US20150344536A1 - Variant of bpifb4 protein - Google Patents

Variant of bpifb4 protein Download PDF

Info

Publication number
US20150344536A1
US20150344536A1 US14/655,651 US201314655651A US2015344536A1 US 20150344536 A1 US20150344536 A1 US 20150344536A1 US 201314655651 A US201314655651 A US 201314655651A US 2015344536 A1 US2015344536 A1 US 2015344536A1
Authority
US
United States
Prior art keywords
seq
fragment
protein variant
bpifb4
position corresponding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US14/655,651
Other languages
English (en)
Inventor
Annibale Alessandro Puca
Carmine Vecchione
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lgv1 Srl
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of US20150344536A1 publication Critical patent/US20150344536A1/en
Assigned to LGV1 S.R.L. reassignment LGV1 S.R.L. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: Puca, Annibale Alessandro, Vecchione, Carmine
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1751—Bactericidal/permeability-increasing protein [BPI]
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4742—Bactericidal/Permeability-increasing protein [BPI]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C12N2015/8518—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a variant of BPIFB4 protein (Bactericidal/Permeability Increasing protein family B, member 4) and to its use for the treatment of pathologies associated with endothelial dysfunction due to impaired eNOS and NO mediated vasodilatation.
  • Human BPIFB4 (also known as C20orf186; RY2G5; LPLUNC4) is a secreted protein member of the BPI/LBP/PLUNC-like family, which has been implicated in host defence processes against bacteria.
  • the protein exists as two different isoforms of different length with aminoacid sequences of 575 (Acc. P-59827-2) and 613 (Acc. EAW76337.1) aminoacids (Bingle C D et al, Biochem Soc Trans. (2011) 39 (4): 977-83; Andrault J.-B et al, Genomics (2003) 82: 172-184; Bingle C. D et al., Hum. Mol. Genet. (2002) 11: 937-943; Bingle C. D et al, Protein Sci. (2004) 13: 422-430).
  • the present inventors have found that the most common haplotype (65% analyzed chromosomes) is the combination AACT that codify for aminoacids Ile229/Asn281/Leu488/Ile494 (INLI); the second most frequent haplotype is the combination GCTC (30% chromosomes contain this haplotype) that codify for aminoacids Va1229/Thr281/Phe488/Thr494 (VTFT) and finally the combination of AATC is represented only in 2% of human Caucasian chromosomes that codify for Ile229/Asn281/Phe488/Thr494 (INFT).
  • the vascular endothelium is formed by a layer of cells located between the vessel lumen and the vascular smooth muscle cells. These cells continuously produce nitric oxide (NO), a soluble gas that is synthesized by the enzyme endothelial nitric oxide synthase (eNOS).
  • NO nitric oxide
  • eNOS endothelial nitric oxide synthase
  • the present inventors have now surprisingly identified that a specific variant of the BPFIB4 protein is associated with exceptional longevity.
  • the inventors have further found that the variant identified is surprisingly able to increase the activation of eNOS and the production of NO in endothelial cells.
  • These biological properties are dependent by the presence in the protein of four specific aminoacids at positions 229, 281, 488 and 494 since replacement of any of these positions with different aminoacids leads to loss of activity of the protein.
  • the present invention provides a protein, which is a variant of a BPIFB4 protein, having an aminoacid sequence with at least 85% homology to the aminoacid sequence of SEQ ID NO: 1, wherein said sequence comprises a Valine at the position corresponding to position 229 of SEQ ID NO: 1, a Threonine at the position corresponding to position 281 SEQ ID NO: 1, a Phenylalanine at a position corresponding to position 488 of SEQ ID NO: 1 and a Threonine at position corresponding to position 494 of SEQ ID NO: 1.
  • Said homology in the aminoacid sequence is preferably of at least 90%, more preferably of at least 95% and even more preferably of at least 99%.
  • the protein of the invention has the aminoacid sequence of SEQ ID NO: 1.
  • the protein of the invention has an aminoacid sequence corresponding to the sequence of SEQ ID NO 1, wherein one or more aminoacids at positions different from positions 229, 281, 488 and 494 of SEQ ID NO 1 have been substituted by a conserved aminoacid.
  • conserved aminoacid it is meant an aminoacid with functionally physicochemical properties equivalent to those of the original aminoacid.
  • the invention further provides a polynucleotide having a nucleotide sequence coding for the above protein and a vector containing said polynucleotide operatively linked to expression control sequences.
  • said polynucleotide has the sequence of SEQ ID NO: 2.
  • the invention also provides the above protein, polynucleotide or vector for use in therapy.
  • object of the invention is the above protein, polynucleotide or vector for use in the prevention, reduction of the risk of, amelioration and/or treatment of endothelial dysfunctions due to a decrease in the activity of eNOS and/or in the production of NO or of pathologies or conditions where it is beneficial to increase the activity of eNOS and/or the production of NO.
  • the above protein, polynucleotide or vector is for use in the prevention, reduction of the risk, amelioration or treatment of a pathology or condition selected from arterial hypertension, atherosclerosis, diabetes mellitus, dyslipidemia, renal failure, metabolic syndrome, stroke, myocardial Infarction, erectile dysfunction, neurodegenerative diseases, multiple sclerosis and cognitive disorders, retinal degeneration, uveoretinitis, vascular retinopathy, cataract and glaucoma, coronary spastic angina, thrombosis, pulmonary hypertension, pre-eclampsia, vasculites, cancer, inflammatory disorders, venus insufficiency, genetic diseases with reduced eNOS activity and NO production, for example MTHFR gene variations, post-exercise fatigue in muscular dystrophy patients.
  • the above protein, polynucleotide or vector is for use as a co-adjuvant in the implantation of one or more stents,
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the protein or polynucleotide of the invention in combination with pharmaceutically acceptable carriers and excipients.
  • FIG. 1 shows the sequence of the pRK5 vector encoding INFT hBPIFB4 (SEQ ID NO: 3) or VTFT hBPIFB4 (SEQ ID NO:1) used in Example 3, with the sequence of the BPIFB4 protein underlined and that of EGFP in italics.
  • FIG. 2 shows detection of green fluorescent protein in mesenteric vessels perfused ex vivo with a plasmid encoding INFT BPIFB4 (left panel) or a control empty pRK5 plasmid (right panel) in Example 3.
  • FIG. 3 represents BPIFB4 protein expression and eNOS activation in mesenteric vessels perfused with empty vector (EV), a plasmid encoding INFT hBPIFB4 or VTFT hBPIFB4.
  • Panel a shows a Western blot of seven pooled experiments and detection of BPIFB4 (top) and P-eNOS S1177 (middle).
  • Panel b shows quantification of BPIFB4 expression and panel c shows quantification of phosphorylation at serine 1177 of eNOS.
  • FIGS. 4 , 5 and 6 panels 4a, 5a and 6a represent KCl induced vasoconstriction observed in Example 3 in mesenteric vessels perfused ex vivo with an empty plasmid pRK5 plasmid (EV/ FIG. 4 a ), a pRK5 plasmid encoding INFT hBPIFB4 (INFT/ FIG. 5 a ), or a pRK5 plasmid encoding VTFT hBPIFB4 (VTFT/ FIG. 6 a ).
  • Panels 4b, 5b and 6b represents Phenylephrine induced vasoconstriction observed in Example 3 in mesenteric vessels perfused ex vivo with an empty plasmid pRK5 plasmid (EV/ FIG. 4 b ), a pRK5 plasmid encoding INFT hBPIFB4 (INFT/ FIG. 5 b ) or a pRK5 plasmid encoding VTFT hBPIFB4 (VTFT/ FIG. 6 b ).
  • Panels 4c, 5c and 6c represent acetylcoline induced vasodilatation observed in Example 3 in mesenteric vessels perfused ex vivo with an empty plasmid pRK5 plasmid (EV/ FIG. 4 c ), a pRK5 plasmid encoding INFT hBPIFB4 (INFT/ FIG. 5 c ) or a pRK5 plasmid encoding VTFT BPIFB4 (VTFT hBPIFB4/ FIG. 6 c ).
  • FIGS. 7 and 8 panels 7a and 8a represent the effect of the eNOS inhibitor L-NAME on acetylcholine-induced relaxation of vessels perfused ex vivo or with an empty pRK5 plasmid (EV/ FIG. 7 a ) or a pRK5 plasmid encoding mutated VTFT hBPIFB4 (VTFT/ FIG. 8 a ).
  • Panels 8b represents recovery of vasorelaxation of vessels from methylenetetrahydrofolate reductase knockout mice (Mthfr +/ ⁇ ) control (Mthfr +/+ ) and knockout mice treated with either empty pRK5 plasmid ((Mthfr+/ ⁇ EV) ( FIG. 7 b ) or a pRK5 plasmid encoding VTFT hBPIFB4 ((Mthfr +/ ⁇ -M) ( FIG. 8 b ).
  • FIG. 9 panel a) shows a RT-PCR demonstrating induction of expression of BPIFB4 by H 2 O 2 in HEK293T cells.
  • Panel b) shows a Western blot of the phosphorylation on eNOS at Ser1177 in HEK293T cells expressing VTFT hBPIFB4 (VTFT) and in cells overexpressing INFT hBPIFB4 (INFT) or those exposed to an empty vector (EV).
  • Panel c) top, shows ⁇ -actin-normalized ODs.
  • a first object of the present invention is a BPIFB4 protein variant, having an aminoacid sequence with at least 85% homology to the aminoacid sequence of SEQ ID NO: 1 and characterised in that said sequence comprises a Valine at the position corresponding to position 229 of SEQ ID NO: 1 (hereinafter referred to as Valine 229), a Threonine at the position corresponding to position 281 SEQ ID NO: 1 (hereinafter referred to as Threonine 281), a Phenylalanine at a position corresponding to position 488 of SEQ ID NO: 1 (hereinafter referred to as Phenylalanine 488) and a Threonine at position corresponding to position 494 of SEQ ID NO: 1 (hereinafter referred to as Threonine 494).
  • Said homology is preferably of at least 90%, more preferably of at least 95%, even more preferably of at least 99%.
  • the aminoacid sequence of the BPIFB4 protein variant of the invention may differ from that of SEQ ID NO: 1 for the presence of additions, deletions or further substitutions of aminoacids.
  • an essential feature of the variant of the invention is that it contains the above said four aminoacids.
  • the above four aminoacids are present at the position that correspond to its original position in SEQ ID NO:1.
  • the above four aminoacids are present in the same position as in SEQ ID NO: 1.
  • the protein of the invention has an aminoacid sequence corresponding to SEQ ID NO 1, wherein one or more aminoacids at positions different from positions 229, 281, 488 and 494 of SEQ ID NO 1 have been substituted by a conserved aminoacid.
  • conserved aminoacid it is meant an aminoacid with functionally physicochemical properties equivalent to those of the original aminoacid.
  • Particularly preferred proteins according to the invention have the aminoacid sequence of known BPIFB4 proteins identified in Homo Sapiens (Acc. N. NP-59827.2; SEQ ID NO: 1 or Acc. N. NP-872325.2, corresponding to a longer isoform), Felis Catus (Acc N. XP003983665.1); Pan Troglodytes (Acc N. XP525303); Samiri boliviensis boliviensis (Acc N. XP-003932113.1); Macaca Mulatta (Acc N. NP-001230192.1); Pan paniscus (Acc N. XP-003814776.1); Otolemur gamettii (Acc N.
  • XP — 003788148.1 Pongo abelii (Acc N. XP-003780649.1); Sarcophilus harrisii (Acc N. XP-003758987.1); Rattus norvegicus (Acc N. NP-001102679.2); Callithrix jacchus (Acc N. XP-003732841.1); Mus musculus (Acc N. NP-001030047.2); Bos taurus (Acc N. XP-003586861.1); Canis lupus familiaris (Acc N. XP-534383.3); Sus scrofa (Acc N.
  • XP-003134448.3 Gallus gallus (Acc No XP-425718), Didelphis virginiana (LOC100032880) or Xenopus (LOC100485776), which has been modified so that it comprises a Valine at the position corresponding to position 229 of SEQ ID NO:1, a Threonine at the position corresponding to position 281 SEQ ID NO: 1, a Phenylalanine at the position corresponding to position 488 of SEQ ID NO: 1 and a Threonine at the position corresponding to position 494 of SEQ ID NO: 1.
  • the BPIFB4 protein variant of the invention has the sequence of SEQ ID NO:1.
  • a protein having such sequence will be hereinafter called VTFT hBPIFB4.
  • a second object of the present invention is a protein having a sequence which consist of the aminoacid sequence of a BPIFB4 protein variant according to the invention linked to an additional aminoacid sequence able to impart to the protein particularly advantageous properties.
  • said additional aminoacid sequence is useful for identifying the BPIFB4 protein variant according to the invention or to target the BPIFB4 protein variant of the invention to a specific organ or tissue.
  • said protein is a chimeric protein.
  • the present inventors have surprisingly found that the above VTFT hBPIFB4 is associated with exceptional longevity in three independent populations.
  • the present inventors have further demonstrated that the beneficial effect of the mutant protein on life expectancy is a consequence of its ability to modulate vascular dysfunctions associated with aging. As demonstrated in the experimental section, this modulation is dependent on the presence of the specific four aminoacids at positions corresponding to positions 229, 281, 488 and 494 of SEQ ID NO:1 in the VTFT hBPIFB4 of the invention.
  • mice mesenteric vessels were perfused ex vivo with an empty plasmid or a plasmids encoding VTFT hBPIFB4 or proteins that differ from VTFT hBPIFB4 in that they show various substitutions at the 4 relevant aminoacids: INFT hBPIFB4, having the aminoacid sequence of SEQ ID NO: 3, which differs from that of VTFT hBPIFB4 in that it contains Isoleucin and an Aspargin at positions 229 and 281, respectively, VNFT hBPIFB4, having the aminoacid sequence of SEQ ID NO: 4, which differs from that of VTFT hBPIFB4 in that it contains an Aspargin at position 281, ITFT hBPIFB4, having the aminoacid sequence of SEQ ID NO: 5, which differs from that of VTFT hBPIFB4 in that it contains Isoleucine at position 229, VTLI hBPIFB4, having the aminoacid sequence of SEQ ID NO:
  • VNFT hBPIFB4, ITFT hBPIFB4, VTLI hBPIFB4 and INLI hBPIFB4 did not show any effect on vascular function and INFT hBPIFB4 strongly inhibited any vascular function, blocking both vasoconstriction and vasodilatation, the VTFT BPIFB4 protein showed a weak effect on inhibition of vasoconstriction and a significant enhancement of vasodilatation. This effect has been demonstrated to be mediated by activation of eNOS through phosphorylation on serine 1177 and it is therefore associated to an increase in the release of NO by endothelial cells. The ability of VTFT hBPIFB4 to induce activation of eNOS has been corroborated in the cell line HEK293T (Example 5).
  • a third object of the present invention is a fragment of the BPIFB4 protein variant of the invention having a sequence comprising the above said Valine 229, Threonine 281, Phenylalanine 488 and Threonine 494.
  • the above said BPIFB4 protein variant, protein or fragment of the invention may advantageously be used in the in the prevention, reduction of the risk of, amelioration and/or treatment of pathological conditions of the endothelium due to decreased production of NO or activity of eNOS or of pathologies or conditions where it is beneficial to increase the activity of eNOS and/or the production of NO.
  • a fourth object of the invention is the above said BPIFB4 protein variant, protein or fragment for use in therapy.
  • the BPIFB4 protein variant, the protein or the fragment of the invention are for use in the prevention, reduction of the risk of, amelioration and/or treatment of an endothelial dysfunction due to release of NO from endothelial cells below the physiological levels or a decrease in the activity of eNOS or in clinical situations wherein it is beneficial to obtain an increase in the activation of eNOS and or in the production of NO.
  • said BPIFB4 protein variant, said protein or said fragment of the invention are for use in the prevention, reduction of the risk, amelioration or treatment of a pathology selected from arterial hypertension, atherosclerosis, diabetes mellitus, dyslipidemia, renal failure, metabolic syndrome, stroke, myocardial infarction, erectile dysfunction, neurodegenerative diseases, multiple sclerosis, cognitive disorders, retinal degeneration, uveoretinitis, vascular retinopathy, cataract, glaucoma, coronary spastic angina, thrombosis, pulmonary hypertension, pre-eclampsia, vasculites, cancer, inflammatory disorders, venus insufficiency, genetic diseases with reduced eNOS activity and NO production, for example MTHFR gene variations.
  • a pathology selected from arterial hypertension, atherosclerosis, diabetes mellitus, dyslipidemia, renal failure, metabolic syndrome, stroke, myocardial infarction, erectile dysfunction, neurodegenerative diseases, multiple sclerosis, cognitive disorders, retinal
  • said BPIFB4 protein variant is for use for the improvement of post-exercise fatigue in muscular dystrophy patients and as a co-adjuvant in the implantation of one or more stents, preferably medicated, for vascular occlusions.
  • the BPIFB4 protein variant, the protein or the fragment according to the invention may be administered to an subject in need thereof, affected by one of the above pathologies or in the above clinical conditions, by oral, nasal, endovenous, topical-, intra- or retro-ocular administration.
  • a fifth object of the invention is a pharmaceutical composition, preferably suitable for oral, nasal-, endovenous topical-, intra- or retro-ocular administration, comprising the BPIFB4 protein variant, the protein or the fragment of the invention in admixture with pharmaceutically acceptable carriers and/or excipients.
  • suitable formulations for the pharmaceutical composition of the invention are well known in the art and are, for example, described in “Remington's Pharmaceutical Sciences Handbook”, Mack Publishing Company, Easton, Pa., last or Babizhayev M A. Drug Testing and Analysis, Volume 4, Issue 6, pages 468-485, June 2012).
  • a particularly suitable pharmaceutical formulation for the administration of the BPIFB4 protein variant, the protein or the fragment according to the invention is based on synthetic copolymers, using polyaminoacidic and polysaccharidic structures, able to form reversible physical complexes with the BPIFB4 protein variant, the protein or the fragment thereof by electrostatic, hydrophobic or other physical interactions, and generate nano-aggregates from which the protein or fragment is released in intact form after administration.
  • a sixth object of the present invention is a polynucleotide, preferably a DNA polynucleotide, coding for the aminoacid sequence of the BPIFB4 protein variant, the protein or the polypeptide according to the present invention.
  • said polynucleotide has a sequence which comprises or consists in SEQ ID NO: 2 or the sequence of a fragment thereof comprising the nucleotides coding for the above said Valine 229, Threonine 281, Phenylalanine 488 and Threonine 494.
  • the above polynucleotide may be used in order to obtain expression of the mutated protein or polypeptide in host cells either in vitro, ex vivo or in vivo by means of a suitable expression vector comprising it.
  • a seventh object of the invention is a vector containing the above said polynucleotide of the invention operatively linked to expression control sequences.
  • the BPIFB4 protein variant, the protein or the fragment of the invention is recombinantly produced in host cells transfected with the above said vector.
  • the vector of the invention in preferably one that it is suitable for high yield production of the protein or polynucleotide.
  • the pcDNATM3.3-TOPO® vector can be used for high level expression of the protein of the invention in adherent mammalian tissue culture cells following transient transfection, or high level expression of secreted protein using the FreeStyleTM MAX CHO and FreeStyleTM MAX 293 systems (Invitrogen INC.)
  • an eight object of the present invention are host cells transfected with the above said vector of the invention.
  • An ninth object of the invention is a method of recombinantly producing the BPIFB4 protein variant, the protein or the fragment according to the invention comprising culturing the above said host cells under conditions allowing expression of the BPIFB4 protein variant, the protein or the fragment and recovering said BPIFB4 protein variant, protein or fragment.
  • the BPIFB4 protein variant, the protein or the fragment of the invention may be expressed in the target tissue following administration, preferably via the endovenous, subcutaneous, intraocular or retroocular route, into a subject in need thereof of a vector according to the present invention, which is suitable to induce expression in said target tissue of the mutated protein or polypeptide.
  • the target tissue may differ depending on the pathology to be treated and may be, for example, the endothelial tissue, the tissue of the liver, heart, kidney, eye or muscle.
  • the vector of the invention is one that is preferably suitable for transfection of the cells of the target tissue of interest following endovenous administration.
  • said vector is a viral vector, preferably an Adenovirus vector, more preferably a vector selected from AAV serotypes 1-9 vectors, on the basis of specificity for the target tissue of interest (Varadi K, et al, Gene Ther. (2012); 19 (8):800-9; Zincarelli C et al, Mol Ther. (2008), 16(6): 1073-80, Diaz-Fernandez Y A et al, Oligonucleotides. 2010; 20(4): 191-8).
  • a tenth object of the invention is the above said polynucleotide or vector of the invention for use in therapy.
  • said polynucleotide or vector is for use in the prevention, reduction of the risk of, amelioration or treatment of an endothelial dysfunction due to release of NO from endothelial cells below the physiological levels or a decrease in the activity of eNOS or in conditions wherein it is beneficial to obtain an increase in the activation of eNOS and or in the production of NO.
  • said polynucleotide or vector is for use in the prevention, reduction of the risk of, amelioration or treatment of a pathology or condition selected from arterial hypertension, atherosclerosis, hypertension, diabetes mellitus, dyslipidemia, renal failure, metabolic syndrome, stroke, myocardial infarction, erectile dysfunction, neurodegenerative diseases, multiple sclerosis cognitive disorders, retinal degeneration, uveoretinitis, vascular retinopathy, cataract, glaucoma, coronary spastic angina, thrombosis, pulmonary hypertension, pre-eclampsia, vasculites, cancer, inflammatory disorders, venus insufficiency, genetic diseases with reduced eNOS activity and NO production, for example MTHFR gene variations.
  • a pathology or condition selected from arterial hypertension, atherosclerosis, hypertension, diabetes mellitus, dyslipidemia, renal failure, metabolic syndrome, stroke, myocardial infarction, erectile dysfunction, neurodegenerative diseases, multiple sclerosis cognitive disorders
  • said polynucleotide or vector is for use for the improvement of post-exercise fatigue in muscular dystrophy patients and as a co-adjuvant in the implantation of one or more stents, preferably medicated, for vascular occlusions.
  • a eleventh object of the invention is a pharmaceutical composition, preferably suitable for endovenous, subcutaneous, intraocular or retroocular administration, comprising a vector according to the invention in admixture with pharmaceutically acceptable carriers and/or excipients.
  • Suitable formulations for the pharmaceutical composition of the invention are well known in the art.
  • polymeric-based nano-systems or polycomplex nanosystems may be used to deliver the vector of the invention (Murano E et al, Nat Prod Commun. (2011), 6(4): 555-72, Moustafine R I et al, Int J Pharm. 2012 Oct. 3).
  • the mean daily dosage of the BPIFB4 protein variant, the protein or the fragment or vector of the invention will depend upon various factors, such as the seriousness of the disease and the conditions of the patient (age, sex and weight).
  • the skilled man may use technical means well known in the art in order to find the correct dosage amount and regime to ensure optimal treatment in each particular pathological condition.
  • GWAS Genome Wide Association Study
  • DNA was extracted from peripheral blood (QIAamp DNA blood midi kit, Qiagen) of the individuals and genotyped with TaqMan probe on ABI 7900HT Real Time PCR (Applied Biosystems). For the screening, the following probes were used:
  • hCV25757827 for rs2070325; hCV958887 for rs571391; hCV28993331 for rs7583529; and hCV3073023 for rs285097.
  • Meta-analysis of association results was performed by the “meta” package implemented in R (http://cran.r-project.org/web/packages/meta/index.html). Positional and functional annotation of the identified SNPs were performed by the SNPNexus on-line resource ( ).
  • Haplotype analyses revealed patterns of strong linkage disequilibrium (LD) within the BPIFB4 genomic locus, delimiting a region that is highly enriched in non-synonymous SNPs (Fig. S1 in the Supplementary Appendix).
  • the rs2070325 variation (Ile229Val) of BPIFB4 tags rs2889732 (Asn288Thr), rs11699009 (Leu488Phe), and rs11696307 (Ile494Thr).
  • the three-dimensional structure of human BPIFB4 was predicted by homology modeling with the program I-TASSER, (REF: Ambrish Roy, Alper Kucukural, Yang Zhang. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols, vol 5, 725-738 (2010).) using as template Protein BPI from PDB (code 1EWF) All models were considered in the visual structural analysis, performed with the program PyMOL Version 1.2r3pre, Schrodinger, LLC (Molecular Graphics System). The above analysis revealed that Ile268Val and Asn320Thr are both located in putative protein-protein interaction site.
  • BPIFB4 wild-type (WT) and mutated (Ile229Val, Asn281Thr, leu488Phe, Ile494Thr) BPIFB4 proteins by homology modeling.
  • WT wild-type
  • mutated BPIFB4 binds lipopolysaccharides in regions that are similar to those of the other two proteins.
  • Our structural analysis revealed that Leu488Phe is located in a lipid-binding pocket whose size is predicted to decrease as a consequence of the mutation.
  • the Ile494Thr mutation is located in a second lipid-binding pocket, whose hydrophobicity is decreased by the substitution. In both cases, the mutation may result in an decreased ability to bind lipids.
  • Ile229Val and Asn281Thr are located far from the lipid-binding sites of the structurally homologous proteins, so they probably affect functions such as interaction with other proteins, rather than lipid binding.
  • Second-order branches of the mesenteric arterial tree of C57BL6 mice were transfected as described previously (Vecchione C et al., J Exp Med 2005; Vol. 201, pages 1217-28).
  • vessels were placed in a Mulvany pressure system filled with Krebs solution to which was added 20 ⁇ g of a pRK5 vector encoding either INFT or VTFT hBPIFB4.
  • An empty plasmid was used as a negative control.
  • Vessels were perfused at 100 mmHg for 1 hour then at 60 mmHg for 5 hours.
  • the efficiency of transfection was evaluated by the presence of green fluorescent protein (GFP) co-expression ( FIG. 2 ) and by Western blotting.
  • GFP green fluorescent protein
  • the membranes were washed three times and then incubated for 1 or 2 h with the secondary antibody (horseradish peroxidase-linked anti-rabbit IgG or anti-mouse IgG, Amersham Life Science) at 1:3000 dilution. The membrane was then washed four times and specific protein bands were detected with ECL Prime chemiluminescent agents (Amersham Life Science). Western blot data were analyzed using ImageJ software (developed by Wayne Rasband, National Institutes of Health, USA) to determine optical density (OD) of the bands. The OD reading was normalized to ⁇ -actin to account for variations in loading.
  • BPIFB4 protein was abundantly detected in vessels after perfusion with either INFT hBPIFB4- or VTFT hBPIFB4-encoding plasmids both wild type and VTFT hBPIFB4 being expressed in comparable amounts. On the contrary, vessels exposed to empty plasmids expressed a low level of native BPIFB4 protein.
  • vessels expressing VTFT hBPIFB4 but not INFT hBPIFB4 showed a string induction of phosphorylation of eNOS on serine 1177, an activation site of the enzyme.
  • Vasoconstriction was assessed with KCl (80 mM) and increasing doses of phenylephrine (from 10 ⁇ 9 M to 10 ⁇ 6 M), as the percentage of lumen diameter change after drug administration.
  • Vascular responses were tested before and after transfection. Endothelium-dependent and independent relaxations were assessed by measuring the dilatatory responses of mesenteric arteries to cumulative concentrations of acetylcholine (from 10 ⁇ 9 M to 10 ⁇ 5 M) and nitroglycerine (from 10 ⁇ 9 M to 10 ⁇ 5 M), respectively, in vessels precontracted with phenylephrine at a dose necessary to obtain a similar level of precontraction in each ring (80% of initial KCl-induced contraction).
  • VTFT hBPIFB4 upon expression of VTFT hBPIFB4 there was a significant enhancement in acetylcholine-induced vessel vasodilatation compared with that observed before transfection ( FIG. 6 c ), but no differences in nitroglycerin-evoked smooth muscle relaxation (data not shown), indicating that this effect is due to an enhancement in endothelial function. No effect on vascular function was observed with VNFT hBPIFB4, ITFT hBPIFB4, VTLI hBPIFB4 and INLI hBPIFB4.
  • HEK293T Human embryonic kidney cells
  • DMEM Dulbecco's modified Eagle's medium
  • fetal bovine serum 10% (v/v) fetal bovine serum and 1% non-essential amino acids at 37% in a 5% CO2 atmosphere.
  • Cells were plated at 0.25 ⁇ 106 per well in six-well plates, and 24 h after plating were transfected using 10 ⁇ l of Lipofectamine 2000 (LifeTechnologies) and 4 ⁇ g of plasmids. After 24 h, cells were serum-starved for 24 h. During serum starvation, transfected cells were treated with 400 ⁇ M H 2 O 2 for 24 h.
  • BPIFB4 Transcription of BPIFB4 was detected by extraction from the cells of total RNA with TRIzol (Ambion), retrotranscription (iScript BioRad). cDNA was amplified with specific primers for BPIFB4 (Fw: CTCTCCCCAAAATCCTCAACA, Rev: AGCCTCTCTGGGACTGGTTC) and GAPDH (Fw: GTGAAGGTCGGAGTCAACG, Rev: GGTGGAATCATATTGGAACATG).
  • BPIFB4 could be induced in HEK293T cells upon exposure to H 2 O 2 : this demonstrates a role of BPIFB4 in the stress response ( FIG. 9 , panel a).
  • BPIFB4 affected stress-mediated phosphorylation of eNOS on serine 1177.
  • HEK293T Human embryonic kidney cells
  • DMEM Dulbecco's modified Eagle's medium
  • fetal bovine serum 10% (v/v) fetal bovine serum and 1% non-essential amino acids at 37° C. in a 5% CO2 atmosphere.
  • Cells were plated at 0.25 ⁇ 106 per well in six-well plates, and 24 h after plating were transfected using 10 ⁇ l of Lipofectamine 2000 (LifeTechnologies) and 4 ⁇ g of plasmids. After 24 h, cells were serum-starved for 24 h. During serum starvation, transfected cells were treated with 400 ⁇ M H 2 O 2 for 24 h.
  • Protein extracts were separated on 10% SDS-PAGE at 100V for 1 h or on 4-12% SDS-PAGE at 100V for 2 h and then transferred to a nitrocellulose or PVDF membrane.
  • the membranes were incubated overnight with the following primary antibodies: anti-phospho-eNOS Ser1177 (Cell Signaling, rabbit mAb, 1:1000), and anti- ⁇ -actin (Cell Signaling, mouse mAb, 1:3000).
  • the membranes were washed three times and then incubated for 1 or 2 h with the secondary antibody (Amersham Life Science horseradish peroxidase-linked anti-rabbit IgG or anti-mouse IgG, 1:3000).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Diabetes (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Endocrinology (AREA)
  • Hematology (AREA)
  • Virology (AREA)
  • Mycology (AREA)
US14/655,651 2012-12-28 2013-12-27 Variant of bpifb4 protein Abandoned US20150344536A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP12425208.1A EP2749569A1 (en) 2012-12-28 2012-12-28 Variant of BPIFB4 protein
EP12425208.1 2012-12-28
PCT/EP2013/078076 WO2014102343A1 (en) 2012-12-28 2013-12-27 Variant of bpifb4 protein

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2013/078076 A-371-Of-International WO2014102343A1 (en) 2012-12-28 2013-12-27 Variant of bpifb4 protein

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/511,051 Continuation US11208447B2 (en) 2012-12-28 2019-07-15 Viral vector suitable for gene therapy encoding a variant of a BPIFB4 protein

Publications (1)

Publication Number Publication Date
US20150344536A1 true US20150344536A1 (en) 2015-12-03

Family

ID=47715813

Family Applications (3)

Application Number Title Priority Date Filing Date
US14/655,651 Abandoned US20150344536A1 (en) 2012-12-28 2013-12-27 Variant of bpifb4 protein
US16/511,051 Active US11208447B2 (en) 2012-12-28 2019-07-15 Viral vector suitable for gene therapy encoding a variant of a BPIFB4 protein
US17/525,531 Active 2034-06-10 US11891421B2 (en) 2012-12-28 2021-11-12 Variant of a BPIFB4 protein

Family Applications After (2)

Application Number Title Priority Date Filing Date
US16/511,051 Active US11208447B2 (en) 2012-12-28 2019-07-15 Viral vector suitable for gene therapy encoding a variant of a BPIFB4 protein
US17/525,531 Active 2034-06-10 US11891421B2 (en) 2012-12-28 2021-11-12 Variant of a BPIFB4 protein

Country Status (8)

Country Link
US (3) US20150344536A1 (ja)
EP (4) EP2749569A1 (ja)
JP (1) JP6461810B2 (ja)
CN (1) CN104955836B (ja)
AU (1) AU2013369292B2 (ja)
CA (1) CA2896166A1 (ja)
ES (2) ES2951691T3 (ja)
WO (1) WO2014102343A1 (ja)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11208447B2 (en) * 2012-12-28 2021-12-28 Roxiant ApS Viral vector suitable for gene therapy encoding a variant of a BPIFB4 protein

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104558144B (zh) * 2014-12-30 2017-08-08 深圳市第二人民医院 鼠Bpifb5突变体、蛋白及其应用
AU2018317807A1 (en) * 2017-08-16 2020-02-06 Roxiant ApS VTFT isoform of a BPIFB4 protein for use in neuronal diseases and injuries

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079269A2 (en) * 2000-04-18 2001-10-25 Merck Patent Gmbh Lipid binding protein 4

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL87737A (en) * 1987-09-11 1993-08-18 Genentech Inc Method for culturing polypeptide factor dependent vertebrate recombinant cells
US6391311B1 (en) * 1998-03-17 2002-05-21 Genentech, Inc. Polypeptides having homology to vascular endothelial cell growth factor and bone morphogenetic protein 1
EP1379654A2 (en) 2001-02-08 2004-01-14 Incyte Genomics, Inc. Intracellular signaling molecules
PT2573170T (pt) 2001-12-17 2018-03-26 Univ Pennsylvania Sequências de um vírus adenoassociado (aav) de serotipo 9, vetor contendo as mesmas, e suas utilizações
DK2292779T3 (en) 2003-09-30 2017-02-27 Univ Pennsylvania ADENOASS-ASSOCIATED VIRUS (AAV) CLADES, SEQUENCES, VECTORS CONTAINING SAME AND APPLICATIONS THEREOF
WO2006022619A2 (en) * 2004-07-22 2006-03-02 Sequenom, Inc. Methods for identifying risk of type ii diabetes and treatments thereof
AU2007266452A1 (en) 2006-05-31 2007-12-06 The Children's Hospital Of Philadelphia Compositions and methods for detection and modulation of T cell mediated immune responses against viral vectors utilized for gene therapy
CN101627121A (zh) * 2006-12-08 2010-01-13 奥斯瑞根公司 作为治疗干预的靶标的miRNA调控基因和路径
EP2019143A1 (en) 2007-07-23 2009-01-28 Genethon CNS gene delivery using peripheral administration of AAV vectors
WO2010071832A1 (en) 2008-12-19 2010-06-24 Nationwide Children's Hospital Delivery of polynucleotides across the blood brain barrier using recombinant aav9
JP2011231022A (ja) * 2010-04-23 2011-11-17 Ajinomoto Co Inc イミダゾロン誘導体
JP6224459B2 (ja) 2011-02-17 2017-11-01 ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア 組織特異性を改変し、ａａｖ９媒介遺伝子導入を改善するための組成物および方法
US20140010861A1 (en) 2012-04-02 2014-01-09 modeRNA Therapeutics Modified polynucleotides for the production of proteins associated with human disease
EP2749569A1 (en) * 2012-12-28 2014-07-02 Annibale Alessandro Puca Variant of BPIFB4 protein

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079269A2 (en) * 2000-04-18 2001-10-25 Merck Patent Gmbh Lipid binding protein 4

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
Brown, Guy C., "Nitric oxide and mitochondria." Fronteirs in Bioscience (2007) 12 p1024-1033 *
Hunt, Jay D. "An introduction to cancer," available online at http://www.medschool.lsuhsc.edu/genetics_center/louisiana/article_cancer.htm, downloaded 18 July, 2016 *
NIH's web page discussing hypercholesterolemia, https://web.archive.org/web/20110514024033/http://ghr.nlm.nih.gov/condition/hypercholesterolemia *
Paget, Douglas S. et al, "Infectibility of endovascular stents following antibiotic prophylaxis or after arterial wall incorporation." Am. J. Surg. (1999) 178 p219-224 *
Plaza-Plaza, Jose Christian et al, "P{harmacolgenetic polymorphisms contributing to toxicity induced by methotrexate in the southern spanish population with rheumatoid arthritis." Omics (2012) 16 p589-595 *
Sunada, Yoshihide et al, "Transgenic mice expressing mutant caveolin-3 show severe myopathy associated with increased nnos activity." Human Mol. Gen. (2001) 10(3) p173-178 *
the myocardial infarction webpage for healthline, http://www.healthline.com/health/acute-myocardial-infarction#Causes3, downloaded 9 Jan, 2017 *
Yampolsky, Lev Y. and Stoltzfus, Arlin, "THe exchangeability of amino acids in proteins." Genetics (2005) 170 p1459-1472 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11208447B2 (en) * 2012-12-28 2021-12-28 Roxiant ApS Viral vector suitable for gene therapy encoding a variant of a BPIFB4 protein

Also Published As

Publication number Publication date
EP3560952B9 (en) 2023-10-04
EP3560952A1 (en) 2019-10-30
JP6461810B2 (ja) 2019-01-30
AU2013369292B2 (en) 2017-12-07
EP2938629B1 (en) 2019-04-10
US11891421B2 (en) 2024-02-06
EP2749569A1 (en) 2014-07-02
US20220169686A1 (en) 2022-06-02
JP2016508036A (ja) 2016-03-17
ES2951691T3 (es) 2023-10-24
ES2731126T3 (es) 2019-11-14
EP4253405A3 (en) 2024-01-03
EP2938629A1 (en) 2015-11-04
CN104955836B (zh) 2019-03-08
EP3560952B1 (en) 2023-06-07
WO2014102343A1 (en) 2014-07-03
CN104955836A (zh) 2015-09-30
US20210017244A1 (en) 2021-01-21
EP3560952C0 (en) 2023-06-07
AU2013369292A1 (en) 2015-07-09
WO2014102343A8 (en) 2018-10-18
EP4253405A2 (en) 2023-10-04
US11208447B2 (en) 2021-12-28
CA2896166A1 (en) 2014-07-03

Similar Documents

Publication Publication Date Title
US11891421B2 (en) Variant of a BPIFB4 protein
EP1836312B1 (en) Methods and compositions for diagnosing age-related macular degenration
US8211853B2 (en) Method of promoting apoptosis of differentiated adipocytes and increasing endogenous expression of SFRP-5 peptide by administration of SFRP-5 peptide
Diekmann et al. Analysis of the reticulon gene family demonstrates the absence of the neurite growth inhibitor Nogo-A in fish
JP5671678B2 (ja) ニューロン生存因子およびその使用
CA2682516A1 (en) Schizophrenia-related isoform of kcnh2 and development of antipsychotic drugs
JP5514443B2 (ja) カテコールアミン調節性タンパク質
US20060154331A1 (en) Erythrocyte differentiation factor, gene encoding same, and methods of use thereof
US8546324B2 (en) Short-form human MD-2 as a negative regulator of toll-like receptor 4 signaling
US11246912B2 (en) Methods for treating and diagnosing metastatic liver cancer
Meester et al. The role of biglycan in the healthy and thoracic aneurysmal aorta
Andreu et al. PALML, a novel paralemmin-related gene mapping on human chromosome 1p21
EP3515470A1 (en) Methods and compositions for inhibiting skin inflammation and determining cancer susceptibility
US20090298918A1 (en) Alternative Splicing Isoform of Lox-I Protein Encoding Gene, and Uses Thereof
Stuermer Analysis of the reticulon gene family demonstrates the absence of the neurite growth inhibitor Nogo-A in fish
US20060148030A1 (en) Nuclear receptor err y 3
WO2002083728A2 (en) Splice variant
EP1728424A2 (en) Method to treat conditions associated with insulin signaling dysregulation
WO2002046420A2 (en) Nebulin-related protein

Legal Events

Date Code Title Description
AS Assignment

Owner name: LGV1 S.R.L., ITALY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:PUCA, ANNIBALE ALESSANDRO;VECCHIONE, CARMINE;REEL/FRAME:040083/0371

Effective date: 20161018

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STCV Information on status: appeal procedure

Free format text: NOTICE OF APPEAL FILED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION