EP1117776A1 - Synthese enzymatique d'adn simple brin - Google Patents

Synthese enzymatique d'adn simple brin

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Publication number
EP1117776A1
EP1117776A1 EP99951989A EP99951989A EP1117776A1 EP 1117776 A1 EP1117776 A1 EP 1117776A1 EP 99951989 A EP99951989 A EP 99951989A EP 99951989 A EP99951989 A EP 99951989A EP 1117776 A1 EP1117776 A1 EP 1117776A1
Authority
EP
European Patent Office
Prior art keywords
sequence
vector
reverse transcriptase
interest
genetic elements
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99951989A
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German (de)
English (en)
Inventor
Michael J. Skillern
Charles A. Conrad
Jonathan F. Elliston
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ingene Inc
CytoGenix Inc
Original Assignee
Ingene Inc
CytoGenix Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ingene Inc, CytoGenix Inc filed Critical Ingene Inc
Publication of EP1117776A1 publication Critical patent/EP1117776A1/fr
Withdrawn legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • 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
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    • A61P11/06—Antiasthmatics
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention relates to the production of single stranded DNA (ssDNA) in yeast, prokaryotic, and eukaryotic cells from a set of genetic elements delivered to the cell by a vector system.
  • the ss DNA is produced in the cell with minimal vector sequences which could interfere with the intended function of the ssDNA in the cell. So far as is known, there is no method for producing single-stranded deoxyribonucleic acid (ssDNA) species in eukaryotic cells which do not contain intervening and/or flanking vector sequences.
  • ssDNA single-stranded deoxyribonucleic acid
  • the scientific and patent literature does include the disclosure of cDNA-producing vectors (see A. Ohshima, et al., 89 Proc. Natl. Acad. Sci.
  • a set of genetic elements for delivery into a cell comprising a nucleic acid construct comprising a sequence of interest, and a primer binding site for a reverse transcriptase located in a 3' position with respect to the sequence of interest.
  • the set of genetic elements of the present invention provides an efficient system for directing the synthesis of a stable, single-stranded nucleic acid sequence, both in vivo and in vitro.
  • the single-stranded nucleic acid sequence may be used to provide a desired effect in a cell, tissue or organism. Because production of the single-stranded nucleic acid sequence of interest takes place within the cell, prior art problems arising from delivery of the single-stranded nucleic acid sequence to the cell are overcome, or at least alleviated.
  • nucleic acid construct Because of the arrangement of the nucleic acid construct, with the primer binding site in a position which is 3' to the sequence of interest, there is no limit to the size or type of sequence of interest that may be produced using the nucleic acid construct of the present invention, and the construct may be easily incorporated into a vector for delivery by any desired route to a target cell.
  • Reverse transcription may be carried out by a reverse transcriptase which is endogenous to the cell (e.g. in the case of infection by human immunodeficiency virus or simian immunodeficiency virus) or the set of genetic elements may, preferably, further comprise a reverse transcriptase gene.
  • the set of genetic elements comprises a reverse transcriptase gene
  • the reverse transcriptase gene is, preferably, polycistronically transcribable with the sequence of interest and primer binding site.
  • the reverse transcriptase gene is located on the same nucleic acid construct as the sequence of interest and primer binding site and, more preferably, the reverse transcriptase gene is located in a 5' position with respect to said sequence of interest and 3' primer binding site.
  • the reverse transcriptase gene may encode reverse transcriptase or a reverse transcriptase/RNAse H polyprotein.
  • the gene encoding reverse transcriptase/RNAse H polyprotein may suitably be derived from Moloney murine leukaemia virus, human immunodeficiency virus, or simian immunodeficiency virus.
  • the primer binding site is, preferably, specific for the reverse transcriptase encoded by the reverse transcriptase gene.
  • the primer binding site is, preferably, specific for an endogenous reverse transcriptase.
  • the primer binding site is complementary to a transfer RNA (tRNA).
  • the set of genetic elements of the present invention also comprises a promoter and, optionally, an enhancer for each of said sequence of interest and/or said reverse transcriptase gene.
  • the promoter and/or enhancer is a eukaryotic promoter and/or enhancer.
  • the promoter may be a constitutive, inducible, wide-spectrum or tissue-specific promoter.
  • the set of genetic elements of the present invention further comprises a polyadenylation tail sequence located in a 3' position with respect to the sequence of interest and 3' primer binding site.
  • the polyA tail provides stability of the mRNA transcript.
  • the sequence of interest is an antisense sequence.
  • the present invention thus, has far reaching uses in the field of antisense therapy, particularly in treating pathological conditions by regulating gene function.
  • the sequence of interest may also be an aptamer (i.e. an oligonucleotide that binds to a non-oligonucleotide target e.g. a protein).
  • an aptamer i.e. an oligonucleotide that binds to a non-oligonucleotide target e.g. a protein.
  • the present invention has far reaching therapeutic uses.
  • the nucleic acid construct is DNA.
  • the set of genetic elements according to any one of the preceding claims is incorporated into at least one vector.
  • sequence of interest and 3' primer binding site may be incorporated into a first vector, with the reverse transcriptase gene incorporated into a second vector.
  • the reverse transcriptase gene, sequence of interest and primer binding site may be incorporated into a single vector.
  • the reverse transcriptase gene is, preferably, located in a 5' position with respect to the sequence of interest and 3' primer binding site.
  • nucleic acid constructs of the present invention are such that they may be incorporated into commercially available delivery vectors for mammalian and human therapeutic purposes, and may be administered by any feasible route, depending on the target cell.
  • a vector which comprises: (a) a primer binding site and an insertion site for a sequence of interest, the primer binding site being located in a 3' position with respect to the insertion site; and
  • the reverse transcriptase gene is located in a 5' position with respect to the insertion site and 3' primer binding site.
  • a vector system which comprises a first vector, comprising an insertion site for a sequence of interest and a 3' primer binding site, and a second vector which comprises a reverse transcriptase gene.
  • the vector or vector system of the present invention is a plasmid or modified viral construct.
  • the reverse transcriptase gene is operably linked to an expression control sequence.
  • the vector or vector systems of the present invention may be advantageously employed to deliver antisense, sense, triplex, or any other single-stranded nucleotide sequence of interest into a cell, using known digestion and ligation techniques to splice the sequence of interest into the vector.
  • the vector or vector system described herein provides all the necessary signalling instructions and enzymatic functions to allow a host cell to produce a single-stranded nucleic acid molecule having a desired sequence.
  • the vector or vectors systems of the present invention may also be designed to allow the primer binding site to be removed and exchanged, so that different primer binding sites can be used, depending upon the requirements of the user and the specificity of the reverse transcriptase being used.
  • a host cell stably transformed or transfected with a vector or vector system of the present invention in particular, a eukaryotic cell stably transformed or transfected with a vector or vector system of the present invention.
  • Eukaryotic cells include yeast or plant cells, or mammalian cells.
  • kit for producing a single stranded nucleic acid sequence comprises a vector or vector system according to the present invention, and a restriction endonuclease for the insertion site.
  • kit for producing a single-stranded nucleic acid sequence comprises a vector or vector system according to the present invention, a container for the vector/vector system, and instructions for use of the vector/vector system.
  • an in vivo or in vitro method of producing a single-stranded nucleic acid sequence of interest comprises the steps of introducing a nucleic acid construct into a target cell, the nucleic acid construct comprising a sequence of interest and a primer binding site located in a 3' position with respect to the sequence of interest, transcribing the nucleic acid construct into mRNA and reverse transcribing the mRNA into cDNA.
  • the method further comprises the step of removing the mRNA from an mRNA/cDNA heteroduplex formed by reverse transcription of the mRNA.
  • Reverse transcription may be carried out either by a reverse transcriptase expressed by a reverse transcriptase gene introduced into the target cell, or by a reverse transcriptase which is endogenous to the target cell (e.g. where the target cell has been infected with human immunodeficiency virus or simian immunodeficiency virus).
  • the mRNA transcript may be removed from the mRNA/cDNA heteroduplex by means of RNAse H.
  • the RNAse H is expressed from a gene encoding a reverse transcriptase/RNAse H polyprotein introduced into the target cell.
  • the method may comprise the further step of isolating the mRNA transcript, mRNA/cDNA heteroduplex and/or single stranded cDNA from the target cell.
  • a single-stranded cDNA transcript an inhibitory nucleic acid molecule, (e.g. an antisense sequence or an aptamer), an mRNA transcript and/or a heteroduplex molecule produced by the in vivo or in vitro method of the present invention.
  • An inhibitory nucleic acid molecule may be single-stranded DNA synthesized from the mRNA transcript, or the mRNA transcript itself, which can specifically bind to a complementary nucleic acid sequence. Such inhibitory nucleic acid molecules are particularly useful for regulating gene function.
  • An inhibitory nucleic acid molecule may also be an oligo-nucleotide that specifically binds to an RNA or DNA-binding protein, or an oligo-nucleotide that binds to a biomolecule, e.g. thrombin, bradykinin or PGF2 ⁇ , which does not normally bind to RNA or DNA.
  • a pharmaceutical composition which comprises a set of genetic elements, a vector or vector system, or a host cell according to the present invention, together with a pharmacologically acceptable adjuvant, diluent or carrier.
  • a set of genetic elements, a vector or vector system, or a host cell according to the present invention for use in therapy, especially for use in delivering an inhibitory nucleic acid molecule to a target cell.
  • the set of genetic elements, vector and vector systems, and host cells of the present invention are particularly useful for alleviating pathological conditions by regulating gene expression.
  • a set of genetic elements, vector or vector system, or host cell according to the present invention for the manufacture of a medicament for alleviating a pathological condition by regulating gene expression, especially for alleviating a pathological condition by delivery of an inhibitory nucleic acid molecule to a target cell.
  • Other uses are also disclosed.
  • the sets of genetic elements, vectors, vector systems and host cells of the present invention may be used for the prophylactic or therapeutic treatment of a wide range of conditions or diseases, particularly conditions or diseases which are caused by abnormal or altered gene expression, or conditions or diseases which may be alleviated by regulating gene expression.
  • the sets of genetic elements, vectors, host cells, kits and methods of the present invention may be used to produce single-stranded nucleic acid molecules or virtually any predefined or desired nucleotide base composition in a host cell, and are adaptable and applicable to any in vivo or in vitro system.
  • the nucleic acid construct of the present invention is an artificially synthesised, recombinant, chimeric and/or heterologous product and the sequence of interest may be foreign to the host cell in which it is introduced.
  • Figure 1A referenced in the following description is a schematic view of a plasmid containing genetic elements encoding the sequence of interest and a primer binding site for reverse transcriptase.
  • Figure IB is a schematic view of a plasmid containing a gene for reverse transcriptase.
  • Figure 1C is a schematic view of a plasmid containing genetic elements encoding the sequence of interest, a primer binding site, and a gene for reverse transcriptase.
  • FIG. 2 is a schematic diagram illustrating one embodiment of the method of the present invention.
  • a vector (as used herein, the term “vector” refers to a plasmid or modified viral construct, or any other suitable vehicle, used to deliver and/or manipulate nucleic acid sequences of interest) was designed to produce ssDNA in vivo.
  • the vector contains all necessary signaling instructions and enzymatic functions to allow the host cell to produce the ssDNA encoding a desired sequence (a "sequence of interest”). Described are a set of genetic elements adapted for delivery into a cell by incorporation into the vector for synthesizing ssDNA in vitro or in vivo.
  • the vector also contains appropriate promoter(s)/enhancer(s). Also described herein is a method to construct a vector into which these genetic elements have been incorporated.
  • the reverse transcriptase gene which is the first component of the cassette
  • the reverse transcriptase gene from the Moloney Murine Leukemia Virus (MoMuLV) was used to advantage in the examples described.
  • Many other retroviral reverse transcriptase genes may be used to advantage in the cassette of the present invention, it being preferred that the reverse transcriptase gene is regulated by an appropriate upstream promoter/enhancer such as the Cytomegalovirus (CMV) or Rouse Sarcoma Virus (RSV) promoter for expression in eukaryotic cells.
  • the reverse transcriptase gene also preferably includes a downstream polyadenylation signal sequence so that the mRNA produced from the reverse transcriptase gene includes a 3' poly(A) tail for mRNA stability.
  • the reverse transcriptase produced in the cell synthesizes a complementary DNA (cDNA) from the primary mRNA transcript transcribed from the template encoding the genetic element that includes the sequence of interest as described below.
  • cDNA complementary DNA
  • the second component included in the cassette encodes a nucleic acid sequence that provides the template for synthesis of ssDNA in target cells. It is this element that includes the sequence of interest.
  • this genetic element is preferably regulated by an appropriate wide spectrum or tissue-specific promoter(s)/enhancer(s), such as the SV-40 promoter, or combination of promoter(s)/enhancer(s), located upstream of the genetic element.
  • tissue-specific or wide spectrum promoters/enhancers, or combinations of promoters/enhancers may be used to advantage to regulate the reverse transcriptase gene and sequence of interest.
  • the promoters/enhancers may be constitutive or inducible and may include the CMV or RSV (non-cell type specific) or GFAP (tissue specific) promoters/enhancers listed here and many other viral or mammalian promoters.
  • Representative promoters/enhancers that are appropriate for use in connection with the present invention may include, but are not limited to, HSVtk (McKnight et al., 217 Science 316, 1982), human beta-globulin (Breathnach et al., 50 Ann. Rev. of Biochem.
  • beta-actin Kawamoto et al., 8 Mol. Cell Biol. 267, 1988
  • rat growth hormone Larsen et al, 83 Proc. Natl. Acad. Sci. U.S.A. 8283, 1986
  • MMTV Human et al., 27 Cell 245 1981
  • adenovirus 5 E2 Imperiale, et al, 4 Mol. Cell. Biol. 875, 1984
  • SV40 Angel et al, 49 Cell 729, 1987
  • a-2-macroglobulin Kunz, et al., 17 Nucl. Acids Res.
  • PBS primer-binding site
  • tRNA transfer RNA
  • any PBS that is matched to the reverse transcriptase that comprises the set of genetic elements may be utilized for this purpose.
  • Multiple copies of the sequences of interest, each with its corresponding PBS, can be incorporated into the vector for delivery to a cell in accordance with the method of the present invention if desired, for example, for use in delivering anti-sense sequences to various regions of a gene within the target cell.
  • the mRNA primary transcript transcribed from the genetic element acts as the template used by the reverse transcriptase described above to synthesize and process the sequence of interest, which as noted above, can be any desired ssDNA.
  • the mRNA primary transcript contains a primer binding site (PBS) downstream to the sequence of interest.
  • PBS primer binding site
  • the PBS is exclusively recognized by a "primer tRNA.”
  • tRNAs are endogenous to cells. Each tRNA has the ability to recognize a unique sequence (i.e., codon) on the mRNA transcript coding for an amino acid, and has the ability to covalently link to a specific amino acid (i.e., the tRNA becomes "charged" when bound to a specific amino acid).
  • a "primer tRNA" when bound to the mRNA transcript PBS and not covalently linked (i.e., "uncharged") with an amino acid may be used to initiate ssDNA synthesis by the reverse transcriptase.
  • the MoMuLV reverse transcriptase used in the examples described herein recognizes and uses an uncharged lysine tRNA that in turn recognizes and binds to its unique sequence in the PBS.
  • each PBS incorporated into the vector must contain the unique sequence recognized by the primer tRNA, and the primer tRNA must be one that is recognized by the particular reverse transcriptase utilized.
  • the vector contain other specialized genetic elements to facilitate the identification of cells that carry the set of genetic elements of the present invention and/or to increase the level of expression of the sequence of interest.
  • the specialized genetic elements include selectable marker genes so that the vector can be transformed and amplified in a prokaryotic system.
  • selectable markers are genes that confer to the bacteria (e.g., E. coli) resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin (neomycin), or tetracycline.
  • the vector contain specialized genetic elements for subsequent transfection, identification and expression in a eukaryotic system.
  • multiple selection strategies e.g., Chinese Hamster Ovarian: CHO
  • multiple selection strategies e.g., Chinese Hamster Ovarian: CHO
  • Selectable markers used in eukaryotic systems include, but are not limited to, resistance markers for Zeocin, resistance to G418, resistance to aminoglycoside antibiotics, or phenotypic selection markers such ⁇ -gal or green fluorescence protein.
  • the linear ssDNA can be formed into an intact stem-loop ssDNA structure by the addition of inverted tandem repeats flanking the sequence of interest that form the "stem" portion after duplex formation.
  • the stem-loop structure can function similarly in many applications as the linear ssDNA form.
  • Such a ssDNA structure may be more resistant to intracellular nucleases by retaining the "ends" of a ssDNA in double stranded form.
  • the stem can be designed to contain a predetermined sequence or sequences (i.e., "aptamers") that are recognized and bound by specific DNA-binding proteins.
  • a stem structure is used in the cell as a competitor to titer out a selected protein(s) that regulate specific gene expression.
  • Adenovirus El a modulates gene expression of several adenoviral and cellular genes by affecting the activity of cell-encoded transcription factors resulting in changing normal cells to transformed cells.
  • the duplex structure of the stem thus functions to "bind up" the factor, preventing the protein from binding a promoter and thus inhibiting the expression of a particular deleterious gene.
  • the duplex stem structure may optionally contain multiple binding sites, for example, sites which are recognized by various transcription factors that actively regulate expression of particular gene.
  • adenovirus El a has been found to repress transcription of the collagenase gene via the phorbol ester-responsive element, a promoter element responsible for the induction of transcription by 12-O-tetradecanolyphorbol 13-acetate (TPA), by a number of other mitogens, and by the ras, os, src, and trk oncogenes.
  • TPA 12-O-tetradecanolyphorbol 13-acetate
  • the mechanism involves inhibition of the function of the transcription factor family AP-1.
  • the present invention is used to construct complex secondary ssDNA structures in the loop portion of the DNA transcript produced in accordance with the present invention.
  • Such secondary structure is engineered to serve any of several functions.
  • the sequence of interest optionally includes (but is not limited to) a sequence which is incorporated into the loop portion of the single-stranded cDNA transcript to form so-called "clover leaf or "crucible” like structures such as those found in the long terminal repeats of adenoassociated virus or in retrotransposons. Under correct circumstances, such structure is integrated in site-specific manner into the host genome.
  • a vector incorporating the set of genetic elements of the present invention is adaptable for incorporation into multiple commercially available delivery vectors for mammalian and human therapeutic purposes, multiple delivery routes are feasible depending upon the vector chosen for a particular target cell.
  • viral vectors are presently the most frequently used means for transforming the patient's cells and introducing DNA into the genome.
  • viral vectors, carrying new genetic information are used to infect target cells removed from the body, and these cells are then re-implanted (i.e., ex vivo).
  • Direct in vivo gene transfer into postnatal animals has been reported for formulations of DNA encapsulated in liposomes and DNA entrapped in proteoliposomes containing viral envelope receptor proteins (Nicolau et al, Proc.
  • the vector incorporating the set of genetic elements of the present invention is advantageously employed to deliver antisense, sense, triplex, or any other single-stranded nucleotide sequence of interest, using known digestion and ligation techniques to splice the particular sequence of interest into the vector in the presence or absence of inverted tandem repeats.
  • Those skilled in the art who have the benefit of this disclosure will also recognize that the above-described signals used for expression within eukaryotic cells may be modified in ways known in the art depending upon the particular sequence of interest. The most likely change is to change the promoter so as to confer advantageous expression characteristics on the sequence of interest in the system in which it is desired to express the sequence of interest.
  • the present invention is also utilized to produce inhibitory nucleic acids for use in therapeutics in vivo or in vitro.
  • Inhibitory nucleic acids may be ssDNA synthesized from the mRNA template or the mRNA template itself, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA— RNA, a DNA— DNA, or RNA-DNA duplex or triplex is formed. More commonly, these nucleic acids are often termed "antisense” because they are usually complementary to the sense or coding strand of the gene, but the "sense" sequence is also utilized in the cell for therapeutic purposes.
  • oligonucleotides that specifically bind to biomolecules that do not normally bind to RNA or DNA has now been demonstrated for a number of biomolecules that vary widely in size, structure and composition. These molecules include: (1) thrombin, a multifunctional regulatory protein that converts fibrinogen to fibrin in the process of clot formation; (2) bradykinin, a nonapeptide kinin involved in blood pressure regulation and implicated in hypotension; (3) PGF2. alpha., a prostaglandin or fatty acid derivative that exhibits hormonal activity. Additionally, the interaction of oligonucleotides with biomolecules whose natural biological function is primarily extracellular has now been demonstrated. U.S. Pat. No. 5,840,867. The term "inhibitory nucleic acids” as used herein, therefore, refers to both "sense” and "antisense” nucleic acids.
  • an inhibitory nucleic acid By binding to the target nucleic acid, an inhibitory nucleic acid inhibits the function of the target nucleic acid.
  • This inhibitory effect results from, for example, blocking DNA transcription, processing or poly(A) addition to mRNA, DNA replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradation.
  • Inhibitory nucleic acid methods therefore encompass a number of different approaches to altering expression of genes.
  • An example of an antiherpes virus inhibitory nucleic acid is ISIS 2922 (ISIS Pharmaceuticals, Carlsbad, CA) which has activity against CMV (see Biotechnology News 14:5).
  • inhibitory nucleic acid therapy approaches can be classified into (1) those that target DNA sequences, (2) those that target RNA sequences (including pre-mRNA and mRNA), (3) those that target proteins (sense strand approaches), and (4) those that cause cleavage or chemical modification of the target nucleic acids.
  • the first approach contemplates several categories. Nucleic acids are designed to bind to the major groove of the duplex DNA to form a triple helical or "triplex" structure. Alternatively, inhibitory nucleic acids are designed to bind to regions of single stranded DNA resulting from the opening of the duplex DNA during replication or transcription. More commonly, inhibitory nucleic acids are designed to bind to mRNA or mRNA precursors.
  • Inhibitory nucleic acids are used to prevent maturation of pre-mRNA. Inhibitory nucleic acids may be designed to interfere with RNA processing, splicing or translation. In the second approach, the inhibitory nucleic acids are targeted to mRNA. In this approach, the inhibitory nucleic acids are designed to specifically block translation of the encoded protein. Using this second approach, the inhibitory nucleic acid can be used to selectively suppress certain cellular functions by inhibition of translation of mRNA encoding critical proteins.
  • an inhibitory nucleic acid complementary to regions of c-myc mRNA inhibits c-myc protein expression in a human promyelocytic leukemia cell line, HL60, which overexpresses the c-myc proto-oncogene.
  • HL60 promyelocytic leukemia cell line
  • inhibitory nucleic acids targeting mRNA have been shown to work by several different mechanisms to inhibit translation of the encoded protein(s).
  • the inhibitory nucleic acids introduced into the cell can also utilize the third approach of designing the "sense" strand of the gene or mRNA to trap or compete for the enzymes or binding proteins involved in mRNA translation, as described in Helene and Toulme. Lastly, the inhibitory nucleic acids is used to induce chemical inactivation or cleavage of the target genes or mRNA. Chemical inactivation occurs by the induction of crosslinks between the inhibitory nucleic acid and the target nucleic acid within the cell.
  • the present invention takes the form of a kit comprised of a plasmid having the above-described reverse transcriptase gene cloned therein as well as a multiple cloning site (MCS) into which the user of the kit inserts a particular sequence of interest, which may or may not include the above-described inverted tandem repeats in accordance with the user's intended result.
  • MCS multiple cloning site
  • the MCS is upstream from the genetic element encoding the primer binding site.
  • the resulting plasmid is then purified from the cell culture in which it is maintained, lyophilized or otherwise preserved for packaging and shipping to the user.
  • the kit preferably also includes the restriction endonuclease(s) for the MCS into which the sequence of interest is to be cloned, the ligases and other enzymes for inserting the sequence of interest into the plasmid, and a map of the plasmid, along with suitable reaction buffers.
  • the plasmid pcDNA3.I/Zeo+ was purchased from Invitrogen Corp. (San Diego, CA) and plasmid PBK-RSV from Statagene (La Jolla, CA). Oligodeoxyribonucleotides (ODN) were synthesized by Midland Certified Reagent Co. (Midland, TX). Polymerase chain reactions (PCR) were carried out using Taq DNA polymerase purchased from Boehringer Mannheim Corp. (Indianapolis, IN) in a Robo-gradient thermal cycler (Stratagene (La Jolla, CA). Restriction endonucleases and T4 DNA ligase were obtained from Boehringer Mannheim Corp. (Indianapolis, IN). The ODNs used are listed in the attached Sequence Listing.
  • the following in vivo experiments were designed to determine whether ssDNA could be produced in intact cells.
  • the plasmid utilized included the RSV promoter.
  • Plasmid Constructs The cloning vector pssXB and the plasmids containing the sequences to be expressed as single- stranded DNA were constructed from a common intermediate construct.
  • the host strain for these manipulations was XLl-Blue MRF' (Stratagene, La Jolla, CA).
  • the vector pcDNA3.1Zeo ⁇ (Invitrogen, San Diego, CA) was digested with the restriction enzymes Nhe I and Apa I.
  • This insert contains the Moloney Murine leukemia virus (MoMuLV) reverse transcriptase promoter region.
  • MoMuLV Moloney Murine leukemia virus
  • the plasmid pssDNA-Express-A (pssXA), containing genes for MoMuLV reverse transcriptase, was constructed from the vector pBK-RSV (Stratagene, La Jolla, CA), also using XL-1 Blue MRF' as the host strain.
  • pBK-RSV Stratagene, La Jolla, CA
  • XL-1 Blue MRF' A mouse cell line expressing MoMuLV was obtained from the American Type Culture Collection (ATCC #CRL-1858).
  • Virus RNA was isolated and reverse transcribed from ODN-RT (-) (Table I).
  • the reverse transcript was then PCR amplified according to the manufacturer's intructions using a kit from Promega (Madison, WI), primers ODN-RT (+) and ODN-RT (-), and digested with Sac I and Hind III (sites for these restriction endonucleases are present in the 5' and 3' primers, respectively).
  • the 2.4 kb product obtained includes the sequence of the MoMuLV genome between positions 2546 and 4908.
  • the mature virus reverse transcriptase peptide is encoded by the sequence between positions 2337 and 4349 (Petropoulos, C.J. Retroviral taxonomy, protein structure, sequences and genetic maps. In: Retroviruses,
  • the pBRK-RSV vector was digested with Xba I and Nbe I, which removes the lac promoter region.
  • the Nhe I end was converted to a Sac I end using the linker formed by annealed oligodeoxynucleotides OD ⁇ - ⁇ >S (+) and ODN-N>S (-).
  • the reverse transcriptase amplimers were ligated through the Hind III sites and this construct was subsequently ligated between the Sac I and Xba I sites of pBK-RSV to give pBK-RSV-RT.
  • the set of genetic elements comprising the present invention are also expressed from a single plasmid made by a fusion of, for instance, the pc3.1DNA/Zeo-derived plasmids and the pBK-RSV-derived plasmids such that fused plasmids encode the ss-cDNA-encoding genetic element, the Mo-MuLV-RT gene, and the PBS.
  • pBK-RSV-RT/MboL is digested with Nsil to release a 5.3kb fragment containing the Mo-MuLV-RT gene with an intervening his-pro linker and associated regulatory elements.
  • the 5.3kb DNA fragment is ligated to a linker containing an internal EcoRI site and digested with EcoRI.
  • the pc3.1/Zeo/N-M and the derivative plasmids containing test sequences are digested with Bglll, which recognizes a unique site on pc3.1DNA/Zeo in the cytomegalovirus enhancer/promoter (P CMV).
  • the Bglll ends are ligated to Seq. ID 15 and Seq. ID 16, which contain an internal EcoRI site.
  • the 5.3kb fragment is ligated to pc3.1/Zeo/N-M and derivatives to generate the plasmid.
  • the ss- cDNA is isolated from cells transfected 48-72-hr earlier using triazol reagent (Gibco Life Technologies, Gaithersburg, MD). Assays for specific ss-cDNA species are carried out by both PCR based assays for internal fragment and by denatured single stranded gel electrophoresis with subsequent nylon blotting and probing with an internal biotin-labeled probe.
  • Example 2 Reverse Transcriptase Activity in Transformed Cells To determine the presence of reverse transcriptase activity in extracts of cells containing the pBK-RSV-RT construct, the following assay is used. This assay relies upon reverse transcriptase activity in protein extracts of transformed cells to produce a DNA copy of the Brome Mosaic Virus RNA genome (Silver, et al, 1993). The replication cycle of this virus does not involve a DNA intermediate, eliminating the possibility that an amplification product could be produced without prior reverse transcription.
  • a method and pharmaceutical preparation for diagnosing and treating pathological conditions related to a dopamine receptor abnormality Abnormal activity of the dopaminergic nervous system has been implicated in a number of motor and behavioral disorders including Parkinson's disease, Huntington's disease, tardive dyskinesia, certain forms of schizophrenia and other dystonias and dyskinesias.
  • Dysfunctions of the dopaminergic system may be caused either by a reduced or increased activity of the dopaminergic system or by the inability of the systems to be modulated by a changing external or internal environment.
  • a plasmid is constructed to include a sequence of interest that generates an antisense oligonucleotide capable of binding specifically to an expression-controlling sequence of a nucleic acid encoding the dopamine receptor.
  • the plasmid is administered under conditions whereby the plasmid enters cells expressing the dopamine receptor and generates the inhibitory nucleotide.
  • the inhibitory nucleotide binds specifically to expression-controlling sequences of such RNA molecules, thereby selectively controlling expression of one or more dopamine receptor subtypes, and alleviating the pathological conditions related to their expression. Efficacy is tested in accordance with the method described in U.S. Patent No. 5,840,708.
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • Kaposi's sarcoma-associated herpes virus is a new human herpes virus (HHV8) believed to cause Kaposi's sarcoma (KS).
  • Kaposi's sarcoma is the most common neoplasm occurring in persons with acquired immunodeficiency syndrome (AIDS). Approximately 15-20% of AIDS patients develop this neoplasm which rarely occurs in immunocompetent individuals. Epidemiologic evidence suggests that AIDS-associated KS (AIDS-KS) has an infectious etiology.
  • Gay and bisexual AIDS patients are approximately twenty times more likely than hemophiliac AIDS patients to develop KS, and KS may be associated with specific sexual practices among gay men with AIDS.
  • KS is uncommon among adult AIDS patients infected through heterosexual or parenteral HIV transmission, or among pediatric AIDS patients infected through vertical HIV transmission.
  • Agents previously suspected of causing KS include cytomegalovirus, hepatitis B virus, human papillomavirus, Epstein-Barr virus (EBV), human herpesvirus 6, human immunodeficiency virus (HIV), and Mycoplasma penetrans.
  • Non-infectious environmental agents, such as nitrite inhalants also have been proposed to play a role in KS tumorigenesis. Extensive investigations, however, have not demonstrated an etiologic association between any of these agents and AIDS-KS.
  • Virion protein 26 is a component of the nucleocapsid structure in most herpes viruses. This structure serves as a delivery mechanism for the viral genome as it is spread from one infected cell to another. As part of the original infecting virus, it is recognized as a major antigen by the immune system and can therefore be used to screen for antibodies to the herpes virus in patient sera and as a vaccine.
  • a plasmid is constructed using the methods described above to include a sequence of interest.
  • the sequence of interest is an isolated nucleic acid molecule which encodes KSHV virion protein 26 or antisense or triplex oligonucleotide molecule as described in U.S. Patent No. 5,840,708.
  • the plasmid is administered under conditions whereby the plasmid enters infected cells and generates the inhibitory nucleotide.
  • the inhibitory nucleotide binds specifically to expression-controlling sequences of such RNA molecules, or encoding sequences, thereby selectively controlling expression of KSHV virion protein 26, and alleviating the pathological conditions related to expression.
  • Inhibitory nucleotides to modulate the expression of IL-8 and/or IL-8 Receptor to control growth, metastasis and/or angiogenesis in tumors are included in the group consisting of IL-8 and/or IL-8 Receptor and/or angiogenesis in tumors.
  • Interleukin-8 (IL-8, neutrophil activating protein- 1, or NAP-1) is a member of C- X-C chemokine family of related cytokines having broad involvement in inflammatory responses, tissue injury, growth regulation and cellular adhesion. Cerretti, D. P., et al., Molecular Characterization of Receptors for Human Interleukin-8, GRO/Melanoma Growth- Stimulatory Activity and Neutrophil Activating Peptide-2, Molecular Immunology, 30(4), 359-367 (1993); and Koch, A.
  • IL-8 has also been shown to have a potent stimulatory effect on angiogenesis. See, e.g., Koch, A. E., Interleukin-8 as a Macrophage- Derived Mediator of Angiogenesis, Science, 258, 1798-1800 (1992).
  • IL-8 is produced by a variety of normal human somatic cells including monocytes/macrophages, dermal fibroblasts, vascular endothelial cells, keratinocytes, and mesangeal cells. Yasumoto, K., et al., Tumor Necrosis Factor Alpha and Interferon Gamma Synergistically Induce Interleukin 8 Production in a Human Gastric Cancer Cell Line Though Acting Concurrently on AP-1 and NF-kB-like Binding Sites of the Interleukin 8 Gene, J. of Biological Chemistry, 267(31), 22506-11 (1992). Apparently, such cells produce IL-8 only when stressed, and not under conditions of normal growth and homeostasis.
  • IL-8 Factors that induce IL-8 production include inflammation, IL-1, TNF, LPS and thrombin. It is also known that IL-8 is commonly secreted by tumor cells. Because of its effects on growth, it is suspected that IL-8 has a significant role in the metastatic spread of melanoma and other cancers.
  • IL-8 is a ligand for cell-membrane IL-8 Receptor, and it is thought that interaction between IL-8 and IL-8 Receptor is required for IL-8 action.
  • Two IL-8 receptor genes have been identified so far, IL-8 Receptor type A and type B. Both genes belong to the so-called seven transmembrane domain, G protein-coupled receptor family.
  • Receptor A has been shown to be activated by IL-8
  • receptor B has been shown to be activated by IL-8 as well as other cytokines belonging to C-X-C family including Melanoma Growth Stimulatory Activity (MGSA).
  • MGSA Melanoma Growth Stimulatory Activity
  • IL-8 Receptor B present in cancer and other tumor cells is not fully elucidated. There is, however, evidence that activation of IL-8R B (1) is involved in the mechanism of growth regulation of melanoma and tumorigenic fibroblasts; (2) is associated with transformation of lung cells by asbestos, and (3) correlates with metastic potential of melanoma.
  • oligonucleotides which modulate expression of either IL8 or IL-8 Receptor in cancers in vivo. It would be particularly advantageous to provide oligonucleotides which are effective against lung cancer and melanoma because each of these cancers produce their own growth factors.
  • SCLC small cell lung carcinoma
  • NSCLC non-small cell lung carcinoma
  • SCLC comprises approximately one-fourth of the cases, expresses neuroendocrine markers, and generally metastasizes early to lymph nodes, brain, bones, lung and liver.
  • NSCLC comprises the majority of the remaining lung tumor types, and includes adeno-carcinoma, squamous cell carcinoma, and large cell carcinoma.
  • NSCLC is characterized by epithelial-like growth factors and receptors, and is locally invasive.
  • Melanoma cells unlike normal melanocytes, can proliferate in the absence of exogenous growth factors. This independence apparently reflects the production of growth factor and cytokines for autocrine growth stimulation, including TGF-.ANG., TGF-, platelet-derived growth factor A and B chains, basic fibroblast growth factor, IL-8, IL-6, IL-1, granulocyte macrophage colony stimulating factor, and MGSA. Guo Y, et al, Inhibition of Human Melanoma Growth and Metastasis in Vivo by Anti-CD44 Monolclonal Antibody. Cancer Res., 54, 1561-1565 (1994).
  • a plasmid is constructed using the methods described above to include a sequence of interest.
  • the sequence of interest is an isolated nucleic acid molecule as described in U.S. Patent No. 5,849,903.
  • the plasmid is administered (e.g., inhalation or direct injection into solid tumors) under conditions whereby the plasmid enters cells and generates the inhibitory nucleotide.
  • the inhibitory nucleotide binds specifically to expression-controlling sequences of such RNA molecules, or encoding sequences, thereby selectively controlling expression of IL-8 receptors, and alleviating the pathological conditions related to expression.
  • Example 7 Antisense oligonucleotide inhibition of cytomegalovirus infection.
  • Cytomegaloviruses are ubiquitous in nature and are the most common causes of intrauterine infection. Congenital infection is common in newborns of infected mothers. In some populations, as much as 10% of children display perinatal infections. In a small percentage of newborns, the infection is virulent, involving multiple organs.
  • a plasmid is constructed using the methods describe above to include a sequence of interest encoding for an inhibitory nucleotide. Oligonucleotides having a sequence of nucleotide bases specifically hybridizable with a selected sequence of a cytomegalovirus DNA or RNA are described in U.S. Patent No. 5,442,049.
  • the plasmid is administered to the patient under conditions whereby the plasmid enters cells and generates the inhibitory nucleotide.
  • the inhibitory nucleotide binds specifically to expression-controlling sequences of such RNA molecules, or encoding sequences, thereby selectively controlling replication of CMV, and alleviating the pathological conditions related to CMV infection.
  • This plasmid is used either prophylactically or therapeutically to reduce the severity of disease caused by CMV.
  • Oligonucleotides specifically hybridizable with RNA or DNA deriving from a gene corresponding to one of the open reading frames UL5. UL8. UL9. UL20. UL27. UL29. UL30. UL42. UL52 and IE175 of herpes simplex virus type 1.
  • Oligonucleotides are designed to be specifically hybridizable with DNA or even more preferably, RNA from one of the species herpes simplex virus type 1 (HSV-1), herpes simplex virus type (HSV-2), cytomegalovirus, human herpes virus 6, Epstein Barr virus (EBV) or varicella zoster virus (VZV).
  • HSV-1 herpes simplex virus type 1
  • HSV-2 herpes simplex virus type
  • cytomegalovirus cytomegalovirus
  • EBV Epstein Barr virus
  • VZV varicella zoster virus
  • a plasmid is constructed using the methods described above to include a sequence of interest.
  • the sequence of interest is an isolated nucleic acid molecule as described in U.S. Patent No. 5,514,577.
  • Tthe plasmid is administered (e.g., inhalation or direct injection into solid tumors) under conditions whereby the plasmid enters cells and generates the inhibitory nucleotide.
  • the inhibitory nucleotide binds specifically to expression-controlling sequences of such RNA molecules, or encoding sequences, from one of the species herpes simplex virus type 1 (HSV-1), herpes simplex virus type (HSV-2), cytomegalovirus, human herpes virus 6, Epstein Barr virus (EBV) or varicella zoster virus (VZV) thereby selectively controlling virus infection, and alleviating the pathological conditions related to infection.
  • HSV-1 herpes simplex virus type 1
  • HSV-2 herpes simplex virus type
  • cytomegalovirus cytomegalovirus
  • human herpes virus 6, Epstein Barr virus (EBV) or varicella zoster virus (VZV) varicella zoster virus
  • the proto-oncogene c-myb is the normal cellular homologue of the avian myeloblastosis virus-transforming gene v-myb.
  • the c-myb gene codes for a nuclear protein expressed primarily in hematopoietic cells. It is a proto-oncogene, that is, it codes for a protein which is required for the survival of normal, non-tumor cells. When the gene is altered in the appropriate manner, it has the potential to become an oncogene.
  • Oncogenes are genes whose expression within a cell provides some function in the transformation from normal to tumor cell.
  • An example is the human c-myb gene which has been isolated, cloned, and sequenced. Majello et al, Proc. Natl. Acad. Sci. U.S.A. 83,
  • a plasmid is constructed using the methods describe above to include a sequence of interest encoding for an inhibitory nucleotide. Oligonucleotides having a sequence of nucleotide bases specifically hybridizable with a selected sequence of the DNA or RNA as are described in U.S. Patent No. 5,098,890.
  • the plasmid is administered to the patient under conditions whereby the plasmid enters cells and generates the inhibitory nucleotide thus acting as an antineoplastic or immunosuppressive agent.
  • inhibitors of ICAM-1, VCAM-1 and ELAM-1 expression would provide a novel therapeutic class of anti-inflammatory agents with activity towards a variety of inflammatory diseases or diseases with an inflammatory component such as asthma, rheumatoid arthritis, allograft rejections, inflammatory bowel disease, various dermatological conditions, and psoriasis.
  • inhibitors of ICAM- 1, VCAM-1, and ELAM-1 may also be effective in the treatment of colds due to rhinovirus infection, AIDS, Kaposi's sarcoma and some cancers and their metastasis.
  • there are no known therapeutic agents which effectively prevent the expression of the cellular adhesion molecules ELAM-1, VCAM-1 and ICAM-1.
  • Monoclonal antibodies may prove to be useful for the treatment of acute inflammatory response due to expression of ICAM-1, VCAM-1 and ELAM-1.
  • the host animal develops antibodies against the monoclonal antibodies thereby limiting their usefulness.
  • monoclonal antibodies are large proteins which may have difficulty in gaining access to the inflammatory site. Soluble forms of the cell adhesion molecules suffer from many of the same limitations as monoclonal antibodies in addition to the expense of their production and their low binding affinity. Thus, there is a long felt need for molecules which effectively inhibit intercellular adhesion molecules.
  • Antisense oligonucleotides avoid many of the pitfalls of current agents used to block the effects of ICAM-1, VCAM-1 and ELAM-1.
  • PCT/US90/02357 discloses DNA sequences encoding Endothelial Adhesion Molecules (ELAMs), including ELAM-1 and VCAM-1 and VCAM- lb.
  • ELAMs Endothelial Adhesion Molecules
  • a number of uses for these DNA sequences are provided, including (1) production of monoclonal antibody preparations that are reactive for these molecules which may be used as therapeutic agents to inhibit leukocyte binding to endothelial cells; (2) production of ELAM peptides to bind to the ELAM ligand on leukocytes which, in turn, may bind to ELAM on endothelial cells, inhibiting leukocyte binding to endothelial cells; (3) use of molecules binding to ELAMS (such as anti-ELAM antibodies, or markers such as the ligand or fragments of it) to detect inflammation; (4) use of ELAM and ELAM ligand DNA sequences to produce nucleic acid molecules that intervene in ELAM or ELAM
  • a plasmid is constructed using the methods describe above to include a sequence of interest encoding for an inhibitory nucleotide for ICAM-1, VCAM-1 or ELAM-1. Oligonucleotides having a sequence of nucleotide bases specifically hybridizable with a selected sequence of ICAM-1, VCAM-1 or ELAM-1 DNA or RNA are described in U.S. Patent No. 5,843,738.
  • the plasmid is administered to the patient under conditions whereby the plasmid enters cells and generates the inhibitory nucleotide.
  • the inhibitory nucleotide binds specifically to expression-controlling sequences of such RNA molecules, or encoding sequences, thereby selectively controlling the expression of ICAM-1, VCAM- 1 or ELAM-1, and alleviating the pathological conditions related to ICAM-1, VCAM-1 and ELAM-1 expression.
  • This plasmid is used either prophylactically or therapeutically to reduce the severity of inflammation caused by ICAM-1, VCAM-1 and ELAM-1.
  • Protein-Binding Oligonucleotides Specifically Bind Target Molecules
  • Tuerk and Gold describe the use of a procedure termed "systematic evolution of ligands by exponential enrichment.”
  • a pool of RNAs that are completely randomized at specific positions is subjected to selection for binding by a desired nucleic acid-binding protein which has been fixed on a nitrocellulose filter.
  • the bound RNAs then are recovered and amplified as double-stranded DNA that is competent for subsequent in vitro transcription.
  • the newly transcribed RNA then is recycled through this procedure to enrich for oligonucleotides that have consensus sequences for binding by the cognate protein.
  • the oligonucleotides so obtained then may be sequenced for further study. Tuerk and Gold applied this procedure to identify RNA oligonucleotides which are bound by the RNA binding region of T4 DNA polymerase.
  • oligonucleotides that specifically bind to biomolecules that do not normally bind to RNA or DNA has now been demonstrated for a number of biomolecules that vary widely in size, structure and composition. These molecules include: ( 1 ) thrombin, a multifunctional regulatory protein that converts fibrinogen to fibrin in the process of clot formation; (2) bradykinin, a nonapeptide kinin involved in blood pressure regulation and implicated in hypotension; (3) PGF2. alpha., a prostaglandin or fatty acid derivative that exhibits hormonal activity. Additionally, the interaction of oligonucleotides with biomolecules whose natural biological function is primarily extracellular has now been demonstrated.
  • a plasmid is constructed using the methods describe above to include a sequence of interest encoding for an aptamer to thrombin.
  • Aptamers having a sequence of nucleotide bases specifically binding to thrombin are described in U.S. Patent No. 5,840,867.
  • the plasmid is administered to the patient under conditions whereby the plasmid enters cells and generates the aptamer.
  • an ex vivo administration is performed where cells are removed from a patient, the plasmid is transfected into the cells, and the cells are then placed back into the patient.
  • the aptamer binds specifically to thrombin, thereby selectively controlling the biological activity of thrombin, and alleviates the pathological conditions related to thrombin' s presence.
  • This plasmid is used either prophylactically or therapeutically.
  • the cassette described herein is described as being made up of three primary components, genetic elements which comprises a sequence of interest and primer binding site, and a reverse transcriptase gene, each of these components being provided with appropriate promoters as described herein.
  • the MoMuLV reverse transcriptase gene described for use as the reverse transcriptase gene of the cassette can be replaced with other reverse transcriptase genes and that promoters other than the CMV promoter may be used to advantage. All such changes and modifications which do not depart from the spirit of the present invention are intended to fall within the scope of the following non-limiting claims.

Abstract

L'invention concerne des méthodes et compositions de production d'ADN complémentaire simple brin grâce à un système vectoriel dans des cellules eucaryotes. Le vecteur contient toutes les instructions de signalisation et le fonctions enzymatiques nécessaires pour permettre à la cellule hôte de produire l'ADN simple brin codant pour une séquence d'acide nucléique voulue (une « séquence intéressante »). L'invention concerne également les composants compris dans le vecteur et destinés à synthétiser l'ADN simple brin in vivo. Ces composants comprennent: (1) un gène de transcriptase inverse, (2) un élément génétique fournissant la matrice de la séquence d'ADN simple brin intéressante et (3) un deuxième élément génétique situé à proximité de l'élément génétique codant pour la séquence intéressante qui fournit le site d'amorçage de la transcription inverse par la molécule de transcriptase inverse. Le vecteur contient également des promoteurs/séquences activatrices appropriées. L'invention concerne enfin une méthode de mise au point d'un vecteur comprenant ces composants.
EP99951989A 1998-10-09 1999-10-12 Synthese enzymatique d'adn simple brin Withdrawn EP1117776A1 (fr)

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CA2346155A1 (fr) 2000-04-20
MXPA01003642A (es) 2003-07-21
KR20010099682A (ko) 2001-11-09
JP2002527061A (ja) 2002-08-27
BR9914772A (pt) 2001-12-11
WO2000022113A9 (fr) 2000-08-24
WO2000022113A1 (fr) 2000-04-20
AU6430599A (en) 2000-05-01

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