin can inhibit endothelial cell (EC) proliferation in vitro and angiogenesis in vivo.
  • recombinant proteins have been shown to inhibit tumor growth and metastases in mouse models when injected at high doses.
  • combination of angiostatin and endostatin has synergistic effect in inhibition of tumor growth and metastases (Bachelot, T. et al., Abstract from AARC, vol 39, March 1998).
  • anti-angiogenic therapy can synergize radiation therapy in a murine tumor model (Mauceri, et al., Na ture, 394:287 (1998)).
  • Anti-angiogenic therapy targets to EC, but not tumor cells.
  • EC is more accessible to systemically deliveried drugs, thus anti-angiogenic therapy is particularly useful in treatment of disseminated cancer.
  • EC is not transformed and anti-angiogenic therapy of an experimental model of cancer does not induce acquired drug resistance. Because angiostatic therapy will require a prolonged maintenance of therapeutic levels in vivo, the continuous delivery of a recombinant protein will be expensive and cumbersome.
  • the present invention relates to gene delivery and gene therapy, and provides novel nucleic acid constructs for expression of anti-angiogenic coding sequences in an organism, preferably a mammal, formulations for delivery that incorporate a nucleic acid construct for expression, and methods for preparing and using such constructs and formulations.
  • this invention relates to plasmid constructs for delivery of therapeutic genes to cells in order to modulate tumor activity.
  • the invention also provides methods of using those constructs (including combination therapy with other treatment methods, such as radiation therapy, or agents, such as cytokines, preferably IL-12), as well as methods for preparing such constructs.
  • the pharmaceutically acceptable, cost effective and highly efficient delivery system presented herein represents an unanticipated improvement over the art.
  • a gene therapy approach utilizing an interactive polymeric gene delivery system that increases protein expression by protecting plasmid DNA (pDNA) from nucleases and controlling the dispersion and retention of pDNA in injected tissues has been employed.
  • PINC polymeric interactive non- condensing
  • These polymeric interactive non- condensing (PINC) systems routinely result in a greater amount of gene expression from tissues as compared to delivery of unformulated plasmid in saline.
  • a plasmid expression system encoding murine IFN ⁇ 4 or IL-12 formulated as a complex with PVP could induce an anti-tumor immune response following direct injection into subcutaneous murine tumors.
  • hlGF- I human insulin-like growth factor-I
  • PINC complex a plasmid that encodes human insulin-like growth factor-I
  • intramuscularly delivered gene can achieve a high and persistent expression of therapeutic product in circulating system, it is conceivable that i.m. delivered cancer gene medicines could be used in treatment of disseminated disease, a major limitation of gene therapy.
  • lipid/DNA By tail-vein injection of lipid/DNA, detectable levels of human growth hormone (hGH) in serum, human factor IX (hFIX) in plasma and chloramphenicol acetyltransferase (CAT) in the lung and liver were observed with positively charged lipid/plasmid complexes prepared from 400 nm extruded liposomes with a cationic lipid to co-lipid ratio of 4:1 mol/mol.
  • hGH human growth hormone
  • hFIX human factor IX
  • CAT chloramphenicol acetyltransferase
  • IL-12 transgene expression in murine lungs following administration of IL-12 plasmid/lipid complexes inhibits the growth of pulmonary metastases of Renca tumors in syngeneic BALB/c mice.
  • tumor EC-specific promoters Use of tumor EC-specific promoters to express anti- angiogenic factors is described herein. At least two strategies were used to select proliferating EC-specific promoters. One is to clone promoters of genes that specifically expressed in tumor EC such as flk-1 and avb3 integrins. Another is to generate chimeric promoters of EC- specific enhancers such as endothelin-1 enhancer and cell- cycle-specific promoters such as cyclin A.
  • the present invention by using prototype anti-angiogenic genes, endostatin and angiostatin, demonstrated that anti- angiogenic gene medicine inhibited growth of solid tumor by either intratumoral or intramuscular injection. Anti- angiogenic gene medicines also inhibit lung metastatic tumors after intramuscular or intravenous delivery of formulated angiostatic genes.
  • the invention features a plasmid that contains a tissue specific element and an anti-angiogenic coding sequence.
  • tissue specific element is specific for endothelial cells and is transcriptionally linked to the anti-angiogenic coding sequence.
  • the plasmid may optionally include transcriptional control sequences such as one or more cytomegalovirus promoter sequences.
  • tissue specific element may include a promoter, preferably selected from the group consisting of ET-1, flk-1, Alpha-V, Beta-3, ICAM-2, eye A, E2F1, and cdc6, or may include an enhancer, preferably selected from the group consisting of CMV, four copies of ET-1, and seven copies of ET-1. Examples of EC tissue specific promoters are described in detail below.
  • the tissue specific element preferably is associated or linked with the anti-angiogenic coding sequence in such a manner that the anti-angiogenic coding sequence expression is enhanced, preferably to a predominant (over 50%) nearly exclusive (about 90% or more) or exclusive level (about 100%) , in the tissue or cells of interest. For example, expression may be enhanced by about two-fold.
  • plasmid refers to a construct made up of genetic material (i.e., nucleic acids) . It includes genetic elements arranged such that an inserted coding sequence can be transcribed in eukaryotic cells. Also, while the plasmid may include a sequence from a viral nucleic acid, such viral sequence does not cause the incorporation of the plasmid into a viral particle, and the plasmid is therefore a non-viral vector. Preferably a plasmid is a closed circular DNA molecule.
  • Cytomegalovirus promoter refers to one or more sequences from a cytomegalovirus which are functional in eukaryotic cells as a transcriptional promoter and an upstream enhancer sequence.
  • the enhancer sequence allows transcription to occur at a higher frequency from the associated promoter.
  • transcriptionally linked means that in a system suitable for transcription, transcription will initiate under the direction of the control sequence (s) and proceed through sequences which are transcriptionally linked with that control sequence (s). Preferably no mutation is created in the resulting transcript, which would alter the resulting translation product.
  • coding region refers to a nucleic acid sequence which encodes a particular gene product for which expression is desired, according to the normal base pairing and codon usage relationships.
  • the coding sequence must be placed in such relationship to transcriptional control sequences (possibly including control elements and translational initiation and termination codons) that a proper length transcript will be produced and will result in translation in the appropriate reading frame to produce a functional desired product.
  • the "anti-angiogenic coding sequence” encodes a product selected from the group consisting of endostatin, angiostatin, thrombspondin-1, p53, IL-12, IFN- alpha, truncated tissue factor, an integrin ⁇ v ⁇ 3 blocking agent, a VHL gene product, a cell cycle-dependant kinase inhibitor, a VEGFr, bFGFr, and a bFGF binding protein and preferably is a synthetic sequence having optimal codon usage (for the organism receiving the plasmid, preferably a human), or semi-optimal codon usage (for the organism receiving the plasmid, preferably a human) , or has the nucleotide sequence of any of the plasmids described herein, more preferably plasmid pES1281, pIP1316, pAS1095 or pAS 1096.
  • An anti-angiogenic coding sequence encodes a product that reduces angiogenesis in the organism of interest, preferably to a significant extent, (for example to an extent that it creates a therapeutic effect) or reduces angiogenesis in an in vitro assay.
  • a particular example of coding regions suitable for use in the plasmids of this invention are the natural sequences coding for anti-angiogenic agents.
  • coding region has a nucleotide sequence which is the same as the natural nucleotide sequence encoding the anti- angiogenic agent.
  • the anti-angiogenic coding sequence is a synthetic sequence utilizing optimal or semi-optimal codon usage.
  • a "sequence coding for a human anti-angiogenic agent" or "a human anti-angiogenic coding sequence” is a nucleic acid sequence which encodes the amino acid sequence of a human anti-angiogenic agent, based on the normal base pairing and translational codon usage relationships. It is preferable that the coding sequence encode the exact, full amino acid sequence of the natural human anti-angiogenic agent, but this is not essential.
  • the encoded polypeptide may differ from the natural human anti-angiogenic agent so long as the polypeptide retains anti-angiogenic activity, preferably the polypeptide is at least 50%, 75%, 90%, or 97% as active as natural human anti-angiogenic agent, and more preferably fully as active as the natural human anti-angiogenic agent.
  • the polypeptide encoded by the anti-angiogenic coding sequence may differ from a natural human anti-angiogenic agent by a small amount, preferably less than a 15%, 10%, 5%, or 1% change. Such a change may be of one of more different types, such as deletion, addition, or substitution of one or more amino acids .
  • transcriptional control sequence refers to sequences which control the rate of transcription of a transcriptionally linked coding region.
  • the term can include elements such as promoters, operators, and enhancers.
  • the transcriptional control sequences will include at least a promoter sequence.
  • the plasmid in preferred embodiments, may also contain a growth hormone 3' untranslated region, preferably from a human growth hormone gene.
  • a "growth hormone 3 1 untranslated region” is a sequence located downstream (i.e., 3') of the region encoding material polypeptide and including at least part of the sequence of the natural 3' UTR/poly(a) signal from a growth hormone gene, preferably the human growth hormone gene. This region is generally transcribed but not translated. For expression in eukaryotic cells it is generally preferable to include sequence which signals the addition of a poly-A tail. As with other synthetic genetic elements a synthetic 3' UTR/poly(A) signal has a sequence which differs from naturally-occurring UTR elements. The sequence may be modified, for example by the deletion of ALU repeat sequences. Deletion of such ALU repeat sequences acts to reduce the possibility of homologous recombination between the expression cassette and genomic material in a transfected cell.
  • the plasmid preferably includes a promoter, a TATA box, a Cap site and a first intron and intron/exon boundary in appropriate relationship for expression of the coding sequence.
  • the plasmid may also include a 5' mRNA leader sequence inserted between the promoter and the coding sequence and/or an intron/5' UTR from a chicken skeletal ⁇ -actin gene.
  • the plasmid may have a nucleotide sequence which is the same as the nucleotide sequence of plasmid any of the plasmids described herein.
  • the plasmid may also include: (a) a first transcription unit containing a first transcriptional control sequence transcriptionally linked with a first 5 ' -untranslated region, a first intron, a first coding sequence, and a first 3'- untranslated region/poly (A) signal, wherein the first intron is between the control sequence and the first coding sequence; and (b) a second transcription unit containing a second transcriptional control sequence transcriptionally linked with a second 5 ' -untranslated region, a second intron, a second coding sequence, and a second 3 ' -untranslated region/poly (A) signal, wherein the second intron is between the control sequence and the second coding sequence; wherein the first and second coding sequences contain a sequence coding for any two different anti-angiogenic agents, preferably angiostatin and endostatin, although other combinations such as IP-10 and endostatin or IP-10 and TSPf are also possible.
  • the present invention also provides plasmids and related products and methods with a tissue specific element and an anti-angiogenic coding sequence, wherein the anti-angiogenic coding sequence encodes a fusion or hybrid peptide or protein.
  • the fusion or hybrid agent may be a fusion product of angiostatin and endostatin. Each portion of the fusion product is preferably anti-angiogenic on its own, and has elevated anti-angiogenic effects when presented as part of a fusion or hybrid product.
  • transcription unit or "expression cassette” refers to a nucleotide sequence which contains at least one coding sequence along with sequence elements which direct the initiation and termination of transcription.
  • a transcription unit may however include additional sequences, which may include sequences involved in post-transcriptional or post- translational processes.
  • the first transcriptional control sequence or the second transcriptional control sequence contain one or more cytomegalovirus promoter sequences.
  • the first and second transcriptional control sequences can be the same or different.
  • a "5' untranslated region” or “5' UTR” refers to a sequence located 3' to promoter region and 5' of the downstream coding region. Thus, such a sequence, while transcribed, is upstream of the translation initiation codon and therefore is not translated into a portion of the polypeptide product.
  • one or more of a promoter, 5' untranslated region (5' UTR), the 3' UTR/poly(A) signal, and introns may be a synthetic sequence.
  • synthetic means that the sequence is not provided directly by the sequence of a naturally occurring genetic element of that type but rather is an artificially created sequence (i.e., created by a person by molecular biological methods) . While one or more portions of such a synthetic sequence may be the same as portions of naturally occurring sequences, the full sequence over the specified genetic element is different from a naturally occurring genetic element of that type. The use of such synthetic genetic elements allows the functional characteristics of that element to be appropriately designed for the desired function.
  • a "synthetic intron” refers to a sequence which is not a naturally occurring intron sequence but which will be removed from an RNA transcript during normal post transcriptional processing.
  • Such introns can be designed to have a variety of different characteristics, in particular such introns can be designed to have a desired strength of splice site.
  • first and second coding regions are coding regions for angiostatin then endostatin in the 5' to 3' direction.
  • a "sequence coding for angiostatin” is a nucleic acid sequence which encodes the human angiostatin as described above, based on the normal base pairing and translational codon usage relationships. The sequence coding for endostatin of similarly defined.
  • the sequence coding for angiostatin is 5 1 to the sequence coding for endostatin.
  • the anti- angiogenic coding sequences may all be on a single transcription unit, that all may be on separate transcription units, or that any two coding sequences may be on one transcription unit and the other coding sequence on another transcription unit (in the case of three coding sequences) .
  • the plasmid may also contain an intron having variable splicing, a first coding sequence, and a second coding sequence, wherein the first and second coding sequences include a sequence coding for any two different anti- angiogenic agents, preferably angiostatin and endostatin.
  • the plasmid also has: (a) a transcriptional control sequence transcriptionally linked with a first coding sequence and a second coding sequence; (b) a 5' -untranslated region; (c) an intron 5 1 to the first coding sequence; (d) an alternative splice site 3' to the first coding sequence and 5 1 to the second coding sequence; and (e) a 3 ' -untranslated region/poly (A) signal.
  • the transcriptional control sequence preferably includes a cytomegalovirus promoter sequence.
  • the plasmid also has: (a) a transcriptional control sequence transcriptionally linked with a first coding sequence, an IRES sequence, a second coding sequence, and a 3 ' -untranslated region/poly (A) signal, wherein the IRES sequence is between the first coding sequence and the second coding sequence; and (b) an intron between the promoter and the first coding sequence; wherein the first and second coding sequences include a sequence coding for any two different anti-angiogenic agents, preferably angiostatin and endostatin.
  • the transcriptional control sequence preferably includes a cytomegalovirus promoter sequence and the IRES sequence preferably is from an encephalomyocarditis virus.
  • a delivery composition or delivery system which includes one or more other components in addition to the nucleic acid sequences.
  • Such a composition can, for example, aid in maintaining the integrity of the DNA and/or in enhancing cellular uptake of the DNA and/or by acting as an immunogenic enhancer, such as by the non-DNA components having an immuno-stimulatory effect of their own.
  • the invention features a composition containing a plasmid as described above and a protective, interactive non-condensing compound (PINC) .
  • PINC protective, interactive non-condensing compound
  • the PINC enhances the delivery of the nucleic acid molecule to mammalian cells in vivo, and preferably the nucleic acid molecule includes a coding sequence for a gene product to be expressed in the cell.
  • the relevant gene product is a polypeptide or protein.
  • the PINC is used under conditions so that the PINC does not form a gel, or so that no gel form is present at the time of administration at about 30-40°C.
  • the PINC is present at a concentration of 30% (w/v) or less. In certain preferred embodiments, the PINC concentration is still less, for example, 20% or less, 10% or less, 5% or less, or 1% or less.
  • compositions differ in compound concentration and functional effect from uses of these or similar compounds in which the compounds are used at higher concentrations, for example in the ethylene glycol mediated transfection of plant protoplasts, or the formation of gels for drug or nucleic acid delivery.
  • the PINCs are not in gel form in the conditions in which they are used as PINCs, though certain of the compounds may form gels under some conditions.
  • the term "protects” or “protective” refers to an effect of the interaction between such a compound and a nucleic acid such that the rate of degradation of the nucleic acid is decreased in a particular environment. Such degradation may be due to a variety of different factors, which specifically include the enzymatic action of a nuclease.
  • the protective action may be provided in different ways, for example, by exclusion of the nuclease molecules or by exclusion of water.
  • Some compounds which protect a nucleic acid and/or prolong the bioavailability of a nucleic acid may also interact or associate with the nucleic acid by intermolecular forces and/or valence bonds such as: Van der Waals forces, ion-dipole interactions, ion-induced dipole interactions, hydrogen bonds, or ionic bonds. These interactions may serve the following functions: (1) Stereoselectively protect nucleic acids from nucleases by shielding; (2) facilitate the cellular uptake of nucleic acid by "piggyback endocytosis" . Piggyback endocytosis is the cellular uptake of a drug or other molecule complexed to a carrier that may be taken up by endocytosis.
  • CV Uglea and C Dumitriu-Medvichi Medical Applica tions of Synthetic Oligomers, In: Polymeric
  • the compounds which protect the nucleic acid and/or prolong the bioavailability of a nucleic acid have amphiphilic properties; that is, have both hydrophilic and hydrophobic regions.
  • the hydrophilic region of the compounds may associate with the largely ionic and hydrophilic regions of the nucleic acid, while the hydrophobic region of the compounds may act to retard diffusion of nucleic acid and to protect nucleic acid from nucleases.
  • the hydrophobic region may specifically interact with cell membranes, possibly facilitating endocytosis of the compound and thereby also of nucleic acid associated with the compound. This process may increase the pericellular concentration of nucleic acid.
  • Agents which may have amphiphilic properties and are generally regarded as being pharmaceutically acceptable are the following: polyvinylpyrrolidones; polyvinylalcohols; polyvinylacetates; propylene glycol; polyethylene glycols; poloxamers (Pluronics) ; poloxamines (Tetronics) ; ethylene vinyl acetates; methylcelluloses, hydroxypropylcelluloses, hydroxypropylmethylcelluloses; heteropolysaccharides
  • polylactic acid polyhydroxybutyric acid
  • copolymer systems such as polyethylene glycol- polylactic acid (PEG-PLA) , polyethylene glycol- polyhydroxybutyric acid (PEG-PHB) , polyvinylpyrrolidone- polyvinylalcohol (PVP-PVA) , and derivatized copolymers such as copolymers of N-vinyl purine (or pyrimidine) derivatives and N-vinylpyrrolidone.
  • PEG-PLA polyethylene glycol- polylactic acid
  • PEG-PHB polyethylene glycol- polyhydroxybutyric acid
  • PVP-PVA polyvinylpyrrolidone- polyvinylalcohol
  • derivatized copolymers such as copolymers of N-vinyl purine (or pyrimidine) derivatives and N-vinylpyrrolidone.
  • not all of the above compounds are protective, interactive, non-condensing compounds as described below.
  • non- condensing means that an associated nucleic acid is not condensed or collapsed by the interaction with the PINC at the concentrations used in the compositions.
  • the PINCs differ in type and/or use concentration from such condensing polymers. Examples of commonly used condensing polymers include polylysine, and cascade polymers (spherical polycations) .
  • the term “enhances the delivery” means that at least in conditions such that the amounts of PINC and nucleic acid is optimized, a greater biological effect is obtained than with the delivery of nucleic acid in saline.
  • the level of expression obtained with the PINC: nucleic acid composition is greater than the expression obtained with the same quantity of nucleic acid in saline for delivery by a method appropriate for the particular PINC/coding sequence combination.
  • the PINC is polyvinyl pyrrolidone (PVP) , polyvinyl alcohol (PVA) , a PVP-PVA co-polymer, N-methyl-2-pyrrolidone (NM2P) , ethylene glycol, or propylene glycol.
  • PVP polyvinyl pyrrolidone
  • PVA polyvinyl alcohol
  • NM2P N-methyl-2-pyrrolidone
  • ethylene glycol or propylene glycol.
  • the nucleic acid is preferably not a viral vector, i.e., the nucleic acid is a non-viral vector.
  • the PINC is bound with a targeting ligand.
  • targeting ligands can be of a variety of different types, including but not limited to galactosyl, residues, fucosal residues, mannosyl residues, carntitine derivatives, monoclonal antibodies, polyclonal antibodies, peptide ligands, and DNA-binding proteins.
  • the targeting ligands may bind with receptors on cells such as antigen- presenting cells, hepatocytes, myocytes, epithelial cells, endothelial cells, and cancer cells.
  • the term "bound with” means that the parts have an interaction with each other such that the physical association is thermodynamically favored, representing at least a local minimum in the free energy function for that association.
  • Such interaction may involve covalent binding, or non-covalent interactions such as ionic, hydrogen bonding, van der Waals interactions, hydrophobic interactions, and combinations of such interactions.
  • the targeting ligand may be of various types, in one embodiment the ligand is an antibody. Both monoclonal antibodies and polyclonal antibodies may be utilized.
  • the nucleic acid may also be present in various forms.
  • the nucleic acid is not associated with a compounds (s) which alter the physical form, however, in other embodiments the nucleic acid is condensed (such as with a condensing polymer) , formulated with cationic lipids, formulated with peptides, or formulated with cationic polymers .
  • the protective, interactive non-condensing compound is polyvinyl pyrrolidone, and/or the plasmid is in a solution having between 0.5% and 50% PVP, more preferably about 5% PVP.
  • the DNA preferably is at least about 80% supercoiled, more preferably at least about 90% supercoiled, and most preferably at least about 95% supercoiled.
  • the invention features a composition containing a protective, interactive non-condensing compound and a plasmid containing an anti-angiogenic coding sequence.
  • the invention provides a composition containing a plasmid of the invention (or a plasmid containing an anti-angiogenic coding sequence) and a cationic lipid, preferably with a neutral co-lipid.
  • the cationic lipid is DOTMA and the neutral co-lipid is cholesterol (chol) .
  • DOTMA is 1,2-di-O- octadecenyl-3-trimethylammonium propane, which is described and discussed in Eppstein et al., U.S. Patent 4,897,355, issued January 30, 1990, which is incorporated herein by reference.
  • other lipids and lipid combinations may be used in other embodiments. A variety of such lipids are described in Gao & Huang, 1995, Gene Therapy 2:710-722, which is hereby incorporated by reference.
  • Other cationic lipid delivery technology is described in Brigham, U.S. Patent 5,676,954, issued October 14, 1997, incorporated herein by reference in its entirety, including any drawings.
  • the charge ratio of the cationic lipid and the DNA is also a significant factor
  • the DNA and the cationic lipid are present is such amounts that the negative to positive charge ratio is between 1:0.1 and 1:10, preferably between 1:0.3 and 1:6, more preferably about 1:3. While preferable, it is not necessary that the ratio be 1:3.
  • the charge ratio for the compositions is between about 1:0.1 and 1:10, more preferably between about 1:0.3 and 1:6.
  • cationic lipid refers to a lipid which has a net positive charge at physiological pH, and preferably carries no negative charges at such pH.
  • An example of such a lipid is DOTMA.
  • neutral co-lipid refers to a lipid which has is usually uncharged at physiological pH.
  • An example of such a lipid is cholesterol.
  • negative to positive charge ratio for the DNA and cationic lipid refers to the ratio between the net negative charges on the DNA compared to the net positive charges on the cationic lipid.
  • the DNA preferably is at least about 80% supercoiled, more preferably at least about 90% supercoiled, and most preferably at least about 95% supercoiled.
  • the composition preferably includes an isotonic carbohydrate solution, such as an isotonic carbohydrate solution that consists essentially of about 10% lactose.
  • the composition the cationic lipid and the neutral co-lipid are prepared as a liposome having an extrusion size of between 100 and 1,000 nanometers, preferably between 200 and 900 nanometers, more preferably about 800 nanometers.
  • the liposomes are prepared to have an average diameter of between about 20 and 800 nm, more preferably between about 50 and 400 nm, still more preferably between about 75 and 200 nm, and most preferably about 100 nm.
  • Microfluidization is the preferred method of preparation of the liposomes.
  • the invention features a composition containing: (a) a first component having a plasmid including an anti-angiogenic coding sequence and a cationic lipid, preferably with a neutral co-lipid, wherein the cationic lipid is DOTMA and the neutral co-lipid is cholesterol, wherein the DNA in the plasmid and the cationic lipid are present in amounts such that the negative to positive charge ratio is between 1:0.1 and 1:10, preferably between 1:0.3 and 1:6, more preferably about 1:3; and (b) a second component including a protective, interactive non-condensing compound, wherein the first component is present within the second component.
  • the invention provides a composition having a protective, interactive non-condensing compound, a first plasmid including a first anti-angiogenic coding sequence, preferably an angiostatin coding sequence and one or more other plasmids independently having a second anti- angiogenic subunit coding sequence, preferably an endostatin coding sequence.
  • the invention features a composition
  • a composition comprising a protective, interactive non-condensing compound, a first plasmid having an angiostatin coding sequence, and one or more other plasmids having an endostatin coding sequence.
  • a composition containing a plasmid with an anti-angiogenic coding sequence and a cationic lipid, preferably with a neutral co-lipid.
  • the invention features a method for making any of the plasmids described above by inserting a tissue specific element and an anti-angiogenic coding sequence into a plasmid.
  • the invention also provides methods of making the compositions described above.
  • the method may involve: (a) preparing a DNA molecule having a transcriptional unit, wherein the transcriptional unit contains an anti-angiogenic coding sequence; (b) preparing a protective, interactive non- condensing compound; and (c) combining the protective, interactive non-condensing compound with the DNA in conditions such that a composition capable of delivering a therapeutically effective amount of an anti-angiogenic coding sequence to a mammal is formed.
  • the DNA molecule is a plasmid, wherein the plasmid includes an anti-angiogenic coding sequence, and more preferably also includes a human growth hormone 3'- untranslated region/poly (A) signal.
  • the method may involve the steps of: (a) preparing a DNA having an anti-angiogenic coding sequence; (b) preparing a cationic lipid, preferably in a mixture with a neutral co- lipid, wherein the cationic lipid is DOTMA and the neutral co- lipid is cholesterol; and (c) combining the cationic lipid with the DNA in amounts such that the cationic lipid and the DNA are present in a negative to positive charge ratio between 1:0.1 and 1:10, preferably between 1:0.3 and 1:6, more preferably about 1:3.
  • the method involves the steps of: (a) preparing a first component having a plasmid containing an anti-angiogenic coding sequence and a cationic lipid, preferably with a neutral co-lipid, wherein the cationic lipid is DOTMA and the neutral co-lipid is cholesterol, wherein the DNA in the plasmid and the cationic lipid are present in amounts such that the negative to positive charge ratio is between 1:0.1 and 1:10, preferably between 1:0.3 and 1:6, more preferably about 1:3; (b) preparing a second component having a protective, interactive non-condensing compound; and (c) combining the first and second components such that the resulting composition includes the first component within the second component .
  • the method involves the steps of: (a) preparing a protective, interactive non-condensing compound, (b) preparing a first plasmid having a first anti- angiogenic coding sequence, preferably an angiostatin coding sequence (c) preparing one or more other plasmids independently having other anti-angiogenic coding sequence, preferably an endostatin coding sequence and (d) combining the protective, interactive non-condensing compound, the plasmid having the first anti-angiogenic coding sequence and the other plasmids .
  • the method of making a composition of the invention may also involve combining a plasmid with an anti-angiogenic coding sequence and a cationic lipid, and preferably also with a neutral co-lipid.
  • the invention provides a method for treatment of a mammalian condition or disease, by administering to a mammal suffering from the condition or disease a therapeutically effective amount of a plasmid as described herein.
  • a “therapeutically effective amount” of a composition is an amount which is sufficient to cause at least temporary relief or improvement in a symptom or indication of a disease or condition. Thus, the amount is also sufficient to cause a pharmacological effect. The amount of the composition need not cause permanent improvement or improvement of all symptoms or indications.
  • a therapeutically effective amount of a cancer therapeutic would be one that reduces overall tumor burden in the case of metastatic disease (i.e., the number of metasteses or their size) or one that reduces the mass of the tumor in localized cancers.
  • the condition or disease preferably is a cancer, such as epithelial glandular cancer, including adenoma and adenocarcinoma; squamous and transitional cancer, including polyp, papilloma, squamous cell and transitional cell carcinoma; connective tissue cancer, including tissue type positive, sarcoma and other (oma's); hematopoietic and lymphoreticular cancer, including lymphoma, leukemia and Hodgkin' s disease; neural tissue cancer, including neuroma, sarcoma, neurofibroma and blastoma; mixed tissues of origin cancer, including teratoma and teratocarcinoma.
  • a cancer such as epithelial glandular cancer, including adenoma and adenocarcinoma
  • squamous and transitional cancer including polyp, papilloma, squamous cell and transitional cell carcinoma
  • connective tissue cancer including tissue type positive, sarcoma and other
  • cancerous conditions that are applicable to treatment include cancer of any of the following: adrenal gland, anus, bile duct, bladder, brain tumors: adult, breast, cancer of an unknown primary site, carcinoids of the gastrointestinal tract, cervix, childhood cancers, colon and rectum, esophagus, gall bladder, head and neck, islet cell and other pancreatic carcinomas, Kaposi's sarcoma, kidney, leukemia, liver, lung: non-small cell, lung: small cell, lymphoma: AIDS-associated, lymphoma: Hodgkin' s disease, Lymphomas: non-Hodgkin' s disease, melanoma, mesothelioma, metastatic cancer, multiple myeloma, ovary, ovarian germ cell tumors, pancreas, parathyroid, penis, pituitary, prostate, sarcomas of bone and soft tissue, skin, small intestine, stomach, testis, thymus, thyroid,
  • the invention provides a method for transfection (i.e., the delivery and expression of a gene to cells) of a cell in situ, by contacting the cell with a plasmid described herein for sufficient time to transfect the cell.
  • Transfection of the cell preferably is performed in vivo and the contacting preferably is performed in the presence of about 5% PVP solution.
  • the invention features a method for delivery and expression of an anti-angiogenic gene in a plurality of cells, by: (a) transfecting the plurality of cells with a plasmid or composition of the invention; and (b) incubating the plurality of cells under conditions allowing expression of a nucleic acid sequence in the vector, wherein the nucleic acid sequence encodes an anti-angiogenic agent.
  • the anti-angiogenic agent is a human anti-angiogenic agent and the cells are human cells and/or the contacting is performed in the presence of an about 5% PVP solution.
  • the invention features a method for treating a disease or condition, by transfecting a cell in si tu with a plasmid or composition of the invention.
  • in situ is meant at the cell's naturally occurring location, which may be in vivo or in vi tro depending upon the cell.
  • in situ transfection for example, is a term used primarily to distinguish from ex vivo transfection.
  • the disease or condition can be a localized disease or condition (e.g., a solid tumor) or a systemic disease or condition, (e.g., a metastatic cancer).
  • the invention features a cell transfected with a plasmid or composition of the invention.
  • the invention features a method for treatment of a mammalian condition or disease, by administering to a mammal suffering from the condition or disease a therapeutically effective amount of a composition described herein.
  • a method for treatment of a mammalian condition or disease involves administering to a mammal suffering from the condition or disease a therapeutically effective amount of a composition of a first plasmid with an angiostatin coding sequence and a second plasmid with an endostatin coding sequence.
  • compositions are useful for delivery of a nucleic acid molecule to cells in vivo
  • the invention provides a composition at an in vivo site of administration. In particular this includes at an in vivo site in a mammal.
  • the nucleic acid molecule includes a sequence encoding a gene product.
  • the site of administration is in an interstitial space or a tissue of an animal, particularly of a mammal .
  • compositions also provides methods for using the above compositions. Therefore, in further related aspects, methods of administering the compositions are provided in which the composition is introduced into a mammal, preferably into a tissue or an interstitial space.
  • the composition is introduced into the tissue or interstitial space by injection.
  • the compositions may also be delivered to a variety of different tissues, but in preferred embodiments the tissue is muscle or tumor.
  • the invention provides methods for treating a mammalian condition or disease by administering a therapeutically effective amount of a composition as described above.
  • the disease or condition is a cancer.
  • Figure 1 shows plasmid maps for pESHOO, pES1062, and pES1281.
  • Figure 2 shows plasmid maps for pAS1095 and pAS1096.
  • Figure 3 shows plasmid information for various endothelial cell-specific constructs.
  • Figure 4 shows a plasmid map for pLC1264.
  • Figure 5 shows luciferase activity and endothelial cell specificity of plasmids of the invention.
  • Figure 6 shows a procedure for multimerization of an endothelial enhancer.
  • Figure 7 shows plasmid maps for pAS1359 and pES1358.
  • Figure 8 shows in vitro expression of bioactive endostatin.
  • Figure 9 shows that endostatin/PVP inhibits Renca tumor.
  • Figure 10 shows that endostatin/PVP induces apoptosis of EC.
  • Figure 11 shows endostatin and angiostatin expresion in serum after intramuscular delivery.
  • Figure 12 shows that endostatin/PVP inhibits sc Renca tumor after im delivery.
  • Figure 13 shows that endostatin/PVP inhibits sc Renca tumor after im delivery.
  • Figure 14 shows endostatin transgene mRNA in lung.
  • Figure 15 shows a mouse cornea angiogenesis assay.
  • Figure 16 shows a preferred codon usage table.
  • This invention concerns expression systems for the delivery and expression of anti-angiogenic coding sequences in mammalian cells, and formulations and methods for delivering such expression systems or other expression systems to a mammal . Therefore, particular genetic constructs are described which include nucleotide sequences coding for anti-angiogenic agents, preferably human endostatin or angiostatin. Such a construct can beneficially be formulated and administered as described herein, utilizing the expression systems of this invention.
  • the plasmid be capable of replication in a cell to high copy number.
  • the production is carried out in prokaryotic cells, particularly including Esherichia coli [ E . coli ) cells.
  • the plasmid preferably contains a replication origin functional in a prokaryotic cell, and preferably the replication origin is one which will direct replication to a high copy number.
  • these elements affect post- transcriptional processing in eukaryotic systems.
  • the use of synthetic sequences allows the design of processing characteristics as desired for the particular application.
  • the elements will be designed to provide rapid and accurate processing.
  • a delivery system For delivery of DNA encoding a desired expression product to a mammalian system, it is usually preferable to utilize a delivery system.
  • a delivery system can provide multiple benefits, notably providing stabilization to protect the integrity of the DNA, as well as assisting in cellular uptake.
  • non-DNA components of the formulation may contribute to an immune system enhancement or activation.
  • components of a delivery system can be selected in conjunction with a particular gene product to enhance or minimize the immuno-stimulatory effect.
  • the plasmids may also include elements for expression of an anti-cancer or anti-tumor agent, such as cytokine, for example IL-12 or one or more subunits thereof.
  • an anti-cancer or anti-tumor agent such as cytokine, for example IL-12 or one or more subunits thereof.
  • a "subunit" of a therapeutic molecule is a polypeptide or RNA molecule which combines with one or more other molecules to form a complex having the relevant pharmacologic activity.
  • complexes include homodimers and heterodimers as well as complexes having greater numbers of subunits.
  • a specific example of a heterodimer is IL-12, having the p40 and p35 subunits.
  • the treatment may involve administration of an anti-angiogenic coding sequence and one or more cytokine or other anti-cancer or anti-tumor coding sequences whether on a single plasmid or on separate plasmids.
  • plasmids may be incorporated into compositions for delivery with a protective, interactive non-condensing compound, a cationic lipid and neutral co-lipid, or both. While these are specific effective examples, other components may be utilized in formulations containing the anti-angiogenic expression vectors of the present invention to provide effective delivery and expression of anti-angiogenic agents or with other coding sequences for which manipulation of the activation of immune system components is desirable.
  • the selection of delivery system components and preparation methods in conjunction with the selection of coding sequences provides the ability to balance the specific effects of the encoded gene products and the immune system effects of the overall delivery system composition.
  • This capacity to control the biological effects of delivery system formulation administration represents an aspect of the invention in addition to the anti-angiogenic agent encoding constructs and specific formulations of delivery systems.
  • Co- selection of the encoded gene product and the delivery system components and parameters provides enhanced desired effects rather than merely providing high level gene expression. In particular, such enhancement is described below for the antitumor effects of the exemplary PVP containing compositions .
  • the antitumor effect may be greater than merely additive (i.e., greater than merely the sum of the antitumor effects of the anti-angiogenic agent alone and IL-12 alone) . Enhancement of immuno-stimulatory effects is also desirable in other contexts, for example, for vaccine applications.
  • the immune system activation be minimized for compositions to be delivered to the lung in order to minimize lung tissue swelling.
  • a useful approach for selecting the delivery system components and preparation techniques for a particular coding sequence can proceed as follows, but is not limited to these steps or step order.
  • Such parameters can, for example, include the amount and ratio of DNA to one or more other composition components, the relative amounts of non-DNA composition components, the size of delivery system formulation particles, the percent supercoiled DNA for circular dsDNA vectors, and the specific method of preparation of delivery system formulation particles. The particular parameters relevant for specific types of formulations will be apparent or readily determined by testing.
  • Plasmid Construct Expression Systems A. Plasmid Design and Construction For the methods and constructs of this invention, a number of different plasmids were constructed which are useful for delivery and expression of sequences encoding anti- angiogenic agents. Thus, these plasmids contain coding regions for anti-angiogenic agents, along with genetic elements necessary or useful for expression of those coding regions .
  • Coding sequences for anti-angiogenic agents were incorporated into an expression plasmid that contains eukaryotic and bacterial genetic elements.
  • Eukaryotic genetic elements include the CMV immediate early promoter and 5' UTR, and a human growth hormone 3' UTR/poly(a) signal, which influence gene expression by controlling the accuracy and efficiency of RNA processing, mRNA stability, and translation.
  • the human growth hormone 3' UTR is from a human growth hormone gene, and preferably includes a poly (a) signal. This sequence can be linked immediately following the natural translation termination codon for a cDNA sequence, genomic sequence, modified genomic sequence, or synthetic sequence coding for anti-angiogenic agent.
  • human growth hormone 3' UTR/poly(a) signal is shown by the human growth hormone 3' UTR (nucleotides 1 - 100) and 3 1 flanking sequence (nucleotides 101 - 191; GenBank accession #J03071, HUMGHCSA) below. 1 GGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGT
  • the 5' and 3' UTR and flanking regions can be further and more precisely defined by routine methodology, e . g. , deletion or mutation analysis or their equivalents., and can be modified to provide other sequences having appropriate transcriptional and translational functions. Construction of plasmid, plasmid backbone, human anti-angiogenic cDNA, and final construct is described below in the examples.
  • vWF Von Willebrand factor
  • the preproendothelin (ET-1) gene promoter is a 119 base- pair ("bp") fragment of human Endothelin gene promoter (-240 to -86) which directs EC-specific expression of CAT when fused to minimal SV40 promoter.
  • Murine ET-1 promoter directs expression of either LUC or lipid-peroxidating enzyme in transgenic mice (Harats et al., JCI 95:1335-44, 1995).
  • expression of the transgenes was not confined to vascular EC, but also present in arteries smooth muscle, and selected epithium.
  • the level of expression ranged from high in arteries to low in veins and capillaries, and there was significant variation in expression both between and withing organs.
  • the Styl (-336 to +23) fragment of intracellular adhesion molecule-2 (ICAM-2) gene promoter has been shown to direct heterologous gene (CD59) expression to kidney and lung vasculature in trangenic mice. It is TATA-less promoter and contains Spl, GATA and ETS binding sites.
  • Alpha v beta 3 integrin is preferentially expressed in tumor endothelium.
  • alpha v beta 3 integrin fibronectin receptor
  • the alpha v beta 3 integrin myronectin receptor
  • the transcription initiation site was mapped 169 bp upstream of ATG site.
  • the 5' flanking region does not contain a TATA box or initiation element, but does contain four Spl, two Ets and one GATA binding site.
  • the 222 bp region of alpha v gene promoter has been shown to exert a strong positive effect on alpha v promoter activity.
  • a 6 kb human genomic DNA fragment containing 2.0 kb of the sequence 5' to the start codon is defined as the human beta 3 gene promoter.
  • the 584-bp fragment 5' to the start codon promotes expression of the CAT reporter gene by 5-fold over promoter-less control CAT construct.
  • This beta 3 promoter lacks TATA and CAAT cis-acting elements, but there are two Spl sites flanking the transcription start site. It has been shown that beta 3 promoter can be upregulated by PMA and retinoic acid, but not by proinflammatory cytokine such as TNF/IFN-gamma.
  • Hypoxia has been shown to be a major mechanism for up-regulation of VEGF and its receptors in vivo . In transient transfection assays, hypoxia led to strong transcriptional activation of the Flt-1 promoter, whereas Flk- 1/KDR transcription was essentially unchanged.
  • a 430-bp region of the Flt-1 promoter is required for transcription in response to hypoxia and this region includes a hypoxia- inducible factor (HIF) consensus sequence.
  • the Endothelin-1 enhancer an endothelial cell-specific regulatory region located between 320 and 364 bp upstream of the transcription initiation site of the mouse endothelin-1 gene, was identified by Bu and Quartermous (J. Biol . Chem . 272:32613-32622, (1997)). Three copies of this enhancer sequence have been shown to activate both the ET-1 promoter and heterologous promoters.
  • Gene expression driven by the cell-cycle-specific promoters cyclin A, E2F1, or cdc ⁇ is regulated in a cell- cycle-dependent fashion and this regulation is primarily at the transcriptional level.
  • the promoters of these genes contain common E2F sites which are responsible for repression in the resting GO (zero) phase, and in some cases for activation in cycling cells.
  • the structure and cell cycle-regulated transcription of the human cyclin A gene is described in Proc. Natl . Acad. Sci . USA, 91:5490-5494.
  • Autoregulatory control of E2F1 expression in response to positive and negative regulators of cell cycle progression is described in Genes & Dev. 8:1514-1525; Williams, R. S., R. V.
  • Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells.
  • some or all of the genetic elements can be synthetic, derived from synthetic oligonucleotides, and thus are not obtained directly from natural genetic sequences. These synthetic elements are appropriate for use in many different expression vectors.
  • a synthetic intron is designed with splice sites that ensure that RNA splicing is accurate and efficient.
  • a synthetic 3' UTR/poly(A) signal is designed to facilitate mRNA 3 1 end formation and mRNA stability.
  • a synthetic 5' UTR is designed to facilitate the initiation of translation. The design of exemplary synthetic elements is described in more detail below.
  • Exemplary synthetic 5 'UTR, intron, and 3 ' UTR/poly (A) signal have the general features shown below: 5' UTR Short.
  • Intron 5' splice site sequence matches consensus.
  • 5' splice site sequence is exactly complementary to 5' end of Ul snRNA.
  • Branch point sequence matches consensus. Branch point sequence is complementary to U2 snRNA.
  • Polypyrimidine tract is 16 bases in length and contains 7 consecutive T's. (The tract preferably contains at least 5 consecutive
  • the 5' untranslated region influences the translational efficiency of messenger RNA, and is therefore an important determinant of eukaryotic gene expression.
  • the synthetic 5 'UTR sequence (UT6) has been designed to maximize the translational efficiency of mRNAs encoded by vectors that express genes of therapeutic interest.
  • the sequence of the synthetic 5' UTR (UT6) is shown below.
  • the Kozak sequence is in boldface and the initiation codon is double underlined.
  • the location of the intron (between residues 48 and 49) is indicated by the triangle and the sequences that form the exonic portion of consensus splice sites are single underlined.
  • the restriction sites for Hindlll and Ncol are overlined.
  • the 5' untranslated region (5' UTR), located between the cap site and initiation codon, is known to influence the efficiency of mRNA translation. Any features that influence the accessibility of the 5' cap structure to initiation factors, the binding and subsequent migration of the 43S preinitiation complex, or the recognition of the initiation codon, will influence mRNA translatability.
  • An efficient 5' UTR is expected to be one that is moderate in length, devoid of secondary structure, devoid of upstream initiation codons, and has an AUG within an optimal local context (Kozak, 1994, Biochi ie 76:815-821; Jansen et al., 1994).
  • a 5' UTR with these characteristics should allow efficient recognition of the 5' cap structure, followed by rapid and unimpeded ribosome scanning by the ribosome, thereby facilitating the translation initiation process.
  • the sequence of the synthetic 5 'UTR was designed to be moderate in length (54 nucleotides (nts) ) , to be deficient in G but rich in C and A residues, to lack an upstream ATG, to place the intended ATG within the context of a optimal Kozak sequence (CCACCATGG) , and to lack potential secondary structure.
  • the synthetic 5' UTR sequence was also designed to lack AU-rich sequences that target mRNAs to be rapidly degraded in the cytoplasm.
  • the synthetic 5' UTR sequence was designed to accommodate an intron with consensus splice site sequences.
  • the intron may, for example, be located between residues 48 and 49 (See intron sequence structure below) .
  • the CAG at position 46-48 is the exonic portion of a consensus 5' splice site.
  • the G at position 49 is the exonic portion of a consensus 3' splice site.
  • the synthetic 5' UTR sequence was designed to begin with a Hindlll site and terminate with a Ncol site.
  • RNA splicing is required for the expression of most eukaryotic genes. For optimal gene expression, RNA splicing must be highly efficient and accurate.
  • a synthetic intron termed OPTIVS8B, was designed to be maximally efficient and accurate .
  • the structure of the exemplary synthetic intron, OPTIVS8 is shown below. Sequences for the 5' splice site (5'ss), branch point (bp) , and 3' splice site (3'ss) are double underlined.
  • the recognition sequences for the restriction enzymes Bbsl and Earl are overlined. The cleavage site for Bbsl corresponds to the 5'ss, and the cleavage site for Earl corresponds to the 3'ss.
  • 5'CAG GTAAGTGTCTTC (77) TACTAACGGTTCTTTTTTTCTCTTCACAG G 3'
  • the branch point is located 24 nts upstream from the 3' splice site.
  • the polypyrimidine tract (Yu) is the major determinant of splice site strength.
  • the length of the polypyrimidine tract was extended to 16 bases, and its sequence was adjusted to contain 7 consecutive T residues.
  • OPTIVS8B was designed with three internal restriction enzyme sites, Bbsl, Nhel, and Earl. These restriction sites facilitate the screening and identification of genes that contain the synthetic intron sequence.
  • Bbsl and Earl sites were placed so that their cleavage sites exactly correspond to the 5'ss (Bbsl) or 3'ss (Earl).
  • the sequence of the polypyrimidine tract was specifically designed to accommodate the Earl restriction site. Inclusion of the Bbsl and Earl sites at these locations is useful because they permit the intron to be precisely deleted from a gene. They also permit the generation of an "intron cassette" that can be inserted at other locations within a gene.
  • the 77 bases between the Bbsl site and the branch point sequence are random in sequence, except for the inclusion of the Nhel restriction site.
  • the 3' ends of eukaryotic mRNAs are formed by the process of polyadenylation. This process involves site specific site RNA cleavage, followed by addition of a poly (A) tail. RNAs that lack a poly(A) tail are highly unstable. Thus, the efficiency of cleavage/polyadenylation is a major determinant of mRNA levels, and thereby, of gene expression levels.
  • 2XPA1 is a synthetic sequence, containing two efficient poly (A) signals, that is designed to be maximally effective in polyadenylation.
  • a poly (A) signal is required for the formation of the 3' end of most eukaryotic mRNA.
  • the signal directs two RNA processing reactions: site-specific endonucleolytic cleavage of the RNA transcript, and stepwise addition of adenylates (approximately 250) to the newly generated 3' end to form the poly (A) tail.
  • a poly (A) signal has three parts: hexanucleotide, cleavage site, and downstream element.
  • the hexanucleotide is typically AAUAAA and cleavage sites are most frequently 3' to the dinucleotide CA (Sheets et al . , 1987).
  • Downstream elements are required for optimal poly (A) signal function and are located downstream of the cleavage site.
  • the sequence requirement for downstream elements is not yet fully established, but is generally viewed as UG- or U-rich sequences (Wickens, 1990; Proudfoot, 1991, Cell 64:671-674; Wahle, 1992, Bioessays 14:113-118; Chen and Nordstrom, 1992, Nucl . Acids Res . 20:2565-2572).
  • Naturally occurring poly (A) signals are highly variable in their effectiveness (Peterson, 1992) .
  • the effectiveness of a particular poly (A) signal is mostly determined by the quality of the downstream element. (Wahle, 1992) .
  • Poly (A) efficiency is important for gene expression, because transcripts that fail to be cleaved and polyadenylated are rapidly degraded in the nuclear compartment. In fact, the efficiency of polyadenylation in living cells is difficult to measure, since nonpolyadenylated RNAs are so unstable. In addition to being required for mRNA stability, poly (A) tails contribute to the translatability of mRNA, and may influence other RNA processing reactions such as splicing or RNA transport (Jackson and Standart, 1990, Cell 62:15-24; Wahle, 1992) .
  • Some eukaryotic genes have more than one poly (A) site, implying that if the cleavage/polyadenylation reaction fails to occur at the first site, it will occur at one of the later sites.
  • a gene with two strong poly (A) sites yielded approximately two-fold more mRNA than one with a single strong poly (A) site (Bordonaro, 1995). These data suggest that a significant fraction of transcripts remain unprocessed even with a single "efficient" poly (A) signal. Thus, it may be preferable to include more than one poly(A) site.
  • the sequence of the exemplary synthetic poly (A) signal is shown below.
  • the sequence is named 2XPA.
  • the hexanucleotide sequences and downstream element sequences are double underlined, and the two poly (A) sites are labeled as pA#l and pA#2. Convenient restriction sites are overlined.
  • the entire 2XPA unit may be transferred in cloning experiments as a Xbal- Kpnl fragment. Deletion of the internal BspHI fragment results in the formation of a 1XPA unit.
  • sequence of the synthetic poly (A) site shown above is based on the sequence of the rabbit ⁇ -globin poly (A) signal, a signal that has been characterized in the literature as strong (Gil and Proudfoot, 1987, Cell 49:399-406; Gil and Proudfoot, 1984, Nature 312:473-474).
  • One of its key features is the structure of its downstream element, which contains both UG- and U-rich domains.
  • a double-stranded DNA sequence corresponding to the IXPA sequence was constructed from synthetic oligonucleotides. Two copies of the IXPA sequence were then joined to form the 2XPA sequence. The sequences were joined in such as way as to have a unique Xbal site at the 5' end of the first poly (A) signal containing fragment, and a unique Kpnl site at the 3' end of the second poly (A) signal containing fragment.
  • nucleotide sequences of several natural human anti- angiogenic coding sequences are known, and are provided below, along with a synthetic sequence which also codes for an anti- angiogenic agent.
  • synthetic sequences which encode the anti-angiogenic agent.
  • Such synthetic sequences have alternate codon usage from the natural sequence, and thus have dramatically different nucleotide sequences from the natural sequence.
  • synthetic sequences can be used which have codon usage at least partially optimized for expression in a human. The natural sequences do not have such optimal codon usage. Preferably, substantially all the codons are optimized.
  • Optimal codon usage in humans is indicated by codon usage frequencies for highly expressed human genes, as shown in Fig. 16.
  • the codon usage chart is from the program "Human_High.cod” from the Wisconsin Sequence Analysis Package, Version 8.1, Genetics Computer Group, Madison, WI .
  • the codons which are most frequently used in highly expressed human genes are presumptively the optimal codons for expression in human host cells, and thus form the basis for constructing a synthetic coding sequence.
  • An example of a synthetic anti- angiogenic coding sequence is shown as the bottom sequence in the table below.
  • Endostatin is a member of the expanding angiogenensis inhibitor family of proteins. It is a 20 KDa C-terminal fragment (184 a. a.) of collagen XVIII and selectively inhibits endothelial cell proliferation in vi tro and angiogenesis in vivo (O'Reilly, M.S. et al., Cell 88, 277-285, 1997).
  • Angiostatin is an internal proteolytic fragment of mature plasminogen (38 Kda and 362 a. a) (O'Reilly, M.S. et al., Cell 79, 315-328, 1994) . It contains four triple loop disulfide- linked structures, known as kringle domains.
  • E. coli expressed rEndostatin or Angiostatin was injected at high doses (10 mg/kg/d for 15-16 days) to achieve 97% inhibition of tumor growth efficacy in preclinical studies. It has been claimed that Angiostatin has a half-life of 2 days in blood.
  • Systemic intravenous administration of ⁇ -actin driven p53 gene complexed to cationic liposomes has been found reduce growth and metastases of a malignant human breast cancer in nude mice (Lesoon-Wood et al .
  • VHL Von Hippel Lindau gene
  • Integrin ⁇ v ⁇ 3 are functionally associated on the surface of angiogenic blood vessels.
  • the RGD-containing peptide ligand for integrin ⁇ v ⁇ 3 can home to tumors when injected intravenously into tumor-bearing mice.
  • This ligand has been recently used to target chemotherapeutic drug to tumor vasculature for cancer treament in mice model (Arap, W et al . , Science, 219 : 311 , 1998).
  • MMP-2 matrix metalloproteinase 2
  • PEX truncated MMP-2
  • Angiogenic factor receptor blockage Receptors for VEGF (fit and flk) and for bFGF (bFGF receptors and bFGF binding proteins) regulate angiogenic signaling of growth factors. Neutrolizing Antibody against the angiogenic factors could block angiogenesis and tumor growth. Alternatively, soluble receptor can compete with wild type receptor in growth factor binding thus block angiogenesis.
  • Cytokine such as IL-12 and IFN- ⁇ and chemokine such as IP-10 have been shown to be potent inhibitors of angiogenesis in addition to their immunoregulatory effects.
  • Thrombosis factor (stimulator of blood coagulation:
  • Tissue factor is the major initiating receptor for the thrombogenic cascades.
  • a truncated TF has been targeted to tumor vasculature by a bispecific antibody and cause tumor infarction in mice (Huang, X. et al . , Science,
  • nucleic acids in many formulations are limited due to degradation of the nucleic acids by components of organisms, such as nucleases. Thus, protection of the nucleic acids when delivered in vivo can greatly enhance the resulting expression, thereby enhancing a desired pharmacological or therapeutic effect. It was found that certain types of compounds which interact with a nucleic acid (e.g., DNA) in solution but do not condense the nucleic acid provide in vivo protection to the nucleic acid, and correspondingly enhance the expression of an encoded gene product .
  • a nucleic acid e.g., DNA
  • PINC systems are non-condensing systems that allow the plasmid to maintain flexibility and diffuse freely throughout the muscle while being protected from nuclease degradation. While the PINC systems are primarily discussed below, it will be understood that cationic lipid based systems and systems utilizing both PINCS and cationic lipids are also within the scope of the present invention.
  • a common structural component of the PINC systems is that they are amphiphilic molecules, having both a hydrophilic and a hydrophobic portion.
  • the hydrophilic portion of the PINC is meant to interact with plasmids by hydrogen bonding (via hydrogen bond acceptor or donor groups) , Van der Waals interactions, or/and by ionic interactions.
  • PVP and N-methyl-2-pyrrolidone (NM2P) are hydrogen bond acceptors while PVA and PG are hydrogen bond donors.
  • PINC polymers There is a linear relationship between the structure of a series of co-polymers of vinyl pyrrolidone and vinyl acetate and the levels of gene expression in rat muscle. The substitution of some vinyl pyrrolidone monomers with vinyl acetate monomers in PVP resulted in a co-polymer with reduced ability to form hydrogen bonds with plasmids.
  • the PINC compounds are generally amphiphilic compounds having both a hydrophobic portion and a hydrophilic portion.
  • the hydrophilic portion is provided by a polar group. It is recognized in the art that such polar groups can be provided by groups such as, but not limited to, pyrrolidone, alcohol, acetate, amine or heterocyclic groups such as those shown on pp. 2-73 and 2-74 of CRC Handbook of Chemistry and Physics (72nd Edition) , David R.
  • Lide editor, including pyrroles, pyrazoles, imidazoles, triazoles, dithiols, oxazoles, (iso) thiazoles, oxadiazoles, oxatriazoles, diaoxazoles, oxathioles, pyrones, dioxins, pyridines, pyridazines, pyrimidines, pyrazines, piperazines,
  • hydrophobic groups which, in the case of a polymer, are typically contained in the backbone of the molecule, but which may also be part of a non-polymeric molecule.
  • hydrophobic backbone groups include, but are not limited to, vinyls, ethyls, acrylates, acrylamides, esters, celluloses, amides, hydrides, ethers, carbonates, phosphazenes, sulfones, propylenes, and derivatives of these groups.
  • the polarity characteristics of various groups are quite well known to those skilled in the art as illustrated, for example, by discussions of polarity in any introductory organic chemistry textbook.
  • nucleic acid/PINC complexes described above for delivery and expression of nucleic acid sequences it is also useful to provide a targeting ligand in order to preferentially obtain expression in particular tissues, cells, or cellular regions or compartments .
  • Such a targeted PINC complex includes a PINC system (monomeric or polymeric PINC compound) complexed to plasmid (or other nucleic acid molecule) .
  • the PINC system is covalently or non-covalently attached to (bound to) a targeting ligand (TL) which binds to receptors having an affinity for the ligand.
  • TL targeting ligand
  • Such receptors may be on the surface or within compartments of a cell.
  • Such targeting provides enhanced uptake or intracellular trafficking of the nucleic acid.
  • the targeting ligand may include, but is not limited to, galactosyl residues, fucosal residues, mannosyl residues, carnitine derivatives, monoclonal antibodies, polyclonal antibodies, peptide ligands, and DNA-binding proteins.
  • Examples of cells which may usefully be targeted include, but are not limited to, antigen-presenting cells, hepatocytes, yocytes, epithelial cells, endothelial cells, and cancer cells .
  • nucleic acid coding sequences can be delivered and expressed using a two-step targeting approach involving a non-natural target for a PINC system or PINC- targeting ligand complex.
  • a PINC-plasmid complex can target a binding pair member which is itself attached to a ligand which binds to a cellular target (e.g., a MAB) . Binding pairs for certain of the compounds identified herein as PINC compounds as identified in Subramanian et al.
  • the PINC can be complexed to a tareting ligand, such as an antibody. That antibody can be targeted to a non- natural target which binds to, for example, a second antibody.
  • murine model systems were utilized based on murine tumor cell lines.
  • the line primarily used was S.C. VII/SF, which is a cell line derived from murine squamous cell carcinoma (S.C. ) .
  • Squamous cell carcinoma of the head and neck begins with the cells lining the oral and pharyngeal cavities. Clinical disease progresses via infiltration and spreads into the underlying tissues and lymphatics.
  • the undifferentiated, in vivo passage tumor line S.C. VII/SF displays this typical growth pattern. In addition, its rapid growth rate provides a relatively short test period for individual experiments.
  • Other murine tumor cell lines include another SCC line KLN- 205, a keratinocyte line 1-7, and a colon adenocarcinoma line MC-38.
  • An optimal model system preferably satisfies the criteria based on having tumor growth rate in vivo (i . e . , tumors are ready for treatment in 4-10 days post implant), invasiveness, and local spread similar to those observed in clinical disease, and providing accessibility for experimental treatment.
  • the SCC VII/SF cell line was utilized as the primary model system cell line. This cell line typically grows rapidly, resulting in death of untreated syngeneic mice 14-17 days after tumor cell implantation.
  • SCCVII cells can be utilized in cell culture to provide an in vitro evaluation of anti-angiogenic agent expression construct and formulation characteristics, such as expression levels and cellular toxicities.
  • the cells can be implanted subcutaneously in mice. This system can be utilized in tests in which accessibility of the implant site is beneficial. As an example, the method was utilized in evaluations of expression efficiencies based on the expression of chloramphenicol acetyltransferase (CAT) . Third, the cells can be implanted transcutaneously into the fascia of digastric muscle.
  • CAT chloramphenicol acetyltransferase
  • the cells can be implanted transcutaneously into digrastric/mylohyoid muscles.
  • the important features of models 3 and 4 are shown in the table below.
  • the tumor size treated in the mouse models is generally 20-50 mm 3 .
  • a 50 mm 3 mouse tumor is approximately equivalent to 150 cc 3 human tumor having an average diameter of about 6.6 cm. This tumor size is approximately 10-fold larger than the size proposed to be treated in the phase I clinical trials. This indicates that the mouse models are strongly biased towards over estimating the expected tumor burden in human patients .
  • expression systems such as those described above provide the potential for expression when delivered to an appropriate location
  • this invention also provides particular formulations which include one or more expression system constructs (e.g., DNA plasmids as described above), and a protective, interactive non-condensing compound.
  • an additional significant factor relating to the plasmid construct is the percentage of plasmids which are in a supercoiled (SC) form rather than the open circular (OC) form.
  • a variety of delivery methods can be used with the constructs and formulations described above, in particular, delivery by injection to the site of a tumor can be used.
  • the submandibular tumor models utilized injection into four quadrants of the tumor being treated.
  • the effects of the administration of the anti-angiogenic formulations described above were evaluated using the S.C. VII mouse tumor models. Plasmid constructs as described above were incorporated in delivery formulations . The formulations were delivered by injection. The effects of the expression of the human anti-angiogenic plasmids in tumor cells on the progress of the mouse tumors demonstrates that such anti- angiogenic expression is effective against such tumors.
  • D. Toxicity Evaluation of Exemplary Formulations The exemplary formulations do not show high cellular toxicity at the concentrations tested, suggesting that the formulations do not significantly kill cells by direct toxic action in vivo.
  • Administration Administration refers to the route of introduction of a plasmid or carrier of DNA into the body.
  • the expression systems constructs and the delivery system formulations can be administered by a variety of different methods.
  • Administration can be directly to a target tissue or by targeted delivery to the target tissue after systemic administration.
  • the present invention can be used for treating disease by administration of the expression system or formulation to the body in order to establishing controlled expression of any specific nucleic acid sequence within tissues at certain levels that are useful for gene therapy.
  • vector vector
  • formulations for delivery are described above.
  • the preferred embodiments are by direct injection using needle injection.
  • any selected vector construct will depend on the particular use for the expression vectors. In general, a specific formulation for each vector construct used will focus on vector uptake with regard to the particular targeted tissue, followed by demonstration of efficacy. Uptake studies will include uptake assays to evaluate cellular uptake of the vectors and expression of the DNA of choice. Such assays will also determine the localization of the target DNA after uptake, and establishing the requirements for maintenance of steady-state concentrations of expressed protein. Efficacy and cytotoxicity can then be tested. Toxicity will not only include cell viability but also cell function.
  • Muscle cells have the unique ability to take up DNA from the extracellular space after simple injection of DNA particles as a solution, suspension, or colloid into the muscle. Expression of DNA by this method can be sustained for several months.
  • DNA vectors Delivery of formulated DNA vectors involves incorporating DNA into macromolecular complexes that undergo endocytosis by the target cell.
  • complexes may include lipids, proteins, carbohydrates, synthetic organic compounds, or inorganic compounds.
  • the complex includes DNA, a cationic lipid, and a neutral lipid in particular proportions.
  • the characteristics of the complex formed with the vector determines the bioavailability of the vector within the body.
  • Other elements of the formulation function as ligand which interact with specific receptors on the surface or interior of the cell. Other elements of the formulation function to enhance entry into the cell, release from the endosome, and entry into the nucleus . Delivery can also be through use of DNA transporters.
  • DNA transporters refers to molecules which bind to DNA vectors and are capable of being taken up by epidermal cells.
  • DNA transporters contain a molecular complex capable of non- covalently binding to DNA and efficiently transporting the DNA through the cell membrane. It is preferable that the transporter also transport the DNA through the nuclear membrane. See, e.g., the following applications all of which
  • a DNA transporter system can consist of particles containing several elements that are independently and non- covalently bound to DNA. Each element consists of a ligand which recognizes specific receptors or other functional groups such as a protein complexed with a cationic group that binds to DNA. Examples of cations which may be used are spermine, spermine derivatives, histone, cationic peptides and/or polylysine. One element is capable of binding both to the DNA vector and to a cell surface receptor on the target cell. Examples of such elements are organic compounds which interact with the asialoglycoprotein receptor, the folate receptor, the mannose-6-phosphate receptor, or the carnitine receptor. A second element is capable of binding both to the DNA vector and to a receptor on the nuclear membrane.
  • the nuclear ligand is capable of recognizing and transporting a transporter system through a nuclear membrane.
  • An example of such ligand is the nuclear targeting sequence from SV40 large T antigen or histone.
  • a third element is capable of binding to both the DNA vector and to elements which induce episomal lysis. Examples include inactivated virus particles such as adenovirus, peptides related to influenza virus hemagglutinin, or the GALA peptide described in the Szoka patent cited above.
  • the lipids may form liposomes which are hollow spherical vesicles composed of lipids arranged in unilamellar, bilamellar, or multilamellar fashion and an internal aqueous space for entrapping water soluble compounds, such as DNA, ranging in size from 0.05 to several microns in diameter.
  • Lipids may be useful without forming liposomes. Specific examples include the use of cationic lipids and complexes containing DOPE which interact with DNA and with the membrane of the target cell to facilitate entry of DNA into the cell. Gene delivery can also be performed by transplanting genetically engineered cells.
  • myoblasts immature muscle cells called myoblasts may be used to carry genes into the muscle fibers.
  • Myoblast genetically engineered to express recombinant human growth hormone can secrete the growth hormone into the animal's blood. Secretion of the incorporated gene can be sustained over periods up to 3 months.
  • Myoblasts eventually differentiate and fuse to existing muscle tissue. Because the cell is incorporated into an existing structure, it is not just tolerated but nurtured. Myoblasts can easily be obtained by taking muscle tissue from an individual who needs gene therapy and the genetically engineered cells can also be easily put back with out causing damage to the patient's muscle. Similarly, keratinocytes may be used to delivery genes to tissues. Large numbers of kera- tinocytes can be generated by cultivation of a small biopsy. The cultures can be prepared as stratified sheets and when grafted to humans, generate epidermis which continues to improve in histotypic quality over many years. The keratinocytes are genetically engineered while in culture by transfecting the keratinocytes with the appropriate vector. Although keratinocytes are separated from the circulation by the basement membrane dividing the epidermis from the dermis, human keratinocytes secrete into circulation the protein produced.
  • the chosen method of delivery should result in expression of the gene product encoded within the nucleic acid cassette at levels which exert an appropriate biological effect.
  • the rate of expression will depend upon the disease, the pharmacokinetics of the vector and gene product, and the route of administration, but should be in the range 0.001-100 mg/kg of body weight /day, and preferably 0.01-10 mg/kg of body weight/day. This level is readily deter inable by standard methods. It could be more or less depending on the optimal dosing.
  • the duration of treatment will extend through the course of the disease symptoms, possibly continuously. The number of doses will depend upon the disease, delivery vehicle, and efficacy data from clinical trials.
  • Promoters Two sets of reporter vectors - CMV enhVpro " and CMV enhVpro " with either CAT (pCT1132 and pCT1133) or LUC (pLC1137 and pLC1138 as reporter gene) were constructed.
  • Sad site is an unique site in thses vectors.
  • the promoter sequence will be amplified from human genomic DNA by PCR with two primers (5' primer, 3' primer with Sacl site) followed by cloning into TA vector.
  • the construct with right orientation (3' is away from the Sacl site on TA vector so that Sacl digest give PCR insert) .
  • the Sacl fragment will be inserted into Sacl site of a vector above.
  • the endothelin enhancer (ETe) was synthesized with overhangs to create Bgl II and Bam HI sites as shown below.
  • gatctGTACTTCATACTTTTCATTCCAATGGGGTGACTTTGCTTCTGGAG aCATGAAGTATGAAAAGTAAGGTTACCCCACTGAAACGAAGACCTCctag
  • DNA fragment was multimerized by ligation at high concentration and digested with Bgl II and Bam HI to eliminate inverted and everted repeats. DNA species containing four and seven tandem copies were gel-purified and inserted into plasmids containing various endothelial-specific or proliferation-specific promoters . Materials and Methods
  • Plasmids containing endothelial-specific promoters were constructed as follows. The minimal promoter sequences of endothelin-1 (ET-1), KDR/flk-1, ICAM-2, ⁇ 3, and ⁇ V, and cell cycle-dependent genes (cyclin A, E2F1, or cdc6) were directly amplified by PCR from human genomic DNA. The amplified promoter sequence was then subcloned into pCR 2.1 (Invitrogen) .
  • the promoter sequence was then subcloned as a Sacl-Sacl fragment into an expression plasmid pLC1136 which contains the luciferase reporter gene, a synthetic intron, and the human growth hormone 3' untranslated region/poly (A) signal to create the promoter specific expression constructs.
  • Plasmids were grown under kanamycin selection in E. coli host strains DH5 ⁇ and purified using alkaline lysis and chromatographic methods. Purified plasmid utilized for injections had the following specifications: ⁇ 50 Eu/mg endotoxin. Endothelial Cell Culture and transfection
  • Human umbilical aortic endothelial cells were grown in six well plates in EBM-1 media (Clonetics, Inc.) supplemented with 5% fetal bovine serum with supplement of endothelial cell growth factors. HUVEC at passages 2-4 were used for transfection. HeLa cells were also grown in 6-well plates in Dulbecco' s modified Eagle's medium supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin. Cultured cells were transfected by DEAE-dextran with 2 ⁇ g of reporter constructs.
  • plasmid pBG0965
  • pBG0965 expressing the ⁇ -galactosidase gene, driven by the cytomeglovirus immediate early promoter
  • Cell extracts were prepared 48 hr after transfection and luciferase and ⁇ -galactosidase assays were performed.
  • the relative luciferase activity was calculated as the ratio of light units to ⁇ -galactosidase units.
  • the corrected light units for HUVECs were divided by the corrected light units for HeLa cells to obtain fold endothelial- specificity.
  • the resulting reporter plasmids containing EC-enhancer and cell- cylce-specific promter into two different endothelial cell lines, HUVEC and BAEC, and compared the activity to that in a non-endothelial cell line, NIH3T3. The reporter activities was assayed in these samples.
  • EC-specific activity analysis The activity of ETe/ETp in HUVEC and HeLa cells and the specificity (EC vs HeLa) are shown in the figures. The data indicated that ETe enhances ETp expression specifically in EC by 10-fold.
  • EC-specific expression of anti-angiogenic genes The coding sequence for endostatin or angiostatin was inserted into vector pLC1265 to generate pES1358 and pAS1359. Thus, the expression of endostatin and angiostatin was driven by ETe/ETp. The specificity of endostatin and angiostatin expression will be determinced.
  • Plasmid construction Plasmids containing an expression cassette for endostatin or angiostatin were constructed as follows.
  • the coding sequences of endostatin is the 184 aa of C-terminal of collagen 18al.
  • Angiostatin is internal fragment (97- 440aa) of human plasminogen (Accession # M74220; Browne, M.J., Chapman, C.G., Dodd, I., Carey, J.E., Lawrence, G.M.P., Mitchell, D., and Robinson, J. H. Expression of recombinant human plasminogen and aglycolplasminiogen in HeLa cells.
  • Endostatin and angiostatin coding sequences were directly amplified by PCR from human liver cDNA (Clontech) using oligonucleotide primers (shown below) , which add a BamHI site at the 5' end and an Xbal site at the 3' end: human angiostatin 5' primer
  • the coding sequence for Igk-HA-endostatin or -angiostatin was then subcloned as an BamHl-Xbal fragment into the expression plasmid containing the cytomegalovirus immediate early promoter, a synthetic intron, and the bovine growth hormone 3' untranslated region/poly (A) signal to create the endostatin or angiostatin expression systems, pESHOO (murine endostatin, mE) , pES1281 (human endostatin, hE) , pAS1095 (human angiostatin kl-k4, hAk4), or pAS1096 (human angiostatin kl-k3, hAk3) .
  • pESHOO murine endostatin, mE
  • pES1281 human endostatin, hE
  • pAS1095 human angiostatin kl-k4, hAk4
  • pAS1096 human angiostatin
  • Plasmid pVC0612 empty plasmid, EP contains expression elements including the cytomegalovirus immediate early promoter and the 3' UTR/poly(A) signal from the bovine growth gene in the pVC0289 backbone described by Alila et al., Human Gene Therapy, 8:1785-1795 (1997). Plasmid pVC0612 was used as a control plasmid in all in vivo experiments. Plasmids for intra-tumoral injection were grown under kanamycin selection in E.
  • Plasmid formulation DNA/PVP complex: Purified expression plasmid and control plasmids were formulated at a concentration of 3 mg DNA/ml in a PINC delivery system as described previously (Mumper et al., Pharmaceutical Research, Vol. 13, No.
  • Positively charged plasmid/lipid complexes were prepared at a 1:3 -/+ charge ratio in 10% (w/v) lactose by mixing the plasmid with the liposomes under controlled conditions (Freimark et al., The Journal of Immunology, 160:4580-4586 (1998)).
  • the mean diameter and zeta potential of the complexes were characterized using dynamic light scattering and Doppler electrophoretic light scattering.
  • the complexation efficiency was determined by agarose gel electrophoresis .
  • Cos-1 cells were cultured in DMEM.
  • Endothelial cells were from Clonetics) and cultured in specified medium according to the Manufacturer.
  • TS/A is a tumor cell line established by Dr. P. Nanni, University of Bologna, Italy, from the first in vivo transplant of a moderately differentiated mammary adenocarcinoma that spontaneously arose in a BALB/c mouse (Nanni et al., Clin . Exp .
  • Tumor cell cultures were maintained in sterile disposable flasks from Corning (Corning, NY) at 37° C in a humidified 5% C0 2 atmosphere, using either RPMI 1640 (Renca, TS/A) or DMEM ( LLC) supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin and 50 ⁇ g/ml gentamycin; all from Life Technologies.
  • Angiosta tin/Endosta tin ELISA Supernatants (1 ml/well) were collected from transfected cells above and levels of endostatin or angiostatin were assayed by enzyme-linked immunosorbent assay (ELISA) .
  • ELISA plates (Falcon flexible PVC #3912) were coated with affinity purified goat anti-PG (plasminogen) IgG (Enzyme Research Laboratories, Inc.) or anti-endostatin (Folkman lab) in 50 mM carbonate buffer (1/500 dilution) at 4° C overnight and blocked with 150 ⁇ l/well of 2% BSA in PBS at room temperature for 4 hr .
  • OPD substrate o-phenylenediamine (Sigma 5 mg tablets) diluted in citrate-phosphate buffer (5.2 M citric acid, 13.8 M Na 2 HP0 4 ; pH 5) with 0.1% H 2 0 was applied and color was developed for 2-5 min. The reaction was stopped by the addition of 50 ⁇ l/well of 2.5 M H 2 S0 4 . Absorbance was determined at 450 nm in an EL340 Microplate reader (Bio-tek Instruments) . Serial two-fold dilutions of plasminogen lysine-binding site I (Sigma) , HA-angiostatin or HA-endostatin from transfected cells was used as a standard.
  • mice Normal 8-week-old female BALB/c or C57bl mice were purchased from Harlan Laboratories, Houston, TX. Mice were maintained on ad libitum rodent feed and water at 23° C, 40% humidity, and a 12-h/12-h light-dark cycle. Animals were acclimated for at least 4 days before the start of the study.
  • mice BALB/c or C57bl mice were challenged s.c. in the middle of the left flank with 30 ⁇ l of a single-cell suspension contained the specified number of cells. Seven days later when the tumor size reached approximately 10 mm 3 , treatments with endostatin/PVP or EP/PVP started and were repeated at 1-2 day intervals for 2 weeks (total of 8 treatments: 4/week) . Tumor volume was measured with electronic caliper in the two perpendicular diameters and in the depth. Measurements of the tumor masses (mm 3 ) were performed twice a week for 40-50 days. All mice bearing tumor masses exceeding 1 cm 3 volume were sacrificed for humane reasons.
  • mice Lung metastases were established in mice by injecting 3 x 10 5 Renca cells in 100 ⁇ L HBSS (Hank's Balanced Salt Solution, without Ca ++ or Mg ++ , Life Technologies) into the tail vein. Animals were warmed using a 150-watt lamp and placed in a mouse restrainer prior to tail vein injection. 4 or 7 day after tumor injection, mice were injected intravenously with endostatin/DC, angiostatin/DC or EP/Dconce a week for 2-3 weeks.
  • the lungs were insufflated with 1-2 mL India ink solution (150 mL distilled H 2 0, 30 L India ink, 4 drops ammonium hydroxide) , using a 22 gauge gavage needle, then fixed with Fekete' s solution (90 mL Formaldehyde, 37% solution, 900 mL 70% EtOH, 45 mL glacial acetic acid) for at least 24 hours. Metastases counts were performed under a dissecting microscope. Survival data were analyzed using the Kaplan-Meier log-ranked test. All other data were analyzed using the Neuman-Keul' s Test on StatMost for Windows software (DataMost Corporation, Sandy, UT) . Data were considered statistically significant if p values were ⁇ 0.05.
  • Subcutaneous tumor (8-12 mm in diameter) were resected aseptically. All necrotic zones were removed and the viable tissue was minced and dissociated with collagen (Type I, 200 U/ml) and Dnase (270 U/ml) (Sigma Chemical Co., St. Louis, MO) . Cells were susupended in DMED with nsupplement and plated at 5-10x106 viable cells/Tl75 flask. After a 3 hr adherence, the culture were rinsed and given fresh medium. 48 h later, te adherent tumor cells were harvested by brief trypsinization, wahsed once with medium and resuspend in HBSS.
  • collagen Type I, 200 U/ml
  • Dnase 270 U/ml
  • mice Aliquotes of 106 cells in 0.1 ml of HBSS were injected subcutaneously. When tumors were 12-15 in diameter, the mice were anesthetized with methoxyflurane . The tumors in one group of mice were surgical excised, and the area closed with metal woud clips. The other group of mice underwent a sham surgical procedure which left the sc tumors intact. The mice were monitored daily and killed 10-14 days after surgery. The lungs were weighed and stained as described above.
  • Intramuscular injection and electropora tion 200-300 ug of DNA/PVP (3mg/ml) were injected into tibialis (25 ul) and gastrocnemia (50 ul) in one mouse leg, 2 min after injection, electroporation (500V/cm, 96 usec with 4 pulses) was applied on the injected leg. Histological analysis : For CD-31, CD3, CD4 , MAC-1
  • Immunostaining Frozen sections were cut at 5 ⁇ M and then subsequently fixed in Acetone for 10 minutes at room temperature. Immunohistochemistry was accomplished utilizing an avidin biotin technique. Endogenous peroxidases were quenched by incubating the sections with a 1% H 2 0 2 solution for 10 minutes at room temperature. Nonspecific binding was blocked with an incubation of 5 minutes at room temperature with PowerBlock (Cat# HK085-5K, Biogenex, San Ramon, CA, USA) . The sections were then incubated with the appropriate dilution of primary antibody for 1 hour at room temperature. Following washes in PBS, the secondary antibody, biotinylated anti-Rat
  • PCNA immunostaining was accomplished utilizing the Mouse to Mouse HRP kit (Cat#MTM001, Scytek, Logan, UT, USA) and following the manufacturer's instructions.
  • HLMEC human lung microvessel nendothelial cells
  • bFGF growth factors
  • Hydron pellet (Hydron; Interferon Sciences) of ⁇ 1 ul (Aluminum sucrose sulfate; Sigma) were formulated containing bFGF at 1 ug/ml and implanted into the cornea of C57bl mouse 0.3-0.5 mm from the limbus. Next day, DNA/PVP was injected im or DNA/DC was injected iv. Angiogenesis was assessed by slit-lamp microscopy on day 3, 4, and 5 after implantation. Maximal angiogenesis was achieved on day 5. Inhibition of angiogenesis by endostatin or angiostatin gene medicine was examined by comparing to control vector.
  • angiostatin precusors are rich in liver. Due to the fact that Angiostatin and Endostatin are internal fragment devoid of their natural secretion sequence, and that there are no commercially available antibodies against angiostatin or endostatin, HA epitope which is 9aa from influenza hemaglutinin protein is tagged to the N-terminus of angiostatin or endostatin so that antibody against this peptide can be used for detection. Ig-kappa signal peptide is added to upstream of HA-angiostatin or -endostatin to direct secretion of the fusion protein.
  • the expression plasmids for HA-tagged mouse endostatin (mE, pESllOO) , HA-free mouse endostatin (mE-HA " , pES1062), HA-human endostatin (hE, pES1281) , HA-human angiostatin kl-k4 (hAk4), HA-angiostatin kl-k3 (hAk3, pAS1096) were constructed and the maps were shown in Fig.l.
  • Expression of bioactive endostatin and angiostatin To assess their expression, the expression plasmids are transfected into cos-1 cells, human endothelial cells (HUVEC) , or Renca tumor cells.
  • the transgene expression was examined for mRNA in cells by RT-PCR and for protein in media by western blotting (Fig 2) .
  • the results showed Endostatin and Angiostatin were transcribed as correct sizes as indicated and no missplicing products were detected.
  • the recombinant protein was present as a single band with an approximate molecular weight of 22 KD (HA-mE) , 21 KD (mE) , 20 KD (HA-hE) or 30 KD (HA-hAk3) .
  • the protein expression was quantitatively assessed an ELISA with anti-Endosattin or plasminogen antibody and anti-HA epitope antibody. The result indicated that mE and hAk3 was about 4 ng/ml in the culture medium.
  • hAk3 was expressed at higher level than hAk4.
  • mE-HA " expression was determined by western blttoing with anti-endosattin antibody.
  • the conditioned media containing endostatin and angiostatin showed strong inhibitory effects on endothelial cell proliferation.
  • Intratumoral injection 24 ug DNA/PVP was injected intratumorally and tumor was harvested at 24 h.
  • the protein expression of endostatin or angiostatin was determined by ELISA.
  • the tumor culture media was also examined for bioactivity of endostatin and angiostatin.
  • Intramuscular delivery 400-420 ⁇ g DNA/PVP was injected intramulscularly followed by electroporation (see Materials and Methods) . Serum was collected at day 2, 5 and 10. The data showed that 3-5 ng/ml of Endostatin or 10-15 ng/ml of Angiostatin in serum was produced on day 5, and electroporation increased the level of trangenes by 3-5-fold. The expression declined on day 10.
  • Intravascular delivery DOTMA: chol formulated endostatin or angiostatin expression plasmid was injected i.v. to normal mice. Expression of endostatin and angiostatin were determined by RT-PCR for mRNA in lung and ELISA for protein expression in serum. 400 nm size particle resulted in higher expression and may have more secondary cytokine effect than the SUV formulation (see in vivo efficacy) .
  • Renca s . c. tumor model with intratumoral injection Intratumoral administration of 24 ⁇ g Endostatin gene/PVP at 4 times/week for two weeks (total 7 treatments) induced complete regression in 7 out of 14 mice (50% regression rate, p ⁇ 0.05). Survival rate was increased from 21% (3/14) in vector/PVP group to 78% (11/14) in Endostatin/PVP group on day 21. Human endostatin with HA-tag was constructed. Intratumoral injection of human endostatin/PVP resulted in 51% tumor growth inhibition in Renca sc tumor model.
  • Endostatin gene medicine The regressed tumors by Endostatin gene medicine remain in a microscopic dormant state by day 29, whereas 6 cycles treatments (400 ⁇ g/mouse/day for 27 days each cycle) with recombinant protein is needed to keep tumor dormancy (Boehm et al., Na ture 390, 404-7).
  • the data presented herein clearly demonstrates that Endostatin gene medicine has strong antitumor activity in mouse tumor model, and may have many advantages over the recombinant protein. Histological analysis of the regressing tumors by Endostatin/PVP treatment showed 3-5-fold reduced vascularization (CD31 staining) , 3- fold increased tumor apoptosis (TdT immunostaining) . No change in tumor proliferation (PCNA staining) and tumor infiltration lymphocytes (CD3, CD4 and Mac-1 staining) .
  • Renca s . c. tumor with systemic delivery Im injection of 120 ⁇ g of Endostatin/PVP followed by electroporation (2x week, switch legs) induced 40% Renca tumor injection. The regressing tumors had reduced vascularization. Repeated experiment showed that 34 % tumor growth inhibition and prolonged survival. However, intravascular delivery of vector or Endostatin/Dotma:chol (SUV, 30 ⁇ g/2x week) didn't show a gene- specific effect, although overall 50-60% reduction of tumor growth was observed. Use of other lipids is expected to yield improved results. Lung metastasis model with systemic delivery: It has been shown that IL-12 at 15 ⁇ g DNA in 400 nm particle induced prolonged survival.
  • Angiostatin can prolong survival by iv or im delivery of the gene medicine. LLC can metastize to lung when sc primary tumor is excised. This lung metastasis model will be established to assess inhibitory effects of lung metastases by anti- angiogenic gene medicines.
  • Mouse cornea assay Mouse cornea angiogenesis was induced by implantating bFGF pellet. Endostatin gene medicine was delivered by either iv of DNA/DC or DNA/PVP on next day (see Materials and Methods) . The results showed Endostatin by iv strongly inhibited bFGF-induced cornea angiogenesis by day 5.
  • New anti -angiogenic genes A double-barrel expression plasmid for endostatin-angiostatin (pAS1254), in which endostatin and angiostatin are driven by separate transcription units, has also been also constructed.
  • IP-10 interferon-inducing factor 10
  • TSP- 1 Thrombospondin-1
  • TSPf An internal fragment
  • IP-10 cDNA was reverse transcribed from published protein sequence with optimal humanized codons. The cDNA was synthesized by Operon and the expression plasmids for IP-10 (pIP-1316) , IP-10/endostatin fusion protein (pIP1311) , and IP-10/TSPf fusion protein have been constructed.
  • IP-10, IP-10/hE fusion protein, and IP-10/TSPf fusion protein were determined by RT- PCR for mRNA and by western blotting for protein. About 150- 250 ng/ml protein was expressed in culture medium. These constructs will be exmained in Renca sc model to see if synergistic anti-tumor efficacy could be achieved.

Abstract

L'invention concerne des procédés d'administration de gènes et de thérapie génique, ainsi que de nouveaux produits de recombinaison d'acides nucléiques servant à exprimer des agents anti-angiogéniques chez un mammifère, des formulations d'administration incorporant un produit de recombinaison d'acides nucléiques d'expression et des procédés de préparation et d'utilisation de ces produits de recombinaison et de ces formulations. Elle concerne, en particulier, des produits de recombinaison de plasmides permettant d'introduire des agents anti-angiogéniques codant des acides nucléiques dans des cellules, dans le but de moduler une activité tumorale, des procédés d'utilisation de ces produits de recombinaison (y compris une thérapie combinée à d'autres agents, tels que des cytokines, de préférence, IL-12), et des procédés de préparation de ces produits de recombinaison.
PCT/US1999/016388 1998-07-27 1999-07-20 Systemes d'administration et plasmides anti-angiogenese et leurs procedes de preparation et d'utilisation WO2000006759A2 (fr)

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