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WO2001053345A1 - Isoforme de vegf 148, variant d'epissage tronque de vegf, heterodimeres de vegf et utilisations therapeutiques de ces derniers - Google Patents

Isoforme de vegf 148, variant d'epissage tronque de vegf, heterodimeres de vegf et utilisations therapeutiques de ces derniers Download PDF

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Publication number
WO2001053345A1
WO2001053345A1 PCT/GB2000/000134 GB0000134W WO0153345A1 WO 2001053345 A1 WO2001053345 A1 WO 2001053345A1 GB 0000134 W GB0000134 W GB 0000134W WO 0153345 A1 WO0153345 A1 WO 0153345A1
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vegf
isoform
glomeruli
exon
expression
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PCT/GB2000/000134
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Steven James Harper
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North Bristol Nhs Trust
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Priority to AU2000230635A priority Critical patent/AU2000230635A1/en
Priority to PCT/GB2000/000134 priority patent/WO2001053345A1/fr
Publication of WO2001053345A1 publication Critical patent/WO2001053345A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • VEGF 148 ISOFORM, A TRUNCATED SPLICE VARIANT OF VEGF, VEGF HETERODIMERS AND THERAPEUTICAL USES THEREOF
  • the present invention relates to method for treating or preventing a disease in a mammalian patient comprising the step of vascular endothelial growth factor (VEGF) heterodimer formation.
  • VEGF vascular endothelial growth factor
  • the present invention also relates to novel VEGF isoforms capable of forming such a heterodimer including a novel truncated splice variant of VEGF, and to nucleotide sequences encoding such VEGF isoforms.
  • VEGF vascular endothelial growth factor
  • VEGF 206 VEGF 189 , VEGF 183; VEGF 165/ VEGF 145 and VEGF 121 ( Figure 1) .
  • Each isoform has distinct properties and patterns of expression (Ferrara et al (1997) Endocr. Rev. 18, 4-25; Houck et al (1991) Mol . Endocrinol . 5, 1806-1814; Poltorak et al (1997) J. Biol . Chem. 272,
  • VEGF vascular endothelial growth factor
  • VEGF has been implicated in the control of normal glomerular permselectivity in health and of proteinuria in glomerular disease (Grone et al (1995) Nephrol . Dial. Transplant., 10, 761-763). Proteinuria is clinically important, because of nephrotic syndrome and because it influences the progression of established glomerular lesions.
  • VEGF is also known to promote vascular endothelial cell proliferation and angiogenesis, which are important components of a variety of pathologies, including tumour growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, hemangiomas, immune rejection of transplanted corneal tissue and other tissues and chronic inflammation.
  • pathologies including tumour growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, hemangiomas, immune rejection of transplanted corneal tissue and other tissues and chronic inflammation.
  • VEGF endothelial proliferative activity
  • Flt-1 fms-like tyrosine kinase-1
  • KDR kinase domain region
  • Neuropilin-1 has also been identified as a VEGF receptor. Neuropilin-1 modulates the interaction of VEGF with KDR and therefore the mitogenic activity of VEGF. It might also be involved in endothelial cell guidance. Neuropilin-1 interacts with exons 6 and 7 of VEGF. (Ortega et al (1999) Frontiers in Bioscience 4:141- 152) .
  • Flt-1 Flt-1
  • US-A-5952199 describes a chimeric receptor protein, which comprises amino acid sequences derived from F T-4 (which is a receptor for the VEGF-related protein VHl.4.5) and amino acid sequences derived from flt-1 or KDR, and which is capable of binding to VEGF and exerting an inhibitory effect thereon.
  • O-A-9816551 describes variant VEGF polypeptides which comprise modifications of at least one cysteine residue in the native VEGF-sequence, thereby inhibiting the ability of the variant polypeptide to dimerise through the formation of disulphide bonds .
  • the VEGF polypeptides are claimed to act as VEGF antagonists by binding to and occupying cell-surface VEGF receptors without inducing a VEGF response .
  • VEGF 165 The role of VEGF 165 in glomerular disease has been investigated using an oligonucleotide-based antagonist with specificity for the VEGF 165 isoform (Ostendorf et al (1999) J. Clin. Invest. 104:913-923).
  • VEGF 148 a new truncated splice variant of VEGF. This form results from a 35bp deletion at the end of exon 7. The deletion changes the reading frame and a premature stop codon results, producing the additional deletion of exon 8.
  • VEGF 148 acts in vivo as a native inhibitor of other VEGF isoforms. Specifically, VEGF 148 is expected to form a heterodimer with a monomer of another VEGF isoform, which renders the other VEGF isoform less potent.
  • VEGF 165 is the most predominant isoform (at least in the kidney) and is believed to be the most active. While a VEGF 165 :VEGF 165 homodimer is a highly potent mitogen, a heterodimer between VEGF 165 and one of the other isoforms (for example, VEGF 148 , VEGF 145 or VEGF 12i ) is expected to have reduced activity. Therefore the balance between the expression of the various isoforms of VEGF in a tissue influences the local spectrum of VEGF dimers, and as a result controls VEGF-receptor stimulation.
  • a method for treating or preventing a disease in a mammalian patient comprising the step of either
  • the VEGF heterodimer comprises a first, native VEGF monomer and a second VEGF monomer, which is different from the first VEGF monomer, but which retains the capacity to dimerise.
  • the first, native VEGF monomer may be any isoform which, when in the ho odimeric form, is capable of stimulating a VEGF receptor (such as Flt-1 or KDR) .
  • VEGF receptor such as Flt-1 or KDR
  • VEGF 121 VEGF 145 , VEGF 165 , VEGF 183/ VEGF 189 , VEGF 206
  • VEGF 165 is the most predominant form in most tissues and also seems to be the most active (at least in the kidney) .
  • the second VEGF monomer may be one of the previously known VEGF isoforms (VEGF 121; VEGF 145 , VEGF 165 , VEGF 183 , VEGF 189 , VEGF 206 ) , as long as it is different from the first VEGF monomer.
  • the second VEGF monomer may comprise a version of one of the previously known VEGF isoforms with one or more sections of sequence deleted or substituted, provided that the polypeptide retains its capacity to dimerise.
  • VEGF 148 is a truncated version of VEGF 165 .
  • the previously known VEGF isoforms have some sections of sequence (notably the section encoded by exons 1-5 and 8) in common, but have one or more sections of sequence which are deleted or substituted (notably in the sections encoded by exons 6 and 7) .
  • the second VEGF monomer would be likely to have a high degree of homology to the amino acid sequence encoded by exons 1 to 5.
  • the novel splice variant VEGF 148 shows homology to the VEGF 206 sequence as far as exons 1-5 are concerned, but lacks exon 6, has a 35bp deletion at the end of exon 7 which produces a frame shift and a premature stop codon at the start of exon 8.
  • the present inventors predict that truncated VEGF isoforms which have a high degree of homology to the amino acid sequence encoded by exons 1 to 5, but which have one or more deletions or substitutions in the amino acid sequence encoded by exon 6 and/or exon 7 and/or exon 8 would retain the capacity to dimerise and, by forming heterodimers, would inhibit VEGF- receptor stimulation.
  • the monomers in the heterodimer are preferably associated by one or more (preferably two) disulphide bonds. Since the residues of the previously known VEGF isoforms which are responsible for dimerisation are believed to be C 51 and C 60 (in exon 3) , the second VEGF monomer is likely to include these residues. Preferably, the second VEGF monomer shows a high degree of homology to the amino acid sequence encoded by exon 3 of the known VEGF isoform sequences . More preferably the second VEGF monomer shows a high degree of homology to the amino acid sequence encoded by exons 1 to 5 of SEQ ID No. 1.
  • such a variant version of a known VEGF isoform has at least 60%, more preferably at least 75%, most preferably at least 90% overall sequence identity with the known VEGF isoform sequence.
  • the second VEGF monomer comprises the sequence shown in SEQ ID No 1 (VEGF 148 ) .
  • heterodimer formation may occur in vivo or in vi tro .
  • pre-formed heterodimers may be administered to the mammalian patient.
  • Pre-formed heterodimers would comprise a first, native VEGF monomer associated with a second VEGF monomer, which is different from the first VEGF monomer, but which retains the capacity to dimerise.
  • VEGF monomers may be associated non covalently, for example by hydrogen bonds, electrostatic forces and/or van der Waals forces.
  • first and second VEGF monomers may be covalently linked, for example they may by a peptide bond in the form of a fusion protein.
  • the second VEGF monomer may be administered to, or caused to be produced in, the mammalian patient, so that heterodimers form in si tu with the first VEGF monomer.
  • the preformed heterodimers or second VEGF monomer could be administered to a mammalian patient in a pharmaceutically acceptable dose or doses by any of the known methods, such as by intervenous, intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-arterial, intrasynovial, intrathecal, oral topical or inhalation routes. Depending on the nature of the disease, intratumoral, peritumoral, intralesional or perilesional routes may also be appropriate .
  • the mammalian patient may be treated such that they produce the second VEGF monomer or "heterodimer" fusion protein endogenously.
  • a nucleotide sequence encoding the second VEGF monomer or "heterodimer" fusion protein may be administered directly to the mammalian patient.
  • such a nucleotide sequence could be ligated into a carrier (such as an expression vector or attenuated virus) for introduction into the mammalian patient.
  • a carrier such as an expression vector or attenuated virus
  • the carrier may have particular qualities such that it is targeted to direct the expression of the polypeptide in a particular tissue, or in response to a particular stimulus (for example, the expression of the polypeptide may be under the control of an inducible promoter) .
  • the method of the present invention is useful in the treatment or many diseases, including tumour growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, hemangiomas, immune rejection of transplanted corneal tissue and other tissues and chronic inflammation.
  • diseases including tumour growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, hemangiomas, immune rejection of transplanted corneal tissue and other tissues and chronic inflammation.
  • diseases including tumour growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, retrolental fibroplasia, neovascular gla
  • Neoplastic disorders which are amenable to treatment include as carcinomas of the breast, lung, kindey, eosophagus, gastric anatomy, colon, rectum, liver, ovary, cervix, endometrium, thecomas, arrhenoblastomas, endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, heptoblastoma, Karposi ' s sarcoma, melanoma, skin carcinomas, hemangioma, caverous hemangioma, hemangioblastoma, pancreas carcinoma, retinoblastoma, astrocytoma, glioblastoma, Scwannoma, oligodendroglioma, medulloblastorma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, lei
  • Non-neoplastic conditions which are amenable to treatment include rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and other retinopathies, neovascular glaucoma, age-related macular degeneration, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, nephrotic syndrome, preclampsia, ascites, pericardial effusion and pleural effusion. From the results on VEGF 148 , the present inventors predict that, in vivo, heterodimer formation is used generally to control and modulate VEGF-receptor stimulation.
  • VEGF-receptor interaction both the heterodimer and the polypeptide will be native or similar to their native forms. Hence, the method of the present invention will be less likely to induce undesirable supplementary immune responses .
  • a VEGF isoform other than VEGF 206 , VEGF 189 , VEGF 183 , VEGF 165 , VEGF 145 and VEGF 121 capable of forming a heterodimer with a native VEGF isoform. The presence of such a heterodimer in vivo inhibits VEGF-receptor stimulation by the native VEGF isoform.
  • the VEGF isoform of this aspect of the invention may comprise a version of one of the previously known VEGF isoforms with one or more sections of sequence deleted or substituted, provided that the polypeptide retains its capacity to dimerise.
  • VEGF 148 is a truncated version of VEGF 165 .
  • the VEGF isoform of this aspect of the invention preferably comprises C 51 and C 60 (which are within the sequence encoded by exon 3) . More preferably the VEGF isoform of this aspect of the invention shows a high degree of homology to the amino acid sequence encoded by exon 3 of the known VEGF isoform sequences . More preferably the VEGF isoform of this aspect of the invention shows a high degree of homology to the amino acid sequence encoded by exons 1 to 5. Most preferably the second VEGF isoform comprises the sequence shown in SEQ ID No 1 (VEGF 148 ) .
  • such a variant version of a known VEGF isoform has at least 60%, more preferably at least 75%, most preferably at least 90% overall sequence identity with the known VEGF isoform sequence .
  • the present invention also provides a heterodimer between a VEGF isoform of the second aspect of the invention and a native VEGF isoform monomer.
  • the present invention also provides a fusion protein, which comprises a VEGF isoform of the second aspect of the invention linked to a native VEGF isoform monomer by a peptide bond.
  • a polypeptide comprising the sequence shown in SEQ ID No:l, or a variant thereof.
  • variant it is intended to include polypeptides which comprise one or more deviations from the described amino acid sequence, but which retain at least some of the properties of VEGF 148 .
  • the deviation may be a substitution, insertion or deletion.
  • the present invention also provides a nucleotide sequence encoding a polypeptide in accordance with the second or third aspects of the invention.
  • the term "nucleotide sequence" is intended to include single stranded and double stranded RNA, DNA and cDNA. It is also intended to include sequences with known modifications to the backbone (such as phosphorothioate linkages) and/or to the side chains.
  • the nucleotide sequence of the present invention may be isolated from its natural source (if it is naturally occurring) , modified from another sequence (for example by digestion and ligation or site-directed mutagenesis) , or amplified by PCR.
  • the present invention also provides an expression vector which comprises such a nucleotide sequence, and a transformed cell expressing such an expression vector. The nucleotide sequence can be expressed and isolated from such a transformant by standard techniques.
  • the nucleotide comprises the sequence shown in SEQ ID No . ID No : 2 or a variant thereof .
  • variant is intended to include sequences which, by virtue of the degeneracy of the genetic code, have one or more substitutions from the sequence shown in SEQ ID No. 2 but which encode the amino acid sequence of SEQ ID No. 1.
  • variant is also intended to include sequences having one or more substitutions, insertions or deletions, which encodes a variant of the amino acid sequence of SEQ ID No. 1.
  • the present invention also provides a nucleotide sequence which encodes a fusion protein, comprising a VEGF isoform of the second aspect of the invention linked to a native VEGF isoform monomer by a peptide bond, an expression vector comprising such a nucleotide sequence and a cell transformed with such an expression vector.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a VEGF isoform as defined in any aspect of the invention, or a nucleotide sequence encoding such a VEGF isoform.
  • the pharmaceutical composition may also comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier examples include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol.
  • Carriers for topical or gel-based forms of chimeric protein include polysaccharides such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxypropylene- block polymers, polyethylene glycol and wood wax alcohols.
  • conventional depot forms are suitably used. Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nose sprays, sublingual tablets, and sustained release preparations.
  • antioxidants e.g., ascorbic acid
  • low molecular weight polypeptides e.g., polyarginine or tripeptides
  • proteins such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyvinylpyrrolidone
  • amino acids such as glycine, glutamic acid, aspartic acid, or arginine
  • monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivates, glucose, mannose, or dextrins
  • chelating agents such as EDTA
  • sugar alcohols such as mannitol or sorbiotol .
  • the sequence may be part of a carrier, such as an expression vector or an attenuated virus.
  • the carrier may have particular qualities such that it is targeted to direct the expression of the polypeptide in a particular tissue, or in response to a particular stimulus (for example, the expression of the polypeptide may be under the control of an inducible promoter) .
  • Figure 1 is a diagram to illustrate how known VEGF splice variants result from differential exon splicing
  • Figure 2 is a diagram to show the positions of external and internal primers.
  • B Sites of primer pairs (a,b and c) used to identify the mRNA of the new splices variant VEGF148 (shown in Table 2) ;
  • Figure 3 is a photograph of agarose gel which shows heterogeneity of VEGF mRNA isoform expression in 11 glomeruli from a single needle biopsy. Size (bp) is indicated on the right of the gel photograph;
  • Figure 4 is a photograph of an agarose gel showing unexpected nested RT-PCR products seen in some glomeruli using internal primer set "a ' .
  • Lanes 1-3 show individual glomeruli from the same needle biopsy, lane 4 and 5 show products from five combined glomeruli, lane 6 is a control (no reverse transcriptase) , lane 7 is a control (beads only) lane 8 is a single glomerulus and lane 9 is peripheral blood mononuclear cells.
  • Figure 5 shows a comparison between the nucleotide and predicted amino acid sequences of VEGF 148 and VEGF 165 ; and Figure 6 is an agarose gel photograph showing expression of VEGF 148 in other tissues.
  • Lanes 1 and 2 show single glomeruli, lane 3 is brain, lane 4, tonsil, lane 5 liver, lane 6 placenta, lane 7 peripheral blood mononuclear cells. Size (bp) is indicated on the left.
  • Table 1 shows the nested PCR sequences used in the example described below;
  • Table 2 shows primer sequences used to identify the mRNA of the new splice variant VEGF 148 ;
  • Table 3 shows the frequency of VEGF mRNA isoform expression in normal glomeruli. Values in parentheses represent ranges for individual tissue samples;
  • Table 4 shows the frequency of VEGF mRNA receptor expression in normal glomeruli. Values in parentheses represent ranges for individual tissue samples.
  • RT-PCR reverse transcription-PCR
  • Nephrectomy tissue was supplied by the Department of Urology, Southmead Hospital, from patients undergoing nephrectomy for polar renal tumour (age range 47-78 years) . All patients were non-diabetic and normotensive, with normal excretory renal function and no urinary sediment. A uniform collection system was used. Immediately after nephrectomy, excess fat was removed from the normal pole by blunt dissection, and several "full-thickness 1 (capsule to deep medulla) needle biopsies were taken.
  • Single subcapsular, mid-cortical and juxta-medullary glomeruli were isolated from needle biopsies. A total of 190 glomeruli were studied from 20 individuals. Glomeruli were collected under an Olympus SZ11 dissection microscope using a pair of fine tweezers ground to a point and a 12 -gauge needle. Only glomeruli visibly isolated from surrounding tissue were used. It became evident with time that glomeruli could be encouraged to spontaneously "pop* out of their Bowman's capsule by gently squeezing the biopsy core with fine tweezers.
  • cDNA-linked Dynabeads were incubated in 50 ⁇ l of TE buffer (10 mM Tris/1 mM EDTA, pH 8) and heated to 95°C for 1 min. This TE buffer was then discarded, removing the RNA that had become detached from the beads. The cDNA- linked Dynabeads were then resuspended in 50 ⁇ l of TE buffer for storage or PCR.
  • TE buffer 10 mM Tris/1 mM EDTA, pH 8
  • Nested PCR for VEGF isoforms and total VEGF cDNA-linked Dynabeads were washed in 10 x PCR buffer (Hybaid Proof 2 buffer) prior to addition to a 25 ul PCR reaction mixture containing external forward and reverse primers (Figure 2; Table 1) (400 nM each), 1 x Proof 2 buffer (including 2.5 mM MgCl 2 ) and 200 ⁇ M dNTP s (Hybaid) .
  • the reaction was "hot started' by addition of 0.75 unit of tag polymerase (Hybaid; PWO proof mix) in a first-round synthesis carried out as follows: 95°C for 7 min ("hot start' after 3 min), 95°C for 1 min, 55°C for 1.5 min and 72°C for 2 min.
  • the cDNA-linked Dynabeads were then removed by pelleting after 2 min at 95°C.
  • the supernatant containing the first strand of cDNA was then amplified using 18 cycles of 95°C for 30s, 55°C for 50s and 72°C for 50s, with a final extension for 5 min at 72 °C.
  • a 1 ⁇ l portion of the first-round PCR was used as a template in the second round of amplification.
  • the reaction mixture was as above, except that either internal forward and reverse primers for VEGF isoforms (spanning exon deletions) or a VEGF common exon were used (Figure 2; Table 1).
  • the final MgCl 2 concentration was 1.5 mM, and 0.75 unit of Tag polymerase was added per reaction.
  • Conditions for the second round were as follows: 25 cycles of 95°C for 30 s, 55°C for 40 s and 72 °C for 40 s, followed by a 5 min extension at 72 °C. All PCR reactions were carried out in a Hybaid thermal cycler.
  • Nested PCR for glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) , CD45 and the VEGF receptors KDR, Flt-1 and sFlt cDNA-linked Dynabeads were washed in lOx PCR buffer contaning 1.5 mM MgCl 2 (Promega) prior to addition to a 25 ul PCR reaction mixture containing eternal forward and reverse primers (Figure 2; Table 1) (400nM each), lx Promega PCR buffer and 200 ⁇ M dNTP s (Hybaid) .
  • the reaction was "hot started' by the addition of 1 unit of Tag polymerase (Promega) in a first-round synthesis carried out as follows: 95 °C for 7 min ("hot start' after 3 min), 95°C for 1 min, 55°C for 1.5 min and 72°C for 2 min.
  • the cDNA-linked Dynabeads were then removed by pelleting after 2 min at 95 °C.
  • the supernatant containing the first strand of cDNA was then amplified used 18 cycles of 95°C for 30s, 55°C for 40s and 72°C for 40s, with a final extension of 5 min at 72 °C.
  • a 1 ⁇ l portion of the first-round PCR was used as template in the second round of amplification.
  • the reaction mixture was as above, except that either internal forward and reverse primers for the appropriate molecule were used and 1 unit of Tag polymerase was added.
  • PCR products were mixed with 3 u.1 of loading dye (0.25% Bromophenol Blue/50% glycerol) and electrophoresed in a 3% (w/v) agarose (Sigma) gel. Bands were visualized by ethidium bromide staining on a UVP dualintensity transilluminator. Nested PCR controls and experimental design
  • Each glomerulus was examined for: (a) "total 1 VEGF, using primers located in exons 2-4 (which are conserved in all VEGF isoforms) , to act as an internal control, (b) VEGF isoforms, (c) VEGF receptors, (d) GAPDH, a housekeeping gene, and (e) CD45, the leucocyte common antigen, to detect any contamination by RNA from trafficking leucocytes. Each reaction included negative controls in which either template or the reverse transcription enzyme was omitted. Any glomerulus that was positive for CD45, and all glomeruli from kidneys subsequently found to be histologically abnormal, were excluded from analysis.
  • VEGF-isoform and VEGF-receptor specific bands were cloned and sequenced to confirm their identity. Multiple clones were sequenced of each of the expected VEGF-isoform and VEGF-receptor PCR products, and each confirmed the published sequences of VEGF 12 ⁇ , VEGF 165 , VEGF 189f Flt-1, KDR and sFlt, as anticipated. Sequencing of unexpected PCR products
  • the unexpected PCR products were ligated into pGEMT vector (Promega) using the manufacturer's instructions.
  • the products of ligation were transformed into competent TOP 10F' Escherichia coli (Invitrogen, Groningen, The Netherlands) according to the recommended protocol, and positive colonies were selected on ampicillin (100 ul/ml) /agar plates.
  • Clones shown to contain VEGF inserts by PCR with M13 primers were purified using a Qiagen QIA PCR Purification kit (Qiagen, Crawley, W.shire, U.K.) and sequenced on an ABI 373 automatic sequencer using fluorescent dye terminators .
  • primer sets b and c were designed (primer sets b and c; Table 2, Figure 2B) .
  • primer set c the band of interest was excised and used as the template in a further PCR reaction.
  • the products were separated on Spredex 600 gels (VH Bio, Newcastle-Upon-Tyne, U.K.), ligated using an Invitrogen TOPO TA cloning kit and transformed and sequenced as described above.
  • VEGF isoform mRNA expression Marked heterogeneity was seen in VEGF isoform and receptor expression in different glomeruli from the same nephrectomy specimen; indeed from the same needle biospy.
  • Figure 3 shows an electophoretic gel of the VEGF mRNA isoforms expressed by 11 glomeruli from the same needle biopsy. These glomeruli were harvested sequentially from the subcortical area to the juxtamedullary zone. The clearly demonstrates that individual glomeruli show different patterns of VEGF mRNA isoform expression.
  • the first glomerulus expressed the three common forms of VEGF mRNA ( Figure 3); however, the VEGF isoform mRNA ( Figure 3); however, the VEGF isoform mRNA expression of the glomerulus in lane 2 (which was anatomically approx. 50 ⁇ m away from the first) was totally different.
  • VEGF 189 was expressed in 63% of glomeruli. Overall, VEGF 165 in 85% and VEGF 121 were expressed in 84%. In addition, unexpected PCR products were consistently seen in a minority of glomeruli ( Figure 4). Subsequent identification of these products revealed the expression of VEGF 145 ( Figure 4, Product D) in 18% of glomeruli, and 27% of glomeruli (range 0-67%) expressed a new splice variant, VEGF 148 ( Figure 4, Product C) . This new splice variant was seen in glomeruli from eight individuals only. VEGF 148 mRNA
  • the sequencing data identified a 35 bp deletion at the end of exon 7. This dictated a frame-shift and a premature stop codon at the start of the short (19- base) exon 8.
  • the protein product of the new mRNA splice variant would be predicted to contain 148 amino acids after signal peptide cleavage, i.e. VEGF 148 (a truncated form of VEGF 165 ) .
  • VEGF 148 a truncated form of VEGF 165
  • the novel sequence was identical in multiple experiments with nine different clones and three sets of primer pairs, in five different glomeruli from four different individuals .
  • the sequences and positions of the three primer pairs are detailed in Table 2 and Figure 2(B) respectively. These were: (a) one pair placed beyond exon deletions to enable all isoforms to be identified, (b) a second pair, the forward primer of which spanned the novel exon boundary of VEGF 148 and (c) a third pair in which the forward primer was positioned in exon 7.
  • KDR KDR
  • s Flt,and 21% range 0- 33%) expressed only one of the three receptors.
  • no mRNA was detected for any of the receptors.
  • KDR was expressed in 59% of glomeruli, Flt-1 in 45% and sFlt in 57%.
  • VEGF-isoform VEGF-receptor mRNA expression in isolated single glomeruli within and between individuals.
  • the scatter of frequencies of particular isoform and receptor mRNA patterns was always normally distributed.
  • No specific patterns of VEGF-isoform or VEGF- receptor expression were apparent when comparing subcortical with juxta-medullary glomeruli.
  • No specific pattern VEGF-isoform expression was convincingly associated with any particular pattern of VEGF-receptor expression.
  • VEGF 146 and VEGF 148 products were on identified in the presence of VEGF 165 .
  • VEGF I2I VEGF I2I
  • VEGF VEGF
  • a and VEGF VEGF
  • VEGF VEGF
  • VEGF, ⁇ VEGF I(S 20 (0-60)
  • VEGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • ⁇ or YEGF ⁇ n vascular endothelial growth factor II
  • Minor pattern VG m and VEGF IN> 4 (0-20) or VEGF I(S ami VEGF,,,) None 7 (0-30)

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Abstract

L'invention concerne une méthode de traitement ou de prévention d'une maladie chez un mammifère, qui consiste soit (i) à induire la formation d'hétérodimères du facteur de croissance vasculaire (VEGF) in vivo soit (ii) à administrer un hétérodimère de VEGF au mammifère. L'invention concerne un variant d'épissage tronqué de VEGF à partir de glomérules humains, à savoir l'isoforme de VEGF 148 qui possède une homologie importante à la séquence d'acides aminés codée par les exons de VEGF 1 à 5, ne possède pas exon 6 et comporte une délétion de 35 bp à l'extrémité d'exon 7 qui produit un déphasage et un codon terminateur prématuré au début d'exon 8.
PCT/GB2000/000134 2000-01-20 2000-01-20 Isoforme de vegf 148, variant d'epissage tronque de vegf, heterodimeres de vegf et utilisations therapeutiques de ces derniers WO2001053345A1 (fr)

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AU2000230635A AU2000230635A1 (en) 2000-01-20 2000-01-20 Vegf148 isoform, a truncated splice variant of vegf. vegf heterodimers and therapeutical uses thereof
PCT/GB2000/000134 WO2001053345A1 (fr) 2000-01-20 2000-01-20 Isoforme de vegf 148, variant d'epissage tronque de vegf, heterodimeres de vegf et utilisations therapeutiques de ces derniers

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WO2003012105A2 (fr) * 2001-08-01 2003-02-13 University Of Bristol Isoforme de facteur de croissance
AU2010212443B2 (en) * 2004-03-12 2013-08-29 Alnylam Pharmaceuticals, Inc. iRNA agents targeting VEGF
EP3216458A1 (fr) 2016-03-07 2017-09-13 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Facteur de croissance endothéliale vasculaire (vegf-a)modifié et son utilisation médicale

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003012105A2 (fr) * 2001-08-01 2003-02-13 University Of Bristol Isoforme de facteur de croissance
WO2003012105A3 (fr) * 2001-08-01 2003-05-08 Univ Bristol Isoforme de facteur de croissance
JP2005502333A (ja) * 2001-08-01 2005-01-27 ユニバーシティ　オブ　ブリストル 増殖因子イソ型
US7820178B2 (en) 2001-08-01 2010-10-26 University of Brisol VEGF isoforms and their use as anti-angiogenic, anti-vasodilatory, anti-permeability and anti-proliferative agents
JP4689956B2 (ja) * 2001-08-01 2011-06-01 ユニバーシティ オブ ブリストル 増殖因子イソ型
US8933211B2 (en) 2001-08-01 2015-01-13 University Of Bristol Growth factor isoform
AU2010212443B2 (en) * 2004-03-12 2013-08-29 Alnylam Pharmaceuticals, Inc. iRNA agents targeting VEGF
AU2010212443A8 (en) * 2004-03-12 2013-09-12 Alnylam Pharmaceuticals, Inc. iRNA agents targeting VEGF
AU2010212443B8 (en) * 2004-03-12 2013-09-12 Alnylam Pharmaceuticals, Inc. iRNA agents targeting VEGF
EP3216458A1 (fr) 2016-03-07 2017-09-13 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Facteur de croissance endothéliale vasculaire (vegf-a)modifié et son utilisation médicale

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