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WO1999010509A1 - Nucleotides inhibiteurs de la destabilisation et de la sequestration de l'arn - Google Patents

Nucleotides inhibiteurs de la destabilisation et de la sequestration de l'arn Download PDF

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Publication number
WO1999010509A1
WO1999010509A1 PCT/US1998/017261 US9817261W WO9910509A1 WO 1999010509 A1 WO1999010509 A1 WO 1999010509A1 US 9817261 W US9817261 W US 9817261W WO 9910509 A1 WO9910509 A1 WO 9910509A1
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polynucleotide
rna
cell
complementary
transcript
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David J. Lipman
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National Institutes of Health NIH
US Department of Health and Human Services
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National Institutes of Health NIH
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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
    • C12N15/11—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
    • 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
    • C12N15/67—General methods for enhancing the expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • the present invention relates to a method of regulating RNA stability.
  • the invention more specifically relates to the use of polynucleotides to increase the stability of RNA transcripts in order to augment expression of the target gene product.
  • Countertranscript i.e., antisense RNA is encoded by the complementary strand of a gene. Countertranscripts are sometimes found in different tissues or developmental stages than their corresponding transcript ( . e. , sense) RNA and thus a regulatory role for endogenous countertranscripts has been proposed. Examples of regulation of gene expression by endogenous countertranscripts have been described for nematode, dictyostelium, and prokaryotes. Moreover, studies on the regulation of the countertranscripts of Wilms tumor suppressor, eif2- a, and myc show that when the sense transcript is up-regulated, the levels of countertranscript decrease, and when the sense transcript is down-regulated, the countertranscript accumulates.
  • Double-stranded RNA adenine deaminase has been implicated in the recognition and destabilization of transcript RNA base-paired with its countertranscript. Kimelman et al, Cell 59:687-696 (1989). The modification efficiency of DSRAD has also been shown to decrease exponentially as the length of RNA duplex drops below 100 base pairs. Thus, the destabilization pathway appears to be sensitive to perturbations in the sequence of conserved regions necessary for transcript-countertranscript interactions.
  • transcript-countertranscript interactions also appear to promote nuclear sequestration of RNA.
  • All known cellular RNA species exit the nucleus through nuclear pore complexes (NPCs). Nakielny et al, RNA Transport in Ann. Rev. Neurosci., 20:269-301 (1997).
  • NPCs nuclear pore complexes
  • proteins associated with the RNA molecule These proteins appear to utilize signal-dependent, receptor mediated pathways to transport RNA molecules from the nucleus to the cytoplasm.
  • RNA which has been processed by 5' capping, 3' polyadenylation and intron removal is transported from the nucleus more efficiently then unprocessed RNA.
  • transcript-countertranscript duplexes promote nuclear accumulation of RNA by interfering with signal-dependent receptor mediated NPC transport and by inhibiting mechanisms required for RNA post-transcriptional processing. Consequently, transcript-countertranscript interactions result in down- regulation of expression of the encoded protein product.
  • the present invention is based on the fundamental counterintuitive discovery that defined polynucleotides can prevent transcript-countertranscript interactions and inhibit the degradation and nuclear sequestration of transcript RNA.
  • the present invention provides methods which can enhance expression of a target protein encoded by transcript RNA.
  • the invention provides a method for the inhibition of transcript RNA destabilization and nuclear sequestration through the use of polynucleotides.
  • the polynucleotides are designed such that they interfere with countertranscript annealing to transcript RNA and, thereby, prevent degradation and nuclear accumulation of the targeted transcript. Consequently, the method of the present invention is unexpectedly useful in enhancing translation and augmenting protein expression.
  • agents in the form of polynucleotides which are effective for enhancing protein expression in target cells and tissues.
  • an expression vector containing in operable linkage a polynucleotide which inhibits destabilization and nuclear sequestration of targeted transcript RNA is provided.
  • the expression vector can be used to stably express polynucleotides in cells or tissue for the purpose of enhancing protein expression.
  • a method of enhancing translation of mRNA in a patient tissue comprising administering to the patient tissue a therapeutically effective amount of a polynucleotide which inhibits destabilization of transcript RNA is provided. The method includes administering the polynucleotide in vivo or ex vivo.
  • FIGURE 1 shows a diagram of the mechanism by which the addition of therapeutic polynucleotides can inhibit the destabilization of RNA transcripts and enhance protein expression.
  • Transcript (T) -countertranscript (C) duplex formation promotes the degradation and nuclear sequestration of transcript RNA.
  • the addition of a polynucleotide complementary to a transcript or countertranscript inhibits transcript RNA degradation and sequestration and promotes increased protein production.
  • the present invention originated from studies on RNA stability and the effect of RNA stability on protein expression.
  • the inventors describe a method for using short polynucleotides to enhance protein expression by preventing transcript-countertranscript interactions which result in long duplex formation.
  • RN As contain long conserved sequences in their untranslated regions as well as highly conserved sequences without silent changes in their protein coding regions. These conserved regions are largely comprised of unique sequence within the genome and appear to be associated with regulation of RNA stability. Further, the inventor has identified these highly conserved sequences in the coding regions and 5' and 3' UTR's of orthologous vertebrate RNAs. A representative sample from a comparison of human and mouse orthologs is shown in Table I. A mechanism is proposed wherein conserved sequences contained in transcript RNA form long, highly complementary duplexes with the corresponding region of the corresponding countertranscript RNA.
  • duplexes are targeted by post-transcriptional regulatory mechanisms which promote long duplex degradation or nuclear sequestration of the transcript- countertranscript duplex, thereby preventing transcript translation and inhibiting protein production. This mechanism lends itself to modulation by compositions which disrupt or interfere with long duplex formation.
  • the present invention relates to a method for enhancing transcript RNA stability in a cell by contacting the cell with a polynucleotide which inhibits transcript RNA degradation.
  • the polynucleotides prevent long transcript- countertranscript duplex formation which results in enhanced transcript RNA stability and cytoplasmic translocation.
  • transcript RNA is RNA which contains nucleotide sequence encoding a protein product.
  • the transcript RNA is messenger RNA (mRNA).
  • mRNA messenger RNA
  • mRNA is a single-stranded RNA molecule that specifies the amino acid sequence of one or more polypeptide chains.
  • transcript RNA can be heterogenous nuclear RNA (hnRNA) or masked RNA.
  • hnRNA represents the primary transcripts of RNA polymerase II and includes precursors of all messenger RNAs from which introns are removed by splicing. hnRNA are extensively processed to give mRNA which is exported to the cytoplasm where protein synthesis occurs.
  • This processing may include the addition of a 5'-linked 7-methyl-guanylate "cap” at the 5' end and a sequence of adenylate groups at the 3' end, the poly A "tail”, as well as the removal of any introns and the splicing together of exons.
  • Mask RNA is any form of mRNA which is present in inactive form. More specifically, masked RNA constitutes a store of maternal information for protein synthesis that is unmasked (derepressed) during the early stages of morphogenesis.
  • Countertranscript is that sequence which is transcribed from the non- coding strand of a gene sequence. Countertranscript RNA can be fully complementary to the transcript RNA. However, it is understood that any countertranscript capable of generating duplex structures with transcript RNA such that the duplex structures result in transcript RNA degradation or sequestration are included in the method of the present invention.
  • transcript RNA degradation is any mechanism which initiates RNA destabilization through the recognition of duplex RNA sequences resulting in the loss of transcript RNA integrity.
  • the mechanism can involve double-strand RNA adenine deaminase (DSRAD), however, any activity or mechanism which acts to degrade or destabilize the transcript RNA is included in the present method.
  • DSRAD double-strand RNA adenine deaminase
  • Transcript RNA sequestration is any mechanism which relies on RNA duplex formation to inhibit or prevent transcript RNA translocation from the nucleus to the cytoplasm. These mechanisms include, but are not limited to, those that prevent interaction of the transcript RNA with export factors required for nuclear pore complex (NPC) mediated transport. Further, duplex formation can prevent normal post- transcriptional RNA processing resulting in the nuclear accumulation of unprocessed transcript. Other mechanisms which promote RNA nuclear sequestration will be known to those of skill in the art.
  • Nucleic acid sequences of the invention can be obtained by several methods.
  • the sequence can be isolated using hybridization or computer-based techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences; 2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features; 3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the DNA sequence of interest; 4) computer searches of sequence databases for similar sequences; and 5) differential screening of a subtracted DNA library. Based upon the size of the human genome, statistical studies show that a DNA segment approximately 14-15 base pairs long will have a unique sequence in the genome.
  • the sequence of a suitable polynucleotide can be obtained, for example, by comparing the sequences of orthologous genes, or the transcripts or countertranscripts of orthologous genes, and identifying highly conserved regions within the orthologous sequences.
  • highly conserved regions contained within orthologs, or transcripts and countertranscripts of orthologs can be used to design polynucleotides useful for inhibiting transcript- countertranscript interactions.
  • an "orthologous sequence" is that in which sequence homology is retained or conserved between species. Two gene sequences from different organisms are orthologs if they derived from the same gene in the closest ancestor to the two organisms.
  • polynucleotides useful for the method of the present invention can be designed such that they contain nucleic acid sequence which is, for example, wholly or partially complementary to conserved sequences identified from orthologous sequences.
  • the polynucleotide is designed such that it is complementary to a coding region or the 5' or 3' UTR of a transcript or countertranscript identified from the aforementioned orthologous sequences.
  • any portion of a gene or transcript or countertranscript can encode sequences which are useful for designing polynucleotides according to the method of the present invention. Similarity in nucleic acid sequences may be determined by procedures and algorithms which are well-known in the art.
  • Such procedures and algorithms include, for example, a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET (Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS, Smith- Waterman algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign, Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence Analysis Package), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC (Sensitive Sequence Comparison
  • the polynucleotide is designed to be complementary to the transcript RNA of a gene sequence. In another aspect of the invention, the polynucleotide is designed to be complementary to the countertranscript RNA of a gene sequence.
  • Polynucleotides useful in the context of the present invention include both short sequences of RNA or DNA, usually 10-50 bases in length as well as longer sequences of RNA or DNA that may exceed the length of the target transcript sequence itself.
  • any polynucleotide which inhibits, abrogates or interferes with endogenous mechanisms for transcript RNA destabilization or sequestration is hereby incorporated in the present method.
  • polynucleotides useful for the present invention are complementary to specific regions of a corresponding target RNA.
  • target RNA can be either the transcript or countertranscript RNA.
  • Hybridization of polynucleotides to their target transcripts can be highly specific as a result of complementary base pairing.
  • a stabilizing RNA polynucleotide can be produced in a cell by introducing a DNA segment that codes for the polynucleotide into the cell.
  • a stabilizing polynucleotide can also be introduced to a cell by adding the polynucleotide to the environment of the cell such that the cell takes up the polynucleotide directly. The latter route is preferred for the shorter polynucleotides of up to about 20 bases in length.
  • long polynucleotides which may exceed the length of the target transcript can be used to prevent transcript RNA destabilization.
  • long polynucleotides can include multiple regions complementary to multiple corresponding regions in a target transcript.
  • one or more highly conserved regions in a transcript or countertranscript can be targeted by a polynucleotide containing regions which are complementary to at least one an preferably more of the highly conserved regions.
  • a single long polynucleotide can prevent transcript RNA destabilization by containing at least one and preferably multiple regions of complementarity of the targeted transcript.
  • a long polynucleotide can contain multiple regions of complementarity to a highly conserved sequence in one or more transcript or countertranscript RNAs.
  • Such a polynucleotide can contain, for example, one or more regions complementary to highly conserved regions in a target transcript RNA and, in addition, one or more regions complementary to highly conserved regions in a second target transcript RNA.
  • a long polynucleotide is useful for the inhibition of transcript destabilization of one or more transcript RNAs.
  • tumorigenic cells can be treated with a vector expressing a polynucleotide with multiple regions of homology to transcripts encoding polypeptides involved in regulating apoptosis.
  • a polynucleotide can inhibit transcript destabilization of one or more different transcripts simultaneously or individually.
  • a polynucleotide can contain regions of complementarity to highly conserved regions in, for example, a transcript encoding the p53 protein and highly conserved regions in, for example, a transcript encoding the c-myc protein. The presence of a single polynucleotide with such regions can inhibit the destabilization of multiple apoptosis- promoting transcripts thereby increasing the cellular concentration of the encoded protein and resulting in programmed cell death in tumorigenic cells so treated.
  • polynucleotides of the present invention are at least 15 bp in length and preferably about 15 to about 100 bp in length. More preferably, the polynucleotides are about 15 bp to about 80 bp in length and even more preferably, the polynucleotides of the present invention are about 15 to about 60 bp in length. Shorter polynucleotides such as 10-to under 15-mers, while offering higher cell penetration, have lower gene specificity. In contrast, while longer polynucleotides of 20-30 bases offer better specificity, they show decreased uptake kinetics into cells.
  • polynucleotide encompasses both oligomeric nucleic acid moieties of the type found in nature, such as the deoxyribonucleotide and ribonucleotide structures of DNA and RNA, and man-made analogues which are capable of binding to nucleic acids found in nature.
  • the polynucleotides of the present invention can be based upon ribonucleotide or deoxyribonucleotide monomers linked by phosphodiester bonds, or by analogues linked by methyl phosphonate, phosphorothioate, or other bonds. They may also comprise monomer moieties which have altered base structures or other modifications, but which still retain the ability to bind to naturally occurring transcript and countertranscript RNA structures.
  • Such polynucleotides may be prepared by methods well-known in the art, for instance using commercially available machines and reagents such as those available from Perkin-Elmer/ Applied Biosystems (Foster City, CA). For example, polynucleotides specific to a targeted transcript are synthesized according to standard methodology.
  • Phosphorothioate modified DNA polynucleotides typically are synthesized on automated DNA synthesizers available from a variety of manufacturers. These instruments are capable of synthesizing nanomole amounts of polynucleotides as long as 100 nucleotides. Shorter polynucleotides synthesized by modern instruments are often suitable for use without further purification. If necessary, polynucleotides may be purified by polyacrylamide gel electrophoresis or reverse phase chromatography. See Sambrook et al, Molecular Cloning: A Laboratory Manual, Vol. 2, Chapter 11, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
  • Phosphodiester-linked polynucleotides are particularly susceptible to the action of nucleases in serum or inside cells, and therefore in a preferred embodiment, the polynucleotides of the present invention are phosphorothioate or methyl phosphonate- linked analogues, which have been shown to be nuclease-resistant. Persons of ordinary skill in this art can easily select other linkages for use in the invention. These modifications also may be designed to improve the cellular uptake and stability of the polynucleotides.
  • An appropriate carrier for administration of a polynucleotide can include, for example, vectors, antibodies, pharmacologic compositions, binding or homing proteins, or viral delivery systems to enrich for the sequence into the target cell or tissue.
  • a polynucleotide of the present invention can be coupled to, for example, a binding protein which recognizes endothelial cells or tumor cells.
  • a polynucleotide of the present invention can be targeted to a recipient cell or tissue such that enhanced expression of, for example, cytokines, transcription factors, G-protein coupled receptors, tumor suppressor proteins and apoptosis initiation proteins can occur.
  • an expression vector containing in operable linkage a polynucleotide which inhibits destabilization or nuclear sequestration of transcript RNA is disclosed.
  • the polynucleotide is an RNA polynucleotide produced by introducing an expression construct into a target cell.
  • the RNA molecule thus produced is chosen to have the capability to hybridize to a target transcript RNA or its countertranscript.
  • the polynucleotides which have the capability to hybridize with RNA targets can enhance expression of corresponding gene products by multiple mechanisms. Such molecules that have this capability can inhibit degradation of the target mRNA and thereby enhance translation.
  • operably linked promoter refers to functional linkage between a promoter sequence and the structural gene regulated by the promoter nucleic acid sequence.
  • the operably linked promoter controls the expression of the polypeptide encoded by the structural gene, or in a preferred embodiment of the invention, the heterologous gene nucleic acid sequences.
  • the orientation or placement of the elements of the vector is not strict, so long as the "operable linkage" requirement is fulfilled for control of and expression of the nucleic acid sequences.
  • a polynucleotide in the form of complementary RNA may be introduced to a cell by its expression within the cell from a standard DNA expression vector.
  • Target DNA sequences can be cloned from standard plasmids into expression vectors, which expression vectors have characteristics permitting higher levels of, or more efficient expression of, the resident polynucleotides.
  • these constructs require a prokaryotic or eukaryotic promoter sequence which initiates transcription of the inserted DNA sequences.
  • a preferred expression vector is one where the expression is inducible to high levels. High levels of expression can be accomplished by the addition of a regulatory region which provides increased transcription of downstream sequences in the appropriate host cell. See Sambrook et al, Vol. 3, Chapter 16 (1989).
  • RNA polynucleotide expression vectors can be constructed using the polymerase chain reaction (PCR) to amplify appropriate fragments from single-stranded cDNA of a plasmid in which target DNA has been incorporated.
  • PCR polymerase chain reaction
  • Polynucleotide synthesis and purification techniques are described in Sambrook et al. and Ausubel et al. (eds.), Current Protocols in Molecular Biology (Wiley Interscience 1987) (hereafter "Ausubel"), respectively.
  • the PCR procedure is performed via well-known methodology. See, for example, Ausubel, and Bangham, "The Polymerase Chain Reaction: Getting Started," in Protocols in Molecular Genetics (Humana Press, 1991).
  • PCR kits can be purchased from companies such as Stratagene Cloning Systems (La Jolla, CA) and Invitrogen (San Diego, CA).
  • cloning vector is a DNA molecule, such as a plasmid, cosmid or bacteriophage, that can replicate autonomously in a host prokaryotic or eukaryotic cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.
  • Cloning vectors are described by Sambrook et al, Ausubel, and Brown (ed.), Molecular Biology LabFax (Academic Press 1991). Cloning vectors can be obtained, for example, from GIBCO/BRL (Gaithersburg, MD), Clontech Laboratories, Inc. (Palo Alto, CA), Promega Corporation (Madison, WI), Stratagene Cloning Systems (La Jolla, CA), Invitrogen (San Diego, CA), and the American Type Culture Collection (Rockville, MD).
  • Cloned polynucleotides are amplified by transforming competent bacterial cells with a cloning vector and growing the bacterial host cells in the presence of the appropriate antibiotic. See, for example, Sambrook et al, and Ausubel. PCR is then used to screen bacterial host cells for the appropriate clones.
  • PCR for bacterial host cells is described, for example, by Hofinann et al, "Sequencing DNA Amplified Directly from a Bacterial Colony," in PCR Protocols and Applications, White (ed.), pages 205-210 (Humana Press 1993), and by Cooper et al, "PCR-Based Full-Length cDNA Cloning Utilizing the Universal-Adaptor/Specific DOS Primer-Pair Strategy," Id. at pages 305- 316.
  • RNA polynucleotides complementary to a target RNA transcript or a target RNA countertranscript such that the polynucleotide prevents duplex formation.
  • a nucleic acid encoding a polynucleotide of the present invention is operably linked to a mammalian-active promoter.
  • a "mammalian active" promoter is a nucleic acid sequence that directs transcription in a mammalian cell (e.g., a promoter of a mammal or a virus that infects a mammal).
  • the promoter can be cell- specific, tissue-specific, or stage-specific in order to express the polynucleotide with increased specificity.
  • Numerous genetic constructs and methods for expressing heterologous genes in cells of mammals are known in the art and are suitable for use in the invention.
  • polynucleotide expression can be accomplished with conventional gene therapy methods, such as those that employ viral vectors (e.g., vectors derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, Sindbis viruses, or adeno-associated viruses).
  • viral vectors e.g., vectors derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, Sindbis viruses, or adeno-associated viruses.
  • the transcriptional and translational regulatory signals preferably are derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression.
  • viral sources such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression.
  • Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.
  • Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al, J. Molec. Appl. Genet. 1 : 273 (1982)); the
  • TK promoter of Herpes virus McKnight, Cell 31: 355 (1982)
  • the SV40 early promoter (Benoist et al, Nature 290: 304 (1981); the Rous sarcoma virus promoter (Gorman et al, Proc. Nat'l Acad. Sci. USA 79: 6777 (1982)); and the cytomegalovirus promoter (Foecking et al, Gene 45: 101 (1980)).
  • a vector for introducing at least one polynucleotide, complementary to a transcript or its countertranscript, into a cell by expression from a DNA is the vector pRc/CMV (Invitrogen, San Diego, CA), which provides a high level of constitutive transcription from mammalian enhancer-promoter sequences.
  • Virus-like vectors usually derived from retroviruses, may prove useful as vehicles for the importation and expression of polynucleotide constructs in human cells.
  • such vectors are non-replicative in vivo, precluding any unintended infection of non-target cells.
  • helper cell lines are provided which supply the missing replicative functions in vitro, thereby permitting amplification and packaging of the vector encoding the polynucleotide.
  • a further precaution against accidental infection of non-target cells involves the use of target cell-specific regulatory sequences. When under the control of such sequences, polynucleotide constructs would not be expressed in normal tissues.
  • a method for increasing cytoplasmic transcript RNA in a subject comprising administering a therapeutically effective amount of a polynucleotide which inhibits degradation or nuclear sequestration of transcript RNA.
  • a pharmaceutical composition containing the polynucleotide of the present invention is administered.
  • Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan and those discussed throughout the specification.
  • the term "subject” means any mammal, preferably a human.
  • the method is useful for the treatment of any condition wherein inhibition of transcript RNA degradation and sequestration would be efficacious.
  • the treatment would be useful in treating disorders in which increased production of a target protein would ameliorate the symptoms of the disorder.
  • tumorigenic cells can be treated with a polynucleotide such that the polynucleotide enhances the production of a protein involved in regulating cellular apoptosis and inhibiting cell growth.
  • proteins would include, but not be limited to, ICE (interleukin lb converting enzyme), BAD, Bax, Bak, NBK, p53, Rb, bcl-x s , c-myc, Fas, TNF, Hid and WAF1.
  • treating means obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder.
  • Treating covers any treatment of a disorder in a mammal, and includes:
  • treatment is intended to mean providing a therapeutically detectable and beneficial effect on a patient afflicted with a disorder the symptoms of which can be ameliorated by administration of a polynucleotide which prevents transcript- countertranscript duplex formation.
  • an effective amount of the vector expressing, for example, a polynucleotide of the invention is generally determined by the physician in each case on the basis of factors normally considered by one skilled in the art to determine appropriate dosages, including the age, sex, and weight of the subject to be treated, the condition being treated, and the severity of the medical condition being treated.
  • compositions of the present invention are advantageously administered in the form of injectable compositions.
  • a typical composition for such purpose comprises a pharmaceutically acceptable solvent or diluent and other suitable, physiologic compounds.
  • the composition may contain polynucleotides and about 10 mg of human serum albumin per milliliter of a phosphate buffer containing NaCl.
  • non-aqueous or aqueous solutions examples include propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
  • Aqueous solutions include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to routine skills in the art.
  • a preferred pharmaceutical composition for topical administration is a dermal cream or transdermal patch.
  • Polynucleotides or their expression vectors may be administered by injection as an oily suspension.
  • Suitable lipopbilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides.
  • polynucleotides or vectors may be combined with a lipophilic carrier such as any one of a number of sterols including cholesterol, cholate and deoxycholic acid.
  • a preferred sterol is cholesterol.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension also contains stabilizers.
  • Liposome encapsulation provides an alternative formulation for the administration of polynucleotides and expression vectors.
  • Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments. See, generally, Bakker-Woudenberg et al, Eur. J. Clin. Microbiol Infect. Dis. 12 (Suppl. 1): S61 (1993), and Kim, Drugs 46: 618 (1993). Liposomes are similar in composition to cellular membranes and as a result, liposomes can be administered safely and are biodegradable.
  • liposomes may be unilamellar or multilamellar, and liposomes can vary in size with diameters ranging from 0.02 ⁇ m to greater than 10 ⁇ m.
  • agents can be encapsulated in liposomes: hydrophobic agents partition in the bilayers and hydrophilic agents partition within the inner aqueous space(s). See, for example, Machy et al, Liposomes in Pharmacology (John Libbey 1987), and Ostro et al, American J. Hosp. Pharm. 46: 1576 (1989).
  • Liposomes can adsorb to virtually any type of cell and then slowly release the encapsulated agent.
  • an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents. Scherphofet ⁇ /., Ann. N. Y. Acad. Sci. 446:368 (1985).
  • reticuloendothelial system After intravenous administration, conventional liposomes are preferentially phagocytosed into the reticuloendothelial system.
  • the reticuloendothelial system can be circumvented by several methods including saturation with large doses of liposome particles, or selective macrophage inactivation by pharmacological means. Claassen et al, Biochim. Biophys. Ada 802:428 (1984).
  • incorporation of glycolipid- or polyethelene glycol-derivatised phospholipids into liposome membranes has been shown to result in a significantly reduced uptake by the reticuloendothelial system. Allen et al, Biochim. Biophys.
  • Polynucleotides and expression vectors can be encapsulated within liposomes using standard techniques.
  • a variety of different liposome compositions and methods for synthesis are known to those of skill in the art. See, for example, U.S. Patent No. 4,844,904, U.S. Patent No. 5,000,959, U.S. Patent No. 4,863,740, and U.S. Patent No. 4,975,282, all of which are hereby incorporated by reference.
  • Liposomes can be prepared for targeting to particular cells or organs by varying phospholipid composition or by inserting receptors or ligands into the liposomes.
  • antibodies specific to tumor associated antigens may be incorporated into liposomes, together with polynucleotides or expression vectors, to target the liposome more effectively to the tumor cells. See, for example, Zelphati et al, Antisense Research and Development ⁇ : 323-338 (1993), describing the use "immunoliposomes" containing polynucleotides for human therapy.
  • the dosage of administered liposome-encapsulated polynucleotides and vectors will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Dose ranges for particular formulations can be determined by using a suitable animal model.
  • Polynucleotides, or vectors containing the polynucleotide of the claimed invention can be administered ex vivo by transfer of genetic material to cells, for example, hematopoietic stem cells located outside a subject.
  • Ex vivo transfer utilizes available techniques known to those skilled in the art to remove cells from a subject and introduce into these cells a therapeutic amount of genetic material.
  • the transduced cells can be implanted back in the original host subject or into a different host, such as a member of the same or a different species from the donor.
  • hematopoietic stem cells can be removed from a subject, treated with the appropriate polynucleotide and re-introduced into the host subject.
  • In vivo transfer utilizes available techniques known to those skilled in the art to introduce into a subject a therapeutic amount of genetic material.
  • tumor cells within an organism can be targeted for therapy by polynucleotides of the claimed invention.
  • Polynucleotides which promote expression of a protein involved in regulating apoptosis for example, can be used to promote programmed cell death in tumorigenic cells.
  • DNA sequences encoding a polynucleotide specific for a transcript or countertranscript can be expressed in vitro by DNA transfer into a suitable host cell.
  • "Host cells” are cells in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
  • the polynucleotide sequences may be inserted into a recombinant expression vector.
  • recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the target genetic sequences.
  • Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.
  • Vectors suitable for use in the present invention include, but are not limited to the T7 -based expression vector for expression in bacteria (Rosenberg, et al, 1987, Gene ,56: 125), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, 1988, J. Biol.
  • the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).
  • a promoter e.g., T7, metallothionein I, or polyhedrin promoters.
  • host-expression vector systems may be utilized to express a portion of the target transcript coding sequence.
  • host-expression vector systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the polynucleotide sequence; yeast transformed with recombinant yeast expression vectors containing the polynucleotide sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the polynucleotide coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the polynucleotide coding sequence; or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses,
  • Mammalian cell systems which utilize recombinant viruses or viral elements to direct expression may be engineered.
  • the coding sequence when using adenovirus expression vectors, the coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • the vaccinia virus 7.5K promoter may be used, (e.g., see, Mackett et al, 1982, Proc. Natl. Acad. Sci. USA 79:7415-7419; Mackett et al, 1984, J. Virol. 49:857-864; Panicali et al, 1982, Proc. Natl. Acad. Sci. USA 79:4927-4931).
  • vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements (Sarver, et al, 1981, Mol Cell. Biol. 1 :486). Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression.
  • These vectors can be used for stable expression by including a selectable marker in the plasmid, such as, for example, the neo gene.
  • the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the polynucleotide sequence in host cells (Cone & Mulligan, 1984, Proc. Natl. Acad. Sci. USA 81:6349- 6353). High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionine IIA promoter and heat shock promoters.
  • host cells can be transformed with the cDNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, etc.), and a selectable marker.
  • expression control elements e.g., promoter, enhancer, sequences, transcription terminators, etc.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al, 1977, Cell ⁇ :223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adenine phosphoribosyltransferase (Lowy, et al, 1980, Cell 22:817) genes can be employed in tk-, hgprt " or aprt " cells respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al, 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al, 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072; neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al, 1981, J Mol. Biol.
  • hygro which confers resistance to hygromycin (Santerre, et al, 1984, Gene 30:147) genes.
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • ODC ornithine decarboxylase
  • Eukaryotic cells can also be cotransformed with DNA sequences encoding the polynucleotide of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein, (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • the introduced sequence is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose.
  • Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • vectors For a mammalian host, several possible vector systems are available for expression of the polynucleotide specific for a targeted transcript.
  • One class of vectors utilize DNA elements which provide autonomously replicating extra-chromosomal plasmids, derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, or SV40 virus.
  • a second class of vectors include vaccinia virus expression vectors.
  • a third class of vectors relies upon the integration of the desired gene sequences into the host chromosome. Cells which have stably integrated the introduced DNA into their chromosomes may be selected by also introducing one or more markers (e.g., an exogenous gene) which allow selection of host cells which contain the expression vector.
  • the marker may provide for prototropy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper or the like.
  • the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
  • the cDNA expression vectors incorporating such elements include those described by Okayama, H., Mol. Cell. Biol, 3:280 (1983), and others. Once the vector or DNA sequence containing the construct has been prepared for expression, the DNA construct may be introduced (transformed) into an appropriate host. Various techniques may be employed, such as protoplast fusion, calcium phosphate precipitation, electroporation or other conventional techniques.
  • the pharmaceutical compositions are preferably prepared and administered in dose units.
  • Solid dose units are tablets, capsules and suppositories.
  • different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate.
  • the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
  • compositions according to the invention are in general administered topically, intravenously, orally or parenterally or as implants, but even rectal use is possible in principle.
  • suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science, 249:1527-1533 (1990), which is incorporated herein by reference.
  • compositions according to the invention may be administered locally or systemically.
  • therapeutically effective dose is meant the quantity of a compound according to the invention necessary to prevent, to cure or at least partially arrest the symptoms of the disease and its complications or enhance protein expression in cells or tissue where it is desirable to do so.
  • dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders.
  • Various considerations are described, e.g., in Gilman et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th ed., Pergamon Press; and Remington 's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa., each of which is herein incorporated by reference.
  • a sequence comparison of human and mouse orthologous mRNA's was performed using an available database of nucleic acid sequences from both organisms.
  • the data shown in Table I includes a representative sample of human mRNA's with long perfect blocks of identity in the coding regions when aligned with orthologous mouse mRNA. These data further indicate highly conserved regions in 5' and 3' untranslated regions (UTR's) of human and mouse orthologs.
  • the conserved regions contained in orthologous sequences can be used to design polynucleotides of the present invention.
  • the polynucleotides are designed such that their sequence is complementary to a highly conserved region in the transcript or countertranscript of an orthologous sequence.
  • the data in Table I can be used to design polynucleotides such that the polynucleotides inhibit transcript RNA degradation and nuclear sequestration by preventing transcript-countertranscript duplex formation between highly conserved regions of orthologous sequences.
  • Administering such a polynucleotide to a subject can serve to enhance the expression of a target protein in that subject for the purpose of treating a disorder.
  • Techniques for administering nucleic acids to a subject are known to those of skill in the art.
  • a polynucleotide of the present invention can be administered to a subject suffering from a cell proliferation disorder such that the polynucleotide increases the expression of proteins involved in regulating apoptosis. Cells treated in such a manner can undergo apoptosis and alleviate symptoms of a cell proliferation disorder.

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Abstract

La présente invention concerne un procédé selon lequel, d'une part des polynucléotides viennent inhiber la déstabilisation de l'ARN, et d'autre part la séquestration nucléaire renforce la translocation cytoplasmique de l'ARN stabilisé et augmente l'expression des protéines.
PCT/US1998/017261 1997-08-22 1998-08-19 Nucleotides inhibiteurs de la destabilisation et de la sequestration de l'arn Ceased WO1999010509A1 (fr)

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WO2001004313A1 (fr) * 1999-07-09 2001-01-18 American Home Products Corporation Methodes et compositions empechant la formation d'un arn aberrant pendant la transcription d'une sequence plasmidique
WO2015023975A1 (fr) * 2013-08-16 2015-02-19 Rana Therapeutics, Inc. Compositions et procédés pour la modulation d'arn
WO2016130943A1 (fr) * 2015-02-13 2016-08-18 Rana Therapeutics, Inc. Oligonucléotides hybrides et leurs utilisations
US12318489B2 (en) 2015-10-26 2025-06-03 Translate Bio Ma, Inc. Nanoparticle formulations for delivery of nucleic acid complexes

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004313A1 (fr) * 1999-07-09 2001-01-18 American Home Products Corporation Methodes et compositions empechant la formation d'un arn aberrant pendant la transcription d'une sequence plasmidique
WO2015023975A1 (fr) * 2013-08-16 2015-02-19 Rana Therapeutics, Inc. Compositions et procédés pour la modulation d'arn
CN105658797A (zh) * 2013-08-16 2016-06-08 Rana医疗有限公司 用于调节rna的组合物和方法
JP2016528897A (ja) * 2013-08-16 2016-09-23 ラナ セラピューティクス インコーポレイテッド Ｒｎａを調節するための組成物および方法
EP3033424A4 (fr) * 2013-08-16 2017-04-19 Rana Therapeutics, Inc. Compositions et procédés pour la modulation d'arn
WO2016130943A1 (fr) * 2015-02-13 2016-08-18 Rana Therapeutics, Inc. Oligonucléotides hybrides et leurs utilisations
US10758558B2 (en) 2015-02-13 2020-09-01 Translate Bio Ma, Inc. Hybrid oligonucleotides and uses thereof
US12318489B2 (en) 2015-10-26 2025-06-03 Translate Bio Ma, Inc. Nanoparticle formulations for delivery of nucleic acid complexes

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