JP2001524828A - Truncated VEGF-related protein - Google Patents

Abstract

(57) SUMMARY The present invention provides a novel truncated vascular endothelial growth factor-related protein (VRP or VRP) useful for stimulating angiogenesis in vitro and in vivo. The present invention also provides a nucleic acid encoding such a novel truncated VRP and a method for producing the truncated VRP. Pharmaceutical compositions comprising truncated VRP and methods of gene therapy using nucleic acids encoding truncated VRP would be useful for treating heart disease and for wound healing.

Description

DETAILED DESCRIPTION OF THE INVENTION                           Truncated VEGF-related proteinField of the Invention   The present invention relates to a novel truncated vascular endothelial growth factor (VEGF) -related protein. Yo More particularly, the present invention relates to N-terminally truncated VEGF that is substantially free of other proteins. Related to related proteins. Such truncated VEGF-related proteins are available in vivo and in vivo. Can be used to promote angiogenesis in vitro.   The present invention provides a nucleic acid encoding such a novel truncated VEGF-related protein, Cells, tissues and animals containing such nucleic acids; therapeutic methods using such nucleic acids; And methods related to all of the foregoing.Background of the Invention   Vascular endothelial growth factor (VEGF), also called vascular permeability factor (VPF), Family of proteins produced by many different cell types in different tissues And act very selectively to stimulate almost only endothelial cells (Ferrara et al.  al.,Endocri.Rev.13: 18-32, (1992); Dvorak et al.,Am.J.Pathol.146: 1029-39,1 995; Thomas,J. Biol. Chem.271: 603-06, 1996). These publications , And all other publications referenced herein are incorporated by reference in their entirety. And is incorporated herein by reference.   VEGF is strongly mitogenic when evaluated in cell culture (Gospodarwi cz et al.,Proc.Natl.Acad.Sco.USA 86: 7311-15, 1989), which is chemotactic (Favard  et al.,Biol.Cell 73: 1-6, 1991). In addition, VEGF is a plasminogen activator Offspring, plasminogen activator inhibitor, and plasminogen activation Factor receptor (Mandriota et al.,J. Biol. Chem.270: 9709-16,1995; Pepper et al., 1 81: 902-06,1991) is also an enzyme system that regulates the infiltration of proliferating capillaries into tissues Collagenase (Unemori et al.,J.Cell.Physiol.153: 557-62,1992) You. VEGF also forms tube-like structures by endothelial cells, an in vitro example of angiogenesis (Nicosia et al.,Am.J.Pathol., 145: 1023-29, 1994).   In vivo, VEGF promotes angiogenesis (Leung et al., Science 246: 1306-09,1989 ), Increases vascular permeability (Senger et al.,Science 219: 983-85, 1983). VEG F is now known as an important physiological regulator of capillary angiogenesis. They are Normal neovascularization during organ growth, including fetal growth (Peters et al.,Pr oc.Natl.Acid.Sci.USA  90: 8915-19, 1993), tissue repair (Brown et al.,J.Exp.Med . 176: 1375-79,1992), menstrual cycle, and pregnancy (Jackson et al.,Placenta 15:34 1-53, 1994, Cullinan & Koos,Endocrinology 133: 829-37, 1993; Kamat et al.,Am. J.Pathol. 146: 157-65, 1995). Embryos lacking one VEGF allele As evidenced by abnormal vascular development and lethality (Carmeliet et al. ,Nature 380: 435-38, 1996), during fetal development, VEGF forms a de novo form of blood vessels from blood islands. Appear to play an integral role in the development (Rasau & Flamme,Ann.Rev.Cell.De v.Biol. 11: 73-92, 1995). In addition, VEGF is used in solid tumors (Potgens et al.,Biol.Chem .Hoppe-Seyler  376: 57-70, 1995), retinopathy (Miller et al.,Am.J.Pathol.145: 57 4-84, 1994; Aiello et al.,N.Engl.J.Med.331: 1480-87, 1994; Adamis et al.,Am .J.Ophthalmol. 118: 445-50, 1994), psoriasis (Detmar at al.,J.Exp.Med.180: 1141- 46, 1994), and rheumatoid arthritis (Fava et al.,J.Exp.Med.180: 341-46,1994 ) Is strongly associated with physiological vascular growth, characteristic of many diseases, including   VEGF expression is determined by hormones (Schweiki et al.,J.Clin.Invest.91: 2235-43,1993) , Growth factors (Thomas,J. Biol. Chem.271: 603-06, 1996), and hypoxic (Schweiki et al.,Nature 359: 843-45, 1992, Levy et al.,J. Biol. Chem.271: 2 746-53, 1996). Positive regulation of VEGF by hypoxia Tissue Increases Oxygenation by Inducing Further Capillary Formation and Resulting Blood Flow This is especially important as a compensation mechanism. This mechanism affects tumors And in retinopathy are thought to contribute to physiological angiogenesis. However, positive regulation of VEGF expression after hypoxia can be attributed to, for example, skin wound healing (Frank et al.,J. Biol. Chem.270: 12607-613, 1995), and coronary ischemia (Banai et al. .,Cardiovasc.Res. 28: 1176-79, 1994; Hashimoto et al.,Am.J.Pathol.267: H1948- H1954, 1994).   VEGF may pharmacologically induce angiogenesis in animal models of vascular ischemia Repeated intramuscular injection of VEGF as demonstrated by ischemia hind limb blood flow measurements. It has been shown that a single bolus injection or intra-arterial bolus can increase accessory branch angiogenesis. Have been demonstrated in a chronic limb ischemia model (Pu, et al.,Circulatio n  88: 208-15, 1993; Bauters et al.,Am.J.Physiol. 267: H1263-71, 1994: Takeshit a et al.,Circulation 90 [part 2], II-228-34, 1994; Bauters et al.,J.Vasc.Sur g. 21: 314-25, 1995; Bauters et al.,Circulation 91: 2802-09,1995: Takeshita et  al.,J.Clin.Invest.93: 662-70, 1994). In this model, VEGF is a basic FGF It has also been shown to act in concert to improve ischemia (Asahara et al. ,Circulation 92: [suppl 2], II-365-71, 1995). VEGF is balloon injured rat neck Promotes repair of the arterial endothelium, thereby inhibiting the physiological thickening of the underlying smooth muscle layer And have been reported to maintain lumen diameter and blood flow (Asahara et a l.,Circulation 91: 2793-2801,1995). VEGF is expressed in dog coronary arteries by EDRF (internal Induces a skin-derived relaxation factor (nitric oxide) -dependent relaxation and is therefore not associated with angiogenesis The second mechanism of the relationship strongly contributes to increased blood flow to the ischemic area (Ku et al.,Am.J.Physiol.265: H586-H592, 1993). at the same time These data suggest that VEGF in wound healing, ischemic disease and restenosis Provide strong evidence of a therapeutic role.   The VEGF family of proteins has at least four members, VEGF-121, VEGF-165, It is composed of VEGF-189 and VEGF-206. VEGF was first characterized Is a 34-45kDa glycoprotein consisting of two identical subunits of 165 amino acid residues (Tischer et al.,Biochem.Biophys.Res.Commun.165: 1198-1206,1989). VEGF-1 65 cDNA is a 26-residue secretion signal that is cleaved when proteins are secreted from cells. A 191-residue amino acid sequence consisting of the peptide sequence and a 165-residue subunit of the mature protein. VEGF-165 binds strongly to heparin, which is at residue 115 (Fig. 1) (Thomas,JB iol.Chem .. 271: 603-06 (1996)). Other members of the VEGF family are 121, 189 And 206 residues (VEGF-121, VEGF-189, and VEGF-206, respectively) Or longer Is a homodimeric protein with buunit (Tischer et al.,J. Biol. Chem.266 : 11947-54,1991; Park et al.,Mol Biol Cell 4: 1317-26 (1993)). VEGF's four The morphology results from alternative splicing of up to eight exons of the VEGF gene ( VEGF-121, exons 1-5, 8; VEGF-165, exons 1-5, 7, 8; VEGF-189, exo 1-5, 6a, 7, 8; VEGF-206, exons 1-5, 6b, 7, 8 (exons 6a and 6b Refers to an alternatively spliced form of the same exon))) (Houck et  al.,Mol.Endocr ..5: 1806.14 (1991)). The VEGF sequence contains eight conserved Fid-forming core cysteine residues are included. All VEGF genes are proteins Encodes a signal peptide that directs E. coli to the secretory pathway. However, VEGF-189 VEGF-121 and -206 remain associated with the extracellular matrix, while VEGF-121 and And -165 alone have been found to be readily secreted from cultured cells. These VE The form of GF is compatible with residues 115-139 of VEGF-189 and -206 (matrix target sequence). It also has a very basic sequence that High affinity with the acidic constituents (Thomas,J. Biol. Chem.271: 603-06 (1996) )).   The mitogenic activity of various VEGF isoforms depends on each isoform. Various. For example, VEGF-121 and VEGF-165 are very similar to endothelial cells Cell mitogenic activity. However, VEGF-189 and VEGF-206 It is only weakly mitogenic (Ferrara et al.,Endocr.Rev.13: 18- 32,1992). A 24-residue "matrix target" sequence common to VEGF-189 and VEGF-206 ( A mutant of VEGF-206 lacking residues 115-139 in FIG. 1 had normal mitogenic activity. As demonstrated by some, the reduced activity of these isoforms Is due to strong binding to cells and the matrix (Ferrara et al.,Endocr. Rev. 13: 18-32, 1992).   The N-terminal fragment of VEGF-165 (VEGF (1-110)) generated by plasmin is VEGF-16 5 and KGF receptor with similar affinity as VEGF-121, but with a C-terminal VEGF fragment (11 1-165) had no binding activity (Key et al.,J. Biol. Chem.271: 7788-9 5,1996). Interestingly, this study showed that VEGF-121 and VEGF-110 Activity is about 110 times weaker than VEGF-165 However, this is due to the C-terminal domain of VEGF-165 in the biological capacity of the VEGF isoform. Suggests a possible main role. The significance of this finding is that endothelial cell proliferation Considering earlier results showing comparable capabilities of VEGF-121 and VEGF-165, It is clear. Furthermore, the functional interaction of VEGF with the KDR receptor is at least partially Is thought to be dependent on cell surface heparan sulfate proteoglycans (C ohen et al.,J. Biol. Chem ..270: 11322-26, 1995; Tessler et al.,J. Biol. Chem.Two 69: 12456-61, 1994). available. In such a situation, the heparan sulfate on the cell surface is VEGF-165 (lacking the heparin binding domain) It is unclear to what extent it modulates the functional interaction of Tessler et al.,J. Biol. Chem.269: 12456-61, 1994; Cohen et al.,J. Biol. Chem.27 0: 11322-26, 1995; Gitas-Goren et al.,J. Biol. Chem.271: 5519-23 (1996)).   VEGF is associated with platelet-derived growth factor (PDGF) (Andersson et al.,Growt h Factors  12: 159-64, 1995). VEGF is a protein derived from the placental growth factor (PIGF) gene Family, PIGF-129 and PIGF-150 (Maglione et al.,Pro c.Natl.Acad.Sci.USA  88: 9267-71,1991;Oncogene8: 925-31, 1993). More recently Identified several additional VEGF-related genes and identified VEGF-B (VEGF-related factor VRF- (Grimmond et al.,Genome Res.6: 122-29, 1996; Olofsson et al .,Proc.Natl.Acad.Sci.USA.93: 2567-81, 1996), VRF-2 (Grimmond et al.,Genom e Res. 6: 122-29, 1996), and VEGF-C (Joukov et al.,EMBO J.15: 290-98,1996 ; Lee et al.,Proc.Natl.Acad.Sci.USA 93: 1988-92, 1996) and VEGF-3 (WO96 / 3 PCT Application No. PCT / US95 / 07283) published on December 12, 1996 as 9421 Was done. Finally, two VEGF-related sequences encoded by the virus were identified, That is, the poxviruses ORF-1 and ORF-2 (Lyttle et al.,J.Virol.68 : 84-92,1994). Except for PDGF, these proteins are called VEGF-related proteins [VRP]. ing. An example arrangement of a VRF is depicted in FIG.   Previously known VRPs and PDGFs are relatively conserved within their sequences. There are eight cysteines that are stored. Therefore, even the conserved cysteine The protein sequence that we want to call is referred to herein as the core sequence, and the first N-terminal of that sequence. The conserved cysteine is referred to herein as "the first cysteine of the core sequence" or Called “first core cysteine”.   Interestingly, the members of the VEGF family are derived from VEGF and PIGF subunits. Can form a heterodimer, such as a heterodimer (DiSalvo et al.,J.Bio l.Chem. 270: 7717-23, 1995; Cao et al.,J. Biol. Chem.271: 3154-62, 1996). VEGF Is very potent in promoting angiogenesis and cell proliferation, while VEGF / PIGF heterodimers are weaker mitogens and PIGF homodimers are Has little or no mitogenic activity (DiSalvo et al.,J.Biol.Che m. 270: 7717-23, 1995; Cao et al.,J. Biol. Chem.271: 3154-62, 1996). For other experiments In this case, VEGF-165 / VEGF-B heterodimer transfects both genes into cells. (Olofsson et al.,Proc.Natl.Acad.Sci. USA 93: 2567-81, 1996).   VEGF has two receptors on endothelial cells, KDR / flk-1 (Terman et al.,Bio chem.Biophys.Res.Commun. 187: 1579-86,1992), and flt-1 (De Vries et al. ,Science 255: 989-91,1992). Alanine scan of charged residues Systematic site-directed mutagenesis of VEGF-165, resulting in residues D63, E64 and And E67 are involved in the binding of VEGF to flt-1, while the basic residues R82, KI84, and And H86 were shown to contribute strongly to binding to KDR (keyt et al.,J. Biol .Chem. 271: 5638-46, 1996).   VRP is composed of a large extracellular portion and seven immunoglobulin-type domains each. A single transmembrane protein with a cytoplasmic part that functions as a tyrosine kinase Bind to one or more of three different endothelial cell receptors And is known. These receptors are known as KDR / flk-1 (Terman et al.,Biochem.Bioph ys.Res.Commun. 187: 1579-86,1992), flt-1 (De Vries et al.,Science255: 989- 91,1992), and flt-4 (Pajusola et al.,Canner Res.52: 5738-43, 1992). . There is a clear selectivity between these receptors and various VEGF ligands, but to date, It has not been completely elucidated. However, VEGF does not have KDR and flt1 (Terman et al.,Growth Factors 11: 187-95, 1994) No (Joukov et al.,EMBO J.15: 290-98, 1996) that VEGF-C binds to flt-4 (Joukov et al.,EMBO J.15: 290-98, 1996) debate whether it also binds to KDR (Joukov et al.,EMBO J.15: 290-98, 1996; Lee et al.,Proc. Natl.Acad.Sci.USA  93: 1988-92, 1996). VEGF-B / VRF-1, VR Receptor specificity for F-2 and virally encoded VRP is currently unknown. I haven't. However, VEGF-B promotes endothelial cell proliferation (Olofsson et al.,Pr oc.Natl.Acad.Sci.USA 93: 2567-81, 1996). Is believed to be the primary cause of the angiogenic response of endothelial cells (Gitay-Goren et al.,J. Biol. Chem.271: 5519-23 (1996)), indicating that VEGF-B can bind to KDR It can be inferred.   Most VRPs are thought to render endothelial cells "potential for angiogenesis" It has been shown to activate known KDR receptors. Evidence of such activity is VEGF-B, which promotes endothelial cell proliferation (Olofsson et al.,Proc.Natl.Acad.Sci.USA . 93: 2567-81, 1996), VEGF-C that promotes endothelial cell migration and proliferation (Joukov et al.,EMBO J . 15: 290-98, 1996; Lee et al.,Proc.Natl.Acad.Sci.USA 93: 1988-92,199 6), and both are encoded by the virus, which have been reported to cause angiogenesis. The known VRP (Lyttle et al.,J.Viril.68: 84-92, 1994) . A notable exception is negligible mitogenic activity on endothelial cells. PIGF isoform homodimer with or without However, PIGF / VEGF heterozygous The dimer still maintains considerable mitogenic activity (DiSalvo et al.,JB iol.Chem. 270: 7717-23, 1995; Cao et al,J. Biol. Chem.271: 3154-62, 1996).   VEGF is expressed in many different tissues. Similarly, the VRP gene can be found in multiple tissues. VEGF-B, and to a lesser extent VRF-2, is expressed in the human heart and skeleton Strongly expressed in muscle (Grimmond et al.,Genome Res.6: 122-29, 1996; Olofsson et al.,Proc.Natl.Acad.Sci.USA93: 2567-81, 1996) is particularly interesting No. In fact, VEGF-B is significantly more strongly expressed in mouse heart tissue than VEGF (Ol ofsson et al.,Proc.Natl.Acad.Sci.USA93: 2567-81, 1996). VEGF-C is also found in a variety of human tissues, most notably in heart and skeletal muscle. (Joukov et al.,EMBO J.15: 290-98, 1996). This expression pattern And the sharp specificity of VRP for endothelial cells indicates that these factors And has a physiological role in angiogenesis. This is myocardium Coronary artery is required for collateral neovascularization to provide adequate capillary supply to It is thought to be related to pathological conditions such as blood. Coronary artery ligation or low acid Transient ischemia induced by elementary conditions causes VE in rat or pig heart Positively regulates GF mRNA in vivo, resulting in hypoxia in cardiac myocytes and smooth muscle cells Has been shown to induce VEGF mRNA in vitro (Hashimoto e t al.,Am.J.Physiol,267, H1948-H1954, 1994; Banai, et al.,Cardiovac.Res.28:11 76-79, 1994; Circulation 90, 649-52, 1994). Strong VEGF and VRP in heart Expression is redundant with the ability to induce neovascularization when needed. May help to ensure a proper regulatory system. Collateral angiogenesis is peripheral (lower extremity) It is also required in ductal ischemia and in cerebral ischemia (stroke). After all, new blood vessels Formation is necessary in post-wound tissue repair. In this connection, VEGF is It is positively regulated in epidermal keratinocytes during skin wound healing (Br own et al.,J.Exp.Med.176: 1375-79,1992) is worthless. did Myocardial infarction, chronic coronary ischemia, chronic lower limb ischemia, wound healing and seizures Treatment of various ischemic conditions in VRPs could be clinically beneficial .Summary of the Invention   The present invention is directed to novel truncated VEGF-related proteins (VRPs), preferably human VRPs. are doing. A preferred use of the truncated VRP and nucleic acid molecule compositions of the present invention is for heart disease. Patients, wounds, or other ischemic conditions to promote angiogenesis in such patients By using such a composition for the purpose of treating by proceeding is there. VRO amino acid sequence is conserved between VRP and VEGF proteins Eight disulfide-forming cysteine residues (core cysteines) are included. VRP Is VEGF-B, VEGF-C, VRF-2, ORF-1, ORF-2, and PIGF But are not limited to these.   The first aspect of the present invention relates to the first system of the core sequence of the subunit. Truncated VRP with a deletion in at least one of the N-terminal amino acid residues of provide. Such a composition would be virtually free of other proteins. Preferably Indicates that cleavage cuts only the N-terminal residues of the mature protein subunit to a minimum. To the residue N-terminal to (before) the first core cysteine residue and Including those that cut all of the N-terminal amino acids of the mature protein to the maximum extent. In a more preferred aspect, the first N-terminal one of the first cysteine in the core sequence. N-terminal amino acid residue of the first cysteine, excluding 5 amino acid residues from All of the groups have been deleted.   Amino acid deletions include the deletion of non-adjacent amino acid residues in the N-terminal sequence. Preferably, though, the deletion is at consecutive amino acid residues. Therefore, In a further aspect, the present invention provides an N-terminal N-terminal side of a VRP subunit sequence. Of amino acid residues of increasing length from to the first cysteine of the core sequence Includes human VRP with sequence deletion.   In a preferred aspect, the present invention relates to VRO, VEGF-B, VRF-2, VEGF-C, VEGF-3, PI Cut GF, poxvirus ORF-1, and poxvirus ORF-2 provide. In such a truncated VRP, each subunit is At least one of the amino acid residues N-terminal to the first cysteine of the core sequence Can also independently have one deletion or only one of the subunits May be deleted.   In certain embodiments, the truncated VRP subunit is the first system of the core sequence. VRP subunits with various numbers of amino acid residues deleted at the N-terminal side of No. In one aspect, the remaining N-terminal residues are contiguous from the N-terminal sequence. Amino acid residues. These contiguous N-terminal residues are all in the N-terminal sequence. However, a continuous sequence starting from the N-terminus of the N-terminal sequence is preferable. , A contiguous array immediately N-terminal to the first cysteine in the core sequence of the VRP subunit. Sequences containing amino acid residues are most preferred. An example of such a most preferred embodiment is shown in FIG. Draw on.   In another embodiment, the first cysteine N in the core sequence of the truncated VRP of the invention is The terminal amino acid residue is an amino acid sequence selected at random, In embodiments, the three amino acid residues are derived from the N-terminal sequence of the full length VRP sequence , Not necessarily contiguous amino acids from the full-length VRP sequence.   Therefore, in one most preferred aspect, the present invention provides a truncated VRP subunit Wherein the amino acid residue at the N-terminal side of the first cysteine of the core sequence is deleted. Provide subunits.   In another aspect, the present invention provides that the amino acid sequence at the N-terminal side of the core sequence is 11 to 20, more preferably 11 to 15, more preferably 6 to 10, most preferably 2 to A truncated VRP subunit comprising 5 amino acid residues is provided.   Preferably, the amino acid sequence at the N-terminal side of the core sequence is the VRP subunit. Includes contiguous amino acid residues immediately N-terminal to the first cysteine of the core sequence. Obedience Therefore, in these preferred embodiments, the truncated VRP is required for the core sequence, the core sequence. Contains the essential C-terminal sequence, and is located in the N-terminal region of the first cysteine in the core sequence. Amino acid sequence immediately N-terminal to the first cysteine of the core sequence of the full-length VRP sequence Of the columns, 11 to 20, more preferably 11 to 15, more preferably 6 to 10, most Preferably it contains 2 to 5 consecutive amino acid residues.   One skilled in the art will recognize that the truncated VRP subunit is N-terminal to the first cysteine in the core sequence. Such a truncated VRP subunit corresponds to The full length VRP subunit is the (X + 1) amino acid N-terminal to the first cysteine in the core sequence. You will understand that this is the case with acid.   In the truncated VRP of the present invention, the two truncated VRP subunits have the same amino acid sequence. A truncated VRP homodimer comprising two truncated VRP subunits of the present invention has The book, wherein the two subunits have different amino acid sequences from each other; Heterodimers consisting of two truncated VRP subunits of the invention are also included.   For purposes of the present invention, N and NN each represent, for example, the first 10-15 amino acids. The term "first N-NN" amino acid is used to indicate the number of amino acids, such as acids The first N-NN amino acids (e.g., the first 10 -15 amino acids). The term N-NN includes signal sequences Deletions at any position from N to NN of the first amino acid after are included. Obedience Thus, in a more preferred aspect, the truncated VRP subunit contains the first 10-15 Amino acids are deleted; more preferably, the first 15-20 amino acids are deleted More preferably, the first 20-25 amino acids have been deleted; most preferably, Contains truncated hVEGFDB protein subunits, missing the first 23-24 amino acids It is.   In another more preferred aspect, the truncated VRP subunit contains the first 10-15 Amino acids are deleted; more preferably, the first 15-20 amino acids are deleted More preferably, the first 20-25 amino acids have been deleted; most preferably, Includes truncated hVRF2 protein subunit, missing the first 23-24 amino acids You.   In another more preferred aspect, the truncated VRP subunit comprises the first 95-10 0 amino acids have been deleted; more preferably, the first 100-105 amino acids have been deleted More preferably, the first 105-110 amino acids have been deleted; most preferably Is a truncated hVEGFC protein subunit lacking the first 108-109 amino acids Is included.   In another more preferred aspect, the truncated VRP subunit comprises the first 16-21 Amino acids are deleted; more preferably, the first 21-26 amino acids are deleted More preferably, the first 26-31 amino acids have been deleted; most preferably, Includes truncated hPIGF protein subunit, missing the first 29-30 amino acids You.   In another more preferred aspect, the truncated VRP subunit contains the first 10-15 Amino acids are deleted; more preferably, the first 15-20 amino acids are deleted More preferably, the first 20-25 amino acids have been deleted; most preferably, Contains truncated hVEGF3 protein subunits, missing the first 23-24 amino acids It is.   In another more preferred aspect, the truncated VRP subunit comprises the first 20-25 Amino acids are deleted; more preferably, the first 25-30 amino acids are deleted More preferably, the first 30-35 amino acids have been deleted; most preferably A truncated pvORF1 protein subunit lacking the first 33-34 amino acids Is included.   In another more preferred aspect, the truncated VRP subunit contains the first 30-35 Amino acids are deleted; more preferably, the first 35-40 amino acids are deleted More preferably, the first 40-45 amino acids have been deleted; most preferably, Contains truncated pvORF2 protein subunit with deletion of first 43-44 amino acids It is. The sequences of some typically preferred truncated VRP subunits are shown in FIG. They are lined up.   The present invention provides a nucleic acid encoding a truncated VRP subunit as described herein. Also provide children. A nucleic acid molecule can be, for example, DNA, cDNA or RNA. The present invention A recombinant DNA vector comprising a nucleic acid molecule encoding a truncated VRP, and such Host cells transformed with the various recombinant DNA vectors are also provided. Is intended for the synthesis of truncated VRP subunits, such as those described herein. Target.   The present invention further provides for truncation at the N-terminus to facilitate expression in a suitable host system. Provided is a nucleic acid molecule encoding a precursor for biosynthesis of a truncated VRP subunit. So Nucleic acid molecules such as those described above have a signal that precedes the truncated subunit at its N-terminus. DNA encoding a null peptide is included. VEGF or VRP signal sequence Will be used to construct a truncated version of VRP containing the appropriate signal peptide U. The human VEGF signal peptide is as follows.   mnfllewvhwslalllylhhakwsqa (I)-[SEQ I.D.NO.40]- Alternatively, the signal peptide shown in FIG. 1 can be used. Preferably, for truncated VRP A specific signal peptide is used for the construct.   After fusing the signal sequence to the truncated VRP, the signal peptide is First or last mature VRP peptide to facilitate cleavage of the peptide. The first two residues, such as proline (P) for hVEGFB or proline-vari May need to be included. Thus, suitable nucleic acid molecules include VEGF-B DNA that encodes the signal sequence for proline (the first of mature VEGF-B) Valine (mature VEGF- VEG optionally followed by a codon for (second residue of B) and truncated at the N-terminus The DNA encoding F-B will follow. The present invention, as known to those skilled in the art, Also provide other suitable signal peptide fusion constructs best suited for mammalian hosts You. Those skilled in the art will recognize that signal peptides include various truncated VRP subunits of the invention. Thus, residues required to facilitate cleavage of the signal peptide in mammalian cells Will be included arbitrarily.   Therefore, the present invention relates to a nucleic acid molecule encoding the truncated VRP subunit, wherein the 5 'end of the nucleic acid molecule is A pair operably linked to a DNA sequence encoding a signal peptide. A recombinant DNA expression vector is provided. The signal peptide is a human VRP signal peptide. Can be Further, the DNA sequence encoding the signal peptide is the DNA sequence At the 3 'end of the sequence, the first amino acid of the mature, uncleaved VRP subunit The residue may be operably linked to the DNA encoding the residue, The 3 ′ end of the DNA encoding the nucleic acid encoding the truncated VRP subunit Operably linked to a molecule. In another aspect, the signal peptide The DNA sequence encoding the tide is at the 3 'end of the DNA sequence at the mature, untruncated position. Operable with DNA encoding the first two amino acid residues of the new VRP subunit And the 3 'end of the DNA encoding the two residues is Operably linked to said nucleic acid molecule encoding type VRP subunit . Accordingly, in a preferred aspect, the present invention further provides the method as described above. At the N-terminus of the truncated VRP subunit, Truncated VRP subunits of the invention comprising the first one or two amino acid residues Also provide. One skilled in the art will recognize that the amino acid sequence N-terminal to the first cysteine of the core sequence is Final number (one or two more to facilitate signal peptide cleavage) First cysteine in the core sequence of the corresponding full-length VRP Of the present invention is at least one less than the number of amino acids on the N-terminal side of It will be understood that such truncated VRP subunits are included.   In another preferred aspect, the invention comprises two truncated VRP subunits. A truncated VRP homodimer or heterodimer, wherein the truncated VRP homodimer or heterodimer is provided. The VRP subunit is a mature uncleaved N-terminal of the truncated VRP subunit. Contains the first one or two amino acid residues of the VRP subunit.   In a preferred aspect, a set encoding the truncated VRP subunit of the present invention is provided. The recombinant nucleic acid molecule operates in a host cell transformed with the vector. It is operatively linked to a possible control arrangement. The present invention also relates to the present invention. Transformed or transfected containing the recombinant DNA vector Provided host cells.   The present invention also provides a transport vector comprising a nucleic acid molecule encoding a truncated VRP of the present invention. Including Such a transport vector may be, for example, a viral vector. No. Such viral vectors are, for example, adenoviral vectors or It is a denovirus-related virus vector. In another aspect of the invention, a nucleic acid Operate a DNA sequence encoding a signal peptide at the 5 'end of the appointment Adenovirus vector comprising a ligated nucleic acid molecule encoding a truncated VRP of the invention Is provided. Preferably, the signal peptide is a VEGF signal peptide. Tide, VEGF-B signal peptide, VRF-2 signal peptide, VEGF -C signal peptide, P1GF signal peptide, VEGF-3 signal peptide Pide, poxvirus ORF-1 signal peptide, and poxvirus It is selected from the group consisting of ORF-2 signal peptide. Preferably, the sig The null peptide is a VEGF-B signal peptide. In a favorable perspective The DNA sequence encoding the signal peptide is located at the 3 'end of the DNA sequence. Movement to the DNA encoding the first amino acid residue of the mature uncleaved VRP subunit. The 3 'end of the DNA operably linked and encoding the residue is It is operably linked to the nucleic acid molecule encoding the truncated VRP. Most favorable In a preferred aspect, the adenovirus vector comprises the truncated VRP subunit of FIG. A nucleic acid molecule encoding the knit.   In a further preferred aspect of the present invention, a recombinant adenovirus vector And injectable adenovirus comprising a pharmaceutically acceptable carrier and a pharmaceutically acceptable carrier A vector preparation is provided, said vector containing no wild-type virus, / Partial adenovirus sequence in which the E1B gene has been deleted, and partial adenovirus Truncated VR driven by a promoter flanked by ills sequences Includes transdin encoding the P subunit. In a preferred point of view, The product has been filtered through a 30 micron filter. To another favorable perspective In this case, the truncated VEGF subunit is the truncated VEGF subunit of FIG. is there. In another preferred aspect, an injectable adenovirus vector preparation Is the CMV promoter, ventricular myocyte-specific promoter, and myosin heavy chain And a promoter selected from the group consisting of promoters.   In another aspect, the invention provides a method for producing a truncated VRP polypeptide. Offer. The method comprises transforming with the recombinant DNA expression vector of the present invention. Or the transfected host cells under appropriate conditions. The polypeptide to express it and isolate the polypeptide from the host cell Including. Next, by giving appropriate conditions, the truncated VRP peptide is converted into a truncated VRP peptide. And fold it into the unit. In mammalian cells, such conditions may affect cells Should be given more naturally. In non-mammalian cells, for example, If appropriate pH, isotonicity and reducing conditions are not provided, as described in No. Most preferably, the present invention provides a method for producing a truncated VRP, In the method, under appropriate conditions, the truncated VRP subunit is converted to VR A second selected from the group consisting of a P subunit and a truncated VRP subunit To form a dimer with the VRP subunit. In a preferred aspect of the present invention A fragment containing two truncated VRP subunits having the same amino acid sequence. A method for producing a truncated VRP homodimer is provided.   In another aspect of the invention, the two VRP subunits have different amino acid sequences. Methods for producing truncated VRP heterodimers having a sequence are provided. Like that A heterodimer has one truncated VRP subunit and one non-truncated V May be composed of RP subunits, or both VRP subunits May be a cutting type. The two subunits are from different VRPs There may be. For example, a heterodimer may contain one VEGF-B subunit and And one truncated VEGF-C subunit, or Both subunits may be truncated.   In another preferred aspect, the present invention provides a truncated VRP subunit of the present invention. Pharmaceutical compositions are provided comprising in a suitable carrier. The present invention provides patients with such Methods for stimulating angiogenesis, including administering the composition.   An endothelial cell may be treated with a compound of the invention, including treating the endothelial cell with a truncated VRP. Methods for stimulating endothelial cell growth or migration in vitro are provided.   The present invention also provides a method of treating a patient suffering from a heart disease. The method The patient is administered a nucleic acid molecule encoding at least one truncated VRP subunit. Providing the nucleic acid molecule with a truncated VRP subunit in the patient. Can be expressed. In another aspect, a treatment comprising a truncated VRP of the invention Stimulate angiogenesis in a patient, including administering a therapeutically effective amount of a pharmaceutical composition A method is provided.   Preferably, the pharmaceutical composition is in a delivery system suitable for treatment. To another favorable perspective In order to enhance the angiogenic effect of the truncated VRP, an enhancer is administered. It is. Such agents include, for example, basic fibroblast growth factor (bFGF ) (FGF-2), acidic FGF (aFGF) (FGF-1), FGF-4, FG F-5, FGF-6, or any FGF or other vasculature that stimulates endothelial cells Sex factors. That is, in one aspect of the present invention, a truncated VR A pharmaceutical composition comprising P and one or more enhancers is provided. Pharmaceutical composition Also, ischemic conditions such as cardiac infarction, chronic cardiac ischemia, chronic lower limb ischemia, stroke, and And peripheral vascular disease. In addition, the present invention Also provided is a method of treating a wound, for example a skin or intestinal wound, using the pharmaceutical composition of the present invention. It is.   In a preferred embodiment, a method is provided for stimulating angiogenesis in a patient. . The method comprises direct intracoronary injection of a delivery vector into one or both coronary arteries. Transporting to the patient's myocardium by the at least one cleavage A nucleic acid molecule encoding a type VRP subunit, wherein said vector is cut in myocardium. A truncated VRP subunit can be expressed.   In another preferred embodiment, the method is for stimulating the development of cardiac collateral vessels. Can be used.   In an even more preferred embodiment, vascular development occurs in patients with peripheral vascular disease. A way is provided to stimulate them. The method may include providing one or both of the patient's peripheral vasculature. The delivery vector by direct intra-femoral injection into the femoral artery Wherein the vector comprises a transdin encoding a truncated VRP subunit. Can express the truncated VRP subunit in the peripheral vasculature; It promotes the development of peripheral blood vessels.   Preferably, the transport vector used in the present invention is a viral transport vector It is. In one preferred aspect, the transport vector is a replication defective adenovirus. It is a vector. In another preferred aspect, the transport vector is an adeno-associated vector. It is an ills vector.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows VEGF-B [SEQ ID NO. 1], VRF-2 [SEQ I D NO. 2], VEGF-C [SEQ ID NO. 3], PIGF (human P IGF-2) [SEQ ID NO. 4], VEGF-3 [SEQ ID NO: . 5], poxvirus ORF-1 [SEQ ID NO. 6] and Virus ORF-2 [SEQ ID NO. 7] is shown. small Letters represent signal peptides that are cleaved from the mature protein. Eight core arrays The stain is underlined. The sequence is described in the following references: VEGF-B: Grimmondo et al. , Genome Res. 6 : 122-29 (1996); Olofsson et al. Proc. N atl. Acad. Sci. U. S. A. 93: 2567-81 (1996); Mouse VEGF-B: Olofsson et al. Proc. Natl. Acad. Sci. U. S. A. 93: 2567-81 (1996); human VR F-2: Grimmnd et al. , Genome Res. 6: 122 -29) (1996); human VEGF-C: Joukov et al. , EM BOJ. 15: 290-98 (1996); Lee et al. , Proc . Natl. Acad. Sci. USA 93: 1988-92 (1996); P1GF: Maglione et al. , Oncogene 8: 925- 31 (1993); Hauser & Weich, Growth Factors 9 : 259-68 (1993); human VEGF3: PCT application PCT / US95 / 0. 7283 (published on 12 December 1996 as WO 96/39421); Coxviruses ORF-1 and ORF-2: Lyttle et al. , J. et al. Virol. 68: 84-92 (1994).   2a-2f show examples of truncated VRP amino acid sequences corresponding to full length (F / L) V Shown below the RP sequence. The amino acid sequence of each truncated form is described below: 2a (F / L) [SEQ ID NO. 34] (1) [SEQ ID NO. 8]; 2a ( 2) [SEQ ID NO. 9]; 2a (3) [SEQ ID NO. 10]; 2a (4) [SEQ ID NO. 11]; 2a (5) [SEQ ID NO. 12]; 2a (6) [SEQ ID NO. 13]; 2b (F / L) [SEQ ID NO. 35]; (1) [SEQ ID NO. 14]; 2b (2) [SE Q ID NO. 15]; 2b (3) [SEQ ID NO. 16]; 2b (4 ) [SEQ ID NO. 17]; 2c (F / L) [SEQ ID NO. 36 ]; (1) [SEQ ID NO. 18]; 2c (2) (SEQ ID NO. 19]; 2c (3) [SEQ ID NO. 20]; 2c (4) [SEQ ID   NO. 21]; 2d (F / L) [SEQ ID NO. 37]; (1) [SE Q ID NO. 22]; 2d (2) [SEQ ID NO. 23]; 2d (3 ) [SEQ ID NO. 24]; 2d (4) [SEQ ID NO. 25]; 2e (F / L) [SEQ ID NO. 38] (1) [SEQ ID NO. 2 6]; 2e (2) [SEQ ID NO. 27]; 2e (3) [SEQ ID NO. 28]; 2e (4) [SEQ ID NO. 29]; 2f (F / L) [S EQ ID NO. 39]; (1) [SEQ ID NO. 30]; 2f (2) [SEQ ID NO. 31]; 2f (3) [SEQ ID NO. 32]; And 2f (4) [SEQ ID NO. 33].Detailed description of the invention Construction of novel truncated VRP sequence   A first aspect of the present invention provides a fragment comprising at least one truncated VRP subunit. It features a cut VRP. For example, FIG. Or analogs or derivatives thereof (including but not limited to) Not necessarily) is substantially the same as the amino acid sequence of VRP Means the VRP subunit that has At least one N-terminal amino acid N-terminal to the first cysteine is deleted You. Sequences that are “substantially similar” to VRP include the eight cysteine core of VEGF-B. An amino acid sequence that is at least 25% homologous to the sequence; And retains VRP activity. “Cut VRP "Subunit" also refers to the N-terminal to the first cysteine of the VEGE core sequence. At least one N-terminal amino acid has been deleted and at least one of the core sequences VRP subunit lacking cysteine (this cysteine is not essential) Also means. Non-essential cysteines are important for maintaining VRP activity A cysteine that is not required. Such non-essential cysteine is PDG It is described in connection with F. (Potgens, et al.J. Biol. Chem . 269: 32879-85 (1994)).   "Identity" means the characteristics of sequences that measure their similarity or relationship. You. Identity is determined by dividing the number of identical residues by the total number of residues and multiplying by 100. Be evaluated. Thus, exactly two copies of the same sequence have 100% identity, Sequences with less conservation and deletions, additions or substitutions have a lower degree of identity. Have one sex. In calculating sequence identity, the two sequences were Compared starting from the boxy end. One skilled in the art goes to determine sequence identity You will find that some computer programs are available.   An analog of a truncated VRP polypeptide or subunit may have a similar amino acid sequence Is a functional equivalent having the relevant truncated VRP polypeptide or Buunit retains one or more activities. “Functional equivalence "Is" one or a particular truncated VRP polypeptide or subunit An analog having an activity that can replace the above activity is meant. Preferred functional equivalent Retains all activities of a particular truncated VRP polypeptide or subunit However, functional equivalents, when measured quantitatively, are, for example, those described herein. Stronger or weaker activity as measured by a VRP functional assay You may have one. In most cases, such truncated VRP polypeptides or Subunit is incorporated into a truncated VRP dimer to measure functional activity Must be rare. Preferred functional equivalents are related truncated VRP polypeptides. More preferably between 1% and 10,000% of the activity of the peptide or subunit Is between 10% and 1000%, and even more preferably between 50% and 200% Have activity between.   The ability of the derivative to retain some activity is described herein. It can be measured using techniques. Derivatives are, for example, phosphorylated, glycosylated Cross-linking, acylation, proteolytic cleavage, to antibody molecules, membrane molecules or other ligands (Ferguson). et al. , 1988,Annu. Rev .. Biochem.57: 285- 320).   Particular types of derivatives or analogs also include deletions, substitutions, additions and amino acid modifications. Also includes amino acid changes such as decoration. "Deletion" refers to a It means the absence of one or more amino acid residues. Related to “addition” The presence of one or more amino acid residues in the polypeptide. Additions and deletions of polypeptides may be made at the amino terminus, at the carboxy terminus, and / or Or may be present inside. Amino acid “modification” refers to the modification of naturally occurring amino acids. It is meant to produce a non-naturally occurring amino acid. “Replace” Refers to the substitution of one or more amino acid residues in a polypeptide for another amino acid residue. Means to replace with a group. Derivatives may have one or more modifications and another type of modification. Combinations of different modifications, including variations, can be included.   The effects of amino acid changes on VRP activity include phosphorylation, glycosylation, and intrachain conjugation. Amino acids in the tertiary structure and active or potential allosteric site May vary depending on factors such as the role of Preferably, they are from the same group as the amino acids to be obtained. To some extent, the following The group contains interchangeable amino acids: the basic amino acids lysine, arginine And histidine; acidic amino acids aspartic acid and glutamic acid; neutral polarity The amino acids serine, threonine, cysteine, glutamine, asparagine and Methionine to a lesser degree; non-polar aliphatic amino acids glycine, alanine, valine, Isoleucine and leucine (however, due to their size, glycine and Alanine is more closely correlated, and valine, isoleucine and leucine Are more closely related); and the aromatic amino acids phenylalanine, Putphan and tyrosine. In addition, although they fall into different categories, Nin, glycine and serine seem to be somewhat interchangeable, and Stain additionally fits into this group or is classified as a polar neutral amino acid .   A preferred derivative is the activity of the relevant truncated VRP polypeptide or subunit. Have one or more amino acid modifications that do not significantly affect VRP activity In truncated VRP polypeptides or subunit regions not required for sex, Amino acids can be deleted, added or substituted with less risk of affecting activity Would. In the region required for VRP activity, large Amino acid modifications are less preferred because of the danger. Such modifications are preserved Should be an objective modification. For example, one or more amino acid residues in the sequence Can be replaced by another amino acid of the same polarity that serves as a functional equivalent.   Conserved regions appear to be more important for protein activity than non-conserved regions. In vitro Using mutagenesis techniques or deletion analysis, and as described in this disclosure VRP activity to determine conserved and non-conserved regions important for VRP activity Standard methods can be used to accomplish this.   Derivatives can be made using standard chemical and recombinant nucleic acid molecule techniques. specific Modifications of the polypeptides may be performed by site-directed mutagenesis and amino acid substitution during solid phase synthesis. Are considered carefully, or are mutations in the host producing the polypeptide. It would be accidental. The polypeptide containing the derivative is Sambrook k et al. , Molecular Cloning, Cold Spri ng Harbor Laboratory Press (1989). Can be obtained using standard techniques such as For example, Sambrook k describes a method for site-directed mutagenesis of cloned DNA. Have been.   “Truncated VRP polypeptide” refers to the fragment of the truncated VRP subunit of the present invention. A polypeptide comprising a amino acid sequence, or as described herein. Meaning their functional analogues or derivatives. The term “truncated VRP polypeptide” "Peptides" also include truncated VRP subunits; the terms subunit and Generally refers to a peptide that is folded into an active three-dimensional structure.   "Truncated VRP" refers to a dimer of two VRP subunits. The two subunits may be derived from two different VRPs, in which case the subunits Both units are truncated VRP subunits. One of the subunits or Both ends may be truncated; the two subunits may also have different N-terminal truncations. You may have a loss.   A truncated VRP, a truncated VRP subunit or a truncated VRP polypeptide is Conveniently, it is concentrated or purified. In the present context the term "enriched Are more specific than normal or diseased cells or cells from which the sequence was taken. If the defined amino acid sequence is the total amino acid sequence present in the cell or solution in question This means that an extremely high fraction (2-5 times) is constituted. This means that By the preferential reduction of the amount of other amino acid sequences present or by the specific amino acid in question Caused by a preferential increase in the amount of acid sequences, or a combination of the two Can be. However, enrichment means that no other amino acid sequence is present Not just because the relative amount of the sequence in question is significantly increased. It should be noted that there is. Here, the term "significant" means that the level of increase is such Is used to indicate that it is useful to those who Generally at least about two times, more preferably at least five times the other amino acid sequence. Means 10 times or more. The term also applies to amino acid sequences of other origins. It doesn't mean it doesn't exist. Amino acid sequences of other origin include: For example, yeast or bacterial genomes, or cloning vectors such as pUC19 Will be included. The term is Applicable only in situations where humans are involved to increase the ratio of nonacid sequences Round.   For some purposes, it is convenient for the amino acid sequence to be in purified form. is there. The term “purified” for a polypeptide refers to absolute purity (eg, Una); rather than the sequence is in its natural environment. Indicates that it is relatively more pure (this level compared to the natural level). Should be at least 10 times greater, in terms of mg / ml). At least It is clear that the purification is about 1 digit, preferably 2 or 3 digits, more preferably 4 or 5 digits. Surely contemplated. The substance is preferably free of contaminants at functionally significant levels , For example, 90%, 95% or 99% purity.   Another aspect of the present invention is to encode a truncated VRP polypeptide or subunit. Featured nucleic acid molecules.   In some situations, such nucleic acid molecules must be concentrated or purified. Is desirable. When the term “enriched” is used with respect to nucleic acid molecules, Are more specific DNA or RNA sequences than in the diseased cells or cells from which the sequences were taken. Significantly higher total DNA or RNA sequences present in the cell or solution in question A large fraction (2-5 times). This exists By the preferential reduction of the amount of other DNA or RNA sequences or by the specific D By preferentially increasing the amount of NA or RNA sequences, or by a combination of the two Can be more awake. However, the enrichment will not affect other DNA or RNA sequences. It does not mean that it does not exist, just that the relative amount of the sequence in question is It should be noted that it has been increased. Where the term "significant" Indicates that the level of increase is useful for those making such an increase And is generally at least about twice as good as other nucleic acids, more preferably Means at least 5 to 10 times or more. This term is also of other origin Does not mean that no DNA or RNA sequence is present. DNA sequences of other origin include, for example, yeast or bacterial genomes, or pUC19. DNA from such a cloning vector would be included. This term is Spontaneous events such as ills infection or the level of one mRNA is Distinct from tumor-type growth as spontaneously increased as compared to a class of mRNA. That is The term is used in situations where a person is involved to increase the proportion of the desired nucleic acid. This only applies.   For some purposes, it is convenient for the nucleotide sequence to be in purified form. It is profitable. The term “purified” with respect to nucleic acid molecules refers to absolute purity (eg, Una); rather than the sequence is in its natural environment. Indicates that it is relatively more pure (this level compared to the natural level). Should be at least 2-5 times greater, in terms of mg / ml).   Nucleic acid molecules can be modified, for example, using oligonucleotide-directed mutagenesis. By decoration, or by sequence deletion using restriction enzymes, or as described herein. As will be constructed from existing VRP nucleotide sequences. Inbit B) Site-directed mutagenesis (Hutchinson et al.,J. Biol . Chem .253: 6551 (1978), Sambrook et al. , Chapter 15, supra), use of a TABR linker (Pharmacia), and Standard recombination techniques for mutagenesis such as PCR-directed mutagenesis are Can be used to create such mutations. Nucleic acid molecules can also be Alternatively, it can be synthesized using an automated DNA synthesizer.   The present invention also preferably relates to recombinant DNA vectors and recombinants in cells or organisms. It also features a DNA expression vector. Recombinant DNA vectors are used in host cells. In a vector containing a promoter effective to initiate transcription of It contains a sequence encoding VRP or a functional derivative thereof. Recombinant DN A vector is a transcription initiation region functional in cells and transcription termination functional in cells Area can be included.   The invention also includes a nucleic acid molecule or a recombinant DNA vector as described above, whereby It also relates to a cell or organism that can express a truncated VRP peptide. Polype Peptides are purified from cells that have been modified to express the polypeptide Will. Genetically engineered cells do not normally produce or usually have low levels If the cell is made to produce a protein that does not Modified to express the peptide ". Eukaryotic or prokaryotic cells To introduce and express genomic, cDNA or synthetic sequences into The method can be easily adapted.   Nucleic acid molecules such as DNA contain transcriptional and translational information, and Sequence is "operably linked" to the nucleotides encoding the polypeptide The polypeptide is said to be "expressible." Although the exact nature of the control regions currently required may vary from organism to organism, In general, in eukaryotes, promoters (instructing the initiation of RNA transcription) and In addition, DNA sequences that will give a signal to initiate synthesis when transcribed into RNA It contains a promoter region containing both. Such areas are usually For the initiation of transcription and translation, such as nucleic acids, capping sequences, CAAT sequences, etc. 5'-noncoding sequences that would be included.   For example, the entire coding sequence of a truncated VRP subunit or a fragment thereof One or more of the following in an appropriate expression vector to allow such expression: (1) exogenous promoter sequence (2) ribosome binding site (3) polyadenylation signal (4) secretion signal. Eukaryote or prokaryote The 5 'untranslated and 3' untranslated regions can be modified to improve expression in the cell; Alternatively, they may encode the same amino acid, but have codons selected in the expression system. And the codons may be modified to be preferred codons. Such suitable code The use of thiol is described, for example, in Grantham et al.Nuc. Acids R es . 9: 43-74 (1981) and Lathe,J. Mol. Biol . , 183: 1-12 (1985), which is incorporated herein by reference in its entirety. It is listed.   Optionally, the 3 'non-coding region of the genomic VRP sequence may be replaced with such a VRP subunit. It will be operably linked to the nucleic acid molecule encoding the knit. this The region is a recombinant D.R. for transcription termination regulatory sequences such as termination and polyadenylation. It may be used in NA vectors. Therefore, the DNA encoding the VRP gene By retaining the 3 'region naturally adjacent to the sequence, the transcription termination signal Provided. Alternatively, it may be replaced with a 3 'region functional in the host cell.   Operable linkage is defined as regulatory DNA sequences and the DNA sought to be expressed. With linkages such that the sequences are linked in such a way as to allow gene sequence expression is there. Two DNA sequences (like a promoter region sequence and a truncated VRP sequence) (A) indicates that if the nature of the connection between the two DNA sequences is Directs transcription of (2) truncated VRP gene sequence without introducing shift mutation Does not interfere with the ability of the promoter region sequence to (3) If it does not interfere with the ability of the truncated VRP gene sequence to be more transcribed, It is called dynamically connected. Therefore, if the promoter region is Once the promoter has achieved transcription of the DNA sequence, it is operably linked to the DNA sequence. Will be. Therefore, to express a truncated VRP gene, an appropriate host More recognized transcription and translation signals are required.Expression and purification of novel truncated VRP sequences   Examples 2 and 3 demonstrate the novel truncated V of the present invention expressed in a baculoylurus system. It describes the expression and purification of RP sequences. Those skilled in the art will appreciate that the truncated VRP Expressed in other cell lines (both prokaryotic and eukaryotic), all of which are It will be appreciated that it is within the scope of the invention. Example 4-6 shows a novel truncated VRP Examples of assays suitable for the functional activity of are provided.   The truncated VRPs of the present invention are expressed in prokaryotic cells (which are Generally very efficient and convenient for production), such cells Since the truncated VRP produced will not be glucosylated, Will have a shorter half-life. The most frequently used prokaryotes Represented by various strains of E. coli. However, other microbes, including other bacterial strains Stocks are also used. Recognized prokaryotic hosts include E. coli, Bacillus, Bacteria such as Streptomyces, Pseudomonas, Salmonella, Serratia, etc. Is mentioned. Prokaryotic hosts contain replicons and control sequences in an expression plasmid. Must match.   In prokaryotic systems, replication sites and controls derived from species compatible with the host A plasmid vector containing the sequence will be used. Suitable plasmid vector Examples of pBR include pBR322, pUC118 and pUC119; Phage or bacteriophage vectors include γgt10 and γg and suitable viral vectors include pMAM-ne o and pKRC and the like. Preferably, the selected vector of the invention is selected Has the ability to replicate in selected host cells.   In prokaryotic cells, a truncated VRP polypeptide or subunit (or In order to express these functional derivatives, a truncated VRP sequence must be It is necessary to operably link to a product promoter. Such a promoter Is constitutive or more preferably adjustable (ie, navigable or suppressed) Noh). Examples of constitutive promoters include the int pro of bacteriophage λ. Motor, the bla promoter of the β-lactamase gene sequence of pBR322, And chloramphenicol acetyltransferase gene of pPR325 Sequence CAT promoter and the like. Examples of inducible prokaryotic promoters Contains the main right and left promoters of bacteriophage λ (P_LAnd P_R), E. coli trp, recA, λacZ, λacI and gal promoters, α Amylase (Ulmaner. Et al.,J. Bacteriol. 16 2: 176-182 (1985)) and the B. subtilis 特異 -28-specific promoter. -(Gilman et at., Gene sequence 32: 11- 20 (1984)), Bacillus bacteriophage promoter (Gryc zan ,: The Molecular Biology of the Ba Cilli, Academic Press, Inc. , NY (1982)), And Streptomyces promoter (Ward et at.,Mol. Ge n. Genet . 203: 468-478 (1986)). Prokaryotes The promoter is Glick (J. Ind. Microbiot. 1: 277- 282 (1987)); Cenatiempo (Biochimie  68: 5 05-516 (1986)); and Gottesman (Ann. Rev .. Genet . 78: 415-442 (1984)).   Proper expression in prokaryotic cells also requires that the gene be sequenced upstream of the coding sequence. The presence of a vosome binding site is required. Such a ribosome binding site For example, Gold et at. (Ann. Rev .. Microbiol. 35: 3 65-404 (1981)). Resources required to start translation Bosome binding sites and other sequences include, for example, such control sequences Nuclei encoding truncated VRP by frame joining of synthetic oligonucleotides Operably linked to an acid molecule. Signal for expression in prokaryotic cells No peptide sequence is required. Control sequences, expression vectors, and transformation methods The choice depends on the type of host cell used to express the gene.   As used herein, “cell”, “cell line” and “cell culture” are used interchangeably. Used, and all such names include their descendants. Therefore, "transformation The terms “body” or “transformed cell” refer to primary cells and the number of passages. Regardless, they include cultures derived from them. Cleavage expressed in prokaryotic cells For the truncated VRP peptide, the N-terminal sequence predicted from the sequence of the expression vector is appropriate. Truncated VRP peptide, and insufficient methionine during bacterial expression Includes a mixture of truncated VRP peptides with N-terminal methionine generated by cleavage. It is. The presence of the N-terminal methionine was not expected to affect biological activity Therefore, both types of truncated VRP peptides are considered to be within the scope of the present invention. You. Due to deliberate or unintentional mutations, the DNA content of all progeny is correct. It should also be understood that they will not be exactly the same. However, as defined The progeny of the mutant has the same functionality as the original transformed cell.   Suitable prokaryotic vectors include plasmids capable of replicating in E. coli (For example, pBR322, ColEl, pSC101, pACYC184, πV Like X). Such plasmids are described, for example, in Sambrook ("Mole cultural Cloning: A Laboratory Manual ", Second Edition, Sambrook, Fritsch, & Maniatis, Ed., Cold Spring Harbor Laboratory, (1989)). Has been stated. Bacillus plasmids include pC194, pC221 and pT12 7 and the like. Such a plasmid is known as Gryczan (The Mole). cultural Bio of the Bacilli, Academi cPress, NY (1982) pp. 307-329). You. Suitable Streptomyces plasmids include plJ101 (Kendall e. t al. ,J. Bacteriol. 169: 4177-4183 (1987) )) And ΦC31 (Chater et al., Actinomycete biology). Sixth International Symposium, Akademiai Kaido, Budapest, Hungary (1986), p. 45-54). Pseudomonas The plasmid was prepared by John et al. (Rev .. Infect. Dis. 8: 693-704 (1986)) and Izaki (Jpn. J. Bacteriol. 33: 729-742 (1978)).   Eukaryotic host cells that may be used in the expression system of the present invention are truncated VRP peptides. It is not strictly limited as long as it is suitable for use in the expression of a tide. Suitable eukaryote Product hosts include, for example, yeast, fungi, insect cells, mammalian cells (in vivo or tissue culture). Education) is included. Mammalian cells that may be useful as hosts include healer cells, VER Cells of fibroblast origin, such as O or CHO-K1, or cells of lymph origin And their derivatives.   Truncated VRPs of the present invention may also be prepared, for example, according to Olofsson, B.A. et al. ,Proc. Natl. Acad. Sci. USA  93: 2576-2581 Expresses Epstein-Barr virus nuclear antigen 1 described in (1996) It will also be expressed in human cells, such as human fetal kidney 293EBNA cells. Fine The vesicles were transfected with the expression vector of Example 2 using calcium carbonate precipitation. The cells are then incubated for at least 48 hours. Truncated VRP peptides Purified from the supernatant as described in Example 3.   In addition, plant cells can also be used as hosts, and cauliflower mosaic virus 35S and 19S, and nopaline synthase promoter and polyadenylate Regulatory sequences that are compatible with plant cells, such as a ruling signal sequence, are available. another Suitable hosts are, for example, insect cells such as Drosophila larvae. With the host When using insect cells, the Drosophila alcohol dehydrogenase Motor can be used. Rubin,Science  240: 1453-14 59 (1988).   Promoters from actively expressed gene sequences encoding glycolytic enzymes And a series of yeast gene sequence expression systems that incorporate Produced in large quantities when grown on a medium rich in glucose. Known glycolytic residues Gene sequences can also provide very efficient transcription control signals. The yeast is It also offers the essential advantage that peptide modifications can be performed. The desired protein in yeast Strong promoter sequences and high copy number plasmids available for white matter production There are a number of recombinant DNA strategies using sumids. Yeast is a cloned mammal A peptide that recognizes a leader sequence on a gene sequence product and carries the leader sequence (ie, Secretion of pre-peptides). For mammalian hosts, expression of truncated VRP peptides Several possible vector systems for are available.   A wide range of transcription and translation regulatory sequences will be used depending on the nature of the host . Transcriptional and translational regulatory signals are those associated with a particular gene sequence. Adenovirus, bovine papillomavirus, cytomegalo with high levels of expression Virus or derived from a viral source such as a simian virus. U. Alternatively, mammalian expression products such as actin, collagen and myosin A promoter from a product may be used. Transcription initiation regulatory signals are Those that can be suppressed or activated will be selected so that expression can be regulated . Temperature sensitive, such that expression can be suppressed or initiated by changing the temperature Signals that are regulated or chemically regulated (such as metabolites) are of interest. I will   Expression of truncated VRP in eukaryotic hosts requires the use of eukaryotic regulatory regions I do. Such regions are generally sufficient to direct initiation of RNA synthesis. Motor region. Suitable eukaryotic promoters include, for example, mouse Promoter of the smetallothionein I gene sequence (Hamer et al.,J. Mol. Appl. Gen . 1: 273-288 (1982)); Herpes The viral TK promoter (McKnight,Cell  31: 355-3 65 (1982)); SV40 early promoter (Benoist et al.). . ,Nature(London) 290: 304-310 (1981)); The mother ga14 gene sequence promoter (Johnston et al.,Pro c. Natl. Acad. Sci. (USA) 79: 6971-6975 (19 82); Silver et al. ,Proc. Natl. Acad. Sci . (USA) 81: 5951-5955 (1984)).   Translation of eukaryotic mRNA is initiated at the first methionine-encoding codon . Thus, DNA encoding a eukaryotic promoter and a truncated VRP The linkage between the sequences does not include an intervening codon that can code for methionine (ie, AUG). It is preferred to ensure that The presence of such codons is a form of the fusion protein (If the AUG codon is in the same reading frame as the truncated VRP coding sequence, ) Or a frameshift mutation (if the AUG codon matches the truncated VRP coding sequence) If not in the same reading frame).   The truncated VRP nucleic acid molecule and the operably linked promoter comprise a linear molecule. Non-replicating DNA (RNA) molecule, or more preferably a covalently closed circular molecule As a transfected eukaryotic or prokaryotic cell. Such a molecule is self Due to the inability to replicate, gene expression occurs by transient expression of the introduced sequence Will. Alternatively, by integration of the introduced DNA sequence into the host chromosome Permanent expression occurs.   A vector that can integrate a desired gene sequence into a host chromosome is used. Will be. Cells in which the introduced DNA has been stably integrated into the chromosome are expressed in an expression vector. One or more markers that allow for the selection of host cells containing the Can be selected. Markers are prototrophy to auxotrophic hosts Imparts biocide resistance (eg, antibiotics) or heavy metals (such as copper) Will. Selectable markers are directly linked to the DNA gene sequence to be expressed. Or can be introduced into the same cell by co-transfection. One Additional elements are required for optimal synthesis of chain binding protein mRNA. These points Elements include splice signals and transcription promoters, enhancers and terminations. Includes sieving signals. CDNA expression vector incorporating such elements Okayama,Molec. Cell. Biol. 3: 280 (19 83).   The introduced nucleic acid molecule is a plasmid or virus capable of autonomous replication in the recipient host Can be imported into vector. A wide range of vectors will be used for this purpose . Factors important in selecting a particular plasmid or viral vector are: : Recognition and selection of cells without vector and recipient cells containing vector Ease of use; desired vector copy number in a particular host; Whether it would be desirable to be able to “reciprocate” the vector between different types of host cells ?   Suitable eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2- Micron circles and the like or derivatives thereof are included. Such a plasmid Are well known in the art (Botstein et al.,Miami Wntr. Symp . 19: 265-274 (1982); Broach, "T. he Molecular Biology of the Yeast Sa ccharomyces: Life Cycle and Inheritan ce ", Cold Spring Harbor Laboratory, Co. ld Spring Harbor, NY, p. 445-470 (1981); Broach,Cell  28: 203-204 (1982); Bollon. et al. ,J. Clin. Hematol. ○ ncol. 10: 39-48 (1980); Maniatis, "Cell Biology: A Comp. rehensive Treatise ", Vol. 3, Gene Seque nce Expression, Academic Press, NY, pp. 563-608 (1980)).   Once the vector or nucleic acid molecule containing the construct has been prepared for expression, D The NA construct may be transformed by a variety of suitable means, including transformation, transfection, co- Conjugation, protoplast fusion, electroporation, particle gun technology , Lipofectin, calcium phosphate precipitation, direct microinjection and And more suitable host cells such as DEAE-dextran transfection Is introduced to Transfection of eukaryotic cell lines using plasmid DNA The most effective method for a given cell will vary for a given cell type. After vector introduction, vector The recipient cells are grown in a selection medium that selects for the growth of the containing cells. Cloned gene Preliminary expression results in the production of truncated VRP or fragments thereof. This is a form Transformed cells themselves or after inducing differentiation of these cells (e.g., (E.g., administration of bromodeoxyuracil to neuroblastoma cells). You. Various incubation conditions are used to form the peptides of the invention it can. The most preferred conditions are those that mimic physiological conditions.   Production of stable transfectants includes, for example, truncated VRP polypeptides or Indicates that the coding sequence for the subunit has been cloned into the multiple cloning site Transformation of a suitable cell line with an eukaryotic expression vector (such as pCEP4) Will be achieved by sfection. These expression vectors are available in various Human cytomegaloie performs high-level transcription of desired DNA molecules in mammalian cells It contains a promoter region such as the Ruth promoter (CMV). in addition, These vectors are useful for the selection of cells stably expressing the DNA molecule of interest. Contains the gene for The selectable marker in the pCEP4 vector is Hygromycin, a metabolic inhibitor added to cultures to kill transfected cells Encodes an enzyme that confers resistance to   Cells that have stably taken up the transfected DNA are as described above. Identified by resistance to selective medium, clonal cell lines develop resistant colonies Would be more manufactured. Expression of truncated VRP DNA by these cells is As assessed by liquid hybridization and Northern blot analysis U.Pharmaceutical compositions and therapeutic uses   One object of the present invention is to provide a truncated VRP in a pharmaceutical composition suitable for therapeutic use It is to be. Accordingly, one aspect of the invention is a pharmaceutical composition comprising a truncated VRP For stimulating angiogenesis in patients by administering a therapeutically effective amount of A law is provided.   "Therapeutically effective amount" means the amount of a compound that produces a desired therapeutic effect in a patient. are doing. For example, in the context of a disease or disorder, one or more of the disease or disorder Reduces some of the symptoms to some extent and is partially or completely associated with the disease or disorder To restore normal or causative physiological or biochemical parameters. You. 0.1 mg / kg to 10 mg when used to treat patients therapeutically Between 0 mg / kg, preferably less than 50 mg / kg, more preferably 10 mg / kg Less than, even more suitably, an amount expected to be less than 1 mg / kg. The amount of the compound depends on the age, size and disease of the patient.   The optimal formulation and mode of administration of the compound of this application to a patient will depend on the particular disease or disorder, The desired effect depends on factors known in the art, such as the type of patient. Realization The compound will typically be used to treat human patients, but other primates Farm animals, such as pigs, cattle and poultry, and sport animals, and horses, I Similar or identical disease in other vertebrates such as pets such as dogs and cats It could also be used to treat a disease.   Suitably, a therapeutically effective amount is provided as a pharmaceutical composition. Pharmaceutical drug or pair An adult is an agent or composition in a form suitable for administration to a multicellular organism, such as a human. Is saying. Suitable forms include, in part, the use or route of administration (eg, oral, transdermal) Or by injection). In such a form, the target cell may be a multicellular host. Or a drug or composition, even if present in the culture medium, can reach target cells To For example, a pharmaceutical agent or composition injected into the bloodstream must be soluble. Must. Other factors are known in the art, and toxicity and drug or composition This includes considerations such as forms that prevent it from exerting its effect.   The claimed composition comprises a pharmaceutically acceptable salt (eg, an acid addition salt) and And / or a complex thereof. Pharmaceutically acceptable salts are those It is a non-toxic salt at the concentrations given. Formulations of such salts may have a physiological effect on the composition By changing the physicochemical properties of the composition without impeding its performance Pharmaceutical use can be facilitated. Examples of useful changes in physical properties include transdermal administration. Lowering the melting point for ease of administration and allowing higher concentrations of drug to be administered Increasing the solubility for ease of use.   Pharmaceutically acceptable salts include sulfates, hydrochlorides, phosphates, sulfonates, Famate, sulfate, acetate, citrate, lactate, tartrate, methanesulfo Phosphate, ethanesulfonic acid salt, benzenesulfonic acid salt, p-toluenesulfonic acid Salts, cyclohexyl sulfonate, cyclohexyl sulfamate and quina Contains acid salts. Pharmaceutically acceptable salts include sulfuric acid, hydrochloric acid, phosphoric acid, sulfonic acid, Sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, methanesulfonic acid, ethanes Sulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfur It can be obtained from acids such as phonic acid, cyclohexylsulfamic acid and quinic acid. it can. Such salts can be, for example, in a solution or medium in which the salt is insoluble, or in water. Of the product in a solvent such as that which is removed under vacuum or by lyophilization. Reacting the isolated acid or base form with one or more equivalents of the appropriate base or acid Or the ion of a salt present in a suitable ion exchange resin by another ion Would be manufactured by replacing with   A carrier or excipient can also be used to facilitate administration of the compound. Carrier Examples of excipients include calcium carbonate, calcium phosphate, lactose, Various sugars, such as glucose or sucrose, or starch forms, cellulose Conductors, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents Medium. The composition or pharmaceutical composition can be administered intravenously, intraperitoneally, subcutaneously and intramuscularly. It can be administered by different routes, including, oral, topical or transmucosal.   Desired isotonicity is sodium chloride or dextrose, boric acid, sodium tartrate Lium, propylene glycol, polyol (mannitol and sorbitol) Or other pharmaceutically acceptable drugs such as other inorganic or organic solutes Achieved using goods. Sodium chloride in buffers containing sodium ions Are particularly preferred.   The compounds of the present invention can be administered in a variety of modes including systemic and local or localized administration. Can be prescribed for feeding. Techniques and formulas are generally used in Remjngton's Ph armaceutical Science, 18th edition, Mack Publis ing Co. , Easton, PA, 1990. Also, Wang, Y .; J. and Hanson, M .; A. "Parentalal Formulation of Proteins and Peptides : Stability and Stabilizers, "Journal of Parental Science and Technology , Technica 1 Report No. 10, Supp. 42: 2S (1 988). Appropriate mode of administration is individualized for each patient Would be best determined by the person.   Injection is suitable for systemic administration, for example, intramuscular, intravenous, intraperitoneal, subcutaneous, sheath For intra- and intraventricular injection, the compounds of the invention may be in liquid solutions, preferably in Hanks' solution. Or a physiologically compatible buffer such as Ringer's solution. Or the original The clear compound is one of the generally accepted definitions of safe under USP standards. It is formulated with one or more excipients (eg, propylene glycol). So These are, for example, non-chemical oils (suitable for sesame oil, peanut oil , Vegetable oils such as olive oil) or other acceptable carriers. Preferably An isotonic buffer, for example, at a pH of about 5.6 to 7.4, suspended in an aqueous carrier. These compositions are sterilized by conventional sterilization techniques, or are sterile filtered. The composition contains the medicaments needed to approximate physiological conditions, such as pH buffering agents. And acceptable auxiliary substances. Useful buffers include, for example, Sodium acetate / acetate buffer. Long time after transdermal injection or delivery Or to deliver a therapeutically effective amount of the formulation into the bloodstream over a period of days. As a depot, a sustained release formulation is used. In addition, the compound is in solid form It may be formulated and redissolved or suspended immediately before use. Lyophilized form is also included You.   Systemic administration can be by transmucosal or transdermal, and this molecule can be administered orally. Wear. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are treated. Used for Such penetrants are generally known in the art, and include, for example, Transmucosal administration includes bile and fusidic acids. In addition, it facilitates penetration For this purpose, a surfactant may be used. Transmucosal administration, for example, by nasal spray Or using suppositories. For oral administration, this molecule is a capsule or tablet And oral formulations such as aqueous preparations. For topical administration, The compounds of the invention may be ointments, salves, gels or the like as generally known in the art. Formulated in creams.   If desired, the solution of the composition can be concentrated with a thickening agent such as methylcellulose. It may be. These may be manufactured in emulsified form (water-in-oil or water-in-oil). oil). For example, acacia powder, nonionic surfactants (such as Tween) or Is an ionic surfactant (alkali polyether alcohol sulfate or sulfone A wide range of pharmaceutically acceptable emulsifiers, including, for example, tritons) Is used.   Compositions useful in the present invention mix the ingredients according to generally accepted methods It is manufactured by For example, the components selected may be a mixer or other standard equipment. To form a concentrated mixture, which is then mixed with water or thickener and possibly Buffers to adjust pH or additional solutes to adjust isotonicity, if any To adjust the final concentration and viscosity.   The amounts of the various compounds of the invention to be administered can be determined in conventional manner. Typically , The therapeutically effective amount depends on the patient's age and size, and the patient's disease or disorder But between about 1 and 3 micromoles of molecules, preferably about 10 nanomoles And between 1 micromolar. Generally, the amount will be the k of the animal to be treated. per g, about 0.1 and 50 mg, preferably between 1 and 20 mg.   For use by a physician, the composition may comprise truncated VRP, VRP polypeptide or VRP. It would be provided in a single dosage form containing one amount of the RP subunit.Gene therapy   Truncated VRP or its gene sequence can be used in gene therapy (Miller,Natur e   357: 455-460 (1992)). . Miller Shows First Practical Results With Practical Approach To Human Gene Therapy States that they have made progress. The basic science of gene therapy is Mulligan,Science   260: 926-931 (1993). Heredity One example of offspring treatment is shown in Example 7, which includes adenovirus-mediated genetics. Explains the use of child therapy.   In another embodiment, the expression vector containing the truncated VRP coding sequence is Inserted into the vesicle and the cells are grown in vitro before being injected or injected into the patient in large quantities. Was entered. In another embodiment, a selected promoter (eg, a strong promoter) is used. Motor segment), the promoter segment is exogenous A cell containing an exogenous truncated VRP in a manner that promotes expression of the truncated VRP gene (Eg, the promoter segment is an exogenous truncated VRP gene) Is introduced into the cell to bind directly to the offspring).   For gene therapy, nucleotide sequences encoding truncated VRP subunits Contains naked nucleic acid molecules encoding rows or truncated VRP subunits Use of adenovirus vectors is included. Or, cut type VRP sub unit Genetically engineered cells containing nucleic acid molecules encoding the knit are injected You. Example 7 used adenovirus vectors to provide angiogenesis therapy 1 illustrates a gene therapy method.   Retrovirus, vaccinia virus, adenovirus, adeno-associated virus , Herpes virus, some RNA viruses or bovine papilloma virus Expression vectors derived from viruses such as The nucleotide sequence (eg, cDNA) encoding the kit into the target cell population Used to deliver to A method well known to those skilled in the art It can be used to construct a containing recombinant viral vector. For example, Ma niatis et al. ,Molecular Cloning: A La boratemanual, Cold Spring Harbor L laboratory, N.W. Y. (1989), and Ausubel et a l. ,Current Protocols in Molecular Bi ology , Green Publishing Associates a nd Wiley Interscience, N.W. Y. (1989) I want to be. Alternatively, the recombinant nucleic acid molecule encoding the protein sequence may Or reconstituted systems, such as liposomes or other liposomes for delivery to target cells. Lipid systems (eg, Felgner et al.,Nature  337: 38 7-8, 1989). In human gene therapy, Several other methods exist for the direct introduction of plasmid DNA into cells, Targeting DNA to receptors by complexing plasmid DNA to white matter include. Miller,Nature  357: 455-60, 1992 Please refer to.   In its simplest form, the microinjection technique allows the Gene therapy is achieved by simply injecting small amounts of DNA. Capecc hi MR,Cell  22: 479-88 (1980). Recombination into cells Once the genes are introduced, they become the normal machinery of the cell for transcription and translation. Will be more recognized and the gene product will be expressed. DNA into more cells Other methods have been tried to introduce it. These methods include: Transfection, where DNA is precipitated with calcium phosphate, It is taken up into cells by pinocytosis (Chen C. and Ok ayama H,Mol. Cell Biol. 7: 2745-52 (1987) ); Electroporation, where cells are exposed to high-voltage pulses to create holes in the membrane (Chu G. et al.,Nucleic Acids Res. , 15: 1311-26 (1987)); Lipofectin / liposome fusion, Here, the DNA is encapsulated in a lipophilic carrier that fuses to the target cells (Felgn er PL. , Et al. ,Proc. Natl. Acad. Sci. USA . 84: 7413-7 (1987)); and DNA bound to small projectiles Bombardment using a method (Yang NS. Et al., Proc. Natl. Ac). ad. Sci. 87: 9568-72 (1990)). Introduce DNA into cells Another method is to bind DNA to chemically modified proteins.   Adenovirus proteins can destabilize endosomes and take up DNA into cells. It has also been shown to promote penetration. Adenovirus DNA complex containing solution Or covalently linked to adenovirus using a protein crosslinker Binding of DNA to polylysine substantially modifies uptake and expression of the recombinant gene. Good. Curell DT et al. ,Am. J. Respir. Cel l. Mol. Biol . , 6: 247-52 (1992).   In addition, adeno-associated virus vectors are used for gene delivery into vascular cells. (Gnatenko, D., et al.).J. of Inves t. Med . 45: 87-97, (1997)).   As used herein, "gene transfer" refers to the transfer of a foreign nucleic acid molecule into a cell. Means how to. Gene transfer is a specific process encoded by a gene. It is usually performed to allow expression of the product. Products include proteins, polypeptides , Antisense DNA or RNA, or enzymatically active RNA Will. Gene transfer can be performed by direct injection into cultured cells or into animals. You can go. Gene transfer generally involves non-specific or receptor-mediated interactions. The process by which nucleic acid molecules come into contact with target cells, through membranes or endocytosis Uptake of nucleic acid molecules into cells and from plasma membranes or endosomes Release of nucleic acid molecules into the cytoplasm is involved. In addition, expression of nucleic acid into the nucleus Binding to appropriate nuclear factors for transfer and transcription of the offspring is required.   As used herein, “gene therapy” is a form of gene transfer and is described herein. Included in the definition of gene transfer as used in the textbook, particularly in vivo or Gene transfer to express a therapeutic product from cells in vitro I have. Gene transfer can also be performed ex vivo on cells (which in turn Implanted), or directly administer the nucleic acid molecule or nucleic acid-protein complex to the patient It can be implemented by doing.   In another preferred embodiment, the nucleic acid comprises a nucleic acid molecule sequence encoding a truncated VRP. Provided, wherein the nucleic acid molecule sequence is only available in certain tissues. Is expressed. A method for achieving tissue-specific gene expression was described on November 3, 1992. International Patent Application No. WO 93/092 filed and published on May 13, 1993 No. 36.   In another preferred embodiment, a method for gene replacement is provided. As used herein As used herein, "gene replacement" can be expressed in vivo in an animal. Supplies a nucleic acid molecule sequence that is lost or missing in the animal It means providing or increasing the function of an endogenous gene.   In all of the vectors set forth above, a further feature of the present invention is that Nucleic acid sequences contained in some or all of the nucleic acid sequences defined above It may include additions, deletions or modifications.Example   To assist in understanding the present invention, the following examples, which illustrate the results of a series of experiments, are provided. include. Experiments on the present invention, of course, prove that the invention is particularly limited. Not currently known but will be developed in the future Such variations of the invention described herein may be made to the book described herein and in the claims that follow. It is considered within the scope of the invention.Example 1 VEGF-B with truncated N-terminal (des (1-20) -p21-VEGF-B (Or cloning of des (2-21) -VEGF-B)   To create a new VEGF-B protein lacking the first 20 amino acids, The formula created the cDNA construct:   DNA encoding human VEGF-B is human heart or skeletal muscle cDNA, Human fetal brain cDNA library, or cDNA from another suitable human tissue source Oligonucleotides corresponding to the published human VEGF-B sequence from samples Amplified by PCR using Using standard molecular biology techniques (Sambro ok et al. , Molecular Cloning, A Labora tory Manual, 2nd ed. , Cold Spring Harb or Laboratory, Cold Spring Harbor NY) 5, human VEGF-B signal sequence at 5 'end, followed by proline codon, maturation The first amino acid residue in VEGF-B, and then the C-terminal 2 of human VEGF-B A codon corresponding to the amino acid from two residues, followed by a D which encodes a stop codon Generate NA fragments. To allow insertion of the DNA into an appropriate expression vector. Appropriate additional non-coding nucleotide sequences were added to the 5 'and 3' '.   In this method, signal peptide cleavage sites are observed in native VEGF-B. Are stored in the same manner as However, in this method, truncated VEGF Changes in the new N-terminal amino acid of -B. des (1-20) -VEGF-B While the standard N-terminal amino acid of is a tyrosine residue: mspllrrlllvallqlartqa [PVSQFDGPSHQKKVVPWIDV]YTRAT, new N-terminal amino acid Lorin and the resulting truncated VEGF-B is des (2-21) -VEGF-B Is equivalent to: mspllrrlllvallqlartqaPTRAT ....   Changes in truncated VEGF-B from natural amino acids (20 amino acid truncated form) Has no effect on the biological activity of truncated VEGF-B. Is expected. The advantage of this method is that the signal peptide sequence is maintained, Therefore, signal peptide efficiency from precursors during protein processing / secretion Is to ensure that the target is cut.   In another embodiment, the truncated VEGF-B, des (1-15) -VEGF- B is constructed by deleting the first 15 amino acids. Residues # 16 and Residue # 1 (new and old N-terminus) is identical (proline), The null peptide cleavage site will be preserved:   One of skill in the art will appreciate that other signal peptides may be used in the present invention. Would. For example, a VEGF-B or VEGF-C signal peptide can be used. Of the truncated proteins to conserve each signal peptide cleavage site. It would require that the first amino acid be alanine or glycine, respectively. A further alternative is to use signal peptide sequences from other known proteins. Some of these are the N-terminal strands of truncated des (1-20) -VEGF-B. Has a cleavage site consistent with rosin.   Another option is to use the N-terminus of the native VEGF-B protein sequence (more than one B) encodes a precursor protein with a cleavage site incorporating two amino acids Will generate a construct. The purpose of this method is to determine if the cleavage site is a signal peptide. This would be to more completely ensure that it is consistent with tidase function. this child Refers to the introduction of two new amino acids at the N-terminus of the truncated VEGF-B sequence. However, it is hoped that such changes will not alter the biological function of the truncated peptide. I will be waiting.       des (1-20) -Explanation for generating VEGF-B-expressing DNA The method described is similar to the VEGF-B mutant with other desired length N-terminal truncations. Useful for generation in style. In addition, the method can be used in other VEGF-related forms and in other species. Also to generate other desired lengths of N-terminal truncations in their isoforms of It is for.Example 2: Expression of N-terminal truncated VEGF-B subunit   The DNA fragment encoding truncated VEGF-B from Example 1 was prepared using a suitable plasmid. Cloned into a sumid vector.   Sf9 (Sporoptera frugiperda) cells encode truncated VEGF-B Baculovirus transport vectors pAcUW51 and And baculovirus (Baculogold, Pharmingen, San) (Diego, CA). Selection of recombinant virus and Plaque purification established using Blue Agar Overlay (Gibco BRL) It is performed according to the established protocol. The storage of lots of recombinant virus Exponentially grown in Sf9 cells using a multiplicity of infection of 0.05. Cutting For expression of type VEGF-B, Sf9 cells growing in serum-free medium (1 × 10⁶ Cells / ml) are infected with the recombinant virus at a multiplicity of 10. Supernatant at 72 hours of infection Soon collected. Baculoy that can be used for expression of truncated VEGF-B of the present invention VEGF expression in Lus-infected insect cells was also described by Fiebich et al. , (E ur .J. Biochem. 211: 19-26, 1993). . In this system, VEGF is produced in high yields, as seen in mammalian cells. It shows that highly efficient glycosylation is achieved. In fact, mammals It is recognized that expression in other systems, including cell expression systems, is considered to be within the scope of the present invention. Will recognize. Using the baculovirus system, the truncated VEGF- Methods for expressing VEGF protein that can be used to express B also include VEGF expression. Documents mentioned, for example, U.S. Patent No. 5,521,073 and O'R eilly et al. , (Baculovirus Expression   Vectors: A Laboratory Manual (WH Fre eman, New York), 1992).   Other expression systems may also be used to express a functionally active truncated VRP. Those skilled in the art will recognize that   Functionally active recombinant VEGF isoforms are capable of homo- and heterozygous PDGF. Previously reported methods for Schmerp et al. (Schneppe et al.,G ene   143, 201-09, 1994). Et al. (Wilting et al.,Dev. Biol. 176,76 -85, 1996), and in yeast (Mohanraj et al.,Bio chem. Biophys. Res. Commun . 215: 750-56,1 995) is expressed.   Yet another VEGF expression method that can be used to express a VRP of the invention is described in the Examples. For example, Jasny,Science  238: 1653, 1987; and Mi ller et al. , "Genetic Engineering", 19 86), Setlow, J. et al. K. et. al. , Eds. , Plenum, Vo l. 8, pp. 277-297).Example 3: Purification of recombinant truncated VRP   Purification of the baculovirus-expressed truncated VEGF-B from the insect cell supernatant of Example 2 Many standard techniques can be used for fabrication. These techniques include ammonium sulfate precipitation. , Acetone precipitation, ion exchange chromatography, size exclusion chromatography -, Hydrophobic interaction chromatography, reverse phase HPLC, concanavalin A Affinity chromatography, isoelectric focusing and isoelectric focusing But not limited to them. Other standard protein purification techniques It will be readily apparent to those skilled in the art. For example, characteristics such as histidine tag and antigen tag A protein having a heterologous tag is a protein containing the tag, Purified as expressed and by tag-directed affinity chromatography Into such VEGF-B DNA in such a manner, It can be produced by genetically manipulating NA. Such methods are within the scope of the present invention. It is considered to be.   A suitable purification method for the truncated form of VEGF-B is described below: on Sf9 cells Kiyoshi is centrifuged at 10,000 rpm for 30 minutes to remove cell debris and virus particles I do. The supernatant is then concentrated and dialyzed against 20 mM Tris (pH 8.3) for 24 hours. I do. The dialysis supernatant was centrifuged again to remove insoluble substances, and Sepharose Q anion was used. Apply to the exchange column. For the protein, a NaCl concentration gradient (0-1 M NaCl) was used. Elution from the column with a concentration gradient elution. The chromatography fraction is Using an antibody that recognizes VEGF-B by acrylamide gel electrophoresis and Analyze by ELISA. The VEGF-B immunoreactive fraction was collected, concentrated, and . Dialyze overnight against 1% trifluoroacetic acid. The material so prepared is La And purified by reverse phase HPLC. Typically, about 2-5 mg of protein And injected onto a preparative C4 column and subjected to Esch et al. ,Meth. Enzymol . 103%, 72-39, 1983. It is eluted with a gradient of acetonitrile in acetic acid. Including truncated VEGF-B The pooled fractions are pooled and stored at -80 degrees Celsius until further use.   Basicity and Hepa of VEGF-Related Protein Subunits and Their Analogs Suitable methods for the purification of the phosphorus-linked N-terminal truncated form include essentially Connolly e. t al. ,J. Biol. Chem. 264: 20017-24, 1989, Gospodarowicz et. al. , (Proc. Natl. Acad .Sci.USA86: 7311-15, 1989) or Plouet et.   al. , (Embo J. 8: 3801-06, 1989). Such as heparin-sepharose affinity chromatography and Exchange chromatography, optionally followed by reverse phase HPLC You.   Purification was performed in a functional assay as described in Examples 4-6.¹²⁵ Using a receptor preparation sample consisting of an I-labeled VRP and a cell or cell membrane sample Follow the elution of VRP-like substances using a number of techniques, including radioreceptor assays. Monitored by traces.   Truncated VRP expressed in yeast or other eukaryotic cell lines such as mammalian cells Will be purified in the same manner.   Truncated VRPs expressed in prokaryotic cells are described, for example, in Schnepe et al.   al. , (Gene  143: 201-09, 1994). Need to perform a refolding step for proper dimerization of subunits It is.Example 4: Receptor binding assay   Binding of the truncated VRP to the VEGF receptor can be evaluated by various methods. useful Methods include endothelial cells or cells transfected artificially with KDR, Is the soluble form of the KDR receptor (eg, KDR / alkaline phosphatase fusion). Synthetic proteins (Gitay-Goren et al.,J. Biol. Chem. 271: b519-23 (1996)). include. A suitable method is described in Terman et al. (Biochem. B iophys. Res. Commun. 187: 1579-86, 1992) More described.   In this method, KDR cDNA was used to inoculate the cells at 1 μg / ml DNA. , Containing 0.5 μg / ml DEAE dextran and 100 μM chloroquine CMT-3 monkeys by DEAE-dextran method incubating with DMEM Transfected into kidney cells. Incubated at 37 ° C for 4 hours Thereafter, the medium is aspirated and the cells are exposed to PBS containing 10% DMSO for 1 minute. continue The cells were washed once with DMEM containing 10% bovine serum and 100 μM ZnCl_TwoAnd And 1 μM CdCl_TwoFor 40 hours at 37 ° C in DMEM / 10% bovine serum containing Incubate.   VEGF-B was prepared by radioiodine using the Iodogen method or the chloramine T method. Is converted to Radiolabeled VEGF-B was applied to a Sephadez G25 column or Is the excess free iodine-1 using gel filtration on a heparin-Sepharose column. 25. Radioactive label¹²⁵The specific activity of the I-VEGF-B analog is typical 10^FiveWill be on the order of cpm / ng. For radioreceptor assays Include CMT-3 (10^FiveCells / well) are seeded in a 12-well plate. After 24 hours, cells were washed twice with PBS, 0.15% gelatin and 25 mM Add 0.5 ml of DMEM (pH 7.4) containing HEPES. 1-500p In the concentration range of M¹²⁵Add I-VEGF-B. Binding experiments determine specific binding Therefore, it is performed in the presence or absence of 0.5 nM unlabeled VEGF-B. At room temperature After a 90 minute incubation, 50 μl of media sample from each well gives free radiation Used to determine the concentration of sex ligand, the wells contain 0.1% BSA Wash three times with ice-cold PBS. Cells are 100 mM containing 1% Triton X100 Wells by incubating for 30 minutes with sodium phosphate, pH 8.0 And the radioactivity of the extract is determined with a gamma counter.Example 5: Mitogenesis assay   The mitogenic activity of truncated VRP on endothelial cells of human or mammalian origin Cell proliferation increases cell number, or radioactive DNA precursor (thymidine incorporation) Or removal of other suitable labeled DNA precursors (bromodeoxyuridine incorporation) Can be determined in a number of different ways, including assays measured by integration. Thread These methods in which cleft-promoting activity is assessed in vivo (eg, endothelial cell mitogenesis) Commonly used to determine cell proliferation (by determining an indicator). These or other methods employed are considered within the scope of the present invention. The preferred method is book It is described in the specification (Bohlen et al.,Proc. Natl. Acad. Sci. USA   81: 5364-68, 1994): 10% calf. Serum and antibiotics (gentamicin 50 μg / ml and fungidine 0.25 μg / ml) and basic fibroblast growth factor (1-10 μg / ml, 48 hours) As a stock culture in the presence of Dulbecco's Modified Eagle's Medium supplemented with The retained bovine aortic arch endothelial cells are passaged weekly at a split ratio of 1:64. Thread For mitogenic activity assays, cell monolayers from passage plates (passage 3-1) 0) is dissociated using trypsin. The cells are then DMEM and the antibiotic Seeded at a density of approximately 8000 cells / well in a 24-well plate where quality is present I do. Sample to be assayed (1-10 μl, DMEM / 0.1% bovine serum Approximately 6 hours after seeding the cells and 48 hours Add again later. After 4 days of culture, the endothelial cells are detached from the plate with trypsin, and Count using a Luther particle counter.   Another mitogenic activity assay is described in Olofsson, B .; et al. ,Proc . Natl. Acad. Sci. USA   93: 2576-81, 1996, Provided. Second passage human umbilical vein endothelial cells (HUVEC) at 10% (volume / Volume) Seed into 96-well plate containing M-199 medium supplemented with fetal calf serum Seeds (4x10 per well)^ThreeCells) and incubate for 24 hours. Cutting type V Cell culture conditioned medium containing RP, in the presence of 1-10 μg / ml heparin, Alternatively, purified truncated VRP is added to the HUVEC and the cells are stimulated for 48 hours. [^ThreeH New cell culture medium containing thymidine (Amersham; 10 μCi / ml) Nutritional conditioned media is added to the cells and stimulation is continued for an additional 48 hours. Wash cells and remove Re Psin-treated and incorporated radioactivity is determined by liquid scintillation counting. You. The activity of the truncated VRP was compared to the activity of the uncleaved VRP.   In another alternative, bovine capillary endothelial cells (BCE) are plated in 24-well plates. And confluent in minimal essential medium (MEM) supplemented with 10% fetal bovine serum. Propagate to an ent. Cells were incubated for 72 hours with M supplemented with 3% fetal bovine serum. The cells were starved in EM and then conditioned medium diluted in serum-free medium was added to the cells. The cells were stimulated for 24 hours. [^ThreeH] Thymidine is included in the last 4 hours of stimulation (1 μCi / ml). Wash cells with PBS, lyse with NaOH and take up The radioactivity determined is determined by liquid scintillation counting. Activity of truncated VRP Was compared to the activity of uncleaved VRP. Bovine fibroblast growth factor (b-FGF) Will be used as another control for mitogenic activity, and Will also be used to determine its enhancing activity.Example 6: Angiogenic activity of truncated VRP   The angiogenic activity of a substance can be determined using various in vivo methods. Commonly used Methods include the chicken chorioallantoic membrane assay, rabbit, rat or mouse Corneal sac assay, Matrigel transplantation assay in mice, rabbit ear ventricular vascularization Raw assay, hamster cheek pouch assay, Hunt-Schilling room model and rat A sponge transplant model may be used. Other assays to evaluate new blood vessel formation It is described in the literature and is considered to be within the scope of the present invention.   A preferred method for indicating truncated VRP angiogenesis is the rabbit corneal sac assay. You. In this assay, about 1-1000 ng of growth factor and carrier Elvax (ethylene vinyl acetic acid) containing a certain amount of rabbit serum albumin A polymer pellet is implanted into a surgical incision in the cornea, Phillips and Knightton,Wound Rep. Reg. 3,533-539 Gimbrone et al., 1995; ,J. Natl. Canc. In st . 52: 413-27, 1974; Risau,Proc. Natl. Ac ad. Sci. USA   83: 3855-59, 1986. Have been. Growth factor-induced intracorneal neovascularization is observed for more than 2 weeks. Cornea pictures To Semi-quantitative analysis is possible with the morphometric and image analysis used.Example 7: Gene transfer-mediated angiogenesis treatment using truncated VRP Cutting type VRP   The truncated VRP was released on September 6, 1996 as WO96 / 26742, for example. Gene transfer mediation as described in PCT / US96 / 02631 Used for angiogenesis treatment.Adenovirus construct   The helper-independent replication-defective human adenovirus 5 system is used for gene transfer. Would. A nucleic acid molecule encoding a truncated VRP subunit may be a CMV promoter. And E1A and E1B genes (essential for viral replication) are removed SV40 polyadenylation signal flanked by partial adenovirus sequences Cloned into the polylinker of the containing plasmid ACCMVPLPA . This plasmid is too large to encapsidate pJM17. Includes the entire human adenovirus 5 genome with an additional 4.3 kb insert Cotransfected into plasmid JM17 and 293 cells (Lipofecti ). Homology rescue recombination involves a transgene lacking the E1A / E1B sequence Resulting in an adenovirus vector that is These recombinants are used in mammalian cells Non-replicating, but transformed with E1A / E1B, these essential gene products Can propagate in 293 cells provided in trans. Transfected fine The vesicles show evidence of the cytopathic effect that normally occurs 10-14 days after transfection Monitored. Show cytopathic effects to identify successful recombinants Cell supernatant from plate with proteinase K (50 mg / ml, 0.5% Containing sodium decyl sulfate and 20 mM EDTA) at 56.degree. Perform phenol / chloroform extraction and precipitate with ethanol. Cutting type CMV promoter and SV for amplifying VRP subunit nucleic acid insert Primers complementary to the 40 polyadenylation sequence (Biotechniques 15: 868-72, 1993) and concomitantly amplifies adenovirus sequences. for Designed primer (Biotechniques  15: 868-72, 1 993), successful recombinants are identified. Successful recombinants It is then plaque purified twice. The viral stock was 10 in 293 cells.^TenFrom 10¹² Propagate to titers in the range of virus particles and run a double CsCl gradient before use. Purify by centrifugation. The system used to generate the recombinant adenovirus was A 5 kb loading limit is imposed on the transgene insert. CMV promoter And the truncated VRP gene driven by the SV40 polyadenylation sequence It is within the embedded constraint. The recombinant vector is plaque purified by a conventional method. occured The viral vector is 10 in 293 cells.^Ten-10¹²Up to titers in the virion range Breed. Cells are infected at 80% confluence and harvested at 36-48 hours You. After a freeze-thaw cycle, cell debris was pelleted by standard centrifugation and Ruth was subjected to dual CsCl gradient ultracentrifugation (discontinuous 1.33 / 1.45 CsCl Concentration gradient; cesium prepared in 5 mM Tris, 1 mM EDTA (pH 7.8); 90,000 xg (2 hours), 105,000 xg (18 hours)) Purified. Prior to in vivo injection, the viral reservoir was G25 Sephadex. Desalted through a Sepharose column such as The virus stock obtained is approximately 10^Ten-10¹²Has a final virus titer in the virion range. Adenowi The virus construct is highly purified to be free of wild-type (potentially replicating) virus. Should be.Porcine ischemia model for angiogenesis   Perform a left thoracotomy on a pig (30-40 kg) under aseptic conditions for instrument use You. (Hammond, et al.J. Clin. Invest. 92:26 44-52 (1993); Roth, et al.J. Clln. Invest . 91: 939-49, 1993). The catheter is placed in the left atrium and aorta Provide a means for measuring local blood flow and monitoring pressure. ECG note A wire is sutured to the left atrium to allow recording and atrial pacing. last Ameloid compact (ameloid), a metal ring containing ameloid substance Place around the left circumflex coronary artery (LCx) (Hammond et al.J . C lin. Invest . 92: 2644-52 (1993)). Stable degree After ischemia appeared, the treatment group consisted of a truncated VRP driven by a CMV promoter. An adenovirus construct containing the gene is received. Control animals were CMV promoters Reporter gene, driven by an adenovirus construct containing lacZ Have undergone gene transfer.   The study was started 35 + 3 days after placing the ameloid, at which time collateral vessels Developmental and pacing-derived dysfunctions are stable (Roth, et al.Am .J. Physiol. 253: 1-11279-1288, 1987, and Roth, et al.Circulation  82: 1778-89). Intention Sensitive animals are hung with a triangular cloth and include the left ventricle (LV), left atrium (LA) and Pressure and electrocardiogram from the aorta and the aorta were recorded online in digital format (rest At rest and atrial pacing at 200 bpm). 2D and M-mode images Was obtained using a Hewlett Packard ultrasound imaging system. Was. Images were recorded on VHS tape from the right parasternal direction to the middle papillary muscle level. image Is recorded in animals at basal state and right atrial pacing (HR = 200 bpm) Was. These studies were performed one day before gene transfer and repeated 14 + 1 days later. Was returned. Velocity-pressure results and left atrial pressure were measured both before and after gene transfer. Should be the same in groups, indicating similar myocardial oxygen demand and loading conditions. Echocardiographic measurements were performed using standardized criteria (Sahn, et al.C irculation   58: 1072, 1978). End-expansion wall thickness (EDWT h) and end-systolic wall thickness (ESWTh) were measured on five consecutive beats and averaged. Percent thickening is calculated as [(EDWTh-ESWTh) / EDWTh] x 100 Is done. Data were analyzed without knowledge of which gene the animal received. Should be. Animals were imaged for two consecutive days to demonstrate the reproducibility of echocardiographic measurements. Images should be taken and show high correlation (r2 = 0.90; p = 0.005) are doing.   35 ± 3 days after ameloid installation, well after ameloid closure, but gene transfer Previously, a contrast echocardiographic study was performed into the left atrium during atrial pacing (200 bprn). The procedure is carried out with an injected control substance (Levovist). Research is gene-guided Repeated 14 ± 1 days after entry. Peak contrast intensity is an objective measure of video intensity. Provided computer-based video analysis program (Color Vue II, Nova Microsonics, Indianapolis, Ind (iana) from video images. In contrast studies, which gene is moving It should be analyzed without knowledge of what was received.   At the completion of the study, the animals are anesthetized and a midline thoracotomy is performed. Isolate the short temporal artery and Insert a neurole and ligate the other large vessels. Heparin (10,0 00IU) and papaverine (60 mg). Induce diastolic cardiac arrest Give potassium chloride to tighten and cross the arteries. Salt solution for craniotemporal crab Pumped through the urea (120 mmHg pressure), thereby perfusing the coronary arteries . Glutaraldehyde solution (6.2) until heart is well fixed (10-15 minutes) 5%, 0.1 M cacodylate buffer) was perfused (120 mmHg pressure). Heart Removed, through left anterior descending branch (LAD), left circumflex (LCx) and right coronary artery Beds were identified using color coding dyes injected in antegrade. Ameloid is closed Tested to confirm the chain. Normally perfused and taken from ischemic area The sample was divided into three parts, and one third of the endocardium and epicardium were embedded in plastic. You. Microscopic analysis to quantify the number of capillaries was performed as previously described. (Mathieu-Costello, et al.Am. J. Physi ol . 359: H204, 1990). Four 1 μm-thick transverse sections were each subsample (Endocardium and epicardium in each area) and at 400X magnification, the number of fibers Point-counting was used to determine the ratio of capillary numbers in the sample. From 20 per subsample Twenty-five high-magnification fields were counted. Within each area, the number of capillaries versus the number of fibers The ratio should be the same for the endocardium and epicardium, and 40-50 fields per area must be averaged to provide wall capillary ratio Absent.   Establishing improved local function and blood flow resulted from transgene expression Therefore, the transgene truncated form in the myocardium from an animal that has received the truncated VRP gene transfer PCR and RT-PCR are used for VRP DNA and mRNA detection. Sense primer for CMV promoter [GCAGAGCTCGTTTA GTGAAC] [SEQ ID NO: 41]; and an internally truncated VRP subunit Amplify the expected 500 bp fragment using antisense primers to the sequence PCR was used to perform For the beginning of the truncated VRP subunit sequence Using the sense primers to amplify the expected 400 bp fragment -PCR was used.   Finally, using a VRP-directed polyclonal antibody and a truncated VRP gene 48 hours of transfection in cells and myocardium from transgenic animals Truncated VRP expression after 14 ± 1 days.   Introduction where the helper-independent replication-defective human adenovirus 5 system contains a vector Used to make a gene. Substances injected in vivo are wild-type (replication Compatibility) Does not contain adenovirus. Therefore, adenovirus infection and heart Inflammatory infiltrates in the gut are minimized. Substance is coronary with coronary catheter Genes can be effectively targeted by direct injection into the lumen of the aortic artery. is there. When delivered in this manner, there is no transgene expression in liver cells. No wax or viral RNA is observed in urine at any time after intracoronary infusion. Would be.   Injection of the construct (4 ml, about 10¹¹Contains adenovirus virions Is performed by injecting 2.0 ml into both the left and right coronary arteries. (Collateral flow to the LCx bed appears to come from both vessels). Hemp animal Get sick, access the artery through the right carotid artery by cutting; a 5F heart sheath is placed. 5F A multipurpose (A2) coronary catheter is used to connect with the coronary arteries. L Cx ameloid closure is confirmed by control injection into the left main coronary artery. Injection Catheter 1 cm into arterial lumen to minimize loss to adjacent aorta A chip is placed. This method was performed on each pig.   Once gene transfer is performed, establish successful gene uptake and expression. Three strategies are used for this. (1) Some constructs are reporter genes (LacZ); (2) myocardium from the bed concerned is sampled; Immunoblotting is performed to quantify the presence of truncated VRP, and And (3) PCR is used to detect truncated VRP mRNA and DNA. It is.   The local contractile function data obtained shows that the control pigs were before gene transfer and 14 ± 1 days. Would show similar pacing-induced dysfunction in later ischemic areas . In contrast, pigs that have received truncated gene transduction have The truncated VRP subunit gene transfer according to the present invention shows increased wall thickening. This shows that there is an improved contraction in the ischemic area during pacing. Normal perfusion Wall thickening in the region (ventricular septum) is common between pacing and Not affected. The percent reduction in function as measured by transthoracic echocardiography Ultrasound microscopy during atrial pacing in one model (Hammond, et al. aI.J. Clin. Invest. 92: 2644, 1993). Is very similar to the percentage decrease Shows the accuracy of Koh.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 9/02 C07K 14/52 9/10 A61K 35/76 17/02 C12N 15/00 ZNAA C07K 14/52 5/00 A C12N 5/10 A61K 37/02 // A61K 35/76 37/24 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH) , GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, B , BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD , SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW

Claims (1)

[Claims] 1. Amino acid residues N-terminal to the first cysteine of the core sequence of the subunit A truncated VRP subunit having at least one deletion of: 2. 2. The truncated VRP subunit according to claim 1, wherein the VRP is human VRP. 3. The VRP is VEGF-B, VRF-2, VEGF-C, PIGF, V EGF-3, poxvirus ORF-1, and poxvirus ORF-2 The truncated VRP subunit according to claim 1, which is selected from the group consisting of: 4. The truncated VRP subunit according to claim 1, wherein the VRP is VEGF-B. To 5. 2. The method of claim 1, wherein said VRP subunit comprises the amino acid sequence of FIG. Disconnect VRP subunit. 6. Amino acids N-terminal to the first cysteine of the core sequence of the subunit 2. The truncated VRP subunit of claim 1, wherein the residue has been deleted. 7. The amino acid sequence at the N-terminal side of the core sequence includes 2-5 amino acid residues, The truncated VRP subunit according to claim 1. 8. The 2-5 amino acid residues are the first of the core sequence of the VRP subunit. Claims comprising the most N-terminal 2-5 contiguous amino acid residues of cysteine. 7. The truncated VRP subunit according to 7. 9. The amino acid sequence at the N-terminal side of the core sequence has 6 to 10 amino acid residues. The truncated VRP subunit according to claim 1, comprising: 10. The 6-10 amino acid residues are the core sequence of the VRP subunit Contains the most N-terminal 6-10 contiguous amino acid residues of the first cysteine of The truncated VRP subunit according to claim 1. 11. The amino acid sequence at the N-terminal side of the core sequence is 11 to 20 amino acids 2. The truncated VRP subunit of claim 1, comprising residues. 12. The 11-20 amino acid residues correspond to the core sequence of the VRP subunit. The most N-terminal 11-20 contiguous amino acid residues of the first cysteine in the sequence The truncated VRP subunit according to claim 1, comprising: 13. A mature uncleaved VRP subunit is added to the N-terminus of the truncated VRP subunit. 2. The truncation of claim 1, further comprising the first one or two amino acid residues of the knit. Type VRP subunit. 14． A truncated VRP comprising two VRP subunits according to claim 13. 15. A truncated VRP comprising two VRP subunits according to claim 1, A truncated VRP wherein the two VRP subunits have the same amino acid sequence. 16. A truncated VRP heterodimer, A first subunit comprising the truncated VRP subunit of claim 1; and V A group consisting of an RP subunit and a truncated VRP subunit according to claim 1. Second subunit including the subunit selected Wherein the second subunit has a different amino acid sequence from the first subunit. A heterodimer characterized by having a row. 17． A nucleic acid molecule encoding the truncated VRP subunit according to claim 1. 18. 18. The nucleic acid molecule according to claim 17, wherein the nucleic acid molecule is a DNA molecule. 19. 18. The nucleic acid molecule according to claim 17, wherein the nucleic acid molecule is an RNA molecule. 20. A recombinant DNA vector comprising the nucleic acid molecule according to claim 17. 21. A recombinant DNA expression vector comprising the nucleic acid molecule according to claim 17. 22. The nucleic acid molecule encodes a signal peptide at the 5 'end of the nucleic acid molecule 22. The recombinant D of claim 21 operably linked to a DNA sequence that NA expression vector. 23. The signal peptide is a VEGF signal peptide, VEGF-B sequence. Signal peptide, VRF-2 signal peptide, VEGF-C signal peptide , VEGF-3 signal peptide, and PIGF signal peptide The recombinant DNA expression vector according to claim 22, which is selected from the group consisting of: 24. The signal peptide is a poxvirus ORF-1 signal peptide. And a poxvirus ORF-2 signal peptide. 23. The recombinant DNA expression vector according to claim 22, which is obtained. 25. The signal peptide is a VEGF-B signal peptide. 23. The recombinant DNA expression vector according to 22. 26. The DNA sequence encoding the signal peptide is the DNA sequence Encodes the first amino acid residue of the mature uncleaved VRP subunit at the 3 'end of Said DNA operably linked to said DNA and encoding said residue Operable at the 3 'end of the nucleic acid molecule encoding the truncated VRP subunit 23. The recombinant DNA expression vector according to claim 22, which is operably linked. 27. The DNA sequence encoding the signal peptide is the DNA sequence The first two amino acid residues of the mature uncleaved VRP subunit at the 3 'end of Operably linked to the DNA to be encoded, and encodes the two residues The nucleic acid wherein the 3 'end of the DNA encodes the truncated VRP subunit 23. The recombinant DNA expression vector of claim 22, operably linked to a molecule. Doctor. 28. The nucleic acid molecule is transferred to a host cell transformed with the vector. And operably coupled to the control arrangement so as to be operable. 3. The recombinant DNA expression vector according to 2. 29. A transform comprising the recombinant DNA expression vector according to claim 21. Transformed or transfected host cells. 30. A transform comprising the recombinant DNA expression vector according to claim 22. Transformed or transfected host cells. 31. A transform comprising the recombinant DNA expression vector according to claim 26. Transformed or transfected host cells. 32. A transport vector comprising the nucleic acid molecule of claim 17. 33. 33. The transfer of claim 32, wherein said transfer vector is a viral transfer vector. Send vector. 34. An adenovirus vector comprising the nucleic acid molecule of claim 17. 35. The nucleic acid molecule encodes a signal peptide at the 5 'end of the nucleic acid molecule. 35. The adenovirus of claim 34, operably linked to a DNA sequence to be encoded. Virus vector. 36. The signal peptide is a VEGF signal peptide, VEGF-B sequence. Signal peptide, VRF-2 signal peptide, VEGF-C signal peptide , 36. The method according to claim 35, which is selected from the group consisting of: and a PIGF signal peptide. Adenovirus vector. 37. The signal peptide is a poxvirus ORF-1 signal peptide. And a poxvirus ORF-2 signal peptide. 36. The adenovirus vector of claim 35, wherein 38. The signal peptide is a VEGF-B signal peptide. 35. The adenovirus vector according to 35. 39. The DNA sequence encoding the signal peptide is the DNA sequence Encodes the first amino acid residue of the mature uncleaved VRP subunit at the 3 'end of Said DNA operably linked to said DNA and encoding said residue Operable at the 3 'end of the nucleic acid molecule encoding the truncated VRP subunit The adenovirus vector of claim 35 operably linked. 40. A filtered, injectable adenovirus vector preparation, wherein the recombinant An adenovirus vector, wherein said vector is free of wild-type virus; Partial adenovirus sequence in which the A / E1B gene has been deleted, and partial adenovirus A promoter driven by a promoter flanked by viral sequences. A transgin encoding the truncated VRP subunit of claim 1; And Pharmaceutically acceptable carrier A preparation comprising 41. The adenovirus vector is filtered through a 30 micron filter. 41. The preparation of claim 40, wherein the preparation has been passed. 42. The promoter is a CMV promoter, a ventricular myocyte-specific promoter. 41. and a myosin heavy chain promoter. An injectable adenoviral vector preparation as described. 43. 22. A method for producing a truncated VRP polypeptide, comprising the steps of: Transformed or transfected with a recombinant DNA expression vector Grown under appropriate conditions so that the polypeptide is expressed. And Isolating said polypeptide from a host cell; A method that includes: 44. A VR comprising at least one truncated VRP subunit according to claim 1. A pharmaceutical composition comprising P in a suitable carrier. 45. A method for stimulating the formation of blood vessels, wherein the method comprises administering to a patient at least one of the preceding claims. A medicament comprising a truncated VRP containing one truncated VRP subunit in a suitable carrier A method comprising administering a composition. 46. A method of stimulating endothelial cell growth or cell migration in vitro, comprising: The endothelial cell comprises at least one truncated VRP subunit of claim 1. Treating with truncated VRP in a suitable carrier. 47. A method of treating a patient suffering from a heart disease, the method comprising: Administering a nucleic acid molecule encoding at least one truncated VRP subunit as described above. The nucleic acid molecule emits a truncated VRP subunit in the patient. A method characterized by what can be represented. 48. A method of stimulating angiogenesis in a patient, comprising the steps of: A truncated VRP containing both one truncated VRP subunit in a suitable carrier A method comprising administering a therapeutically effective amount of a pharmaceutical composition. 49. 49. The method of claim 48, further comprising a delivery system suitable for treating the pharmaceutical composition. Method. 50. Administering a potentiator to enhance the angiogenic effect of the truncated VRP. 49. The method of claim 48, further comprising: 51. 51. The method of claim 50, wherein said enhancer is an angiogenic FGF. 52. The enhancer is FGF-1, FGF-2, FGF-4, FGF-5, and 52. The method of claim 51, wherein the method is selected from the group consisting of: FGF-6. 53. A truncation comprising at least one truncated VRP subunit according to claim 1. A medicament comprising type VRP and one or more enhancers in a pharmaceutically acceptable carrier. Drug composition. 54. 54. The pharmaceutical composition according to claim 53, wherein said enhancer is an angiogenic FGF. 55. Wherein said enhancer is FGF-1, FGF-2 in a pharmaceutically acceptable carrier, The method of claim 1, wherein the member is selected from the group consisting of FGF-4, FGF-5, and FGF-6. 54. The pharmaceutical composition according to 54. 56. A method of treating a patient suffering from an ischemic condition, comprising: Also includes a truncated VRP containing one truncated VRP subunit in a suitable carrier. A method comprising administering a therapeutically effective amount of a pharmaceutical composition. 57. Administering an agent that enhances the therapeutic effect of the truncated VRP subunit. 57. The method of claim 56, further comprising: 58. The enhancer is FGF-1, FGF-2, FGF-4, FGF-5, and 58. The method of claim 57, wherein the method is selected from the group consisting of: FGF-6. 59. The ischemic conditions include heart infarction, chronic coronary ischemia, chronic lower limb ischemia, stroke, 57. The method of claim 56, wherein the method is selected from the group consisting of: and peripheral vascular disease. 60. A method of treating a patient suffering from a wound, wherein the method comprises treating at least one of the wounds. Therapeutic comprising truncated VRP containing two truncated VRP subunits in a suitable carrier A method comprising administering an effective amount of a pharmaceutical composition. 61. 5. A method for increasing vascular permeability, wherein at least one of the methods according to claim 1 is used. Therapeutically containing a truncated VRP containing a truncated VRP subunit in a suitable carrier A method comprising administering an effective amount of a pharmaceutical composition. 62. A method of stimulating angiogenesis in a patient, the method comprising administering a delivery vector to the patient. Direct delivery to the myocardium by intracoronary injection into one or both coronary arteries Wherein the vector comprises at least one truncated VRP subtype according to claim 1. A nucleic acid molecule encoding a truncated VR in the myocardium. A method characterized by being capable of expressing a P subunit. 63. 7. The transport vector is a replication defective adenovirus vector. 2. The method according to 2. 64. A method for stimulating the development of cardiac collateral vessels in patients with myocardial ischemia Transported to the patient's myocardium by direct intracoronary injection into one or both coronary arteries Transporting a vector, said vector comprising a truncated VRP subunit. Expressing truncated VRP subunits that contain encoding nucleic acid molecules in myocardium And thereby promoting the development of collateral vessels. . 65. 7. The transport vector is a replication defective adenovirus vector. 4. The method according to 4. 66. A method of stimulating vascular development in a patient with peripheral vascular disease, By direct intra-femoral injection of the delivery vector into one or both femoral arteries Transporting to the patient's peripheral vasculature, wherein said vector comprises a truncated VRP subunit. A truncated VRP cell in the peripheral vasculature, comprising Buunit can be expressed, thereby promoting peripheral vascular development. And the method characterized by the above. 67. 7. The transport vector is a replication defective adenovirus vector. 6. The method according to 6.