WO1997042211A1 - GENE MAMMALIEN Scm A EFFET SUPPRESSEUR DE TUMEUR - Google Patents

GENE MAMMALIEN Scm A EFFET SUPPRESSEUR DE TUMEUR Download PDF

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Publication number
WO1997042211A1
WO1997042211A1 PCT/US1997/007575 US9707575W WO9742211A1 WO 1997042211 A1 WO1997042211 A1 WO 1997042211A1 US 9707575 W US9707575 W US 9707575W WO 9742211 A1 WO9742211 A1 WO 9742211A1
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seq
mammauan
sem
sequence
leu
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PCT/US1997/007575
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English (en)
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Filippo Randazzo
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Chiron Corporation
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Priority to EP97926403A priority Critical patent/EP0915904A4/fr
Priority to AU31175/97A priority patent/AU3117597A/en
Priority to JP09540119A priority patent/JP2000510693A/ja
Publication of WO1997042211A1 publication Critical patent/WO1997042211A1/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
    • C07K14/4745—Cancer-associated SCM-recognition factor, CRISPP
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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

Definitions

  • the invention relates to a gene, mammalian sex comb on midleg (mammalian Son), implicated in proliferative disorders, including malignancies, and in developmental processes.
  • Cancer and malignancy therapies have included treatment with chemical toxins, radiation, and surgery.
  • Genes known to be over-expressed or underexpressed in cancer are used for diagnosis of the disease and evaluation of a patient's progression with the disease and treatment.
  • transcription factors direct development along a particular pathway by activating genes of a differentiated phenotype. Differentiation can involve not only changes in patterns of expressed genes, but also involve the maintenance of those new patterns.
  • an isolated mammalian Sem (mammalian Sem) polypeptide comprising a sequence of at least 54 consecutive amino acids of a sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO.4, and SEQ ID NO. 6.
  • an isolated nucleic acid molecule is provided.
  • the nucleic acid molecule encodes a polypeptide having a sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO.4, and SEQ ID NO. 6.
  • an isolated nucleic acid molecule is provided which comprises at least 30 contiguous nucleotides selected from the group of sequences consisting of SEQ ID NO: 1, SEQ ID NO:3, AND SEQ ID NO: 5.
  • an antibody preparation is provided.
  • the antibodies specifically bind to an mammalian Sem polypeptide, and do not bind specifically to other mammalian proteins.
  • a method of treating a neoplasm comprises: contacting a neoplasm with an effective amount of a therapeutic agent comprising a mammalian Sem polypeptide which comprises a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, and SEQ ID NO: 6, whereby growth of the neoplasm is arrested.
  • a method of inducing cell differentiation comprises: contacting a progenitor cell with a human Sem (hScm) polypeptide which comprises a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, and SEQ ID NO: 6, whereby differentiation of the cell is induced.
  • hScm human Sem
  • a method of regulating cell growth comprises: contacting a cell whose growth is uncontrolled with a human Sem (hScm) polypeptide which comprises a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, and SEQ ID NO: 6, whereby growth of the cell is regulated.
  • hScm human Sem
  • a pharmaceutical composition comprising an effective amount of a therapeutic agent comprising a mammalian Sem polypeptide which comprises a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, and SEQ ID NO: 6, and a pharmaceutically acceptable carrier.
  • a therapeutic agent comprising a mammalian Sem polypeptide which comprises a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, and SEQ ID NO: 6, and a pharmaceutically acceptable carrier.
  • Another aspect of the invention is a method of diagnosing neoplasia.
  • the method comprises: contacting (a) a tissue sample suspected of neoplasia isolated from a patient with (b) an mammalian Sem gene probe comprising at least 12 nucleotides of a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, wherein a tissue which underexpresses mammalian Sem or expresses a variant mammalian Sem is categorized as neoplastic.
  • a method of diagnosing neoplasia comprises: contacting PCR primers which specifically hybridize with an mammalian Sem gene sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, with nucleic acids isolated from a tissue suspected of neoplasia; amplifying mammalian Son sequences in the nucleic acids of the tissue; and detecting a mutation in the amplified sequence, wherein a mutation is identified when the amplified sequence differs from a sequence similarly amplified from a normal human tissue.
  • a method of diagnosing neoplasia comprises: contacting a bDNA probe with nucleic acids isolated from a tissue suspected of neoplasia, wherein the bDNA probe specifically hybridizes with an mammalian Son gene sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5; detecting hybrids formed between the bDNA probe and nucleic acids isolated from the tissue; and identifying a mutation in the nucleic acids isolated from the tissue by comparing the hybrids formed with hybrids similarly formed using nucleic acids from a normal human tissue.
  • a method of diagnosing neoplasia comprises: contacting a tissue sample suspected of being neoplastic with an antibody selected from the group consisting of: one which specifically binds to wild-type mammalian Sem as shown in SEQ ID NO:2, 4, or 6, or one which specifically binds to an expressed mammalian Sem variant; detecting binding of the antibody to components of the tissue sample, wherein a difference in the binding of the antibody to components of the tissue sample, as compared to binding of the antibody to a normal human tissue sample indicates neoplasia of the tissue.
  • Another aspect of the invention is yet another method of diagnosing neoplasia.
  • the method comprises: contacting RNA from a tissue suspected of being neoplastic with PCR primers which specifically hybridize to an mammalian Sem gene sequence as shown in SEQ ID NO: 1, 3, or 5, or a bDNA probe which specifically hybridizes to said sequence;
  • nucleic acid molecules which can be used in regulating a heterologous coding sequence coordinately with hScm. These sequences include the 5' untranslated region of an hScm gene, the 3' untranslated region of an hScm gene, the promoter region of an hScm gene, and an intron of an hScm gene.
  • Also provided by the present invention is a method of identifying modulators of hScm function comprising: contacting a test substance with a human cell which comprises an hScm gene or a reporter construct comprising an hScm promoter and a reporter gene; quantitating transcription of hScm or the reporter gene in the presence and absence of the test substance, wherein a test substance which increases transcription is a candidate drug for anti-neoplastic therapy.
  • a method of diagnosis of neoplasia comprises: contacting a tissue sample suspected of neoplasia isolated from a patient with an mammalian Sem gene probe comprising at least 12 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, wherein a tissue which overexpresses mammalian Sem or expresses a variant mammalian Sem is categorized as neoplastic.
  • a method of dysregulating cell growth comprises: contacting a cell whose growth is controlled with a mammalian Sem polypeptide which comprises a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, and SEQ ID NO: 6, whereby growth of the cell is dysregulated.
  • a method of diagnosing neoplasia comprises: contacting RNA from a tissue suspected of being neoplastic with PCR primers which specifically hybridize to an mammalian Sem gene sequence as shown in SEQ ID NO: 1, 3, or 5, or a bDNA probe which specifically hybridizes to said sequence;
  • RNA as compared to a normal human tissue indicates neoplasia.
  • nucleic acid molecules which can be used in regulating a heterologous coding sequence coordinately with mammalian Sem. These sequences include the 5' untranslated region of an mammalian Sem gene, the 3' untranslated region of an mammalian Sem gene, the promoter region of an mammalian San gene, and an intron of an mammalian Sem gene.
  • Also provided by the present invention is a method of identifying modulators of mammalian Sem function comprising: contacting a mammalian cell which comprises an mammalian Sem gene or a reporter construct comprising an mammalian Sem promoter and a reporter gene with a test substance; quantitating transcription of mammalian Sem or the reporter gene in the presence and absence of the test substance, wherein a test substance which decreases transcription is a candidate drug for anti-neoplastic therapy.
  • Mammalian Sem the mammalian sex comb on midleg (mammalian Sem), that operates to regulate protein expression in mammals, particularly humans.
  • Mammalian Sem may operate by controlling homeotic gene expression.
  • control by this gene involves multiprotein complexes capable of negative regulation of transcription.
  • the polypeptides of the invention include the splice variant polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, which contain different domains of the mammalian Sem gene.
  • the nucleic acid molecules (SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5) encoding the mammalian Sem polypeptides have been cloned from human cells.
  • the polynucleotide of SEQ ID NO: 1 encodes the polypeptide of SEQ ID NO: 2
  • the polynucleotide of SEQ ID NO: 3 encodes the polypeptide of SEQ ID NO: 4
  • the polynucleotide of SEQ ID NO: 5 encodes the polypeptide of SEQ ID NO: 6.
  • Polypeptides comprising at least 6, 10, 20, 30, 40, 50, 54, 60, 65, or 75 amino acids of mammalian Sem are useful as immunogens for raising antibodies and as competitors in immunoassays. They can also be used to purify antibodies. Nucleic acid molecules of at least 15, 20, 30, 40, or 50 contiguous nucleotides are useful as probes for use in diagnostic assays. Both human and murine Sem, and their coding sequences, are provided herein.
  • the domains of mammalian Sem protein which appear to be most conserved are those found in the following locations in each of the isoforms of the human proteins.
  • isoform 1 amino acid SEQ ID NO: 4
  • the conserved domains are at aa 1 to 80, aa 93 to 128, aa 135 to 142, aa 144 to 166, and aa 527 to 565.
  • W amino acid SEQ ID NO: 4
  • the following short segments appear to be well conserved, although they are short: aa 170 to 177, aa 261 to 266, and aa 460 to 467.
  • isoform 2 amino acid SEQ ID NO: 6
  • the conserved domains are: aa 201 to 287, aa 311 to 336, aa 345 to 373, aa 550 to 589, aa 625 to 710, aa 823 to 894, aa 940 to 984, and aa 2170 to 2210.
  • the human Sem gene has been mapped to chromosome lp34. This was accomplished by FISH mapping. Intriguingly, loss of heterozygosity (LOH) for well differentiated gastric cancer and for colon cancer map to this region.
  • LHO heterozygosity
  • Mammalian Sem is implicated in development, by contributing to the
  • mammalian Sem can be used therapeutically to change the gene expression pattern and thus the phenotype of a cell.
  • mammalian Sem can be used to direct differentiation of a progenitor cell.
  • inhibition of mammalian Sem will direct a differentiated cell to become less differentiated, i.e., to alter its pattern of gene
  • Proliferative indications for which an mammalian Scm-based therapeutic agent can be used include, restinosis, benign prostatic hyperplasia, uterine fibroids, retinopathy, psoriasis, keloids, arthritis, wound healing, and premalignant lesions including for example, intestinal polyps, cervical dysplasia, and myeloid dysplasia.
  • Neoplasias that may be treatable with an mammalian Scm-based therapeutic agent, include, but are not limited to, lung carcinoma, colorectal adenocarcinoma, leukemia, Buri ⁇ tt's lymphoma and melanoma.
  • the coding region of mammalian Sem can be used for expression of mammalian Sem and for development of mammalian Sem variants for therapeutic
  • Mammalian Sem coding sequence can be used as a probe for diagnosis of disease or biological disorder where overexpression of mammalian Sem occurs, such as, for example, in cancers such as lung carcinoma, colorectal adenocarcinoma, lymphatic cancer, promyelocytic leukemia, Burkitt's lymphoma, and myeloma.
  • the 5' untranslated and 3' untranslated regions of mammalian Sem can also be used diagnostically to the same effect as the mammalian Sem coding sequence, for example, the 5' untranslated region can be isolated and used to probe tissue, for example, lung tissue, where lung carcinoma is suspected.
  • mammalian Sem has been shown to be upregulated in lung carcinoma
  • probing with any portion of the mammalian Sem gene can identify the upregulation of mammalian Sem in the tissue, as an aid to making a diagnosis.
  • diagnostic probes may also be used for continued monitoring of a diagnosed patient, for signs of improvement after and during treatment, and for indications of progression of the disease.
  • Mammalian Sem genes can be cloned and isolated by probing genomic DNA with the coding region of mammalian Sem, or by probing genomic DNA with any probe-length piece (at least 12 nucleotides) of mammalian Sem DNA.
  • a PI clone of genomic DNA containing hScm Human Genome Sciences #11267, CMCC #4737 has been deposited at the American Type Culture Collection, Rockville, MD.
  • the genomic DNA can be subcloned into a cloning vector, for example a cosmid vector, for sequencing and assembly of the entire gene sequence.
  • the promoter region of mammalian Sem is useful for expression of mammalian Sem in a gene therapy protocol, and for further analysis of mammalian Sem gene function and regulatory control.
  • Knowledge of promoter region sequences specific for binding transcriptional activators that activate the mammalian Sem promoter can facilitate improved expression of mammalian Sem for therapeutic purposes.
  • the mammalian Sem promoter region may be useful for tissue specific expression of heterologous genes, such as, for treatment of lung carcinoma or colorectal adenocarcinoma.
  • the region immediately 5' of the coding region of mammalian Sem can be used, for example, as a diagnostic probe for cancer or a developmental disorder associated with aberrant mammalian Sem activity.
  • the full length gene, or such non-coding regions of it as the promoter and the 5' or 3' untranslated regions can be isolated by probing genomic DNA with a probe comprising at least about 12 nucleotides of mammalian Sem cDNA, and retrieving a genomic sequence that hybridizes to one of these sequences.
  • the 5' untranslated end and the promoter regions for example, can be cloned by PCR cloning with random oligonucleotide and a 5' portion of the known coding sequence.
  • the polypeptides of the invention can further be used to generate monoclonal or polyclonal antibodies.
  • Monoclonal antibodies are prepared using the method of Kohler and Milstein, as described in Nature (1975) 256: 495-96, or a modification thereof.
  • Antibodies to mammalian Sem can be used therapeutically. They are desirably compatible with the host to be treated.
  • the antibodies can be human monoclonal antibodies or humanized antibodies, as the term is generally known in the art.
  • single chain antibodies may be used for therapy.
  • Antibodies may act to antagonize or inhibit the polypeptide activity of mammalian Sem, and are also useful in diagnosing a condition characterized by mammalian Sem expression or over- expression, such as, for example, a malignancy condition. Similarly, underexpression can be detected using such antibodies bind specifically to mammalian Sem but not to other human proteins. More preferred is the situation where the antibodies are human species mammalian Scm-specific.
  • mammalian Sem can be accomplished by any expression system appropriate for the purpose and conditions presented. Some exemplary expression systems are listed below. Where mammalian Sem itself is used as a therapeutic, the polypeptide can be expressed and subsequently administered to a patient.
  • a gene encoding at least a functional portion of mammalian Sem can be administered to a patient for expression in the patient.
  • Recombinant mammalian Sem may be used as a reagent for diagnostic methods for diagnosis of cancer or a developmental disorder. It may also be used as a therapeutic for inducing differentiation in a population of progenitor cells.
  • Recombinant mammalian Sem can also be used to develop modulators of mammalian Sem for achieving a desired therapeutic effect.
  • Construction and expression of any of the recombinant molecules of the invention can be accomplished by any expression system most appropriate for the task, including, for example, an expression system described below.
  • polypeptides of the invention can be expressed in any expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems. Expression systems in bacteria include those described in Chang et al., Nature (1978) 275: 615, Goeddel et al , Nature (1979) 281: 544, Goeddel et al, Nucleic Acids Res. (1980) 8: 4057, EP 36,776, U.S.
  • Constructs including an mammalian San coding sequence or constructs including coding sequences for modulators of mammalian Sem can be administered by a gene therapy protocol, either locally or systemically. These constructs can utilize viral or non-viral vectors and can be delivered in vivo or ex vivo or in vitro. Expression of such coding sequence can be driven by endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.
  • GDVs Gene delivery vehicles
  • a polynucleotide sequence of the invention can be administered either locally or systemically in a GDV.
  • These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.
  • the invention includes gene delivery vehicles capable of expressing the contemplated polynucleotides.
  • the gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vectors.
  • the viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picomavirus, poxvirus, togavirus viral vector. See generally, Jolly, Cancer Gene Therapy 1:51-64 (1994); Kimura, Human Gene Therapy 5:845-852 (1994), Connelly, Human Gene Therapy 6:185-193 (1995), and Kaplitt, Nature Genetics 6:148-153 (1994).
  • 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. 53:160, 1985) polytropic retroviruses (for example, MCF and MCF-MLV (see Kelly, J. Vir. 45:291, 1983), 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. 53:160, 1985
  • polytropic retroviruses for example, MCF and MCF-MLV (see Kelly, J. Vir. 45:291, 1983
  • spumaviruses and lentiviruses See RNA
  • retroviral gene therapy vector may be derived from different retroviruses.
  • retroviral 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 U.S. 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.
  • 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 U.S. 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 Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotiieliosis Virus and Rous Sarcoma Virus.
  • Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe, J. Virol. 19:19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC No. 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 No. 415,731; EP No. 345,242; PCT Publication Nos.
  • Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above-referenced documents and in PCT Patent Publication Nos. 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.
  • 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 No. 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 124:257-262 (1993).
  • Another example of such an AAV vector is psub201. See Samulski, J. Virol. 61:3096 (1987).
  • Another exemplary AAV vector is the Double-D LTR 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.
  • AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhance and albumin promoter and directs expression predominantly in the liver. Its structure and how to make it are disclosed in Su,Human Gene Therapy 7:463-470 (1996). Additional AAV gene therapy vectors are described in U.S. Patent Nos. 5,354,678; 5,173,414; 5,139,941; and 5,252,479.
  • the gene therapy vectors of the invention also include herpes vectors.
  • herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Patent No. 5,288,641 and EP No. 176,170 (Roizman).
  • Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in PCT Patent No. WO 95/04139 (Wistar Institute), pHSVlac described in Geller, Science 241:1667-1669 (1988) and in PCT Patent Publication Nos.
  • Alpha virus gene therapy vectors may be employed in this invention.
  • Preferred alpha virus vectors are Sindbis viruses vectors.
  • 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 U.S. Patent Nos. 5,091,309 and 5,217,879, and PCT Patent Publication No. WO 92/10578. 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 PubUcation Nos.
  • WO 94/21792, WO 92/10578, and WO 95/07994, and U.S. Patent Nos. 5,091,309 and 5,217,879 are employable.
  • alpha viruses may be obtained from depositories or coUections such as the ATCC in Rockville, Maryland or isolated from known sources using commonly available techniques.
  • alphavirus vectors with reduced cytotoxicity are used (see co-owned U.S. Serial No. 08/679640).
  • DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. See PCT Patent Publication No. WO 95/07994 for a detailed description of eukaryotic layered expression systems.
  • the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.
  • Other viral vectors suitable for use in the present invention include those derived from poUovirus, for example ATCC VR-58 and those described in Evans, Nature 339:385 (1989), and Sabin, J. Biol.
  • SV40 virus for example ATCC VR-305 and those described in Mulligan, Nature 277:108 (1979) and Madzak, J Gen Vir 73:1533 (1992); influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in U.S. Patent No. .5,166,057 and in Enami, Proc. Natl. Acad. Sci. 87:3802-3805 (1990); Enami and Palese, J. Virol. 65:2711-2713 (1991); and Luytjes, CeU 59:110 (1989), (see also McMicheal., New England J. Med.
  • 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; 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 virus, 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; Triniti virus, for example ATCC VR-469; Una virus, for example, ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62
  • compositions of this invention into ceUs is not limited to the above mentioned viral vectors.
  • Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to kiUed adenovirus alone, for example see U.S. Serial No. 08/366,787, filed December 30, 1994, and Curiel, Hum Gene Ther 3:147-154 (1992) ligand linked DNA, for example, see Wu, J. Biol. Chem. 264:16985-16987 (1989), eukaryotic cell deUvery vehicles ceUs, for example see U.S. Serial No.
  • sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to ceU targeting ligands such as asialoorosomucoid, as described in Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987), insulin as described in Hucked, Biochem. Pharmacol. 40:253-263 (1990), galactose as described in Plank, Bioconjugate Chem 3:533-539 (1992), lactose or transferrin. Naked DNA may also be employed.
  • synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to ceU targeting ligands such as asialoorosomucoid, as described in Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987), insulin as described in Hucked, Bio
  • Exemplary naked DNA introduction methods are described in PCT Patent PubUcation No. WO 90/11092 and U.S. Patent No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into ceUs 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.
  • Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120, PCT Patent PubUcation Nos. WO 95/13796, WO 94/23697, and WO 91/144445, and EP No. 524,968.
  • the nucleic acid sequences can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to ceU targeting Ugands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin.
  • synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to ceU targeting Ugands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin.
  • Other delivery systems include the use of Uposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters.
  • Non-viral deUvery suitable for use includes mechanical deUvery systems such as the approach described in Woffendin et al., Proc. Natl. Acad. Sci. USA 91(24):11581-11585 (1994).
  • the coding sequence and the product of expression of such can be deUvered through deposition of photopoiymerized hydrogel materials.
  • Other conventional methods for gene deUvery that can be used for deUvery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Patent No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Patent No. 5,206,152 and PCT Patent Publication No. WO 92/11033.
  • Exemplary liposome and polycationic gene deUvery vehicles are those described in U.S. Patent Nos. 5,422,120 and 4,762,915, in PCT Patent PubUcation Nos. WO 95/13796, WO 94/23697, and WO 91/14445, in EP No. 524,968 and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco, Szoka, Biochem. Biophys. Acta. 600:1 (1980); Bayer, Biochem. Biophys. Acta. 550:464 (1979); Rivnay, Meth. Enzymol. 149:119 (1987); Wang, Proc. Natl. Acad. Sci. 84:7851 (1987); and Plant, Anal. Biochem. 176:420 (1989).
  • Test compounds can be tested as candidate modulators by testing the abiUty to increase or decrease the expression of mammaUan Sem.
  • the candidate modulators can be derived from any of the various possible sources of candidates, such as for example, libraries of peptides, peptoids, smaU molecules, polypeptides, antibodies, polynucleotides, smaU molecules, antisense molecules, ribozymes, cRNA, cDNA, polypeptides presented by phage display. Described below are some exemplary and possible sources of candidates, including synthesized libraries of peptides, peptoids, and small molecules.
  • the exemplary expression systems can be used to generate cRNA or cDNA libraries that can also be screened for the ability to modulate mammalian Sem activity or expression.
  • Candidate molecules screened for the ability to agonize mammaUan Sem expression or activity may be useful for inducing differentiation in a population of progenitor ceUs.
  • SmaU molecules can be screened for the abiUty to either affect mammaUan Sem expression or affect mammaUan Sem function by enhancing or interfering in mammalian Scm's ability to interact with other molecules that mammaUan Sem normaUy interacts with in mammalian Scm's normal function.
  • Mammalian Sem peptide modulators are screened using any available method.
  • the assay conditions ideally should resemble the conditions under which the mammaUan Sem modulation is exhibited in vivo, that is, under physiologic pH, temperature, ionic strength, etc.
  • Suitable antagonists will exhibit strong inhibition of mammaUan Sem expression or activity at concentrations that do not cause toxic side effects in the subject.
  • a further alternative agent that can be used herein as a modulator of mammalian Sem is a small molecule antagonist.
  • SmaU molecules can be designed and screened from a pool of synthetic candidates for abiUty to modulate mammalian Sem.
  • smaU molecules including peptide analogs and derivatives, that can act as inhibitors of proteins and polypeptides. Libraries of these molecules can be screened for those compounds that inhibit the activity or expression of mammaUan Sem.
  • ribozymes can be screened in assays appropriate for ribozymes, taking into account the special biological or biochemical nature of ribozymes. Assays for affecting mammaUan Sem expression can measure mammaUan San message or protein directly, or can measure a reporter gene expression which is under the control of an mammaUan Sem promoter and/or 5' untranslated region (UTR).
  • UTR 5' untranslated region
  • MammaUan Sem or a modulator of mammaUan Sem can be administered to a patient exhibiting a condition characterized by abnormal cell proUferation, in which aberrant mammaUan Sem gene expression is implicated, particularly excessive mammaUan Sem activity, or excessive activity controUed or induced by mammaUan Sem activity.
  • the modulator can be incorporated into a pharmaceutical composition that includes a pharmaceuticaUy acceptable carrier for the modulator.
  • Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are weU known to those of ordinary skill in the art.
  • PharmaceuticaUy acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the Uke; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the Uke.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the Uke
  • organic acids such as acetates, propionates, malonates, benzoates, and the Uke.
  • PharmaceuticaUy acceptable carriers in therapeutic compositions may contain Uquids such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the Uke, may be present in such vehicles.
  • the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; soUd forms suitable for solution in, or suspension in, Uquid vehicles prior to injection may also be prepared.
  • Liposomes are included within the definition of a pharmaceuticaUy acceptable carrier.
  • the term "Uposomes" refers to, for example, the liposome compositions described in U.S. Patent NO: 5,422,120, WO 95/13796, WO 94/23697, WO 91/14445 and EP 524,968 Bl.
  • Liposomes may be pharmaceutical carriers for the peptides, polypeptides or polynucleotides of the invention, or for combination of these therapeutics.
  • Any therapeutic of the invention including, for example, polynucleotides for expression in the patient, or ribozymes or antisense oUgonucleotide, can be formulated into an enteric coated tablet or gel capsule according to known methods in the art. These are described in the foUowing patents: US 4,853,230, EP 225,189, AU 9,224,296, AU 9,230,801, and WO 92144,52. Such a capsule is administered o ⁇ dly to be targeted 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, is measured in the plasma and blood, for example by antibodies to the expressed or non-expressed proteins.
  • Administration of a therapeutic agent of the invention includes administering a therapeutically effective dose of the therapeutic agent by a means considered or empiricaUy deduced to be effective for inducing the desired effect in the patient. Both the dose and the administration means can be determined based on the specific quaUties of the therapeutic, the condition of the patient, the progression of the disease, and other relevant factors.
  • Administration of the therapeutic agents of the invention can include, local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration.
  • the therapeutics of the invention can be administered in a therapeutically effective dosage and amount, in the process of a therapeuticaUy effective protocol for treatment of the patient.
  • the initial and any subsequent dosages administered will depend upon the patient's age, weight, condition, and the disease, disorder or biological condition being treated. Depending on the therapeutic, the dosage and protocol for administration will vary, and the dosage wiU also depend on the method of administration selected, for example, local or systemic administration.
  • the dosage can be in the range of about 5 ⁇ g to about 50 ⁇ g/kg of patient body weight, also about 50 ⁇ g to about 5 mg/kg, also about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg.
  • vectors containing expressible constructs including mammaUan San coding sequences or modulator coding sequences, or non-coding sequences can be administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol, also about 500 ng to about 50 mg, also about 1 ug to about 2 mg of DNA, about 5 ug of DNA to about 500 ug of DNA, and about 20 ug to about 100 ug during a local administration in a gene therapy protocol, and for example, a dosage of about 500 ug, per injection or administration.
  • Non-coding sequences that act by a catalytic mechanism may require lower doses than non-coding sequences that are held to the restrictions of stoichiometry, as in the case of, for example, antisense molecules, although expression Umitations of the ribozymes may again raise the dosage requirements of ribozymes being expressed in vivo in order that they achieve efficacy in the patient. Factors such as method of action and efficacy of transformation and expression are therefore considerations that will effect the dosage required for ultimate efficacy for DNA and nucleic acids.
  • microgram ( ⁇ g) amounts per kUogram of patient may be sufficient, for example, in the range of about 1 ⁇ g/kg to about 500 mg/kg of patient weight, and about 100 ⁇ g/kg to about 5 mg/kg, and about 1 ⁇ g/kg to about 50 ⁇ g/kg, and, for example, about 10 ug/kg.
  • the potency also affects the dosage, and may be in the range of about 1 ⁇ g/kg to about 500 mg/kg of patient weight, and about 100 ⁇ g/kg to about 5 mg/kg, and about 1 ⁇ g/kg to about 50 ⁇ g/kg, and a usual dose might be about 10 ug/kg.
  • a therapeutic agent for a condition in which increased expression of mammaUan Sem is impUcated for example, in the case of promyelocytic leukemia, chronic myelogenous leukemia, lymphoblastic leukemia, Buri ⁇ tt's lymphoma, colorectal adenocarcinoma, lung carcinoma, melanoma, and lymphoma, can be preceded by diagnosis of the condition using an mammaUan San probe, generated from any portion of the mammaUan Sem gene, and probing the suspect tissue.
  • bDNA technology using bDNA probes to mammaUan San gene sequences or mammaUan San mRNA sequences may be used, as described in WO 92/02526 or U.S. 5,451,503, and U.S. 4,775,619.
  • treatment can include administration of mammaUan San polynucleotides or anti-sense oUgonucleotide by a gene therapy protocol, or by administration by other means including local or systemic administration, of an mammalian Sem modulator, for example an mammaUan San- specific ribozyme, or a geneticaUy altered mammaUan Sem variant, for example a dominant negative mammalian Sem, or a small molecule or peptide or peptoid mammaUan Sem modulator, or any combination of these potential therapeutics.
  • the patient can be subsequently monitored by periodic reprobing of the affected tissue with an mammaUan Sem probe.
  • mammaUan Sem upregulation or enhancement of mammaUan Sem function may have therapeutic application.
  • increasing mammaUan Sem expression or enhancing mammaUan Sem function may help to suppress the tumors.
  • effecting mammaUan Sem upregulation or augmentation of mammaUan Sem activity may suppress metastases.
  • nucleic acid molecule or a “polynucleotide,” as used herein, refers to either RNA or DNA molecule that encodes a specific amino acid sequence or its complementary strand. Nucleic acid molecules may also be non-coding sequences, for example, a ribozyme, an antisense oligonucleotide, or an untranslated portion of a gene.
  • a polynucleotide may include, for example, an antisense oligonucleotide, or a ribozyme, and may also include such items as a 3' or 5' untranslated region of a gene, or an intron of a gene, or other region of a gene that does not make up the coding region of the gene.
  • the DNA or RNA may be single stranded or double stranded.
  • Synthetic nucleic acids or synthetic polynucleotides can be chemically synthesized nucleic acid sequences, and may also be modified with chemical moieties to render the molecule resistant to degradation. Synthetic nucleic acids can be ribozymes or antisense molecules, for example.
  • Modifications to synthetic nucleic acid molecules include nucleic acid monomers or derivative or modifications thereof, including chemical moieties.
  • phosphothioates can be used for the modification.
  • a polynucleotide derivative can include, for example, such polynucleotides as branched DNA (bDNA).
  • a polynucleotide can be a synthetic or recombinant polynucleotide, and can be generated, for example, by polymerase chain reaction (PCR) amptif ⁇ cation, or recombinant expression of complementary DNA or RNA, or by chemical synthesis.
  • MammaUan Sem polynucleotides contain at least 95% and preferably at least 97% identity to either mouse or human hScm sequences.
  • the term "functional portion of” as used herein refers to a portion of an mammaUan Sem wUd-type molecule which retains at least 50% of activity of mammalian Sem. It also encompasses a portion of an mammaUan San gene having single base substitutions, deletions, or insertions that have no adverse effect on the activity of the molecule. Truncations of mammaUan Sem, fragments of Sem, and combinations of fragments of Sem, which retain at least 50% activity are contemplated. Such portions of hScm may also be fused to other proteins, such as in a gene fusion.
  • modulate refers to the abiUty of a molecule to alter the function or expression of another molecule.
  • modulate could mean, for example, inhibit, antagonize, agonize, upregulate, downregulate, induce, or suppress.
  • a modulator has the capabUity of altering function of its target.
  • Such alteration can be accompUshed at any stage of the transcription, translation, expression or function of the protein, so that, for example, modulation of mammaUan Sem can be accompUshed by modulation of the DNA, RNA, and protein products of the gene. It assumed that modulation of the function of the target, for example, mammaUan Sem, wiU in turn modulate, alter, or affect the function or pathways leading to a function of genes and proteins that would otherwise associate, and interact, or respond to, mammaUan Sem.
  • a "maUgnancy” includes any proUferative disorder in which the ceUs proUferating are ultimately harmful to the host. Cancer is an example of a proUferative disorder that manifests a maUgnancy. Neoplasia is the state of ceUs which experience uncontroUed cell growth, whether or not malignant.
  • the term "regulatory sequence” as used herein refers to a nucleic acid sequence encoding one or more elements that are capable of affecting or effecting expression of a gene sequence, including transcription or translation thereof, when the gene sequence is placed in such a position as to subject it to the control thereof.
  • Such a regulatory sequence can be, for example, a minimal promoter sequence, a complete promoter sequence, an enhancer sequence, an upstream activation sequence ("UAS"), an operator sequence, a downstream termination sequence, a polyadenylation sequence, an optimal 5' leader sequence to optimize initiation of translation, and a Shine-Dalgarno sequence.
  • the regulatory sequence can contain a combination enhancer/promoter element.
  • the regulatory sequence that is appropriate for expression of the present construct differs depending upon the host system in which the construct is to be expressed. Selection of the appropriate regulatory sequences for use herein is within the capability of one skiUed in the art.
  • such a regulatory sequence can include one or more of a promoter sequence, a ribosomal binding site, and a transcription termination sequence.
  • such a sequence can include one or more of a promoter sequence and/or a transcription termination sequence. If any necessary component of a regulatory sequence that is needed for expression is lacking in the polynucleotide construct, such a component can be supplied by a vector into which the polynucleotide construct can be inserted for expression.
  • Regulatory sequences suitable for use herein may be derived from any source including a prokaryotic source, an eukaryotic source, a virus, a viral vector, a bacteriophage or from a linear or circular plasmid.
  • a regulatory sequence is the human immunodeficiency virus ("HIV") promoter that is located in the U3 and R region of the HIV long terminal repeat (“LTR").
  • the regulatory sequence herein can be a synthetic sequence, for example, one made by combining the UAS of one gene with the remainder of a requisite promoter from another gene, such as the GADP/ADH2 hybrid promoter.
  • protein refers herein to the expression product of a polynucleotide construct of the invention as defined above.
  • the terms further include truncations, variants, aUeles, analogs and derivatives thereof.
  • mammaUan Sem polypeptides possess one or more of the bioactivities of the mammaUan Sem protein, such as those discovered herein. This term is not Umited to a specific length of the product of the mammaUan San gene.
  • polypeptides that are identical or contain at least 85%, and more preferably 90%, and most preferably 95% identity with the mammaUan Sem protein or the mature mammaUan Sem protein, wherever derived, from human or nonhuman sources are included within this definition of the mammaUan Sem polypeptide. Also included, therefore, are aUeles and variants of the product of the mammaUan San gene that contain amino acid substitutions, deletions, or insertions.
  • amino acid substitutions can be conservative amino acid substitutions or substitutions to eUminate non-essential amino acid residues such as to alter a glycosylation site, a phosphorylation site, an acetylation site, or to alter the folding pattern by altering the position of the cysteine residue that is not necessary for function, etc.
  • Conservative amino acid substitutions are those that preserve the general charge, hydrophobicity/hydrophUicity and/or steric bulk of the amino acid substituted, for example, substitutions between the members of the foUowing groups are conservative substitutions: Gly/ Ala, Val/Ile/Leu, Asp/Glu, Lys/ Arg, Asn/Gln, Ser/Thr/Cys and Phe/Trp/Tyr.
  • Analogs include peptides having one or more peptide mimics, also known as peptoids, that possess mammaUan Sem protein-Uke activity. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as weU as other modifications known in the art, both naturally occurring and nonnaturally occurring.
  • the term "mammaUan Sem” also may include post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, myrstylations, farnesylations, palmitoylations and the Uke.
  • polypeptide fragment refers to a polypeptide sequence that does not encode the fuU length of a protein but that is identical to a region of the protein. The fragment is designed to retain the functional aspect of the region of the polypeptide from which it is derived. Two fragments can cooperate to provide function. Two distinct polypeptide fragments of the same gene may represent expressed spUce variants of that gene, although functionality and expression of the polypeptide spUce variant products may occur in similar biological conditions, and may be related, at least in part, in function.
  • derivative as used herein in reference to a polypeptide or a polynucleotide means a polypeptide or polynucleotide that retains at least 50% ofthe functionaUty of the polypeptide or polynucleotide to which it is a derivative. They may be variously modified by nucleotide or amino acid deletions, substitutions, insertions or inversions by, for example, site directed mutagenesis of the underlying nucleic acid molecules. Derivatives of a polypeptide or polynucleotide may also be fragments or combinations of fragments thereof. In any case, a derivative, or a fragment, retains at least some, and preferably aU of the function of the polypeptide from which it is derived.
  • an "isolated polypeptide” or “isolated polynucleotide” as used herein refers to a polypeptide or polynucleotide, respectively, produced in vivo or in vitro in an environment manipulated by humans using state of the art techniques of molecular biology, biochemistry and gene therapy.
  • an isolated polypeptide can be produced in a ceU free system by automated peptide or polypeptide synthesis, in heterologous host ceUs transformed with the nucleic acid sequence encoding the polypeptide and regulatory sequences for expression in the host ceUs, and in an animal into which the coding sequence of the polypeptide has been introduced for expression in the animal.
  • a polypeptide or polynucleotide is "isolated" for purposes herein to the extent that it is not present in its natural state inside a ceU as a product of nature.
  • isolated polypeptides or polynucleotides can be 10% pure, 20% pure, or a higher degree of purity, such as 50%, 75%, 85%, or 90%.
  • condition refers to a particular state of molecular and cellular systems in a biological context.
  • a biological context includes any organism considered to have Ufe, and for the purposes of this invention includes but is not Umited the foUowing organisms or groups: animals, mammals, humans, and vertebrates.
  • a biological condition can include, for example, a disease or a medical condition that may or may not be characterized by identifiable symptoms or indicators.
  • a "condition characterized by abnormal ceU proUferation" is most likely a cancer condition, but may also be a condition arising in the development of an organism.
  • modulator as used herein describes any moiety capable of changing the endogenous activity or a polypeptide.
  • Modulatory activities can include, for example, modulation at the level of transcription, translation, expression, secretion, or modulation of polypeptide activity inside or outside a cell.
  • Modulation can include, for example, inhibition, antagonism, and agonism, and modulation can include, for example, modulation of upstream or downstream effects that effect the ultimate activities in a pathway, or modulation of the configuration of a polypeptide such that its activity is altered.
  • Modulation can be transitory or permanent, and may be a dose dependent effect.
  • the term "inhibitor” for use herein can be any inhibitor of a polypeptide activity.
  • the category includes but is not Umited to any of the herein described antagonists of mammaUan Sem.
  • the inhibitor of mammaUan Sem can be an antibody- based mammaUan Sem antagonist, or a polypeptide fragment thereof, a peptide mammaUan Sem antagonist, a peptoid mammalian Sem antagonist, or a smaU molecule mammaUan Sem antagonist.
  • the polypeptide inhibitor can be one screened from a cDNA, cRNA, or phage display Ubrary of polypeptides.
  • the inhibitor can be a polynucleotide, such as, for example a ribozyme or an antisense oUgonucleotide, or can be derivatives of these. It is expected that some inhibitors will act at transcription, some at translation, and some on the mature protein. However, the use and appropriateness of such inhibitors of mammalian Sem for the purposes of the invention are not limited to any theories of mechanism of action of the inhibitor. It is sufficient for purposes of the invention that an inhibitor inhibit the activity of mammaUan Sem.
  • antagonist refers to a molecule that inhibits or blocks the activity of a polypeptide, either by blocking the polypeptide itself, or by causing a reduced expression of the polypeptide by either blocking transcription of the gene encoding the polypeptide, or by interfering with or destroying a transcription or translation product of the gene.
  • An antagonist may be, for example, a small molecule, peptide, peptoid, polypeptide, or polynucleotide.
  • the polynucleotide may be, for example, a ribozyme, an antisense oUgonucleotide, or a coding sequence.
  • agonist refers to a molecule that mimics the activity of the target polypeptide.
  • an agonist could mimic the transcriptional negative regulation capability of mammaUan Sem.
  • An agonist may be, for example a smaU molecule, peptide, peptoid, polypeptide, or polynucleotide.
  • composition refers to a composition for administration of a therapeutic agent, such as antibodies or a polypeptide, or inhibitors or genes and other therapeutic agents tisted herein, in vivo, and refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be admimstered without undue toxicity.
  • a therapeutic agent such as antibodies or a polypeptide, or inhibitors or genes and other therapeutic agents tisted herein
  • an effective amount refers to an amount that is effective to induce a desired effect.
  • the effective amount is that amount that will accomplish a therapeutic goal, for example, tumor regression, tumor marker reduction, or a positive indication from other indicia of cancer that indicates a reduction or growth slowing of cancer ceUs.
  • the therapeutic agent is, for example, an antagonist of mammaUan Sem
  • the effective amount of the antagonist would be an amount that antagonizes mammaUan Sem activity among a population of ceUs. The amount that is effective depends in part upon the indicia selected for determining effectiveness, and depends upon the effect sought.
  • An administration of a therapeutic agent of the invention includes administration of a therapeuticaUy effective amount of the agent of the invention.
  • therapeuticaUy effective amount refers to an amount of a therapeutic agent to treat or prevent a condition treatable by administration of a composition of the invention. That amount is the amount sufficient to exhibit a detectable therapeutic or preventative or ameliorative effect. The effect may include, for example, treatment or prevention of the conditions Usted herein.
  • the precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition being treated, recommendations of the treating physician, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation.
  • Administration can include administration of a polypeptide, and causing the polypeptide to be expressed in an animal by administration of the polynucleotide encoding the polypeptide.
  • a "recombinant vector” herein refers to any vector for transfer or expression of the polynucleotides herein in a cell, including, for example, viral vectors, non-viral vectors, plasmid vectors and vectors derived from the regulatory sequences of heterologous hosts and expression systems.
  • the term "in vivo administration” refers to administration to a mammal of a polynucleotide encoding a polypeptide for expression in the mammal.
  • direct in vivo administration involves transfecting a mammal's ceU with a coding sequence without removing the ceU from the mammal.
  • direct in vivo administration may include direct injection of the DNA encoding the polypeptide of interest in the region afflicted by the malignancy or proliferative disorder, resulting in expression in the mammal's ceUs.
  • Ex vivo administration refers to transfecting a ceU, for example, a ceU from a population of ceUs that are maUgnant or proliferating, after the ceU is removed from the mammal. After transfection the cell is then replaced in the mammal.
  • Ex vivo administration can be accompUshed by removing ceUs from a mammal, optionally selecting for ceUs to transform, (i.e. ceUs that are maUgnant or proUferating) rendering the selected ceUs incapable of repUcation, transforming the selected ceUs with a polynucleotide encoding a gene for expression, (i.e. mammaUan Sari), including also a regulatory region for faciUtating the expression, and placing the transformed ceUs back into the mammal for expression of the mammaUan San.
  • a polynucleotide encoding a gene for expression i.e. mammaUan Sari
  • BiologicaUy active refers to a molecule that retains a specific activity.
  • a biologicaUy active mammaUan Sem polypeptide for example, retains the activity including for example the control of a homeotic gene or group of homeotic genes.
  • “Mammalian cell” as used herein refers to a subset of eukaryotic ceUs useful in the invention as host cells, and includes human cells, and animal cells such as those from dogs, cats, cattle, horses, rabbits, mice, goats, pigs, etc.
  • the ceUs used can be geneticaUy unaltered or can be genetically altered, for example, by transformation with appropriate expression vectors, marker genes, and the like.
  • MammaUan ceUs suitable for the method of the invention are any mammaUan ceU capable of expressing the genes of interest, or any mammaUan ceUs that can express a cDNA Ubrary, cRNA Ubrary, genomic DNA Ubrary or any protein or polypeptide useful in the method of the invention.
  • MammaUan ceUs also include ceUs from ceU Unes such as those immortalized ceU Unes available from the American Type Culture CoUection (ATCC).
  • ceU Unes include, for example, rat pheochromocytoma ceUs (PC 12 cells), embryonal carcinoma cells (P19 ceUs), Chinese hamster ovary (CHO) ceUs, HeLa ceUs, baby hamster kidney (BHK) ceUs, monkey kidney cells (COS), human hepatoceUular carcinoma ceUs (e.g., Hep G2), human embryonic kidney cells, mouse sertoli cells, canine kidney cells, buffalo rat liver ceUs, human lung ceUs, human tiver ceUs, mouse mammary tumor ceUs, as weU as others. Also included are hematopoetic stem ceUs, neuronal stem cells such as neuronal sphere ceUs, and embryonic stem ceUs (ES ceUs).
  • a smaU molecule modulator of mammaUan Sem is identified and incorporated into a pharmaceutical composition including a liposomal-based pharmaceuticaUy acceptable carrier for administration to a cancer patient for controlling the expression or activity of mammaUan Sem in the patient.
  • Administration the composition is achieved by injection into the tumor tissue.
  • the patient is monitored for reduction of mammaUan Sem activity as a diagnostic marker evaluating the effectiveness of the treatment.
  • Example.2 A population of progenitor ceUs are treated with a functional portion of recombinant mammaUan Sem polypeptide and induced to differentiate. The process is reversed by administering to the population of cells an inhibitor of mammaUan Sem activity.
  • Example 3 Northern blots of mRNA isolated from various tissues were probed with mammalian San cDNA for an analysis of the expression differential of mammaUan San in normal and cancerous tissues, using standard techniques for accomplishing the hybridizations.
  • the normal tissues probed were human adult heart, skeletal muscle, pancreas, prostate, testes, ovary, colon, thymus, brain, placenta, lung, liver, kidney, peripheral leukocytes, and spleen.
  • the tissue specific expression of mammaUan San in normal human adult tissue indicated abundant mammaUan San transcript in human heart, skeletal muscle, pancreas, and testes.
  • transcript A somewhat less abundant amount of transcript was present in human prostate, ovary, colon, thymus, brain, placenta, lung, Uver, and kidney, and the transcript was virtually undetectable in human leukocytes, and undetectable in the human spleen tissue probed.
  • mammaUan San transcripts were present at an abundantly high level in the foUowing human cancer ceU lines: promyelocytic leukemia HL-60, HeLa ceU S3, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Buri ⁇ tt's lymphoma Raji, colorectal adenomcarcinoma SW480, lung carcinoma A549, and melanoma G361.
  • San transcript was also abundantly high in lung carcinoma tissue, colorectal adenocarcinoma tissue, and iymphocytic cancer tissues.
  • the mammaUan Sem transcript was approximately 4 to 4.2 kUobases in size for aU hybridizations. Hybridizations were conducted using stringent conditions and a standard hybridization protocol for accomplishing Northern blot hybridizations.
  • Transcript levels were controUed for by probing with actin probe on the same blots probed with mammaUan San coding sequence.
  • SEQ ID NOS: 1, 3, and 5 are human cDNA sequences for Sem isoforms
  • SEQ ID NOS: 2, 4, and 6 are translated human amino acid sequences for the Sem isoforms
  • SEQ ID NO: 7 is the mouse cDNA for Sem
  • SEQ ID NO: 8 is the translated mouse amino acid sequence for Sem
  • ADDRESSEE Chiron Corporation
  • STREET 4560 Horton Street
  • CTAAGGTCCC TCTATTTATT TCTCAACCCT GGCTGGCCTC TCACCAGGAG TTTAGGCTGA 2160 ATGCCTTCCA CGTGATGGAG GAAAAGGCCA ACTCTGTCCT GGTCTTGCTG TGGCACCCCA 2220
  • MOLECULE TYPE DNA (genomic)
  • CTACAGCCAC CTCTTGGATT TCGGCTGAAT GCGTCTTCTT GGCCCATGTT CCTTTTGAAG 840
  • CTGAGTGGCC GGGACCCCTC CTCGTGGACA GTCGAGGATG TGATGCAGTT TGTCCGGGAA 2160 GCTGATCCTC AGCTTGGACC CCACGCTGAC CTGTTTCGCA AACACGAGAT CGATGGCAAG 2220
  • MOLECULE TYPE DNA (genomic)
  • GAACATCAAC CAGGGCAGAG GGGGCGTAAA CCAGGAAAGA AGCGGGGCCG GACACCCAAG 1200
  • MOLECULE TYPE DNA (genomic)
  • ACCTTCCCAC AACTTCTTCA AAATGGGAAT GAAGTTAGAA GCTGTAGACA GAAAGAACCC 900
  • CAACCACGAC AGGTACCTAC CAGGTGAAAC CTTTGTCCTG GGGAATAGCC TGGCCCGGTC 1860

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  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un gène mammalien Scm (Sex Comb on Midleg) et des séquences d'acides aminés codés par ce gène mammalien Scm. De tels gènes mammaliens Scm et produits géniques conviennent particulièrement aux applications diagnostics et thérapeutiques dans le cas des troubles proliférants et de troubles du développement. Les gènes de l'invention permettent d'identifier des modulateurs du gène mammalien Scm. Ces modulateurs sont utilisables dans le cadre d'une thérapie anticancéreuse ou du traitement d'un trouble du développement. Le Scm convient également à l'induction de la différenciation dans une population de cellules souches.
PCT/US1997/007575 1996-05-06 1997-05-06 GENE MAMMALIEN Scm A EFFET SUPPRESSEUR DE TUMEUR WO1997042211A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP97926403A EP0915904A4 (fr) 1996-05-06 1997-05-06 GENE MAMMALIEN (Scm) A EFFET SUPPRESSEUR DE TUMEUR
AU31175/97A AU3117597A (en) 1996-05-06 1997-05-06 Mammalian sex comb on midleg (mammalian (scm)) acts as a tumor suppressor
JP09540119A JP2000510693A (ja) 1996-05-06 1997-05-06 哺乳動物の中肢性コーム（哺乳動物Ｓｃｍ）は腫瘍サプレッサーとして作用する

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
US1687696P 1996-05-06 1996-05-06
US1729496P 1996-05-13 1996-05-13
US2045096P 1996-06-18 1996-06-18
US3299496P 1996-12-16 1996-12-16
US3509097P 1997-01-14 1997-01-14
US60/032,994 1997-01-14
US60/020,450 1997-01-14
US60/016,876 1997-01-14
US60/017,294 1997-01-14
US60/035,090 1997-01-14

Publications (1)

Publication Number Publication Date
WO1997042211A1 true WO1997042211A1 (fr) 1997-11-13

Family

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Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US1997/007575 WO1997042211A1 (fr) 1996-05-06 1997-05-06 GENE MAMMALIEN Scm A EFFET SUPPRESSEUR DE TUMEUR
PCT/US1997/007574 WO1997042218A1 (fr) 1996-05-06 1997-05-06 GENE MAMMALIEN Scm A EFFET ONCOGENE

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/US1997/007574 WO1997042218A1 (fr) 1996-05-06 1997-05-06 GENE MAMMALIEN Scm A EFFET ONCOGENE

Country Status (4)

Country Link
EP (2) EP0915904A4 (fr)
JP (2) JP2000513212A (fr)
AU (2) AU2996797A (fr)
WO (2) WO1997042211A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0960120A1 (fr) * 1996-05-06 1999-12-01 Chiron Corporation GENE MAMMALIEN (Scm) A EFFET ONCOGENE
EP1066311A1 (fr) * 1998-03-31 2001-01-10 Genzyme Corporation Compositions et methodes d'identification de cellules de tumeur du poumon
US6203989B1 (en) 1998-09-30 2001-03-20 Affymetrix, Inc. Methods and compositions for amplifying detectable signals in specific binding assays

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993016178A2 (fr) * 1992-02-12 1993-08-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Sequences caracteristiques du produit de transcription des genes humains
EP0915904A4 (fr) * 1996-05-06 2001-05-02 Chiron Corp GENE MAMMALIEN (Scm) A EFFET SUPPRESSEUR DE TUMEUR

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHENG N N, ET AL.: "INTERACTIONS OF POLYHOMEOTIC WITH POLYCOMB GROUP GENES OF DROSOPHILA MELANOGASTER", GENETICS, GENETICS SOCIETY OF AMERICA, AUSTIN, TX, US, vol. 138, 1 December 1994 (1994-12-01), US, pages 1151 - 1162, XP001007246, ISSN: 0016-6731 *
See also references of EP0915904A4 *
SOTO MARTH C ET AL: "Comparison of germline mosaics of genes in the polycomb group of Drosophila melanogaster.", GENETICS, GENETICS SOCIETY OF AMERICA, AUSTIN, TX, US, vol. 140, no. 1, 1 January 1995 (1995-01-01), US, pages 231 - 243, XP002161968, ISSN: 0016-6731 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0960120A1 (fr) * 1996-05-06 1999-12-01 Chiron Corporation GENE MAMMALIEN (Scm) A EFFET ONCOGENE
EP0960120A4 (fr) * 1996-05-06 2001-05-02 Chiron Corp GENE MAMMALIEN (Scm) A EFFET ONCOGENE
EP1066311A1 (fr) * 1998-03-31 2001-01-10 Genzyme Corporation Compositions et methodes d'identification de cellules de tumeur du poumon
EP1066311A4 (fr) * 1998-03-31 2002-04-10 Genzyme Corp Compositions et methodes d'identification de cellules de tumeur du poumon
US6203989B1 (en) 1998-09-30 2001-03-20 Affymetrix, Inc. Methods and compositions for amplifying detectable signals in specific binding assays
US6806047B2 (en) 1998-09-30 2004-10-19 Affymetrix, Inc. Methods and compositions for amplifying detectable signals in specific binding assays

Also Published As

Publication number Publication date
EP0915904A4 (fr) 2001-05-02
AU2996797A (en) 1997-11-26
EP0960120A1 (fr) 1999-12-01
WO1997042218A1 (fr) 1997-11-13
EP0960120A4 (fr) 2001-05-02
JP2000513212A (ja) 2000-10-10
EP0915904A1 (fr) 1999-05-19
AU3117597A (en) 1997-11-26
JP2000510693A (ja) 2000-08-22

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