Gelsolin is a 731 amino acid protein that, in the presence of Ca2, causes gels fonned 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 fonns the gel. See Alberts, Molecular Biology of the Cell (3rd Edition) pp. 837 (Garland Publishing, N.Y. 1994) and Sun, JBiol Chem 269 (1994) 9473-79. Human gelsolin protein has been found in two fonts: extracellularly in plasma and intracellularly in the cytoplasm. The two forms are derived from a single gene by alternative splicing. See Kwiakowski, J Cell Biol 106 (1988) 375-84. Each fonn contains six homologous domains responsible for function.

The cytoplasmic fonn exists with all five of its cysteine residues as free thiols, and the plasma forn1 exists with three of its five cysteine residues as free thiols. See Wen, Biochem 35 (1996) 9700-9709. The extracellular form has been found to sever actin filaments. See U.S. Patent No. 5,464,817. Gelsolin production has been found to be down-regulated in tumors and in several types of transfonned cells. Gelsolin protein and RNA are markedly decreased in human breast cancer cells compared to normal mammary epithelial cells. See Asch, Cancer Res 56 (1996) 4841-45. Gelsolin appears to be a tumor suppressor in human urinary bladder cancer cells. See Tanaka, Cancer Res 553 (1995) 228-32.

We have now discovered that 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-tenninal cleavage product induces apoptosis in mammalian cells.

SUMMARY In one aspect, 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.

In another aspect, 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. In yet another aspect, 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 Cay independent manner.

Phannaceutical 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.

DETAILED DESCRIPTION To identify substrates of caspase-3, we screened translation products of small murine cDNA pools for sensitivity to cleavage by caspase-3 and isolated and sequenced the cDNA clone encoding the cleaved protein. One such protein, gelsolin, was also shown to be rapidly cleaved in cells undergoing apoptosis. Further, a reduced rate of apoptosis in neutrophils from gelsolin lu1ockout mice compared with wild type neutrophils has been observed. The N-tenninal gelsolin cleavage product depolymerizes actin filaments. These results implicate N-terminal caspase-3 cleavage products of gelsolin as effectors in the breakdown of the cytoskeleton during apoptosis, and ultimately as effectors of apoptosis.

Murine gelsolin is predicted to be essentially functionally equivalent to human gelsolin because the two proteins are 94.40/o 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.

It has also been found that caspase-8 (also known as FLICE) 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- terminal 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. We have found that caspase-8 also has the ability to cleave gelsolin at the same location as caspase- 3.

Thus, in one aspect, 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. By "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. Thus, 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 structul-e, 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 detennination may be employed. Altenzatively, a TUNEL assay may be employed. See Strater, JHistochem Cytochel71 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. Ir7zmurzol Methods 119 (1989) 203.

The apoptosis inducing gelsolin fragments of the invention can be constructed employing kiown recombinant DNA techniques or by enzymatic cleavage of the full length gelsolin protein (SEQ ID NO: 5) by caspase-3 or caspase-8.

In another aspect, 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-tenninal amino acid sequence fragments described herein. For instance, 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. These 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+ 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.

It is understood that the 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 lu1own techniques, including for example, in vitro mutagenesis and primer repair. In addition, 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 M13 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 tennination 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 lmown in the art. Introduction of a termination signal can be accomplished, for example, using stop sequences in single or multiple reading frames. With respect to deletion mutations, it is contemplated that 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 CW independent apoptopic activity following the methods described in Example 3.

In a preferred embodiment, 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. Such 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.

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. Sci.(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, JBacteriol (1983) 153:163; Kurtz, Mol Cell Biol (1986) 6: 142; Kunze, J Basic Microbiol. (1985) 25: 151; Gleeson, J Gen. Microbiol (1986) 132: 3459, Roggenkamp, Mol Gen Genet (1986) 202 :302) Das, JBacteriol (1984) 158: 1165; De Louvencourt, JBacteriol (1983)154: 737, Van den Berg, Bio/Technology (1990)8:135; I (unze, J Basic Microbiol (1985)25:141; Cregg, Mol Cell Biol (1985)5: 3376, US Patent No. 4,837,148, US Patent No. 4,929,555; Beach and Nurse, Nature (1981) 300: 706; Davidow, Cur Genet (1985) 10: 380, Gaillardin, Curr Genet (1985) 10: <BR> <BR> <BR> <BR> 49, Ballance, Biochenz Biophys Res Commun (1983) 112: : 284-289; Tilburn, Gene (1983) 26:205-221, Yelton, Proc Natl Acad Sci (1984) 81: 1470-1474, Kelly and Hynes, EMBO J(1985) 4: 475479; EP Patent Publication 244,234, and PCT Patent Publication WO 91/00357.

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, JGen Virol (1988)69: 765-776, Miller, Ann Rev Microbiol (1988) 42:177, Carbonell, Gene (1988) 73: 409, Macda, 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. Numerous baculoviral strains, variants and corresponding permissive insect host cells employable in caning out this invention are described in Luckow, Bio/Technology (1988) 6. 47-55 and in Miller, Genetic Engineering (Setlow, J.K. et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279.

Exemplary mammalian expression systems are described in Dijkeman, EMBOJ(1985) 4: 761, German, 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: 44, Barnes and Sato, Annl Biochem (1980)102255 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.

In another aspect, 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. By "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 Fg/ml salmon sperm DNA and 15% fonnamide at 68 degrees C. See Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 2d ed.

Altematively, the gene delivery vehicle may contain a DNA sequence coding for an <BR> <BR> <BR> <BR> 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. Especially preferred is 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.

In yet another aspect, 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. These gene delivery vehicles containing such full length gelsolin DNA sequences 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, picornavirus, poxvirus, togavirus or an alpha viral vector. See generally, Jolly, Cancer Gene Therapy J (1994) 51-64, Kimura, Small Gene Therapy 5 (1994) 845-852, Conelly, Human Gene Therapy 6 (1995) 185-193 and Kaplitt, Nature Genetics 6 (1994) 148-153.

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 NZB91 (see O'Neill, J. Vir. 53 (1985) 160) polytropic retroviruses (for example, MCF and MCF-MLV (see Kelly, J. Voir. 45(1983)291), spumaviruses and lentiviruses. See RNA tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.

Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Vies, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.

These recombinant 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 known 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.

Preferably, 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 Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis 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. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such 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 teclmiques.

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. 07/800,921 and in Vile, CancerRes 53 (1993) 3860- 3864, Vile, CancerRes 53 (1993) 962-967, Ram, CancerRes 53 (1993) 83-88, Takamiya, JNeurosci Res 33 (1992) 493-503, Baba, JNeurosurg 79 (1993) 729-735, Mann, Cell 33 (1983)153, Cane, Proc NatlAcad Sci 81 (1984) 6349 and Miller, Human Gene Therapy 1 (1990).

Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkuer, 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. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. 3 (1992)147-154 may be employed.

The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors. 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 1 8 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-l 9, 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. How to make the 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 SSV9AFABTKneo, 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). Additional exemplary 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.

We also contemplate that 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. Serial No.08/198,450 and in PCT Patent Publications WO 94/21792, WO 92/10578, WO 95/07994, U.S. Patent Nos. 5,091,309 and 5,217 879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Maryland or isolated from lu1own sources using commonly available techniques.

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. Preferably, the eukaryotic layered expression systems of the invention are derived from alphaviruses vectors and most preferably from Sindbis viral vectors.

Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin, J. Biol. Standardization 1(1973)115; hinovirus, 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 NatlAcad Sci 86 (1989) 317, Flexner, Ann NYAcad Sci 569 (1989) 86, Flexner, Vaccine 8 (1990)17; in US Patent Nos.

4,603,112 and U.S. 4,769,330 and in PCT Patent Publication WO 89/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan, Nature 277 (1979) 108 and Madzak, J Gen Vir 73 (1992)1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in US Patent No. 5,166,057 and in Enami, Proc Natl Acad Sci 87 (1990) 3802-3805, Enami and Palese, J Virol 65 (1991) 2711-2713 and Luytjes, Cell 59(1989)110, (see also McMicheal., NE JMed 309 (1983) 13, and Yap, Nature 273 (1978) 238 and Nature 277 (1979) 108); human immunodeficiency virus as described in EP Patent Publication 0386882 and in Buchschacher , J. Vir. 66 (1992) 2731; 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 virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fart 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 virus, for example ATCC VR-580 and ATCC VR-1244; Ndmnu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre, Proc Soc Exp Bioliwed 121:190 (1966).

Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. As mentioned above, other gene delivery vehicles, methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlined to killed adenovirus 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, JBiol 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. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in US Patent No. 5,149,655, ionizing radiation as described in US Patent No. 5,206,152 and in PCT Patent Publication WO 92/11033, nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip, Mol Cell Biol 14 (] 994) 2411- 2418 and in Voffendin, Proc Natl Acad Sci 91 (1994) 1581-585.

As described in co-owned US Serial Number 60/023,867 on non-viral delivery, the 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. See Wu and Wu, J. Biol.

Chem. 262 (1987) 4429-4432, Hucked, Biochem Pharmacol 40 (1990) 253-263 and Plank, Bioconjugate Chem 3 (1992) 533-539.

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 viruses in particles. Such carriers are well known 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. A thorough discussion of phannaccutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

Phannaceutically 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.

Such pharmaceutical 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.

Such fonnulations comprise a therapeutically effective amount of a vector or virion in admixture with a phannaceutically 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 serum albumin, citrate, acetate, Ringer's and hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, val;ine, leucine, polyvinylpyrrolidone, polyethylene and glycol. Preferably, the formulation is stable for at least six months at 4 degrees C. A particularly preferred composition comprises a vector or recombinant virus in 10 mg/ml mannitol, 1 mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl. In this case, since 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 fonnulated into an enteric coated tablet or gel capsule according to known 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. In in vivo administration, pharmaceutical 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. In 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. Alternatively or in addition, 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. Typically, 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. Alternatively, 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 tumour.

Tablets or capsules can be administered orally for delivery to the jejunum.

At 1 to 4 days following oral administration 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 tumour, 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 107 to about 1010 c.f.u. A preferred dose is about 108 to 109 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.

The pharmaceutical compositions of the invention may be administered in combination with other therapeutic agents, such as other anti-tumor or immunomodulatory agents or probe 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.

The pharmaceutical 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.

Thus, in yet another aspect, 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. By "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.

In a particular embodiment, 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. When used in conjunction with an apoptosis inducing agent, the pharmaceutical composition preferably will be administered first, before the apoptosis inducing agent is administered. While not being bound by theory, it is thought that by priming gelsolin deficient tumor cells with a full-length gelsolin amino acid sequence, such cells will exhibit increased sensitivity to apoptosis-inducing agents such as chemotherapeutic drugs. 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 - P-D-arabinafuranasylcytasine, doxorubucin and vinblastine which are known to induce apoptosis in tumor cells, thereby increasing the therapeutic ratio of the drugs toward gelsolin-deficient cancers.

In addition, knowledge of gelsolin's involvement in apoptotic pathways, and the ability of N-terminal gelsolin amino acid sequences to cause apotosis presents the opportunity to use gelsolin as a template for probe design, and to use the presence or absence of cleavage products of gelsolin or gelsolin activity as a basis for diagnosing apaptosis. For example, lower than normal levels of gelsolin have been observed in cancer cells, as described in Tanaka, Can Res 55 (1995) 3228, and Kozimaki, Hokkaido Igalca Zasshi 71 (1996) 133.

Thus, 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 apaptosis. In such assays, 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. By "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. By "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 immunohistachemical 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.

For example, where gelsolin is down regulated as compared to controls, a lower level of probe to transcript hybridization will be observed. Alternatively, 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. 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. 4,775,619. 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.).

Additional tests may employ the use of antibodies. Because 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 J307 (1995) 245 and Mashima, Oncogene 14 (1997) 1007. Briefly, 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. 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.

The invention will now be exemplified by reference to the following, non- limiting, examples.

Example 1: Isolation of Caspase-3 Cleavage Products We screened for substrates of caspase-3 in small pools of 35S-methionine labeled proteins made by in vitro translation of murine cDNA. After in vitro translation, the reaction mix was divided into two parts, one half was treated with caspase-3 inactivated by adding the irreversible peptide fluaromethylketane inhibitor, DEVD-fink, and the other half was treated with active caspase-3. The reaction products were resolved by SDS-PAGE, and targets of caspase-3 were identified by comparing the pattern of labeled proteins in the lanes treated with active caspase-3 and inactive caspase-3. In the majority of the translated cDNA pools, no caspase-3 sensitive bands were observed.

However, in three pools a 65 l<Da protein band had a reduced intensity and a new band was seen at 48 ludo, indicating that the 65 kDa protein was cleaved by caspase-3 to generate the 48 kDa fragment.

To avoid false positives, we titrated the amount of enzyme used based on the activity required to cleave 80 % of a baculovirus protein p35 translation product that is a good substrate of caspase-3. A total of 1600 murine cDNA pools representing approximately 160, 000 independent cDNA clones were screened. Five pools contained products cleavable by caspase-3. The cDNAs from these positive pools were used to transform E. coli, and the cDNA clones from single colonies were screened again to identify the cDNA corresponding to the target protein. The cDNA from three of the positive pools were shown by DNA sequencing and PCR analyses to be clones corresponding to the partial nucleotide sequence of gelsolin from residues 141-731.

The efficiency of cleavage of gelsolin compared well with that of p35.

Gelsolin was also cleaved by cell extracts prepared from apoptotic cells, but not cleaved by cell extracts prepared from normal cells.

Example 2: Characterization of the cleavage To further characterize the cleavage of gelsalin, we tested the ability of purified recombinant caspase-3 to cleave the purified, full-length, recombinant murine gelsolin protein produced in bacteria following the method of Witke, Cell 81 (1995) 41- <BR> <BR> <BR> <BR> 51. . 13.5 micrograms of full length murine gelsolin was incubated with 0. 15 micrograms of caspase-3 and buffer A (6 mM Tris-HCl, pH 7.5, 1.2 mM CaCl2, 5 mM DTT, 1.5 mM MgCl2, 1 mM KCl) in a reaction volume of 25 microliters at 37 degrees for 30 minutes.

At time intervals of 0.1, 10 and 30 minutes, two microliter aliquots of this reaction mixture were inactivated in SDS-PAGE sample buffer and subsequently resolved on an SDS polyacrylamide gel and stained with Coomassie blue. At time 0, a single strong band is seen at 86 kD, representing the full length gelsalin. At 0.1 minute, a band is seen at 86 kD and two faint bands at 52 and 48 kD are seen. At 10 and again at 30 minutes, the band at 86 kD has disappeared and two strong bands at 52 and 48 kD are seen, indicating the N- terminal and C-terminal gelsolin fragments.

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 ofthe cleavage product. The cleavage site, between residues Asp 352 and Gly 353 of mouse gelsalin, fits the apparent requirements for efficient cleavage by caspase-3: aspartate residues at P1 and P4 and a small side chain residue at P1'. 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.

We used an inducible system to test whether gelsolin is cleaved when cells undergo apoptosis employing the protocols described in Chu, Proc Natl Acad Sci 92 (1995) 11894-98. Briefly, 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. Cleavage of gelsolin (into the predicted 48 and 45 kD bands) is observed 30 min after inducing apoptosis apparently preceding the cleavage of poly-ADP ribose polymerase (PARP) a well characterized apoptotic substrate of caspase. Further, both gelsolin and PARP cleavage are blocked by the cell-permeable inhibitor of caspase-3, zVAD-fmk. The cleavage observed here is specific to PARP (poly- ADP-ribose polymerase) and gelsalin. Another cytaskeletal protein, filamin, is not cleaved in the same assay.

The results suggest that gelsolin is cleaved by a caspase-3-like enzyme in vivo, and this cleavage is an early step in Fas-mediated apoptasis.

Example 3: The cleavage product induces apoptosis in neutrophils Neutrophils purified from human blood express high levels of gelsolin and undergo spontaneous apaptasis. We used purified human neutrophils to determine if gelsolin is cleaved during apoptasis. Human neutrophils were isolated using the neutrophil isolation medium (Cardinal Associates) and resuspended in RPMI with 10% fetal calf serum. See Martin, JExp Med 182 (1995) 1545-56. Equal numbers of cells were used to prepare cell lysates. The lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-gelsolin monoclonal antibody (Sigma Chemicals), which detects only one of the cleaved fragments, the 48 Kd fragment corresponding to amino acids 353- 731. In untreated neutrophils that undergo apoptosis spontaneously, there is a decrease in the levels of gelsolin with time and the appearance of a 48 kDa fragment after incubation.

A similar size (48 kDa) fragment was observed when purified gelsolin was cleaved with caspase-3.

Neutrophil apoptosis can be enhanced by cross-linking of Fas with anti-Fas antibody or by treatment with TNFa 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 TNFa 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.

These observations suggest that the cleavage of gelsolin observed in vitro also occurs in the normal course of apoptosis in neutrophils.

Example 4: The cleavage product severs actin polymers To determine the functional significance of gelsolin cleavage we examined the activities of cleaved and native gelsolin using pyrene-actin fluorometry following the protocol of Kwaitkowski, J Cell Biol 108 (1989) 1717-26. The cytoskeleton analyses were conducted as follows: pyrene-labeled actin (0.5 mM) and gelsolin (20 nM) cut or uncut with caspase-3 was incubated in buffer F(2 mM Tris-Cl, pH 7.5,0.5 mM ATP. 0.2 mM CaCl2, 0.2 mM DTT, 2 mM MgCl2 and 150 mM KCl). The changes in pyrene fluorescence over time was quantified using a standard fluorometer as described Kwaitkowski, supra. As shown previously, native gelsolin severs actin polymers in a Ca2+ dependent manner, whereas we observed that caspase-3 cleaved gelsolin severed actin polymers independent of Ca2+. In addition, in co-incubation experiments, the cleaved gelsolin preferentially severed actin filaments rather than binding to monomeric actin, suggesting that cleaved gelsolin would sever actin filaments in vivo.

We next used permeablized cells to determine the ability of cleaved gelsolin to depolymerize the cytaskeletal actin filaments. Gsn-' fibroblasts from gelsolin knockout mice (see Witke, Cell 81 (1995) 41-51) were permeablized with ethanol and incubated with gelsolin( mM) or cleaved gelsolin as described in Huckriede, Cell Motil Cytoskeleton 1 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 KCl, 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 Ca2+ regulated as described in Kwiatkowski, J Cell Bio.

108 (1989)1717-26. To determine if the N-terminal fragment of gelsolin generated by caspase-3 cleavage could depolymerize actin, we microinjected DNA encoding the N- terminal fragment (amino acids 1 through 352) or the C-tenninal fragment (amino acids 353 through 731) into fibroblasts. RF 52 rat embryo fibroblasts (see Joneson, Science 271 (1996) 810) were seeded on acid-treated glass coverslips. 70% density cells were microinjected with plasmid DNA pGG-NT (encoding residues 1 through 352 of gelsolin and tagged with EYMPME epitope at the C-tenninus 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.

Expression of the N-terminal fragment caused a rapid depolymerization of the actin cytoskeleton whereas expression of the C-terminal fragment had no effect on actin filaments.

Example 5: Construction of a gene therapy vector containing a caspase-3 cleavage product Adenoviral vectors expressing the N-tenninal amino acid residues of gelsolin from amino acid 1 through amino acid 352 or expressing the native, full length gelsolin amino acid sequence (amino acids 1 through 731) were constructed following the methods of Becker, Methods Cell Biol 43 (1994) 161-89 and each tested for apopotic activity in various tumor cells.

In A7 melanoma cells, upon direct microscopic observation, the adenovirus vector expressing the N-tenninal gelsolin fragment caused rapid cell death, whereas the full length construct had no effect. These results were confirmed using a TUNEL assay.

See Strater, supra. Similar results were observed with the same vector in other cell types, including M2 melanoma cells and NIH3T3 cells.

Example 6: Absence of the cleavage product decreases the rate of neutrophil cell death in gelsolin null mice To examine the effects of caspase-cleaved gelsolin in vivo, we compared the rate of cell death in neutrophils isolated from gelsolin null mice (gsn4) with cells from wild type litter mates. Mice were injected intraperitonealy with thioglycolate, and an inflammmatory exudate that consisted primarily of neutrophils (>95 %) was collected after 5 hr. The neutrophils were incubated with TNF (10 ng/ml) and cycloheximide (10 mg/ml) or with anti-Fas antibodies (500 ng/ml). The rate of cell death as measured by DNA fragmentation was monitored by the TUNEL assay (see Strater, supra). The results are shown below.

TNF + Cvcloheximide Treatment Time (hr.) Wild Type Gsw' 0 5.4 5.7 4 36.8 8.6 8 87.6 15.7 12 90.1 72.6 Fas Treatment Time (hr.) Wild Type Gsn4 0 5.5 5.7 2 37.6 8.7 4 49.6 1.8 6 91.7 72.6 The rate of cell death after treatment with Fas and TNF/cycloheximide was slower in the Gsn-/-neutrophils compared to the Gsn+/+ neutrophils, particularly at early time points. These observations were confirmed by direct DNA analysis, which showed slower DNA fragmentation in Gsn-1 neutrophils. These results appear to explain a previously unexplained observation - - gelsolin null mice contain twice the number of neutrophils as wild type mice.

The lack of gelsolin decreases the rate of neutrophil cell death in gelsolin null mice, supporting the possibility that caspase-3 activation of gelsolin's actin depolymerizaton activity is important for rapid apoptasis.

Any deposit referenced herein is provided as convenience to those of skill in the at, and is not an admission that a deposit is required under 35 U.S.C. 112. The nucleic acid sequence of the deposit, as well as the amino acid sequence of the polypeptide encoded thereby, are incorporated herein by reference and should be referred to in the event of an error in the sequence described herein. A license may be required to make, use, or sell the deposited materials, and no such license is granted hereby. All patents, patent applications, patent publications and scientific articles mentioned in this specification are hereby incorporated by reference for the substance of what they disclose.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Koths, Kirston Kothakota, Srinivas Williams, Louis T.

Reinhart, Christoph (ii) TITLE OF INVENTION: APOPTOSIS-INDUCING GELSOLIN SEQUENCES (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Chiron Corporation Intellecutal Property - R440 (B) STREET: PO Box 8097 (C) CITY: Emeryville (D) STATE: CA (E) COUNTRY: USA (F) ZIP: 94662-8097 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: MS-DOS/WINDOWS95 (D) SOFTWARE: PatentIn Release &num 1.0, Version &num 1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: To Be Assigned (B) FILING DATE: MAY 19, 1997 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Cserr, Luann (B) REGISTRATION NUMBER: 31,822 (C) REFERENCE/DOCKET NUMBER: 1385.001 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 510-834-1448 (B) TELEFAX: 510-839-7810 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 352 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: M V V S H P E F L K A G K E P G 1 5 10 15 L Q I W R V E K F D L V P V P T 20 25 30 <BR> <BR> <BR> <BR> <BR> <BR> <BR> N L Y G D F F T C D A Y V I L K 35 40 45 T V Q L R N G N L Q Y D L H Y W 50 55 60 <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> L G N E C S Q D E S G A A A I F 65 70 75 80 <BR> <BR> <BR> <BR> <BR> <BR> <BR> T V Q L D D Y L N G S A V Q H R 85 90 95 E V Q G F E S T F L G Y F K S 100 105 110 <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> G L K Y K K G G V A S G F K H V 115 120 125 V P N E V V V Q R L F Q V K G R 130 135 140 R V V R A T E V P V S W E S F N 145 150 155 160 N G D C F I L D L G N N I H Q W 165 170 175 C G S N S N R Y E R L K A T Q V 180 185 190 <BR> <BR> <BR> <BR> <BR> <BR> <BR> S K G I R D N S R S G R A R V H 195 200 205 <BR> <BR> <BR> <BR> <BR> <BR> <BR> V S E E G T E P E A M L Q V L G 210 215 220 P K P A L P A G T E D T A K E D 225 230 235 240 A A N R K L A K L Y K V S N G A 245 250 255 <BR> <BR> <BR> <BR> <BR> <BR> <BR> G T M S V S L V A D E N P F A Q 260 265 270 <BR> <BR> <BR> <BR> <BR> <BR> <BR> G A L K S E D C F I L D H G K D 275 280 285 G K I F V W K G K Q A N T E E R 290 295 300 K A A L K T A S D F I T K M D Y 305 310 315 320 P K Q T Q V S V L P E G G E T P 325 330 335 L F K Q F F K N W R D P D Q T D 340 345 350 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1056 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (B) STRAIN: (ix) FEATURE: (A) NAME/KEY: (B) LOCATION: (D) OTHER INFORMATION: N-terminal human gelsolin (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ATGGTGGTGG AACACCCCGA GTTCCTCAAG GCAGGGAAGG AGCCTGGCCT GCAGATCTGG 60 CGTGTGGAGA AGTTCGATCT GGTGCCCGTG CCCACCAACC TTTATGGAGA CTTCTTCACG 120 GGCGACGCCT ACGTCATCCT GAAGACAGTG CAGCTGAGGA ACGGAAATCT GCAGTATGAC 180 CTCCACTACT GGCTGGGCAA TGAGTGCAGC CAGGATGAGA GCGGGGCGGC CGCCATCTTT 240 ACCGTGCAGC TGGATGACTA CCTGAACGGC CGGGCCGTGC AGCACCGTGA GGTCCAGGGC 300 TTCGAGTCGG CCACCTTCCT AGGCTACTTC AAGTCTGGCC TGAAGTACAA GAAAGGAGGT 360 GTGGCATCAG GATTCAAGCA CGTGGTACCC AACGAGGTGG TGGTGCAGAG ACTCTTCCAG 420 GTCAAAGGGC GGCGTGTGGT CCGTGCCACC GAGGTACCTG TGTCCTGGGA GAGCTTCAAC 480 AATGGCGACT GCTTCATCCT GGACCTGGGC AACAACATCC ACCAGTGGTG TGGTTCCAAC 540 AGCAATCGGT ATGAAAGACT GAAGGCCACA CAGGTGTCCA AGGGCATCCG GGACAACGAG 600 CGGAGTGGCC GGGCCCGAGT GCACGTGTCT GAGGAGGGCA CTGAGCCCGA GGCGATGCTC 660 CAGGTGCTGG GCCCCAAGCC GGCTCTGCCT GCAGGTACCG AGGACACCGC CAAGGAGGAT 720 GCGGCCAACC GCAAGCTGGC CAAGCTCTAC AAGGTCTCCA ATGGTGCAGG GACCATGTCC 780 GTCTCCCTCG TGGCTGATGA GAACCCCTTC GCCCAGGGGG CCCTGAAGTC AGAGGACTGC 840 TTCATCCTGG ACCACGGCAA AGATGGGAAA ATCTTTGTCT GGAAAGGCAA GCAGGCAAAC 900 ACGGAGGAGA GGAAGGCTGC CCTCAAAACA GCCTCTGACT TCATCACCAA GATGGACTAC 960 CCCAAGCAGA CTCAGGTCTC GGTCCTTCCT GAGGGCGGTG AGACCCCACT GTTCAAGCAG 1020 TTCTTCAAGA ACTGGCGGGA CCCAGACCAG ACAGAT 1056 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 379 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: G L G L S Y L S S H I A N V E R 1 5 10 15 V P F D A A T L H T S T A M A A 20 25 30 Q H G M D D D G T G Q K Q I W R 35 40 45 I E G S N K V P V D P A T Y G Q 50 55 60 F Y G G D S Y I I L Y N Y R H G 65 70 75 80 G R Q G Q I I Y N W Q G A Q S T 85 90 95 Q D E V A A S A I L T A Q L D E 100 105 110 E L G G T P V Q S R V V Q G K E 115 120 125 P A H L M S L F G G K P M I I Y 130 135 140 K G G T S R E G G Q T A P A S T 145 150 155 160 <BR> <BR> <BR> <BR> <BR> <BR> R L F Q V R A N S A G A T R A V 165 170 175 <BR> <BR> <BR> <BR> <BR> <BR> E V L P K A G A L N S N D A F V 180 185 190 <BR> <BR> <BR> <BR> <BR> <BR> L K T P S A A Y L W V G T G A S 195 200 205 <BR> <BR> <BR> <BR> <BR> <BR> E A E K T G A Q E L L R V L R A 210 215 220 <BR> <BR> <BR> <BR> <BR> <BR> Q P V Q V A E G S E P D G F W E 225 230 235 240 A L G G K A A Y R T S P R L K D 245 250 255 <BR> <BR> <BR> <BR> <BR> <BR> K K M D A H P P R L F A C S N K 260 265 270 <BR> <BR> <BR> <BR> <BR> <BR> I G R F V I E E V P G E L M Q E 275 280 285 D L A T D D V M L L D T W D Q V 290 295 300 <BR> <BR> <BR> <BR> <BR> <BR> F V W V G K D S Q E E E K T E A 305 310 315 320 L T S A K R Y I E T D P A N R D 325 330 335 <BR> <BR> <BR> <BR> <BR> <BR> R R T P I T V V K Q G F E P P S 340 345 350 <BR> <BR> <BR> <BR> <BR> <BR> F V G W F L G W D D D Y W S V D 355 360 365 P L D R A M A E L A A 370 375 379 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1137 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (B) STRAIN: (ix) FEATURE: (A) NAME/KEY: (B) LOCATION: (D) OTHER INFORMATION: C-terminal human gelsolin (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GGCCTGGGCT TGTCCTACCT TTCCAGCCAT ATCGCCAACG TGGAGCGGGT GCCCTTCGAC 60 GCCGCCACCC TGCACACCTC CACTGCCATG GCCGCCCAGC ACGGCATGGA TGACGATGGC 120 ACAGGCCAGA AACAGATCTG GAGAATCGAA GGTTCCAACA AGGTGCCCGT GGACCCTGCC 180 ACATATGGAC AGTTCTATGG AGGCGACAGC TACATCATTC TGTACAACTA CCGCCATGGT 240 GGCCGCCAGG GGCAGATAAT CTATAACTGG CAGGGTGCCC AGTCTACCCA GGATGAGGTC 300 GCTGCATCTG CCATCCTGAC TGCTCAGCTG GATGAGGAGC TGGGAGGTAC CCCTGTCCAG 360 AGCCGTGTGG TCCAAGGCAA GGAGCCCGCC CACCTCATGA GCCTGTTTGG TGGGAAGCCC 420 ATGATCATCT ACAAGGGCGG CACCTCCCGC GAGGGCGGGC AGACAGCCCC TGCCAGCACC 480 CGCCTCTTCC AGGTCCGCGC CAACAGCGCT GGAGCCACCC GGGCTGTTGA GGTATTGCCT 540 AAGGCTGGTG CACTGAACTC CAACGATGCC TTTGTTCTGA AAACCCCCTC AGCCGCCTAC 600 CTGTGGGTGG GTACAGGAGC CAGCGAGGCA GAGAAGACGG GGGCCCAGGA GCTGCTCAGG 660 GTGCTGCGGG CCCAACCTGT GCAGGTGGCA GAAGGCAGCG AGCCAGATGG CTTCTGGGAG 720 GCCCTGGGCG GGAAGGCTGC CTACCGCACA TCCCCACGGC TGAAGGACAA GAAGATGGAT 780 GCCCATCCTC CTCGCCTCTT TGCCTGCTCC AACAAGATTG GACGTTTTGT GATCGAAGAG 840 GTTCCTGGTG AGCTCATGCA GGAAGACCTG GCAACGGATG ACGTCATGCT TCTGGACACC 900 TGGGACCAGG TCTTTGTCTG GGTTGGAAAG GATTCTCAAG AAGAAGAAAA GACAGAAGCC 960 TTGACTTCTG CTAAGCGGTA CATCGAGACG GACCCAGCCA ATCGGGATCG GCGGACGCCC 1020 ATCACCGTGG TGAAGCAAGG CTTTGAGCCT CCCTCCTTTG TGGGCTGGTT CCTTGGCTGG 1080 GATGATGATT ACTGGTCTGT GGACCCCTTG GACAGGGCCA TGGCTGAGCT GGCTGCC 1137 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 731 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: <BR> <BR> <BR> <BR> <BR> <BR> M V V E H P E F L K A G K E P G 1 5 10 15 L Q I W R V E K F D L V P V P T 20 25 30 <BR> <BR> <BR> <BR> <BR> N L Y G D F F T G D A Y V I L K 35 40 45 <BR> <BR> <BR> <BR> <BR> T V Q L R N G N L Q Y D L H Y W 50 55 60 <BR> <BR> <BR> <BR> <BR> L G N E C S Q D E S G A A A I F 65 70 75 80 <BR> <BR> <BR> <BR> <BR> T V Q L D D Y L N G R A V Q H R 85 90 95 <BR> <BR> <BR> <BR> <BR> E V Q G F E S A T F L G Y F K S 100 105 110 <BR> <BR> <BR> <BR> <BR> G L K Y K K G G V A S G F K H V 115 120 125 <BR> <BR> <BR> <BR> <BR> V P N E V V V Q R L F Q V K G R 130 135 140 R V V R A T E V P V S W E S F N 145 150 155 160 <BR> <BR> <BR> <BR> <BR> N G D C F I L D L G N N I H Q W 165 170 175 <BR> <BR> <BR> <BR> <BR> C G S N S N R Y E R L K A T Q V 180 185 190 S K G I R D N E R S G R A R V H 195 200 205 V S E E G T E P E A M L Q V L G 210 215 220 P K P A L P A G T E D T A K E D 225 230 235 240 <BR> <BR> <BR> <BR> <BR> <BR> A A N R K L A K L Y K V S N G A 245 250 255 <BR> <BR> <BR> <BR> <BR> <BR> G T M S V S L V A D E N P F A Q 260 265 270 <BR> <BR> <BR> <BR> <BR> <BR> G A L K S E D C F I L D H G K D 275 280 285 <BR> <BR> <BR> <BR> <BR> <BR> G K I F V W K G K Q A N T E E R 290 295 300 K A A L K T A S D F I T K M D Y 305 310 315 320 P K Q T Q V S V L P E G G E T P 325 330 335 <BR> <BR> <BR> <BR> <BR> <BR> L F K Q F F K N W R D P D Q T D 340 345 350 <BR> <BR> <BR> <BR> <BR> <BR> G L G L S Y L S S H I A N V E R 355 360 365 V P F D A A T L H T S T A M A A 370 375 380 <BR> <BR> <BR> <BR> <BR> <BR> Q H G M D D D G T G Q K Q I W R 385 390 395 400 <BR> <BR> <BR> <BR> <BR> <BR> I E G S N K V P V D P A T Y G Q 405 410 415 F Y G G D S Y I I L Y N Y R H G 420 425 430 <BR> <BR> <BR> <BR> <BR> <BR> G R Q G Q I I Y N W Q G A Q S T 435 440 445 <BR> <BR> <BR> <BR> <BR> <BR> Q D E V A A S A I L T A Q L D E 450 455 460 <BR> <BR> <BR> <BR> <BR> <BR> E L G G T P V Q S R V V Q G K E 465 470 475 480 <BR> <BR> <BR> <BR> <BR> <BR> P A H L M S L F G G K P M I I Y 485 490 495 K G G T S R E G G Q T A P A S T 500 505 510 R L F Q V R A N S A G A T R A V 515 520 525 <BR> <BR> <BR> <BR> <BR> E V L P K A G A L N S N D A F V 530 535 540 <BR> <BR> <BR> <BR> <BR> <BR> L K T P S A A Y L W V G T G A S 545 550 555 560 <BR> <BR> <BR> <BR> <BR> E A E K T G A Q E L L R V L R A 565 570 575 <BR> <BR> <BR> <BR> <BR> Q P V Q V A E G S E P D G F W E 580 585 590 <BR> <BR> <BR> <BR> <BR> A L G G K A A Y R T S P R L K D 595 600 605 K K M D A H P P R L F A C S N K 610 615 620 <BR> <BR> <BR> <BR> <BR> I G R F V I E E V P G E L M Q E 625 630 635 640 D L A T D D V M L L D T W D Q V 645 650 655 <BR> <BR> <BR> <BR> <BR> F V W V G K D S Q E E E K T E A 660 665 670 L T S A K R Y I E T D P A N R D 675 680 685 <BR> <BR> <BR> <BR> <BR> R R T P I T V V K Q G F E P P S 690 695 700 <BR> <BR> <BR> <BR> <BR> F V G W F L G W D D D Y W S V D 705 710 715 720 P L D R A M A E L A A 725 730 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2193 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (B) STRAIN: (ix) FEATURE: (A) NAME/KEY: (B) LOCATION: (D) OTHER INFORMATION: full length human gelsolin (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATGGTGGTGG AACACCCCGA GTTCCTCAAG GCAGGGAAGG AGCCTGGCCT GCAGATCTGG 60 CGTGTGGAGA AGTTCGATCT GGTGCCCGTG CCCACCAACC TTTATGGAGA CTTCTTCACG 120 GGCGACGCCT ACGTCATCCT GAAGACAGTG CAGCTGAGGA ACGGAAATCT GCAGTATGAC 180 CTCCACTACT GGCTGGGCAA TGAGTGCAGC CAGGATGAGA GCGGGGCGGC CGCCATCTTT 240 ACCGTGCAGC TGGATGACTA CCTGAACGGC CGGGCCGTGC AGCACCGTGA GGTCCAGGGC 300 TTCGAGTCGG CCACCTTCCT AGGCTACTTC AAGTCTGGCC TGAAGTACAA GAAAGGAGGT 360 GTGGCATCAG GATTCAAGCA CGTGGTACCC AACGAGGTGG TGGTGCAGAG ACTCTTCCAG 420 GTCAAAGGGC GGCGTGTGGT CCGTGCCACC GAGGTACCTG TGTCCTGGGA GAGCTTCAAC 480 AATGGCGACT GCTTCATCCT GGACCTGGGC AACAACATCC ACCAGTGGTG TGGTTCCAAC 540 AGCAATCGGT ATGAAAGACT GAAGGCCACA CAGGTGTCCA AGGGCATCCG GGACAACGAG 600 CGGAGTGGCC GGGCCCGAGT GCACGTGTCT GAGGAGGGCA CTGAGCCCGA GGCGATGCTC 660 CAGGTGCTGG GCCCCAAGCC GGCTCTGCCT GCAGGTACCG AGGACACCGC CAAGGAGGAT 720 GCGGCCAACC GCAAGCTGGC CAAGCTCTAC AAGGTCTCCA ATGGTGCAGG GACCATGTCC 780 GTCTCCCTCG TGGCTGATGA GAACCCCTTC GCCCAGGGGG CCCTGAAGTC AGAGGACTGC 840 TTCATCCTGG ACCACGGCAA AGATGGGAAA ATCTTTGTCT GGAAAGGCAA GCAGGCAAAC 900 ACGGAGGAGA GGAAGGCTGC CCTCAAAACA GCCTCTGACT TCATCACCAA GATGGACTAC 960 CCCAAGCAGA CTCAGGTCTC GGTCCTTCCT GAGGGCGGTG AGACCCCACT GTTCAAGCAG 1020 TTCTTCAAGA ACTGGCGGGA CCCAGACCAG ACAGATGGCC TGGGCTTGTC CTACCTTTCC 1080 AGCCATATCG CCAACGTGGA GCGGGTGCCC TTCGACGCCG CCACCCTGCA CACCTCCACT 1140 GCCATGGCCG CCCAGCACGG CATGGATGAC GATGGCACAG GCCAGAAACA GATCTGGAGA 1200 ATCGAAGGTT CCAACAAGGT GCCCGTGGAC CCTGCCACAT ATGGACAGTT CTATGGAGGC 1260 GACAGCTACA TCATTCTGTA CAACTACCGC CATGGTGGCC GCCAGGGGCA GATAATCTAT 1320 AACTGGCAGG GTGCCCAGTC TACCCAGGAT GAGGTCGCTG CATCTGCCAT CCTGACTGCT 1380 CAGCTGGATG AGGAGCTGGG AGGTACCCCT GTCCAGAGCC GTGTGGTCCA AGGCAAGGAG 1440 CCCGCCCACC TCATGAGCCT GTTTGGTGGG AAGCCCATGA TCATCTACAA GGGCGGCACC 1500 TCCCGCGAGG GCGGGCAGAC AGCCCCTGCC AGCACCCGCC TCTTCCAGGT CCGCGCCAAC 1560 AGCGCTGGAG CCACCCGGGC TGTTGAGGTA TTGCCTAAGG CTGGTGCACT GAACTCCAAC 1620 GATGCCTTTG TTCTGAAAAC CCCCTCAGCC GCCTACCTGT GGGTGGGTAC AGGAGCCAGC 1680 GAGGCAGAGA AGACGGGGGC CCAGGAGCTG CTCAGGGTGC TGCGGGCCCA ACCTGTGCAG 1740 GTGGCAGAAG GCAGCGAGCC AGATGGCTTC TGGGAGGCCC TGGGCGGGAA GGCTGCCTAC 1800 CGCACATCCC CACGGCTGAA GGACAAGAAG ATGGATGCCC ATCCTCCTCG CCTCTTTGCC 1860 TGCTCCAACA AGATTGGACG TTTTGTGATC GAAGAGGTTC CTGGTGAGCT CATGCAGGAA 1920 GACCTGGCAA CGGATGACGT CATGCTTCTG GACACCTGGG ACCAGGTCTT TGTCTGGGTT 1980 GGAAAGGATT CTCAAGAAGA AGAAAAGACA GAAGCCTTGA CTTCTGCTAA GCGGTACATC 2040 GAGACGGACC CAGCCAATCG GGATCGGCGG ACGCCCATCA CCGTGGTGAA GCAAGGCTTT 2100 GAGCCTCCCT CCTTTGTGGG CTGGTTCCTT GGCTGGGATG ATGATTACTG GTCTGTGGAC 2160 CCCTTGGACA GGGCCATGGC TGAGCTGGCT GCC 2193