WO2002068629A2 - Constructions d'adn pour expression cytoplasmique et mitochondriale et procedes de production et d'utilisation de ces constructions - Google Patents

Constructions d'adn pour expression cytoplasmique et mitochondriale et procedes de production et d'utilisation de ces constructions Download PDF

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WO2002068629A2
WO2002068629A2 PCT/US2002/000543 US0200543W WO02068629A2 WO 2002068629 A2 WO2002068629 A2 WO 2002068629A2 US 0200543 W US0200543 W US 0200543W WO 02068629 A2 WO02068629 A2 WO 02068629A2
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nucleic acid
molecule
sequence
acid molecule
acid sequence
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PCT/US2002/000543
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WO2002068629A3 (fr
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Cahterine J. Pachuk
Daniel Edward Mccallus
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Wyeth
Satishchandran, Chandrasekhar
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Publication of WO2002068629A3 publication Critical patent/WO2002068629A3/fr

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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor

Definitions

  • the invention relates generally to delivery vehicles, and more particularly, to nucleic acid vectors useful for delivering a selected moiety to host cells.
  • the invention further provides a composition containing a nucleic acid molecule of the invention and a physiologically compatible carrier.
  • the invention provides a method of expressing a heterologous nucleic acid sequence in the cytoplasm of a host cell in vitro, ex vivo or in vivo by delivering a DNA molecule of the invention to the cell.
  • the method involves delivering a DNA molecule containing a polyadenine sequence of sufficient length to provide mRNA expression of the heterologous nucleic acid sequence and a Kozak sequence, and expressing the sequences in the presence of an mRNA capping enzyme or co-expressing with an mRNA capping enzyme.
  • a heterologous nucleic acid sequence of the invention is co-expressed with a mitochondrial RNA polymerase, preferably without a functional mitochondrial targeting sequence.
  • the heavy strand promoter is located within SEQ ID NO: 1: 5'-GAACCAACCAAACCCCAAAGACA - 3' and the light strand promoter is located within SEQ ID NO:2
  • these promoters may be modified, if needed or desired, including allelic or polymorphic variants.
  • any functional mitochondrial promoters may be selected, including mitochondrial promoters derived from other eukaryotic species, including plants.
  • Still another viral family desirable for use in targeting antigens for inducing immune responses in humans and non-human animals is the togavirus family, which includes the genera alphavirus, which include Sindbis viruses, RossRiver virus, and Venezuelan, Eastern & Western Equine encephalitis, and rubivirus, including Rubella virus.
  • the flaviviridae family includes dengue, yellow fever, Japanese encephalitis, St. Louis encephalitis and tick borne encephalitis viruses.
  • the family filoviridae which includes hemorrhagic fever viruses such as Marburg and Ebola virus may be a suitable source of antigens.
  • the paramyxovirus family includes parainfluenza Virus Type 1, parainfluenza Virus Type 3, bovine parainfluenza Virus Type 3, rubulavirus (mumps virus, parainfluenza Virus Type 2, parainfluenza virus Type 4, Newcastle disease virus (chickens), rinderpest, morbillivirus, which includes measles and canine distemper, and pneumovirus, which includes respiratory syncytial virus.
  • the influenza virus is classified within the family orthomyxovirus and is a suitable source of antigen (e.g., the HA protein, the Nl protein).
  • the herpesvirus family includes the sub-family alphaherpesviridae, which encompasses the genera simplexvirus (HSVI, HSVII), varicellovirus (pseudorabies, varicella zoster) and the sub-family betaherpesviridae, which includes the genera cytomegalovirus (HCMV, muromegalovirus) and the sub-family gammaherpesviridae, which includes the genera lymphocryptovirus, EBV (Burkitts lymphoma), infectious rhinotracheitis, Marek's disease virus, and rhadinovirus.
  • HSVI simplexvirus
  • varicellovirus pseudorabies, varicella zoster
  • betaherpesviridae which includes the genera cytomegalovirus (HCMV, muromegalovirus)
  • the sub-family gammaherpesviridae which includes the genera lymphocryptovirus, EBV (Burkitts
  • the pox virus family includes the sub-family chordopoxviridae, which encompasses the genera orthopoxvirus (Variola (Smallpox) and Vaccinia (Cowpox)), parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, and the sub-family entomopoxviridae.
  • the hepadnavirus family includes the Hepatitis B virus.
  • One unclassified virus which may be suitable source of antigens is the Hepatitis delta virus.
  • Still other viral sources may include avian infectious bursal disease virus and porcine respiratory and reproductive syndrome virus.
  • the alphavirus family includes equine arteritis virus and various Encephalitis viruses.
  • mycoplasma and chlamydial infections include: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal chlamydial infections.
  • Pathogenic eukaryotes encompass pathogenic protozoans and helminths and infections produced thereby include: amebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans; Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis; schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm) infections.
  • Desirable RNA molecules may include tRNA, dsRNA, ribosomal RNA, catalytic RNAs, and antisense RNAs.
  • a useful RNA sequence is a sequence which extinguishes expression of a targeted nucleic acid sequence in the treated animal.
  • T cell mediated autoimmune diseases include Rheumatoid arthritis (RA), multiple sclerosis (MS), Sj ⁇ gren's syndrome, sarcoidosis, insulin dependent diabetes mellitus (EDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease and ulcerative colitis.
  • TCRs T cell receptors
  • RA T cell receptors
  • TCRs T cell receptors
  • RA T cell receptors
  • TCRs T cell receptors
  • RA several specific variable regions of TCRs which are involved in the disease have been characterized. These TCRs include V-3, V-14, V-17 and V ⁇ -17.
  • delivery of a nucleic acid sequence that encodes at least one of these polypeptides will elicit an immune response that will target T cells involved in RA.
  • MS several specific variable regions of TCRs which are involved in the disease have been characterized.
  • B cell mediated autoimmune diseases include Lupus (SLE), Grave's disease, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosis and pernicious anemia.
  • Some aspects of the present invention relate to gene therapy; that is, to compositions for and methods of introducing into the cells of an individual nucleic acid molecules comprising exogenous copies of genes which either correspond to defective, missing, non-functioning or partially functioning genes in the individual or which encode therapeutic polypeptides, i.e., polypeptides whose presence in the individual will eliminate a deficiency in the individual and/or whose presence will provide a therapeutic effect on the individual thereby providing a means of delivering the polypeptide alternative to protein administration.
  • therapeutic nucleic acid sequences include a gene which encodes dystrophin or a functional fragment thereof, cystic fibrosis transmembrane receptor (CFTR), omithine transcarbamylase (OTC), and Factor VHI.
  • heterologous nucleic acid sequences may be selected for use in constructs and methods of the invention.
  • polyadenine sequence or “polyadenine tract” means a string of adenine residues carried on the nucleic acid molecule of the invention. It may be desirable to include a polyadenylation signal in the nucleic acid molecules as well as a polyadenine sequence or tract. While a polyadenylation signal triggers the addition of adenine residues at the 3' end of mRNAs only in the nucleus and not in the cytoplasm, eucaryotic translation machinery recognizes a polyadenylation signal in combination with a polyadenine sequence in mRNAs to be translated, resulting in greater translation efficiency.
  • polyadenylation signals are located 5' to the adenine residues.
  • a polyadenylation sequence may include splice donor and acceptor sites.
  • the polyadenylation sequence generally is inserted following the heterologous nucleic acid sequences.
  • a bicistronic plasmid there will be a polyadenine sequence located 3' to each coding sequence. More preferably, there will be a polyadenylation signal, followed by a polyadenine tract, located at the extreme 3' end of each 3' UTR.
  • the polyadenylation signal is derived from a viral, mammalian, or other source.
  • the nucleic acid molecule of the invention may further contain a transcription terminator, which is suitably located 3' to the polyadenine sequences.
  • Suitable transcription terminators may be readily selected from among bacterial terminators (Mermalaeva et al., "Prediction of transcription terminators in bacterial genomes", J. Mol Biol, 301(l):27-33 (2000); Henkin, "Transcription termination control in bacteria", Curr. Opin.
  • the nucleic acid molecule of the invention contains a ribonucleolytic cleavage site followed by a pause site terminator.
  • the cleavage site and pause site are separated by a spacer of about 20 to 200 nt in length. However, larger or smaller spacers may be utilized.
  • the nucleic acid molecule of the invention contains a Kozak sequence. Typically, this sequence is typically located at -6 to +1, with reference to the start codon (ATG) of the heterologous nucleic acid sequence. See, e.g., M. Kozak, Nucleic Acids Res., 12(2):867-872 (1954), which identifies a consensus sequence, SEQ ID NO:3: CC(A/G)CC[AUG](G) for eukaryotic translations initiation sites.
  • picornavirus proteases may be more desirable in constructs in which the nucleic acid molecule will be used to induce an immune response (or cell death), because in certain circumstances they may enhance such a response.
  • it may be desirable to place the picornavirus protease under the control of a relatively weak promoter such as, the adeno-associated virus (AAV) inverted terminal repeats (ITRs).
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • mRNA capping enzymes or another capping sequence may be co-expressed with the heterologous nucleic acid sequence. Such sequences may be carried on the nucleic acid molecule or co-expressed from a separate molecule or by the host cell.
  • the capping enzyme or capping sequence is operably linked to regulatory sequences which direct its expression and which are distinct from the regulatory sequences expressing the heterologous nucleic acid sequence.
  • a preferred viral mRNA capping enzyme is the vaccinia capping enzyme, e.g., Moss et al, Proc. Natl. Acad._Sci. USA, 90(7):2860-2864 (1993) and J.
  • eukaryotic initiation factor e_F4G protein
  • eIF4G is a cellular ribosomal cap binding protein which associates with other eukaryotic initiation factors and the ribosome and is involved in the translation process.
  • Truncated e_F4G which consists of the C-terminal half of the protein, continues to associate with the ribosome, but this truncated complex no longer translates capped mRNA (Sonnenberg et al, Mol Cell.
  • a mt RNA polymerase specificity factor which increases the affinity of mt RNA polymerase for the mt promoter may be co-expressed, either alone or in combination with the mt RNA polymerase.
  • MTF1 yeast mitochondrial RNA polymerase specificity factor
  • This mitochondrial RNA polymerase specificity factor has a mitochondrial targeting leader sequence, which can be deleted or left intact, or both.
  • the nucleic acid molecule may be provided with sequences which will facilitate targeting of the molecule to the mitochondria.
  • sequences which will facilitate targeting of the molecule to the mitochondria are particularly desirable where external means of targeting the molecule to the mitochondria such as a gene gun or a mitochondria disruption agent will not be used.
  • sequences which may be used for targeting the molecule of the invention to the mitochondria include a trafficking signal such as the mitochondrial RNA polymerase, or sequences encoding a protein which is imported into the mitochondria (e.g., during nuclear division) to which the heterologous nucleic acid sequences may be linked.
  • the mitochondrial promoter is used for driving expression of the heterologous nucleic acid sequence and one may select a second mitochondrial promoter for driving expression of another product carried on the molecule which is to be co-expressed.
  • the second mitochondrial promoter may be selected without regard to the selection of the first mitochondrial promoter.
  • the first mitochondrial (mt) promoter (which is operably linked to a first heterologous nucleic acid sequence) is a heavy strand mt promoter
  • the second mt promoter may be a light strand mt promoter.
  • a molecule of the invention may have a transcription unit in which the promoter driving expression of the product to be co-expressed with the heterologous nucleic acid sequence is a non-mitochondrial promoter.
  • the promoter driving expression of the product to be co-expressed with the heterologous nucleic acid sequence is a non-mitochondrial promoter.
  • high-level constitutive expression will be desired.
  • Examples of useful constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, Cell, 41:521-530 (1985), the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl ⁇ promoter (Invitrogen).
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • Inducible promoters regulated by exogenously supplied compounds, are also useful and include, the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (International Application WO 98/10088); the ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al, Science, 268:1766-1769 (1995), Harvey et al, Curr. Opin. Chem.
  • MT zinc-inducible sheep metallothionine
  • Dex dexamethasone-inducible
  • nucleic acid molecule includes a coding sequence operably linked to a tissue-specific promoter.
  • Suitable cells include, without limitation, cells such as CHO, BHK, MDCK, and various murine cells, e.g., 10T1/2 and WEHI cells, African green monkey cells, suitable primate cells, e.g., VERO, COS1, COS7, BSC1, BSC 40, and BMT 10, and human cells such as WI38, MRC5, A549, human embryonic retinoblast (HER), human embryonic kidney (HEK), human embryonic lung (HEL), TH1080 cells.
  • Other suitable cells may include NIH3T3 cells (subline of 3T3 cells), HepG2 cells (human liver carcinoma cell line), Saos-2 cells (human osteogenic sarcomas cell line), HuH7 cells or HeLa cells (human carcinoma cell line).
  • compositions of the invention contain nucleic acid molecules of the invention, or mixtures thereof, in an amount of about 1 ng to about 100 mg nucleic acids, and are formulated according to the mode of administration to be used and the desired effect, e.g., immunogenic or therapeutic.
  • the nucleic acid molecules are the sole active component of the compositions.
  • suitable inert components in such compositions may include carriers (e.g., water, saline), preservatives, stabilizers (e.g., gelatin and albumin), and the like.
  • an isotonic formulation is preferably used.
  • additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose.
  • isotonic solutions such as phosphate buffered saline are preferred.
  • the compositions of the invention contain molecules which co-express sequences that facilitate or enhance expression of the heterologous nucleic acid sequence in the cytoplasm or mitochondria.
  • Such molecules may encode the capping enzyme, mitochondrial RNA polymerase, or picornavirus protease, or other desirable sequences for co-expression which are discussed herein.
  • compositions of the invention such that they contain the nucleic acid molecules of the invention in a mixture containing other active components.
  • a cytokine e.g., IL-12 (Genetics Institute, Cambridge, MA)
  • an immunosuppressant e.g., cyclosporin A
  • a vasoconstriction agent is added to the formulation.
  • the nucleic acid molecule of the invention may be formulated into a composition which further contains a co-agent which facilitates uptake of DNA molecules by a cell.
  • CpG oligonucleotides may be included, either as a separate component as described in published International Application WO 96/02555, or incorporated within the nucleic acid molecule for delivering a heterologous nucleic acid sequence. See, published International Application No. WO 97/28259.
  • an immunomodulating protein may be used as a co-agent.
  • Preferred compositions that facilitate uptake of the DNA molecules of the invention by a cell are selected from the group consisting of: cationic lipids, liposomes and local anesthetics.
  • multiple co-agents are used.
  • the nucleic acid molecule of the invention is formulated with bupivacaine and compounds that display a functional similarity to bupivacaine in order to facilitate uptake of the nucleic acid molecule and, thus, expression of the heterologous nucleic acid sequence.
  • Bupivacaine-HCl is chemically designated as 2-piperidinecarboxamide, l-butyl-(2,6- dimethylphenyl)monohydro-chloride monohydrate and is widely available commercially for pharmaceutical uses from many sources including Astra Pharmaceutical Products Inc. (Westboro, Mass.) and Sanofi Winthrop Pharmaceuticals (New York, N.Y.).
  • Bupivacaine is commercially formulated with and without methylparaben and with or without epinephrine. Any such formulation may be used. It is commercially available for pharmaceutical use in concentrations of 0.25%, 0.5% and 0.75% which may be used in the present invention. According to the present invention, about 250 ⁇ g to about 10 mg of bupivacaine is administered. In some embodiments, about 250 ⁇ g to about 7.5 mg is administered. In some embodiments, about 0.50 mg to about 5.0 mg is administered. In some embodiments, about 1.0 mg to about 3.0 mg is administered. In some embodiments about 5.0 mg is administered. Alternative concentrations which elicit desirable effects may be prepared. Alternatively, other local anesthetics may be used as facilitators. Examples of suitable anesthetics include, without limitation, mepivacaine, lidocaine, procaine, carbocaine and methyl bupivacaine, other similarly acting compounds may be used.
  • the nucleic acid molecule is delivered in the form of a DNA plasmid.
  • the nucleic acid molecules of the invention are useful for ex vivo transduction of target cells.
  • ex vivo therapy involves removal of a population of cells containing the target cells, transduction of the cells in vitro, and then reinfusion of the transduced cells into the human or veterinary patient.
  • ex vivo transduction is particularly desirable when the target cells are dendritic cells or macrophages and/or when the heterologous nucleic acid sequence being delivered is highly toxic, e.g., in the case of some genes used in the treatment of cancer.
  • any suitable route of administration of the nucleic acid molecule of the invention may be used, including, direct delivery to the target organ, tissue or site, intranasal, inhalation, intravenous, intramuscular, subcutaneous, intradermal, vaginal, rectal, and oral administration. Routes of administration may be combined within the course of repeated therapy or immunization.
  • the gene gun is available commercially, e.g., from PowerJect and may be used according to manufacturer's instructions.
  • the nucleic acid molecule is most desirably delivered in the form of a DNA plasmid.
  • the nucleic acid molecule of the invention is delivered by a route of administration selected from the group consisting of: intramuscularly, intranasally, intraperitoneally, subcutaneously, intradermally, or topically or by lavage to mucosal tissue selected from the group consisting of vaginal, rectal, nasal, pulmonary, urethral, buccal, sublingual, or any other mucosal route.
  • Preferred routes of administration include intradermal, transdermal, subcutaneous, intraperitoneal, intramuscular, inhalation and oral.
  • Modes of administration include, but are not limited to, intravenous lines, syringes, needleless injectors, and nebulizers and other inhalation devices.
  • the invention further provides a method of treating a human or non-human animal by delivering a nucleic acid molecule according to the invention expressing the molecule in a sufficient amount to providing a therapeutic benefit to the animal.
  • the method involves delivering a nucleic acid molecule of the invention to a host cell comprising a mitochondrial promoter operably linked to a heterologous nucleic acid sequence, wherein the mitochondrial promoter directs expression of the nucleic acid sequence in the cytoplasm of a host cell.
  • bupivacaine is co-administered with a pharmaceutical composition of the invention either before, simultaneously with, or after the pharmaceutical composition. More preferably, the nucleic acid of the invention is formulated together with bupivacaine or other local anaesthetic facilitators. For example, in some embodiments about 50 ⁇ l to about 2 ml, preferably 50 ⁇ l to about 1500 ⁇ l and more preferably about 1 ml of 0.5% bupivacaine-HCl and 0.1% methylparaben in an isotonic pharmaceutical carrier.
  • about 50 ⁇ l to about 2 ml, preferably 50 ⁇ l to about 1500 ⁇ l and more preferably about 1 ml of 0.5% bupivacaine-HCl in an isotonic pharmaceutical carrier is administered at the same site as the pharmaceutical composition either before, simultaneously with, or after the vaccine is administered. See, Ciccarelli et al, International Application No. WO 98/48780.
  • the individual is first subjected to bupivacaine injection prior to delivery of the pharmaceutical composition of the invention. That is, for example, up to about a week to ten days prior to intramuscular injection of the pharmaceutical composition of the invention, the individual is first injected with bupivacaine.
  • the individual prior to immunization, is injected with bupivacaine about 1 to 5 days before administration of the pharmaceutical composition. In some embodiments, prior to immunization, the individual is injected with bupivacaine about 24 hrs before administration of the pharmaceutical composition. Alternatively, bupivacaine can be injected either simultaneously, minutes before or after immunization.
  • the bupivacaine is administered after administration of the pharmaceutical composition. For example, up to about a week to ten days after administration of the pharmaceutical composition, the individual is injected with bupivacaine. In some embodiments, the individual is injected with bupivacaine about 24 hrs after immunization. In some embodiments, the individual is injected with bupivacaine about 1 to 5 days after vaccination. In some embodiments, the individual is administered bupivacaine up to about a week to ten days after immunization.
  • co-expressed molecules may include the mitochondrial RNA polymerase (with or without the mitochondrial targeting sequences), a mitochondrial polymerase specificity factor, a picornavirus ribosomal cap binding protein protease or an eIF4G protein, such as are discussed in more detail above.
  • Other desirable co- expressed molecules may include those which facilitate targeting of the molecule through the use of a trafficking signal such as the mitochondrial RNA polymerase, or sequences encoding a protein which is imported into the mitochondria (e.g., during nuclear division) to which the heterologous nucleic acid sequences may be linked.
  • a mitochondria disruption agent Hoke et ⁇ l, J. Biol.
  • the heavy and light strand minimal and maximal human mitochondrial promoters were cloned into pNASS ⁇ (HG. 1) obtained from Clontech (Palo Alto, CA).
  • the promoter elements were cloned into the Not I site at nucleotide position 200 of pNASSB. The Not I end was blunted prior to all clonings.
  • the cloned promoters are positioned just upstream from the encoded ⁇ -galactosidase gene.
  • oligonucleotides were synthesized by Retrogen (San Diego, CA). The oligonucleotides were cloned into the Not I site using chain reaction cloning (CRC), a directional cloning technique (Pachuk et al, 'Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments ", Gene, 243: 19-25 (2000); U.S. Patent 6,143,527). The sequences of the oligonucleotides encoding the four human mitochondrial promoters and the CRC primers used to clone the oligonucleotides are listed below.
  • a cytoplasmic polyadenylation signal and terminator for mitochondrial polymerase (Kruse et al, "Termination of transcription in human mitochondria: identification and purification of a DNA binding protein factor that promotes termination " Cell, 58: 391-397 (1989) was cloned into one subset of vectors, such that there is a vector with a mitochondrial promoter and a cytoplasmic polyadenylation signal and a vector with only the mitochondrial promoter.
  • the top strand and the bottom strand of the cytoplasmic polyadenylation signal were synthesized as oligonucleotides.
  • the oligonucleotides were cloned into the Not I site (located at position 3700 on the pNASSB vector - see FIG. 1). The Not I site was blunted prior to cloning. Cloning was directional using the CRC technique.
  • sequences of the promoter oligonucleotides and the CRC oligonucleotides used in the ligation reaction are indicated below. Sequences of the cytoplasmic polyadenylation signal oligonucleotides and the CRC oligonucleotides used to clone these oligos are also indicated below.
  • SEQ ID NO:8 Promoter oligonucleotide A.
  • SEQ ID NO:9 Promoter oligonucleotide B
  • SEQ ID NO: 12 Promoter oligonucleotide A. 5' GGGTGACTGTTAAAAGTGCATACCGCCAAAAGATAAAATTTGAA 3'
  • SEQ ID NO: 14 CRC oligonucleotide 5'. 5' CGGAATTGTACCCGCGGCCGGGTGACTGTTAAAAGTGC 3'.
  • SEQ ID NO: 18 CRC oligonucleotide 5'
  • SEQ ID NO: 19 CRC oligonucleotide 3'
  • SEQ ID NO:20 Promoter oligonucleotide A 5' GAGCCAACCAAACCCCAAAGACA 3'
  • SEQ ID NO: 22 CRC oligonucleotide 5'
  • SEQ ID NO: 25 Polyadenylation oligonucleotide B. S'GCGATTACCGGGCTCTGCCATCTTAACATTTTTTTTTTT ⁇
  • SEQ ID NO: 26 CRC oligonucleotide 5'
  • Example 2 Expression from Nucleic Acid Molecule
  • the constructs bearing the human light strand minimal and maximal promoter elements, polyadenylation signal and mitochondrial polymerase termination signal were tested for their ability to promote expression in a tissue culture system.
  • the negative control in the experiments was the parental pNASSB vector depicted in FIG. 1.
  • This vector does not contain a promoter and hence the encoded ⁇ -galactosidase gene should not be expressed. Expression of ⁇ -gal from this vector would be indicative of a cryptic promoter in the backbone.
  • a proprietary vector containing the ⁇ -gal gene under the control of the HCMV immediate early promoter was included as a positive control.
  • Human rhabdomyosarcoma cells seeded into six-well plates and cultured with DMEM (10% FBS) were transfected with the experimental and control plasmids using lipofectamine (Gibco-BRL) as the transfecting agent. All transfections were done using a total of 2.5 ⁇ g DNA per transfection according to the manufacturers directions. At 48 hours post transfection, media was removed from the cells, the cells washed with 1 x PBS and cell lysates harvested in a detergent lysis buffer.
  • lysis buffer 300 mM NaCl, 150 mM Tris-Cl pH 7.6, 0.5% Triton X, 0.5% deoxycholate
  • lysis buffer 300 mM NaCl, 150 mM Tris-Cl pH 7.6, 0.5% Triton X, 0.5% deoxycholate
  • Lysates were aspirated into microfuge tubes and either assayed immediately or stored at -80 °C.
  • Assays were conducted in microtiter plates using a modification of the ⁇ - galactosidase assay procedure described in Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition, Eds: Sambrook, Fritsch and Maniatis, Book 3, page 16.66, Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York.
  • the only modification is that the reading was done in kinetic mode at 405 nm as opposed to an endpoint analysis.
  • Kinetic mode readings were taken every minute for 90 minutes.
  • the Vmax of the reaction was divided by the protein concentration of that sample (Vmax/protein concentration) and this value was used as the expression value.
  • the results are as follows: The negative control gave a baseline value of 0.53 (a value indicating no activity). The positive control gave a value of 1.5.
  • the light strand maximal promoter construct gave a value of 1.11.
  • the light strand minimal construct gave a value of 0.82.
  • the results suggest that the light strand promoter constructs are able to drive expression of the downstream reporter gene.
  • ELISA Microtiter (96 well) plates are coated overnight with purified HSV gD protein 0.4 ⁇ g ml. Plates are washed 3X with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS/Tw-20) and then blocked for 1 hr (at room temperature) with 4% bovine serum albumin (BSA). Mouse sera are diluted serially in PBS/Tw-20 and 50 ⁇ l are added to each well. The plates are incubated at room temperature for 2 hours. Plates are then washed 4X with PBS/Tw-20 and 50 ⁇ l of a 1 :2000 dilution of peroxidase-conjugated anti- mouse IgG is added.
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • Plates are incubated at room temperature for 1 hr. Plates are washed as above and substrate (3,3',5,5' - tetramethylbenzidine [TMB]-H 2 O ). Color is developed for 30 min. And the plate is then read at 450nm on a Emax microplate reader.
  • substrate 3,3',5,5' - tetramethylbenzidine [TMB]-H 2 O ). Color is developed for 30 min. And the plate is then read at 450nm on a Emax microplate reader.
  • Lymphoproliferation is performed on single cell spleen cell cultures as described in Kruisebeek, Shevach Proliferative assays for T -cell function (in:Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W, editors. Current protocols in immunology. USA: John Wiley and Sons, 1994. P.3.12.1-3.12.14 (1994).
  • Cells from spleen are enriched for CD4 + or CD8 + T-cells by depleting the alternate subset using the MiniMACS magnetic separation system (Miltenyi Biotic, Auburn, CA). Results are expressed as a mean stimulation index (SI).
  • Cytoplasmic RNA is prepared from these cells and cDNA is prepared using a primer specific for the 3' untranslated region of mtTFl. Following production of the cDNA, both the entire gene and a version of the gene lacking the mitochondrial signal sequence (containing the initial ATG codon, but lacking the next 41 codons) are amplified. These PCR products are then cloned into the TA cloning vector (pCRH; Invitrogen, Carlsbad, CA). The presence of the correct insert in the proper orientation is determined by restriction digestions followed by agarose gel electrophoresis.
  • the genes for the full-length and N-terminal-truncated mtTFA are then transferred to a mammalian expression plasmid containing a kanamycin resistance gene (described in U.S. Patent 5,851,804).
  • the presence of the correct inserts in the proper orientation is determined by restriction digestions followed by agarose gel electrophoresis. Plasmids containing the full-length and N-terminal-truncated versions of mtTFA are then sequenced to confirm the identity of the genes.
  • SEQ ID NO: 29 Reverse Transcription oligo: 5' -TGA-ACA-CAT-CTC-AAT-CTT- CTA-CTT-3'
  • SEQ ID NO:30 5' PCR oligo (full-length): 5' -GGA-GCG-ATG-GCG-TTT-CTC- CGA-AGC-3'
  • SEQ ID NO: 32 3'PCR oligo: 5' -tta-aca-ctc-ctc-agc-acg-ata-ttt-3'
  • mtRPOL mitochondrial RNA polymerase

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Abstract

La présente invention concerne des compositions et des procédés permettant d'acheminer des séquences d'acides nucléiques hétérologues dans les cellules d'une personne. L'utilisation de promoteurs mitochondriaux permet de favoriser l'expression de polypeptides voulus à l'intérieur des cellules de personnes traitées. L'invention concerne également des compositions et des procédés qui peuvent être employés pour traiter et immuniser des personnes contre des pathogènes. L'invention concerne enfin des compositions et des procédés destinés au traitement de personnes par administration de séquences d'acides nucléiques hétérologues voulues au moyen de la thérapie génique.
PCT/US2002/000543 2001-01-09 2002-01-09 Constructions d'adn pour expression cytoplasmique et mitochondriale et procedes de production et d'utilisation de ces constructions WO2002068629A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015057319A1 (fr) * 2013-10-17 2015-04-23 Clontech Laboratories, Inc. Procédés pour ajouter des adaptateurs à des acides nucléiques et compositions pour leur mise en œuvre
US9410173B2 (en) 2012-10-24 2016-08-09 Clontech Laboratories, Inc. Template switch-based methods for producing a product nucleic acid
EP3091083A1 (fr) * 2015-05-07 2016-11-09 Latvian Biomedical Research and Study Centre Kit de détection d'une mutation ou d'un polymorphisme dans l'adn mitochondrial humain
US9719136B2 (en) 2013-12-17 2017-08-01 Takara Bio Usa, Inc. Methods for adding adapters to nucleic acids and compositions for practicing the same

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WO1998033927A1 (fr) * 1997-01-31 1998-08-06 E.I. Du Pont De Nemours And Company Plantes transformees genetiquement presentant une resistance aux herbicides inhibant la biosynthese des porphyrinogenes
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9410173B2 (en) 2012-10-24 2016-08-09 Clontech Laboratories, Inc. Template switch-based methods for producing a product nucleic acid
US11001882B2 (en) 2012-10-24 2021-05-11 Takara Bio Usa, Inc. Template switch-based methods for producing a product nucleic acid
WO2015057319A1 (fr) * 2013-10-17 2015-04-23 Clontech Laboratories, Inc. Procédés pour ajouter des adaptateurs à des acides nucléiques et compositions pour leur mise en œuvre
US10781443B2 (en) 2013-10-17 2020-09-22 Takara Bio Usa, Inc. Methods for adding adapters to nucleic acids and compositions for practicing the same
US10941397B2 (en) 2013-10-17 2021-03-09 Takara Bio Usa, Inc. Methods for adding adapters to nucleic acids and compositions for practicing the same
US10954510B2 (en) 2013-10-17 2021-03-23 Takara Bio Usa, Inc. Methods for adding adapters to nucleic acids and compositions for practicing the same
US9719136B2 (en) 2013-12-17 2017-08-01 Takara Bio Usa, Inc. Methods for adding adapters to nucleic acids and compositions for practicing the same
US10415087B2 (en) 2013-12-17 2019-09-17 Takara Bio Usa, Inc. Methods for adding adapters to nucleic acids and compositions for practicing the same
US11124828B2 (en) 2013-12-17 2021-09-21 Takara Bio Usa, Inc. Methods for adding adapters to nucleic acids and compositions for practicing the same
EP3091083A1 (fr) * 2015-05-07 2016-11-09 Latvian Biomedical Research and Study Centre Kit de détection d'une mutation ou d'un polymorphisme dans l'adn mitochondrial humain

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