Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
  • C07K14/4747—Apoptosis related proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
  • C12N2310/111—Antisense spanning the whole gene, or a large part of it
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
  • C12N2310/121—Hammerhead
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
  • C12N2310/122—Hairpin
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

• Guanine can be modified to result in 8-bromo-guanine, 8-fluoroguanine, 2-amino-purine, hypozanthine (inosine), 7-deaza-guanine or 6-thio-guanine.
• Uracil can be modified to result in 3-methyl-uracil, 5,6-dihydro-uracil, 4-thio-uracil, thymine, 5- bromo-uracil, 5-iodo-uracil or 5-fluoro-uracil.
• nucleic acid or “nucleic acid molecule” refers to deoxyribonucleotides or ribonucleotides, oligomers and polymers thereof, in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. For example, as disclosed herein, such analogues include those with substitutions, such as methoxy, at the 2-position of the sugar moiety. Unless otherwise indicated by the context, the term is used interchangeably with gene, cDNA and mRNA encoded by a gene.
• the compounds containing the amino acid sequences described above can be used to inhibit induction of apoptosis in a cell. Alternatively, these compounds can be sued to identify agents that promote induction of apoptosis in a cell.
• the diagnostic methods described herein are applicable to the identification of cancer cells resistant to apoptosis induction present in, for example, solid tumors (carcinomas and sarcomas) such as, for example, breast cancer, ovarian cancer and prostate cancer.
• Such methods include the detection of a nucleic acid encoding a molecular product having an RST identified herein as involved in inhibiting apoptosis induction.
• the apoptosis induction regulator source is combined with a nucleic acid element or protein modulated by an apoptosis induction regulator as described above and incubated in the presence or absence of a test compound.
• the expression levels or activity of apoptosis induction or the reporter gene in the presence of the test compound is compared with that in the absence of the test compound.
• Those test compounds which provide an increase in expression levels or activity of apoptosis induction or the reporter gene of at least about 20% are considered to be apoptosis induction regulator activators, or agonists, and are potential therapeutic compounds for the treatment of neoplastic diseases such as cancer.
• test compounds for the above-described assays can be any substance, molecule, compound, mixture of molecules or compounds, or any other composition which is suspected of being capable of inhibiting apoptosis induction regulator activity in vivo or in vitro, for example, compounds with cell proliferation- inhibiting activity.
• the test compounds can be macromolecules, such as biological polymers, including proteins, polysaccharides and nucleic acids.
• Sources of test compounds which can be screened for apoptosis induction regulator inhibitory activity include, for example, libraries of small organic molecules, peptides, polypeptides, DNA, and RNA. Additionally, test compounds can be pre-selected based on a variety of criteria.
• test compounds can be selected as having known inhibition or enhancement activity with respect to cell proliferation.
• the test compounds can be selected randomly and tested by the screening methods of the present invention.
• Test compounds can be administered to the reaction system at a single concentration or, alternatively, at a range of concentrations to determine, for example, the optimal modulatory activity toward the apoptosis induction regulator.
• the "randomness" of the plasmid library was evaluated as described in WO 00/05415 to Barber et al.
• the frequencies of the four nucleotides, with 95% confidence limits, in the random positions were calculated to be G: 22.3 ⁇ 6.1, A: 31.9 ⁇ 7.0, T: 27.3 ⁇ 7.8 and C: 18.01 ⁇ 15.1. Since the expected frequency for each base is 25%, each base appears to be randomly represented (except for C, which may be slightly lower than expected). These variations most likely result from the unbalanced incorporation of nucleotides during the chemical synthesis of the oligonucleotides and could reduce the complexity of the library.
• This example describes a method for identifying ribozymes involved in apoptosis induction.
• the pLPR-library vector described in Example 1 and a control vector, pLPR-TL3, were used to transduce DLD-1 colon carcinoma cells (ATCC, Bethesda MD).
• the control vector differs from the pLPR-library vector (see Figure 2) in having an HCV ribozyme control gene in place of the ribozyme library gene.
• the transduction medium was removed by aspiration and replaced with growth medium containing puromycin (2 ⁇ g/ml).
• growth medium containing puromycin (2 ⁇ g/ml).
• cells were re-fed with media containing 2 ⁇ g/ml puromycin.
• the cells were maintained in selection medium for 10-14 days in order to select for stable integration of the retroviral vector. During the course of this selection the cells were re- fed every tliree days.
• the resulting bacterial colonies were pooled and purified DNA was used in a triple transfection protocol (as described above in Example 1) to produce retroviral vector. Individual colonies were also sequenced by the standard dideoxy method using a vector primer 5'-CTGACTCCATCGAGCCAGTGTAGAG-3' (SEQ ID NO: 16).
• DLD-1 cells were grown to about 70% confluency in T75 flasks (about 5 x 10 6 cells). The media was then removed and replaced with 0.8 ml of serum free Opti-MEM media (GIBCO). The cells were incubated for four hours at 37°C with complexes containing a lipid-plasmid DNA complex.
• the 2.3 kb contig (SEQ ID NO: 150) was then used as the query sequence for a BLAST search to see if the contig could be extended further. This searched yielded the EST fragment hv79F02 (GenBank Accession No. BE327693) (SEQ JD NO: 151) wliich overlapped the 2.3 kb contig and extended the sequence about 300 more bases at the 5' end yielding a contig of about 2.6 kb (SEQ ID NO: 152). This contig was then used as the query sequence to search the human genome sequence at NCBI. The results of this search indicated that the contig hybridized with greater than 98% identity to a region tentatively assigned to cliromosome 1.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• General Health & Medical Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biochemistry (AREA)
• Wood Science & Technology (AREA)
• Biophysics (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Microbiology (AREA)
• Toxicology (AREA)
• Gastroenterology & Hepatology (AREA)
• Public Health (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Enzymes And Modification Thereof (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Publications (2)

Family

ID=23118085

Family Applications (1)

Country Status (6)

Families Citing this family (3)

Family Cites Families (1)

• 2002
  • 2002-05-14 WO PCT/US2002/015198 patent/WO2002092840A2/en not_active Ceased
  • 2002-05-14 EP EP02741703A patent/EP1474175A4/en not_active Withdrawn
  • 2002-05-14 AU AU2002314780A patent/AU2002314780A1/en not_active Abandoned
  • 2002-05-14 CA CA002446991A patent/CA2446991A1/en not_active Abandoned
  • 2002-05-14 JP JP2002589706A patent/JP2005501524A/ja active Pending
  • 2002-05-14 US US10/478,019 patent/US20040248830A1/en not_active Abandoned

Non-Patent Citations (6)

Also Published As

Similar Documents

Legal Events