WO1998052597A1 - Sequences de gelsoline induisant l'apoptose - Google Patents

Sequences de gelsoline induisant l'apoptose Download PDF

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WO1998052597A1
WO1998052597A1 PCT/US1998/010181 US9810181W WO9852597A1 WO 1998052597 A1 WO1998052597 A1 WO 1998052597A1 US 9810181 W US9810181 W US 9810181W WO 9852597 A1 WO9852597 A1 WO 9852597A1
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gelsolin
apoptosis
sequence
cells
amino acid
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PCT/US1998/010181
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English (en)
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Kirston Koths
Srinivas Kothakota
Louis T. Williams
Christoph Reinhart
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Chiron Corporation
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Priority to JP55050798A priority Critical patent/JP2002511748A/ja
Priority to AU75784/98A priority patent/AU7578498A/en
Priority to EP98923505A priority patent/EP1003543A4/fr
Publication of WO1998052597A1 publication Critical patent/WO1998052597A1/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
    • C07K14/46—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
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • cysteinyl protease ced 3 is required for apoptosis, the programmed killing of cells. See Ellis, Ann Rev Cell Biol 7 (1991) 663-98, Seimiya, J Biol Chem 272 (1997) 4631-36 and Vaux, Proc Natl Acad Sci 93 (1996) 2239- 44. Eleven mammalian homologs of ced 3 have been identified. Of these, caspase-3 (also known as CPP32, apopain or YAMA) exhibits the highest degree of sequence and functional similarity to ced 3. In mammals, caspase-3 appears to be required for certain apoptopic pathways.
  • Actin cleavage activity accompanies the development of apoptosis and the cleavage activity is inhibited by ICE/ced-3 family protease inhibitors.
  • Caspase-3 was reported to be a major component responsible for the cleavage of actin itself. See Chen, Cancer Res 56 (1996)24-29. Actin function can also be regulated by depolymerization via gelsolin as enzymatic activity that is deregulated by caspase-3 cleavage during apoptosis, as described below.
  • the relationship between gelsolin activation by caspase 3 and the control of apoptosis has, until now, escaped researchers.
  • Gelsolin is a 731 amino acid protein that, in the presence of Ca 2 ⁇ , causes gels formed from purified actin filaments and filamin to change to a more fluid state.
  • the gelsolin protein severs the actin filaments and caps the exposed plus end, breaking up the cross-linked network of actin filaments and filamin which forms the gel.
  • Human gelsolin protein has been found in two forms: extracellularly in plasma and intracellularly in the cytoplasm. The two forms are derived from a single gene by alternative splicing.
  • gelsolin is a substrate for caspase-3. Cytoplasmic gelsolin is cleaved by caspase-3 at amino acid number 352, resulting in a 352 amino acid N-terminal cleavage product and a 379 amino acid C-terminal cleavage product. The N-terminal cleavage product induces apoptosis in mammalian cells.
  • the invention provides novel, isolated, N-terminal gelsolin amino acid sequence fragments. These fragments comprise the amino acid sequence of SEQ ID NO: 1, from amino acids 1 through 352 and analogs, derivatives and/or variants of that sequence having amino acid substitution(s), deletion(s) and/or addition(s), which analogs, derivatives and/or variants possess the ability to induce apoptosis in mammalian cells as determined by a DNA fragmentation assay.
  • the invention provides novel, isolated nucleic acid sequences encoding the apoptosis inducing gelsolin amino acid fragments of the invention.
  • These nucleic acid sequences comprise the nucleotide sequence of SEQ ID NO: 2, from nucleotide 1 through 1056.
  • the invention provides gene delivery vehicles containing a DNA sequence coding for an apoptosis inducing gelsolin amino acid sequence comprising the DNA sequence of SEQ ID NO: 2, from nucleotide 1 through nucleotide 1056 and analogs, derivatives and/or variants of that sequence that are (a) hybridizable under stringent conditions to the sequence of SEQ ID NO: 2, from nucleotide 1 through nucleotide 1056, or which would hybridize under such conditions but for the degeneracy of the genetic code, and (b) code for an amino acid sequence that exhibits the ability to induce apoptosis in cells in a Ca ++ independent manner.
  • compositions containing the above mentioned proteins or gene delivery vehicles are another aspect of the invention, as are methods of diagnosing abnormal apoptopic conditions, methods of making the proteins and methods of treating biological or medical conditions employing the proteins or gene therapy vehicles.
  • Murine gelsolin is predicted to be essentially functionally equivalent to human gelsolin because the two proteins are 94.4% identical and 97% similar (i.e., differ in conservative amino acids only) at the amino acid level, have conserved structural repeats and motifs and are conserved at the caspase-3 cleavage site. Both the human and murine gelsolin proteins generate the predicated size protein cleavage fragments from caspase-3 cleavage at this site. (See Example 3 employing human gelsolin.) Accordingly, it is within the level of skill in the art to employ either the human or murine gelsolin protein or nucleic acid, or any similarly homologous mammalian gelsolin protein or nucleic acid sequence in this invention.
  • caspase-8 also known as FLICE
  • caspase-8 is implicated in initiating apoptosis. See, Muzio, Cell 85 (1996) 817 and Boldin, Cell 85 (1996) 803.
  • Caspase-8 which is believed to be activated following binding to Fas or tumor necrosis factor receptors that have bound their respective ligands, initiates the caspase protease cascade, including activation of caspase-3.
  • Activated caspase-3 then cleaves the N- te ⁇ ninal region of gelsolin from the native molecule to generate an active and Ca +2 independent N-terminal gelsolin fragment capable of inducing apoptosis in cells.
  • caspase-8 also has the ability to cleave gelsolin at the same location as caspase- 3.
  • the invention provides novel, isolated, N-terminal gelsolin amino acid sequence fragments.
  • These fragments comprise the amino acid sequence of SEQ ID NO: 1, from amino acids 1 through 352 and analogs, derivatives and/or variants of that sequence having amino acid substitution(s), deletion(s) and/or addition(s), which analogs, derivatives and/or variants possess the ability to induce apoptosis.
  • isolated we mean substantially free from other proteins or nucleic acid sequences with which the subject protein or the subject nucleic acid sequence is typically found in its native, i.e., endogenous state.
  • amino acid sequences that are identical or substantially identical (i.e., contain at least 70%, more preferably 80%, and most preferably 90% sequence homology) to the N-terminal gelsolin sequence (SEQ ID NO: 1) of the invention and which possess the ability to induce apoptosis in a Ca++ independent manner are included within this definition.
  • the amino acid substitutions can be conservative amino acid substitutions or substitutions to replace non-essential amino acid residues, to alter a glycosylation site, a phosphorylation site, an acetylation site, or to alter the protein's tertiary structure, for example by altering the position of a cysteine residue not necessary for function.
  • Conservative amino acid substitutions are those that preserve the general charge, hydrophobicity -hydrophilicity and/or steric bulk. For example, substitutions between the members of the following groups are conservative substitutions: Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln, Ser/Thr/Cys and Phe/Trp/Tyr. These analogs may also include amino acids with substituted linkages and non-naturally occurring amino acids. Such modifications are well known in the art. Whether a particular analog, derivative or variant possesses the ability to induce apoptosis may be determined using a variety of well known methods. For example, a DNA fragmentation assay in which measurement is based on gel-based molecular weight determination may be employed.
  • TUNEL assay may be employed. See Strater, J Histochem Cytochem 44 (1996)1497-99. Other assays that find use, alone or in conjunction with DNA fragmentation assays, in the determination of whether an analog, derivative or variant possesses the requisite apoptotic activity include assays for cell viability such as the MTT assay described in Hansen, J. Immunol Methods 119 (1989) 203.
  • the apoptosis inducing gelsolin fragments of the invention can be constructed employing known recombinant DNA techniques or by enzymatic cleavage of the full length gelsolin protein (SEQ ID NO: 5) by caspase-3 or caspase-8.
  • the invention provides novel, isolated nucleic acid sequences encoding the apoptosis inducing gelsolin amino acid fragments of the invention.
  • the isolated gelsolin nucleic acid sequences of the invention are those encoding the novel N-terminal amino acid sequence fragments described herein.
  • these nucleic acid sequences can comprise the nucleotide sequence of SEQ ID NO: 2, from nucleotide 1 through 1056. This sequence has been deposited with the American Type Culture Collection, Rockville, Maryland, USA, in a CMV vector, as ATCC Accession Number 98312.
  • nucleic acid sequences also include analogs, derivatives and/or variants of that sequence that are (a) hybridizable under stringent conditions to the sequence of SEQ ID NO: 2 or which would hybridize under such conditions but for the degeneracy of the genetic code, and (b) code for an amino acid sequence that exhibits the ability to induce apoptosis in cells in a Ca +i independent manner.
  • allelic variations naturally occurring base changes in the species population that may or may not result in an amino acid change
  • in the DNA sequences encoding gelsolin amino acid sequence fragments exhibiting Ca " independent apoptopic activity are also included in this invention, as are analogs or derivatives thereof.
  • nucleic acid sequences of this invention may exclude some or all of the signal and/or flanking sequences. Accordingly, the nucleic acid sequences of this invention may contain modifications in the non-coding sequences, signal sequences or coding sequences, based on deliberate modification. Using the gelsolin nucleotide sequence, it is within the skill in the art to obtain other modified DNA sequences: the sequences can be truncated at their 3' termini and/or their 5' termini, individual nucleotides can be manipulated while retaining the original amino acid(s), or nucleotides may be modified so as to modify the amino acid sequence.
  • Nucleotides can be substituted, inserted or deleted by known techniques, including for example, in vitro mutagenesis and primer repair.
  • short, highly degenerate oligonucleotides derived from regions of imperfect amino acid conservation can be used to identify new members of related families.
  • RNA molecules, transcribed from a DNA of the invention as described above, are an additional aspect of the invention.
  • DNA molecules encoding native gelsolin may be obtained (1) by cloning in accordance with the published methods, (ii) from the deposited plasmid or (iii) by synthesis, for example, using overlapping synthetic oligonulceotides based on the published sequences which together span the desired coding region.
  • Analogs, derivative or variant DNA sequences of this invention may be produced synthetically or by conventional site-directed mutagenesis of a DNA sequence encoding a native gelsolin sequence.
  • Such mutagenesis methods include the Ml 3 system of Zoller and Smith, Nucleic Acids Res. 10 (1982) 6487-6500; Methods Enzymol. 100 (1983) 468-500; and DNA 3 (1984) 479-88, using single stranded DNA and include the method of Morinaga Bio/Technology (1984) 636-39 using heteroduplexed DNA.
  • Mutations in the DNA sequences of the invention can include introduction of a termination signal, the deletion of part of the coding sequence, the introduction of one or more nucleotides that change the reading frame and point mutations or site specific deletion(s) or substitutions(s) using techniques already l ⁇ iown in the art.
  • Introduction of a termination signal can be accomplished, for example, using stop sequences in single or multiple reading frames.
  • the length of the deletion whether single or multiple, will preferably total at least about 30 bases in length. Deletions of nucleotides that encode amino acids not conserved between gelsolin species are preferred.
  • Such modified analogs, derivatives or variants can be tested for their ability to induce Ca ++ independent apoptopic activity following the methods described in Example 3.
  • the nucleic acid sequence of the invention comprises an antisense sequence of the nucleic acid sequences encoding the apoptosis inducing nucleotides sequences of the invention, and more particularly an antisense sequence of at least about 15 to 20 contiguous nucleotides of the sequence 1 through 1056 of SEQ ID NO: 2.
  • antisense sequences may be employed to prevent or delay apoptosis induced cell death, by preventing new gelsolin synthesis.
  • Such sequences may find utility in the treatment of diseases involving inappropriate apoptosis, for example, in certain autoimmune diseases such as Hashimoto's Thyroiditis and juvenile diabetes, in neurodegenerative diseases such as Alzheimer's disease and Huntington's disease and in alopecia and related conditions.
  • autoimmune diseases such as Hashimoto's Thyroiditis and juvenile diabetes
  • neurodegenerative diseases such as Alzheimer's disease and Huntington's disease and in alopecia and related conditions.
  • the nucleic acid sequences of the invention can be made employing standard recombinant DNA techniques.
  • the nucleic acid sequences encoding the apoptosis inducing gelsolin amino acid sequences of the invention, or the antisense sequences can be expressed in any expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems.
  • Exemplary bacterial expression systems include those described in Chang, Nature (1978) 275: 615, Goeddel, Nature (1979) 281: 544, Goeddel, Nucleic Acids Res. (1980) 8: 4057, EP Patent Publication 036776, US Patent No. 4,551,433, deBoer, Proc. Natl. Acad. Scz.(1983) 80: 21-25, and Siebenlist, Cell (1980) 20: 269.
  • Exemplary yeast expression systems include those described in Hinnen, Proc Natl Acad Sci (1978) 75: 1929; Ito, J Bacteriol (1983) 153: 163; Kurtz, Mol Cell Biol (1986) 6: 142; Kunze, J Basic Microbiol. (1985) 25: 141 ; Gleeson, J Gen.
  • Exemplary insect cell expression systems include those described in US Patent No. 4,745,051, Friesen (1986) "The Regulation of Baculovirus Gene Expression” in: The Molecular Biology of Baculoviruses (W. Doerfler, ed.), EP Patent Publications 0127839 and 0155476, Vlak, J Gen Virol (1988) 69: 165-116, Miller, Ann Rev Microbiol (1988) 42: 111, Carbonell, Gene (1988) 73: 409, Maeda, Nature (1985) 315: 592-594, Lebacq-Verheyden, Mol Cell Biol (1988) 8: 3129; Smith, Proc Natl Acad Sci (1985) 82: 8404, Miyajima, Gene (1987) 58: 273; and Martin, DNA (1988) 7/99.
  • Exemplary mammalian expression systems are described in Dijkema, EMBO J( ⁇ 985) 4: 761, Gorman, Proc Natl Acad Sci (1982b) 79: 6777, Boshart, Cell (1985) 41: 521 and in US Patent No. 4,399,216.
  • Other features of mammalian expression can be facilitated as described in Ham and Wallace, Meth Enzymology (1979) 58: AA, Barnes and Sato, Anal Biochem (1980) 702: 255, US Patent No. 4,767,704, No. 4,657,866, No. 4,927,762, No. 4,560,655, PCT Patent Publications WO 90/103430 and WO 87/00195.
  • the invention provides gene delivery vehicles containing a DNA sequence coding for an apoptosis inducing gelsolin amino acid sequence comprising the DNA sequence of SEQ ID NO: 2, from nucleotide 1 through nucleotide 1056 and analogs, derivatives and/or variants of that DNA sequence that are (a) hybridizable under stringent conditions to the sequence of SEQ ID NO: 2, from nucleotide 1 through nucleotide 1056, or which would hybridize under such conditions but for the degeneracy of the genetic code, and (b) code for an amino acid sequence that exhibits the ability to induce apoptosis in cells in a Ca ⁇ + independent manner.
  • stringent conditions we mean conditions of high stringency, for example 6 X SSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, 100 ⁇ g/ml salmon sperm DNA and 15% formamide at 68 degrees C.
  • the gene delivery vehicle may contain a DNA sequence coding for an antisense sequence of the DNA sequence of SEQ ID NO: 2, specifically at least about 15 to 20 contiguous nucleotides from nucleotide 1 through nucleotide 1056.
  • a gene delivery vehicle containing a DNA sequence coding for an apoptosis inducing gelsolin amino acids sequence comprising from amino acid 1 through amino acid 352 of SEQ ID NO: 1.
  • the invention provides gene delivery vehicles containing a DNA sequence coding for the full length gelsolin amino acid sequence (SEQ ID NO: 5) comprising, preferably, the DNA sequence of SEQ ID NO: 6, from nucleotide 1 through nucleotide 2193 and analogs, derivatives and/or variants of that sequence.
  • SEQ ID NO: 5 the DNA sequence coding for the full length gelsolin amino acid sequence
  • SEQ ID NO: 6 the DNA sequence of SEQ ID NO: 6
  • analogs, derivatives and/or variants of that sequence may be employed as tumor cell apoptosis sensitisors, as discussed in greater detail below.
  • the gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, a semiliki forest viral, astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, pico ⁇ iavirus, poxvirus, togavirus or an alpha viral vector.
  • AAV a retroviral, adenoviral, adeno-associated viral
  • Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9.1 (see O'Neill, J. Vir. 53 (1985) 160) polytropic retroviruses (for example, MCF and MCF-MLV (see Kelly, J. Vir. ⁇ 5(1983)291), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.
  • xenotropic retroviruses for example, NZB-X1, NZB-X2 and NZB9.1 (see O'Neill, J. Vir. 53 (1985) 160
  • polytropic retroviruses for example, MCF and MCF-MLV (see Kelly, J. Vir. ⁇ 5(1983)291)
  • retroviral gene therapy vector may be derived from different retroviruses.
  • retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.
  • Retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see US Serial No. 07/800,921, filed November 29, 1991). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle. See, US Serial No. 08/445,466 filed May 22, 1995. It is preferable that the recombinant viral vector is a replication defective recombinant virus.
  • Packaging cell lines suitable for use with the above-described retrovirus vectors are well l ⁇ iown in the art, are readily prepared (see US Serial No. 08/240,030, filed May 9, 1994; see also WO 92/05266), and can be used to create producer cell lines (also termed vector cell lines or "VCLs") for the production of recombinant vector particles.
  • the packaging cell lines are made from human parent cells (e.g., HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.
  • Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Vims, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendothehosis Virus and Rous Sarcoma Virus.
  • Particularly preferred Murine Leukemia Viruses include 4070A and 1504 A (Hartley and Rowe, J Virol 19 (1976) 19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No.
  • Retroviruses may be obtained from depositories or collections such as the American Type Culture Collection (“ATCC”) in Rockville, Maryland or isolated from known sources using commonly available techniques.
  • ATCC American Type Culture Collection
  • Exemplary known retroviral gene therapy vectors employable in this invention include those described in GB 2200651, EP 0415731 , EP 0345242, WO 89/02468; WO 89/05349, WO 89/09271, WO 90/02806, WO 90/07936, WO 90/07936, WO 94/03622, WO 93/25698, WO 93/25234, WO 93/11230, WO 93/10218, WO 91/02805, in U.S. Patent No. 5,219,740, No. 4,405,712, No. 4,861,719, No. 4,980,289 and No. 4,777,127, in U.S. Serial No.
  • Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner, Biotechniques 6 (1988) 616, and Rosenfeld, Science 252 (1991) 431, and PCT Patent Publications WO 93/07283, WO 93/06223, and WO 93/07282.
  • Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in PCT Patent Publications WO 94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95/11984, WO 95/00655, WO 95/27071, WO 95/29993, WO 95/34671, WO 96/05320, WO 94/08026, WO 94/11506, WO 93/06223, WO 94/24299, WO 95/14102, WO 95/24297, WO 95/02697, WO 94/28152, WO 94/24299, WO 95/09241, WO 95/25807, WO 95/05835, WO 94/18922 and WO 95/09654.
  • administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Then 3 (1992)147-154 may be employed.
  • the gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors.
  • AAV adenovirus associated virus
  • Leading and preferred examples of such vectors for use in this invention are the AAV-2 basal vectors disclosed in Srivastava, PCT Patent Publication WO 93/09239.
  • Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides.
  • the native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (i.e., there is one sequence at each end) which are not involved in HP formation.
  • the non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position.
  • Other employable exemplary AAV vectors are pWP-19, pWN-1 , both of which are disclosed in Nahreini, Gene 124 (1993) 257-262.
  • Another example of such an AAV vector is psub201. See Samulski, J. Virol. 61 (1987) 3096.
  • Another exemplary AAV vector is the Double-D ITR vector.
  • Double D ITR vector is disclosed in U.S. Patent No. 5,478,745. Still other vectors are those disclosed in Carter, U.S. Patent No. 4,797,368 and Muzyczka, U.S. Patent No. 5,139,941, Chartejee, U.S. Patent No. 5,474,935, and Kotin, PCT Patent Publication WO 94/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTRneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and how to make it are disclosed in Su, Human Gene Therapy 7 (1996) 463-470. Additional AAV gene therapy vectors are described in US Patent No. 5,354,678, No. 5,173,414, No 5,139,941, and No 5,252,479.
  • the gene therapy vectors of the invention also include herpes vectors.
  • Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Patent 5,288,641 and EP 0176170 (Roizman).
  • herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in PCT Patent Publication WO 95/04139 (Wistar Institute), pHSVlac described in Geller, Science 241 (1988) 1667-1669 and in WO 90/09441 and WO 92/07945, HSV Us3::pgC-lacZ described in Fink, Human Gene Therapy 3 (1992) 11- 19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.
  • alpha virus gene therapy vectors may be employed in this invention.
  • Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. patents 5,091,309, 5,217,879, and WO 92/10578 are exemplary. More particularly, those alpha virus vectors described in U.S. Serial No. 08/405,627, filed March 15, 1995 and U.S.
  • DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acid sequences of the invention. See WO 95/07994 for a detailed description of eukaryotic layered expression systems.
  • the eukaryotic layered expression systems of the invention are derived from alphaviruses vectors and most preferably from Sindbis viral vectors.
  • viral vectors suitable for use in the present invention include those derived from polio virus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin, J. Biol. Standardization 1 (1973) 115; rhinovirus, for example ATCC VR-1110 and those described in Arnold, J Cell Biochem (1990) L401 ; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR- 2010 and those described in Fisher-Hoch, Proc Natl Acad Sci 86 (1989) 317, Flexner, Ann NY Acad Sci 569 (1989) 86, Flexner, Vaccine 8 (1990) 17; in US Patent Nos. 4,603,112 and U.S.
  • measles virus for example ATCC VR-67 and VR-1247 and those described in EP Patent Publication 0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR- 1240; Cabassou vims, for example ATCC VR-922; Chikungunya vims, for example ATCC VR-64 and ATCC VR-1241 ; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo vims, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371 ; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; T ⁇ niti virus, for example ATCC VR-469;
  • compositions of this invention into cells is not limited to the above mentioned viral vectors.
  • other gene delivery vehicles, methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovims alone, for example see US Serial No. 08/366,787, filed December 30, 1994 and Curiel, Hum Gene Ther 3 (1992) 147-154, ligand linked DNA, for example see Wu, J Biol Chem 264 (1989) 16985-16987, eucaryotic cell delivery vehicles cells, for example see US Serial No.08/240,030, filed May 9, 1994, and US Serial No.
  • nucleic acid sequences of the invention can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with syntietic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin.
  • syntietic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin
  • cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin.
  • Naked DNA vectors may be employed. Exemplary naked DNA introduction methods are described in PCT Patent Publications WO 90/11092 and WO 93/03709 and US 5,580,859 and in Roussel., Nature 325 (1987) 549. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.
  • Exemplary liposomes and polycationic gene delivery vehicles are those described in US Patent No. 5,422,120 and No. 4,762,915, in PCT Patent Publications WO 95/13796, WO 94/23697, and WO 91/14445, in EP Patent Publication 0524968 and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco, Szoka, Biochem Biophys Acta 600 (1980) 1, Bayer, Biochem Biophys Acta 550 (1979) 464, Rivnay, Meth Enzymol 149 (1987) 119, Wang, Proc Natl Acad Sci 84 (1987) 7851, Plant, Anal Biochem 176 (1989) 420.
  • the amino acid sequences and/or the gene delivery vehicles of the invention can be incorporated into pharmaceutical compositions for administration to mammalian patients, particularly humans.
  • the amino acid sequences and/or gene delivery vehicles can be formulated in non-toxic, inert, pharmaceutically acceptable aqueous carriers, preferably at a pH ranging from 3 to 8, more preferably ranging from 6 to 8.
  • Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive vimses in particles. Such carriers are well l ⁇ iown to those of ordinary skill in the art.
  • Pharmaceutically acceptable salts can be used, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • Pharmaceutically acceptable carriers may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • compositions may comprise the gene delivery vehicle, for example the vector or virion containing the nucleic acid encoding the therapeutic molecule, dissolved in an aqueous buffer having a acceptable pH upon reconstitution.
  • formulations comprise a therapeutically effective amount of a vector or virion in admixture with a pharmaceutically acceptable carrier and/or excipient, for example saline, phosphate buffered saline, phosphate and amino acid, polymers, polyols, sugar, buffers, preservatives and other proteins.
  • Exemplary amino acids, polymers and sugars and the like are octylphenoxy polyethoxy ethanol compounds, polyethylene glycol monostearate compounds, polyoxyehtylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human semm albumin, citrate, acetate, Ringer's and hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, val;ine, leucine, polyvinylpyrrolidone, polyethylene and glycol.
  • the formulation is stable for at least six months at 4 degrees C.
  • a particularly preferred composition comprises a vector or recombinant vims in 10 mg/ml mannitol, 1 mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl.
  • the recombinant vector represents approximately 1 g of material, it may be less than 1% of high molecular weight material, and less than 1/160,000 of the total material (including water).
  • This composition is stable at -70 degrees C for at least six months.
  • These pharmaceutical compositions comprise yet another aspect of the invention.
  • the pharmaceutical composition can be prepared as a liquid solution or a solid form (e.g., lyophilized) which can be resuspended in a solution prior to administration.
  • the pharmaceutical composition can be formulated into an enteric coated tablet or gel capsule according to l ⁇ iown methods in the art. See for example US Patent No. 4,853,230, EP Patent Publication 0225189, AU Patent Publications 9,224,296 and 9,230,801, and PCT Patent Publication WO 92144,52. Administration may be in vivo or ex vivo.
  • composition containing the gene delivery vehicle can be directly introduced into the patient for expression in the region of the target cells by injection into a body space or an organ or, for example, in the case of a tumor, in the region of the tumor. See US Patent Nos. 5,137,510, 5,213,570, and 5,269,326.
  • ex vivo administration the cells of a patient are removed and transfected with a gene delivery vehicle of the invention. The transfected cells are then reintroduced into the patient and the nucleotide sequence expressed. Cells may be removed from a selected tumor or from an affected organ.
  • the gene delivery vehicle may be transfected into non-tumorigenic cells that have been removed from the patient, including for example, cells from the skin (dermal fibroblasts), or from the blood (e.g., peripheral blood leukocytes) and the cells returned to the patient. If desired, particular fractions of cells such as a T cell subset or stem cells may also be specifically removed from the blood. See, for example, PCT Patent Publication WO 91/16116. Gene delivery vehicles may then be contacted with the removed cells utilizing any of the above-described techniques and the cells tested for incorporation of the gene delivery vehicle, followed by the return of the cells in which the vehicle has been incorporated to the mammal, preferably to or within the vicinity of a tumor.
  • In vivo administration may be by the traditional direct routes, such as buccal/sublingual, rectal, oral, nasal, topical, (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intramuscular, intraperitoneal, subcutaneous, intraocular, intranasal or intravenous, or indirectly.
  • Non-parenteral, i.e., intestinal, routes of administration are specifically contemplated by the invention.
  • the pharmaecutical compositions are prepared as injectables, in liquid solutions or suspensions or in solid forms suitable for solution or suspension in liquid vehicles prior to injection.
  • Injection of the pharmaceutical compositions may be via a variety of routes (e.g., intravenously ("i.v.”), or subcutaneously (“s.c”), intramuscularly (“i.m.”) or preferably when used to treat carcinoma, directly into the tumor site.
  • Direct administration of the injectable pharmaceutical composition may be by various known methods. For example, a small metastatic lesion may be located, and the pharmaceutical composition injected several times in several different locations within the body of tumor. In another example, an organ of the body can be targeted with gelsolin or an gelsolin-derived therapeutic agent for pro-apoptotic or anti-apoptotic effect.
  • arteries which serve a tumor may be identified, and the pharmaceutical composition injected into such an artery, in order to deliver the pharmaceutical composition directly into the tumor.
  • a tumor which has a necrotic center may be aspirated, and the pharmaceutical composition injected directly into the now empty center of the tumor.
  • Tablets or capsules can be administered orally for delivery to the jejunum.
  • expression of the polypeptide, or inhibition of expression by, for example a ribozyme or an antisense oligonucleotide can be measured in the plasma and blood, for example by antibodies to the expressed or non-expressed proteins.
  • the pharmaceutical composition may be directly administered to the surface of the tumor, for example, by application of a formulation containing the gene delivery vehicle, for example, a recombinant retroviral vector containing a DNA sequence of the invention.
  • the dosage regimen will be determined by the attending physician or veterinarian considering various factors known to modify the action of drugs such as, for example, the physical condition of the patient, the severity of the condition, body weight, sex, diet, time of administration and other clinical factors. Generally, the regimen should be in the range of about 10 7 to about 10 10 c.f.u. A preferred dose is about 10 8 to 10 9 c.f.u. Initially, three or four doses are administered at one to four week intervals each. Subsequently, one or two dose booster shots may be given six to twelve months after the end of the initial dosing, and thereafter annually. However, the number of doses administered may vary, depending on the above mentioned factors.
  • compositions of the invention may be administered in combination with other therapeutic agents, such as other anti-tumor or immunomodulatory agents or pro- or anti- apoptotic compositions.
  • Co-administration can be simultaneous, achieved for example by placing nucleotides encoding the agents in the same vector, or by putting the agents, whether nucleotide, polypeptide, or other drug, in the same pharmaceutical composition, or by administering the agents in different pharmaceutical compositions injected at about the same time in substantially the same location. Further, the co-administration can include subsequent administrations as is necessary, for example, repeat in vivo direct injection administrations.
  • compositions of the invention can be used to treat biological or medical conditions in which it is desirable to kill cells by inducing apoptosis, for example in malignant or cancerous conditions, especially those in which the cancerous cells are deficient in gelsolin protein such as in breast cancer or in bladder cancers, in autoimmune diseases such as arthritis, in restenosis, benign prostatic hyperplasia, retinopathy, psoriasis and keloids, in uterine fibroids, in wound healing, in pre-malignant lesions including for example, intestinal polyps, cervical dysplasia, and myeloid dysplasia and in cellular hyperproliferation conditions.
  • autoimmune diseases such as arthritis, in restenosis, benign prostatic hyperplasia, retinopathy, psoriasis and keloids
  • in uterine fibroids in wound healing, in pre-malignant lesions including for example, intestinal polyps, cervical dysplasia, and myeloid dysplasia and in cellular hyperproliferation conditions.
  • the invention provides a method for the treatment of such biological or medical conditions comprising administering to a mammalian patient, preferably a human patient, an apoptosis-inducing amount of a pharmaceutical composition of the invention.
  • apoptosis-inducing amount we mean an amount capable by itself or in conjunction with a secondary therapeutic agent of substantially increasing the amount and/or rate of apopotic cell death in diseased tissue relative to apopotic cell death in normal tissue, resulting in improvement in the treatment profile in hyperproliferative conditions in mammals, especially in humans, in the relative absence of death of disease-free tissue.
  • the invention comprises a method of sensitizing tumor cells to apoptosis comprising administering to tumor cells that are gelsolin deficient a pharmaceutical composition containing a full-length gelsolin amino acid sequence, or an analog, derivative and/or variant of that sequence having amino acid substitution(s), deletion(s) and/or addition(s), which analog, derivative and/or variant possesses the ability to induce apoptosis.
  • the pharmaceutical composition may be administered alone, or used in conjunction with a apoptosis inducing agent such as radiation or chemotherapy.
  • a apoptosis inducing agent such as radiation or chemotherapy.
  • the pharmaceutical composition preferably will be administered first, before the apoptosis inducing agent is administered.
  • the pharmaceutical composition may comprise a gene delivery vehicle containing a DNA sequence encoding a full length gelsolin amino acid sequence, as is described above, followed by administration of one or more chemotherapeutic drugs such as etoposide, camptothecin, 1- ⁇ -D-arabinofuranosylcytosine, doxombucin and vinblastine which are known to induce apoptosis in tumor cells, thereby increasing the therapeutic ratio of the dmg(s) toward gelsolin-deficient cancers.
  • chemotherapeutic drugs such as etoposide, camptothecin, 1- ⁇ -D-arabinofuranosylcytosine, doxombucin and vinblastine which are known to induce apoptosis in tumor cells, thereby increasing the therapeutic ratio of the dmg(s) toward gelsolin-deficient cancers.
  • the nucleic acid and amino acid sequences of the invention may be employed in assays for diagnosing biological conditions in mammals characterized by abnormal gelsolin expression and potential resistance to apoptosis.
  • a probe complementary to mRNA encoding a fragment of a gelsolin amino acid sequence of the invention is contacted with RNA isolated from tissue or cells suspected of abnormal gelsolin expression.
  • Relative expression levels of gelsolin may be determined by quantitative PCR (see Heid, Genome Res 6 (1996) 986-94) or using a bDNA assay.
  • abnormal expression we mean over expression, under expression, or expression of nonfunctional or atypically functional gelsolin protein, as compared to normal gelsolin expression in the same cell type from normal individuals.
  • gelsolin deficient we mean having a cellular expression level significantly lower than that of comparable cells types from normal, healthy individuals when analyzed by Northern, Western or immunohistochemical assays as described in Asch, Cancer Res 56 (1996) 4841 and in Tanaka, Cancer Res 55 (1995) 3228. More specifically, if more than half of the cells are weakly immunoreactive, or if Western analysis using anti-gelsolin antibody shows over about five to ten fold less gelsolin, or if Northern analysis shows over about five to ten fold less gelsolin mRNA, the cells are considered gelsolin deficient. Diagnosis of gelsolin deficiency in certain tissue samples may permit diagnosis of disease, facilitate therapeutic treatment of disease (including gene therapy) and/or allow monitoring of therapeutic efficacy of a given treatment modality.
  • PCR primers or bDNA (branched DNA) probes specific for a gelsolin nucleotide sequence may be used instead of probe-length amino acid fragments and quantitative or qualitative levels of gelsolin RNA may be detected by PCR amplification, RT-PCR (reverse transcription - polymerase chain reaction) or bDNA probe detection.
  • RT-PCR reverse transcription - polymerase chain reaction
  • bDNA probe detection Such a system provides a sensitive method of detection of gelsolin transcription and activity as a means of diagnosing conditions in which abnormal levels in gelsolin expression or activity are implicated.
  • bDNA is described in PCT Patent Publication WO 92/02526 or US Patent No. 5,451,503, and No.
  • RT-PCR is described in Sambrook, Molecular Cloning: a Laboratory Manual, 2d edition (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) (1989), and Ausubel, Current Protocols in Molecular Biology (1994), (Greene Publishing Associates and John Wiley & Sons, New York, N.Y.).
  • gelsolin cleavage at the caspase-specific cleavage site of amino acid 352 is correlated with and precedes apoptopic cell death
  • the presence or absence of apoptosis in cells may be determined by methods that detect the gelsolin cleavage event, including detection of the cleavage fragments or the appearance of neoepitopes generated by the new N- and/or C- terminus.
  • Antibodies to a gelsolin N-terminal neoepitope or C-terminal epitope for example neopitopes comprising the amino acids 346 through 352 of SEQ ID NO: 1 or 1 through 7 of SEQ ID NO: 3 respectively, can be made and employed in a variation of the methods described for the detection of aggrecan cleavage in Bayne, Arthritis and Rheum 38 (1995) 1400; Sakiyama, Int J Cancer 66 (1996) 768; Fossang, J Clin Invest 98 (1996) 2292; Lark, Biochem J 307 (1995) 245 and Mashima, Oncogene 14 (1997) 1007.
  • human tissue or fluids are isolated and treated with cell-lysing methods such as ELISA- compatible detergents in the presence of protease inhibitors, including those that inhibit caspases, and analyzed by methods described by the cited authors, such as ELISA, using anti-neoepitope antibodies.
  • ELISA enzyme-activated immunosorbent assay
  • Detection of gelsolin cleavage products also can be accomplished by standard Western analysis of the same samples, using polyclonal, monoclonal, or neoepitope antibodies. The design of such assays is well within the level of skill in the art.
  • Caspase-3 was able to cleave the murine homolog rapidly, indicating that murine gelsolin is a good substrate for caspase-3.
  • Protein micro-sequencing of the murine cleavage fragment by Edman degradation was used to determine the N-terminal sequence of the cleavage product.
  • the cleavage site between residues Asp 352 and Gly 353 of mouse gelsolin, fits the apparent requirements for efficient cleavage by caspase-3: aspartate residues at PI and P4 and a small side chain residue at PI'. Because the residues at the cleavage site are conserved among the mouse, human and porcine gelsolin proteins, this cleavage site was predicted to occur in human and porcine proteins at the corresponding residues. Cleavage of gelsolin by caspase-3 also was found to result in dissociation of the 45 and 48 kD cleavage products, as assayed by HPLC size exclusion chromatography.
  • the assay uses cells that express a chimeric receptor composed of the extracellular domain and the transmembrane domains of murine CD4 and the cytoplasmic domain of Fas. In this assay, apoptosis is rapidly induced by antibody crosslinking of the extracellular CD4 domains.
  • Example 3 The cleavage product induces apoptosis in neutrophils
  • Neutrophil apoptosis can be enhanced by cross-linking of Fas with anti-Fas antibody or by treatment with TNF and cycloheximide.
  • Increasing the rate of neutrophil apoptosis by treatment with anti-Fas antibodies (CH 11 from Upstate Biotechnology, Lake Placid, NY) or the addition of TNF ⁇ and cycloheximide (10 micrograms/ml TNF and 10 mM cyclohexamide) resulted in a corresponding increase in the rate of gelsolin cleavage and appearance of the 48 kD fragment.
  • Gsn "7" fibroblasts from gelsolin knockout mice were permeablized with ethanol and incubated with gelsolin ( mM) or cleaved gelsolin as described in Huckriede, Cell Motil Cytoskeleton 16 (1990) 229-38. The cells were fixed in 4 % formaldehyde and 0.32 M sucrose for 20 minutes.
  • the cells were washed with cytoskeletonal buffer (10 mM MES, pH 6.1 , 138 mM KC1, 2 mM EGTA) and stained by incubation in 1 mg/ml TRITC- phalloidine in TBS-T(0.15 M NaCl, 0.02 M Tris-Cl, 0.1 % Triton X-160) for 15 minutes.
  • the cell were washed in TBS-T, rinsed in water, then mounted, and visualized using a fluorescence microscope.
  • Cleaved gelsolin could depolymerize the actin cytoskeleton in a calcium- independent manner whereas uncut gelsolin was inactive in the presence of EGTA.
  • the severing activity of gelsolin has been previously localized to the N- terminal 160 amino acid residues of the protein and gelsolin fragments of lengths 1-260 through 1-720 are known to be Ca 2+ regulated as described in Kwiatkowski, J Cell Bio. 108 (1989)1717-26.
  • RF 52 rat embiyo fibroblasts see Joneson, Science 271 (1996) 810) were seeded on acid-treated glass coverslips.
  • 70%o density cells were microinjected with plasmid DNA pGG-NT (encoding residues 1 through 352 of gelsolin and tagged with EYMPME epitope at the C-terminus or with pGG-CT encoding residues 353 through 731 and tagged with EYMPME epitope at the C-terminus) at 0.1 microgram/ml in 100 mM Hepes, pH 7.4.
  • the actin filiments were visualized with TRITC-phalloidin as described in Joneson, supra.
  • Example 5 Construction of a gene therapy vector containing a caspase-3 cleavage product
  • Adenoviral vectors expressing the N-terminal amino acid residues of gelsolin from amino acid 1 through amino acid 352 or expressing the native, full length gelsolin amino acid sequence were constmcted following the methods of Becker, Methods Cell Biol 43 (1994) 161-89 and each tested for apopotic activity in various tumor cells.
  • MOLECULE TYPE DNA (genomic) (ill)
  • HYPOTHETICAL NO (lv)
  • ANTI- SENSE NO
  • GCCCATCCTC CTCGCCTCTT TGCCTGCTCC AACAAGATTG GACGTTTTGT GATCGAAGAG 840
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI- SENSE NO
  • ORIGINAL SOURCE
  • GAGACGGACC CAGCCAATCG GGATCGGCGG ACGCCCATCA CCGTGGTGAA GCAAGGCTTT 2100

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Abstract

Fragments isolés de séquences d'acides aminés de gelsoline à terminaison N qui présentent une activité apoptique et peuvent être utilisés en thérapie génique pour le traitement des états pathologiques malins ou à hyperprolifération et dans des applications diagnostiques pour identifier la régulation à la hausse ou à la baisse de la gelsoline.
PCT/US1998/010181 1997-05-19 1998-05-18 Sequences de gelsoline induisant l'apoptose WO1998052597A1 (fr)

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WO2000063250A1 (fr) * 1999-04-15 2000-10-26 Merck Frosst Canada & Co. Anticorps reconnaissant l'app clivee par les caspases et leurs procedes d'utilisation
EP1116029A1 (fr) * 1998-09-24 2001-07-18 Promega Corporation Anticorps marqueurs d'apoptose et methodes d'utilisations
EP1694866A2 (fr) * 2003-12-12 2006-08-30 Bayer Pharmaceuticals Corporation Profils d'expression geniques et procedes d'utilisation
WO2007079312A2 (fr) * 2005-12-02 2007-07-12 Regents Of The University Of Colorado Compositions et procédés de traitement d’affections pathologiques médiées par l'actine
US9012610B2 (en) 2009-06-29 2015-04-21 Hytest Ltd. Diagnostic kit for IGFBP-4 proteolytic fragments in a patient sample

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WO1991015770A1 (fr) * 1990-04-11 1991-10-17 The General Hospital Corporation Emplois therapeutiques de composes de liaison d'actine

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DOSAKA-AKITA H., ET AL.: "FREQUENT LOSS OF GELSOLIN EXPRESSION IN NON-SMALL CELL LUNG CANCERS OF HEAVY SMOKERS.", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 58., no. 02., 15 January 1998 (1998-01-15), US, pages 322 - 327., XP002910321, ISSN: 0008-5472 *
KOTHAKOTA S., ET AL.: "CASPASE-3-GENERATED FRAGMENT OF GELSOLIN: EFFECTOR OF MORPHOLOGICAL CHANGE IN APOPTOSIS.", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 278., no. 5336., 10 October 1997 (1997-10-10), US, pages 294 - 298., XP002910320, ISSN: 0036-8075, DOI: 10.1126/science.278.5336.294 *
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POLTSCH M. C., ET AL.: "CHARACTERIZATION OF THE ENDOGENOUS DEOXYRIBONUCLEASE INVOLVED IN NUCLEAR DNA DEGRADATION DURING APOPTOSIS (PROGRAMMED CELL DEATH).", EMBO JOURNAL., OXFORD UNIVERSITY PRESS, SURREY., GB, vol. 12., no. 01., 1 January 1993 (1993-01-01), GB, pages 371 - 377., XP002910319, ISSN: 0261-4189 *
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1116029A1 (fr) * 1998-09-24 2001-07-18 Promega Corporation Anticorps marqueurs d'apoptose et methodes d'utilisations
EP1116029A4 (fr) * 1998-09-24 2004-12-15 Promega Corp Anticorps marqueurs d'apoptose et methodes d'utilisations
WO2000063250A1 (fr) * 1999-04-15 2000-10-26 Merck Frosst Canada & Co. Anticorps reconnaissant l'app clivee par les caspases et leurs procedes d'utilisation
EP1694866A2 (fr) * 2003-12-12 2006-08-30 Bayer Pharmaceuticals Corporation Profils d'expression geniques et procedes d'utilisation
EP1694866A4 (fr) * 2003-12-12 2007-12-26 Bayer Pharmaceuticals Corp Profils d'expression geniques et procedes d'utilisation
WO2007079312A2 (fr) * 2005-12-02 2007-07-12 Regents Of The University Of Colorado Compositions et procédés de traitement d’affections pathologiques médiées par l'actine
WO2007079312A3 (fr) * 2005-12-02 2008-02-21 Univ Colorado Compositions et procédés de traitement d’affections pathologiques médiées par l'actine
US9012610B2 (en) 2009-06-29 2015-04-21 Hytest Ltd. Diagnostic kit for IGFBP-4 proteolytic fragments in a patient sample
US9964549B2 (en) 2009-06-29 2018-05-08 Hytest Ltd. Method for detection of IGFBP-4 fragments

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