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WO2000022114A1 - PRODUCTION D'ADN SIMPLE BRIN $i(IN VIVO) - Google Patents

PRODUCTION D'ADN SIMPLE BRIN $i(IN VIVO) Download PDF

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Publication number
WO2000022114A1
WO2000022114A1 PCT/US1999/023936 US9923936W WO0022114A1 WO 2000022114 A1 WO2000022114 A1 WO 2000022114A1 US 9923936 W US9923936 W US 9923936W WO 0022114 A1 WO0022114 A1 WO 0022114A1
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WO
WIPO (PCT)
Prior art keywords
sequence
nucleic acid
interest
reverse transcriptase
tandem repeat
Prior art date
Application number
PCT/US1999/023936
Other languages
English (en)
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WO2000022114A9 (fr
Inventor
Charles A. Conrad
Original Assignee
Ingene, 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
Priority to AU62988/99A priority Critical patent/AU6298899A/en
Priority to CA002345936A priority patent/CA2345936A1/fr
Priority to EP99950296A priority patent/EP1119615B1/fr
Priority to DK99950296T priority patent/DK1119615T3/da
Priority to MXPA01003643A priority patent/MXPA01003643A/es
Priority to BR9914773-4A priority patent/BR9914773A/pt
Application filed by Ingene, Inc. filed Critical Ingene, Inc.
Priority to JP2000576005A priority patent/JP2004503203A/ja
Priority to DE69933382T priority patent/DE69933382T2/de
Publication of WO2000022114A1 publication Critical patent/WO2000022114A1/fr
Publication of WO2000022114A9 publication Critical patent/WO2000022114A9/fr
Priority to IL142492A priority patent/IL142492A/en
Priority to US11/586,797 priority patent/US20070160581A1/en

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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention relates to a stable DNA construct, conveniently referred to as a cassette, into which a nucleic acid sequence is incorporated for use as a template for subsequent production of that sequence in a prokaryotic or eukaryotic host cell, and a vector systems for expression of that sequence within eukaryotic host cells without (or with minimal) flanking sequences.
  • the cassette includes inverted an tandem repeat which forms the stem of a stem-loop intermediate that functions in vivo to cause expression of the sequence as a single stranded DNA (ssDNA) sequence, referred to as the sequence of interest.
  • the vector system of the present invention removes contiguous plasmid (or other vector sequences) from the ssDNA sequence of interest either by stem-loop formation with subsequent termination of a reverse transcription reaction by the stem or by cleavage of the stem-loop intermediate.
  • the ssDNA that is produced by this method can be designed so that it is complimentary to any endogenous nucleic acid sequence target.
  • 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. USA 1016-1020 (1992); S. Inouye, et al, 3 Current Opin. Genet. Develop. 713-718 (1993); O. Mirochnitchenko, et al, 269 J. Biol. Chem. 2380-2383 (1994); J.-R. Mao, et al, 270 J. Biol. Chem.
  • ssDNA can be (but is not limited to) an inhibitory nucleic acid such as an antisense sequence for binding to mRNA in an anti-sense fashion to down regulate a gene product or a viral gene product of interest or for binding to duplex (native
  • ssDNA produced in this manner may also function to disrupt one or more of many highly regulated cell functions.
  • the ssDNA tails of telomeric repeats may be altered by the production of ssDNA which has identical or complimentary nucleotide base composition to the sequence of the native DNA in the telomeric repeats or other regulatory sequence.
  • a cassette, or nucleic acid construct comprised of a nucleic acid sequence which is comprised of a sequence of interest flanked by inverted tandem repeats, a gene encoding an RNA-dependent DNA polymerase and a gene encoding a restriction endonuclease.
  • the cassette also preferably includes a gene encoding an RNase H and either constitutive or inducible eukaryotic promoter(s)/enhancer(s) for the RNA- dependent DNA polymerase and restriction endonuclease genes.
  • the invention also contemplates that the cassette is incorporated into a plasmid and that the plasmid is incorporated into a suitable host cell.
  • the present invention comprises a method of producing single- stranded DNA in vivo comprising the steps of transcription and translation of a cassette comprising am RNA-dependent DNA polymerase gene and a sequence of interest in a eukaryotic cell and converting the mRNA transcript of the sequence of interest to cDNA with the polymerase produced by the RNA-dependent DNA polymerase gene with simultaneous digestion of the mRNA component template with an RNase H expressed enzyme.
  • the sequence of interest also includes an inverted tandem repeat.
  • the cassette may also include a restriction endonuclease gene which, when transcribed and translated, produces a restriction endonuclease which linearizes the transcript of the sequence of interest by cutting the ss-DNA at a restriction endonuclease site formed when the inverted tandem repeat causes the transcript to form a stem-loop intermediate.
  • a restriction endonuclease gene which, when transcribed and translated, produces a restriction endonuclease which linearizes the transcript of the sequence of interest by cutting the ss-DNA at a restriction endonuclease site formed when the inverted tandem repeat causes the transcript to form a stem-loop intermediate.
  • the present invention comprises a method for producing a single-stranded ohgonucleotide in a target cell.
  • this method is intended to deliver an anti-sense sequence.
  • the method is used to deliver triplex-forming sequences or sequences which are recognized and bound by specific DNA-binding proteins, or other nucleic acids and/or proteins which function in cellular metabolism and/or replication.
  • the method comprises the encoding of the ohgonucleotide into a complementary sequence of interest in a cassette which includes a gene encoding for an RNA-dependent DNA polymerase which preferably includes an RNase H gene and an inducible or constitutive eukaryotic promoter/enhancer appropriate for that polymerase/RNase H gene.
  • the cassette includes a gene encoding a restriction endonuclease (RE) and, in the preferred embodiment, an appropriate promoter/enhancer for that RE gene.
  • the cassette further comprises an inverted tandem repeat and, when assimilated into the target cell, the cassette (including the sequence of interest and the inverted tandem repeats) is transcribed by the cell under the control of the promoter(s)/enhancer(s).
  • the normal function of the target cell causes the resulting mRNA transcript of the polymerase and RE genes to be translated, providing all that is needed for production of ss-DNA from the mRNA transcript of the sequence of interest.
  • the RNA-dependent DNA polymerase produced from the cassette converts the mRNA transcript of the sequence of interest and inverted tandem repeats to ss-cDNA, the ss-cDNA forms a stem-loop intermediate as the nucleotide bases comprising the inverted tandem repeats pair up, and the restriction endonuclease produced from the RE gene in the cassette digests the double- stranded portion of the stem-loop intermediate to "free" the single stranded DNA ohgonucleotide from the loop portion of the stem-loop intermediate.
  • Multiple systems can be used to deliver the cassette to the target cell to direct the synthesis of ssDNA within the cell, including plasmid or plasmid-based vector systems or viral based vector systems, and these systems are adapted for that purpose in accordance with standard delivery techniques currently known to the skilled practitioner.
  • These systems include, but are not limited to, viral based systems such as adenovirus, adenoassociated virus, retroviral vectors, and conjugate vectors using double stranded plasma DNA based transfection systems.
  • a nucleic acid construct which comprises an inverted tandem repeat, a primer binding site for a reverse transcriptase located in a 3' position with respect to the inverted tandem repeat, and a sequence of interest located either between the inverted tandem repeat, or between the inverted tandem repeat and the 3' primer binding site.
  • the nucleic acid construct of the present invention allows a single-stranded nucleic acid sequence to be reliably and stably produced in a target cell.
  • the inverted tandem repeat may be used to create a nucleic acid stem-loop intermediate to produce and/or control the production of ssDNA in vivo, with reduced or eliminated contiguous nucleotide sequences.
  • 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.
  • the inverted tandem repeat is, preferably, capable of forming a stem-loop intermediate, with said sequence of interest in the loop, and said inverted tandem repeat forming the stem.
  • an advantage of this stem-loop structure is that the sequence of interest can be released from the loop as a single-stranded cDNA with reduced or eliminated flanking and/or intervening vector sequences, by cutting the loop where it joins at the stem.
  • the ssDNA, thus, obtained is free or substantially free of any intervening and/or flanking nucleotide sequences, e.g. vector nucleotide sequences, that could interfere with the intended function of the ssDNA product.
  • a sequence of interest is located between the inverted tandem repeat, a second sequence of interest may further be provided between the inverted tandem repeat and the 3' primer binding site.
  • This embodiment of the invention has the advantage that the inverted tandem repeat can be selected to produce stem-loops of different stabilities, whereby variable amounts of read-through of the mRNA transcript, or early termination of transcription, can be achieved, thereby providing a method of regulating the production of two or more sequences of interest by secondary folding of the intermediate mRNA transcript.
  • the inverted tandem repeat comprises one or more specific enzyme recognition sequence(s). More preferably, the specific enzyme recognition sequence comprises a restriction endonuclease site.
  • the restriction endonuclease site may either be recognised by an endogenous restriction endonuclease or the nucleic acid construct of the present invention may further comprise a gene encoding a restricting endonuclease for that restriction endonuclease site.
  • the nucleic acid construct further comprises a restriction endonuclease gene
  • the restriction endonuclease gene is, preferably, located in a 5' position with respect to the inverted tandem repeat.
  • the specific enzyme recognition sequence may, for example, include either Hind III and Not I, Hind III, or Not I restriction sites
  • the specific enzyme recognition sequence may be any one of a restriction endonuclease type I, endonuclease type II, endonuclease type III, eukaryotic receptor recognition, prokaryotic receptor recognition, promoter, promoter/enhancer and T-overhang PCR site, or combinations thereof.
  • the inverted tandem repeat is, preferably, capable of forming a stable stem-loop, an unstable stem-loop, or a stem-loop of intermediate stability.
  • the inverted tandem repeats are selected to form a stable stem-loop in the mRNA transcript as a means of causing premature termination of reverse transcription, so that if a sequence of interest is located in the mRNA transcript between the start site for reverse transcription and the stem-loop structure, ssDNA substantially free of contiguous vector sequences can again be produced.
  • the inverted tandem repeat(s) of the present invention allow the use of a stem- loop structure as a vehicle for removing unwanted contiguous nucleotide sequences, for example, either by causing premature termination of reverse transcription from the mRNA transcripts or by cutting away unwanted sequences from the cDNA via restriction endonuclease sites in the inverted tandem repeats.
  • the inverted tandem repeat may comprise one or more eukaryotic, prokaryotic, and or/viral protein DNA binding sites.
  • the stem-loop structure of the present invention thus, has the further advantage that it can also serve as a functional entity in itself, e.g. to protect the ssDNA in the loop from degradation by intracellular nucleases, or to carry out certain applications via functional elements (e.g. protein binding sites) in the inverted tandem repeats.
  • the inverted tandem repeat acts in c/s-orientated fashion.
  • the primer binding site may be specific for an endogenous reverse transcriptase
  • the nucleic acid construct may further comprise a gene encoding a reverse transcriptase.
  • the reverse transcriptase gene is, preferably, located in a 5' position, with respect to the inverted tandem repeat.
  • the nucleic acid construct may further comprise a gene encoding a reverse transcriptase/RNAse H polyprotein.
  • the reverse transcriptase/RNAse H polyprotein gene is, preferably, located in a 5' position with respect to the inverted tandem repeat.
  • the reverse transcriptase/RNAse H polyprotein gene may 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 or reverse transcriptase/RNAse H polyprotein encoded by their respective genes.
  • the nucleic acid of the present invention further comprises a promoter and, optionally, an enhancer for each of said first of second sequence of interest, said restriction endonuclease, said reverse transcriptase and/or said reverse transcriptase/RNAse H gene.
  • the promoter and/or enhancer is a eukaryotic promoter/enhancer.
  • the promoter may be a constitutive, inducible wide-spectrum or tissue-specific promoter.
  • the nucleic acid construct of the present invention further comprises a polyadenylation tail sequence located in a 3' position with respect to the 3' primer binding site.
  • the polyA tail provides for stabilisation of the mRNA transcript.
  • the first or second sequence of interest includes a sequence which encodes a single-stranded DNA having enzymatic activity.
  • a single stranded DNA having enzymatic activity comprises the sequence 5' GGCTAGCTACAACGA-3', which has RNAse activity.
  • This sequence is flanked in both the 5' and 3' directions by one or more sequence(s) which are complementary to a target mRNA species, for example, h-ras, c-raf kinase, antisense sequence to pleiotrophin angiogenic growth factor, or the tat region of simian immunodeficiency virus.
  • the primer binding site is complementary to a transfer RNA (tRNA).
  • tRNA transfer RNA
  • the nucleic acid construct is DNA.
  • the nucleic acid construct 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.
  • mRNA transcript of the nucleic acid construct of the present invention is also provided.
  • an mRNA transcript comprising a sequence of interest flanked by an inverted tandem repeat, and further comprising a primer binding site located 3' to the inverted tandem repeat.
  • an mRNA transcript comprising an inverted tandem repeat and a primer binding site located 3' to the inverted tandem repeat, and a sequence of interest located between the inverted tandem repeat and the 3' primer binding site.
  • an mRNA transcript comprising a first sequence of interest flanked by an inverted tandem repeat, a primer binding site located 3' with respect to the inverted tandem repeat, and a second sequence of interest between the inverted tandem repeat and 3' primer binding site.
  • a single stranded DNA transcript of the mRNA of the present invention is also provided.
  • 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 an inverted tandem repeat, a primer binding site for a reverse transcriptase located in a 3' position with respect to the inverted tandem repeat, and an insertion site for a sequence of interest between the inverted tandem repeat, or between the inverted tandem repeat and the primer binding site.
  • the vector comprises a first insertion site between the inverted tandem repeat and a second insertion site between the inverted tandem repeat and the 3' primer binding site.
  • This allows a user to insert any desired sequence of interest into either, or both, the insertion sites. Because of the arrangement of the nucleic acid construct, with the primer binding site in a position which is 3' to the inverted tandem repeat, there is no limit on the size or type of sequence of interest that may be inserted into the insertion site.
  • the vector further comprises a gene encoding a reverse transcriptase, or a gene encoding a reverse transcriptase/RNAse H polyprotein.
  • the reverse transcriptase or reverse transcriptase/RNAse H polyprotein gene is located in a 5' position with respect to the inverted tandem repeat.
  • a vector system which comprises a first vector according to the present invention, (i.e. a vector which comprises an inverted tandem repeat, a primer binding site for a reverse transcriptase located in a 3' position with respect to the inverted tandem repeat, and an insertion site for O 00/22114 a sequence of interest between the inverted tandem repeat, or between the inverted tandem repeat and the primer binding site) and a second vector, which comprises a gene encoding a reverse transcriptase, a reverse transcriptase/RNAse H polyprotein, or a reverse transcriptase/RNAse H polyprotein linked via a proline rich linker to a restriction endonuclease.
  • a first vector according to the present invention i.e. a vector which comprises an inverted tandem repeat, a primer binding site for a reverse transcriptase located in a 3' position with respect to the inverted tandem repeat, and an insertion site for O 00/22114 a sequence of interest between the inverted tandem repeat, or between the
  • 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.
  • the vector or vector system of the present invention is a plasmid or modified viral construct.
  • the reverse transcriptase, reverse transcriptase/RNAse H polyprotein, or reverse transcriptase/RNAse H/restriction endonuclease 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.
  • a host cell stably transformed or transfected with a vector or vector system according to the present invention.
  • the host cell may be a eukaryotic or prokaryotic cell.
  • Eukaryotic cells include yeast, plant and mammalian cells.
  • Prokaryotic cells include bacterial cells.
  • a 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 each insertion site.
  • kits for producing a single-stranded nucleic acid sequence which kit 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 which method comprises the steps of introducing a nucleic acid construct according to the present invention into a target cell, transcribing the nucleic acid construct into mRNA and reverse transcribing the mRNA transcript into cDNA.
  • the method further comprises the step of removing the mRNA transcript from an mRNA cDNA heteroduplex formed by reverse transcription of the mRNA
  • Reverse transcription may be carried out either by a reverse transcriptase which is endogenous to the target cell, or the method of the present invention may further comprise the step of introducing a gene encoding a reverse transcriptase, or a gene encoding a reverse transcriptase/RNAse H polyprotein, into the target cell.
  • the method of the present invention further comprises the step of linearising the cDNA transcript of the sequence of interest by cutting the cDNA stem loop structure formed by the inverted tandem repeat where the loop structure joins the stem.
  • the method further comprises the step of introducing a gene encoding a restriction endonuclease into the target cell.
  • a gene encoding a restriction endonuclease By providing one or more restriction endonuclease sites in the inverted tandem repeat, the restriction endonuclease expressed by the restriction endonuclease gene can then be used to cut the cDNA stem loop structure.
  • cutting of the cDNA stem loop structure may rely on restriction endonucleases which are endogenous to the target cell.
  • the restriction endonuclease may also be provided by the step of introducing a gene encoding a reverse transcriptase/RNAse H polyprotein linked via a proline rich linker to a restriction endonuclease into the target cell.
  • the method may further comprise the step of isolating the mRNA transcript, mRNA cDNA heteroduplex and/or single- stranded cDNA from the target cell.
  • a single-stranded cDNA transcript e.g. an inhibitory nucleic acid molecule (e.g. an antisense sequence or aptamer), a single-stranded DNA having enzymatic activity, (e.g. RNAse activity), an mRNA transcript and/or a heteroduplex molecule produced by the methods of the present invention.
  • an inhibitory nucleic acid molecule e.g. an antisense sequence or aptamer
  • a single-stranded DNA having enzymatic activity e.g. RNAse activity
  • an mRNA transcript and/or a heteroduplex molecule produced by the methods 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 may be "sense” or “antisense” sequences, and 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 nucleic acid construct, vector or vector system, or host cell according to the present invention, together with a pharmacologically acceptable adjuvant, diluent or carrier.
  • nucleic acid construct, vector or vector system, or host cell for use in therapy, especially for use in delivering an inhibitory nucleic acid molecule to a target cell.
  • the nucleic acid construct, vector or vector system and host cell of the present invention are particularly useful for alleviating a pathological condition by regulating gene expression.
  • nucleic acid construct, vector or vector system, or host cell 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.
  • 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 cell in which it is produced.
  • Figure 1 is a schematic illustration of the production of ss-cDNA in a host cell in accordance with the present invention.
  • Figure 2 is a schematic illustration of the stem-loop intermediate formed by the method illustrated in Fig. 1.
  • Figure 3A is a schematic representation of the binding of an antisense sequence produced in accordance with the present invention which incorporates the "10-23 DNA enzyme" binding to a target mRNA and the subsequent cleavage of the target mRNA.
  • Figure 3B is an enlarged representation of the binding of the antisense sequence of Fig. 3A to the target mRNA showing the interaction of the 10-23 DNA enzyme (included in a generalized inhibitory nucleic acid sequence) with the cleavage site of the target mRNA.
  • Figure 4 is a schematic map of the plasmid pssDNA-Express A constructed in accordance with the present invention.
  • Figures 5 A, 5B, and 5C represent a schematic map of the plasmid pssDNA- Express-B constructed in accordance with the present invention, an enlarged portion of the plasmid pssDNA-Express B, and the sequence of the insert region of plasmid pssDNA-Express B, respectively.
  • Figures 6A and 6B represent the sequence of the insert regions between the Apa I and Nhe I sites of the plasmids pTest and pTelo constructed from the plasmid pssDNA- Express-B in accordance with the present invention.
  • Figure 7A is a schematic map of the starting plasmid pcDNA3JZeo + (Invitrogen, Inc.) for plasmid pcDNA3JZeo + /NM-link2-gag/pssDNA-Express-4B constructed in accordance with the present invention.
  • Figure 7B is a schematic map of pcDNA3JZeo + /NM-link2-gag (also designated pssDNA-Express-4B).
  • Figure 7C is a schematic representation of the cloning sites, inverted repeats, and PBS region (arrow) for pcDNA3JZeo7NM-link2-gag/pssDNA-Express-4B.
  • Figure 7D is a sequence map of the cloning sites, inverted repeats, and PBS region (arrow) for pcDNA3JZeo7NM-link2- gag/pssDNA-Express-4B.
  • Figure 8A is a schematic map of the plasmid pssDNA-Express A constructed in accordance with the present invention.
  • Figure 8B is a schematic map of the plasmid pssDNA-Express-A after deletion of the Mbo II gene by digesting with Xma I and Sac II.
  • Figure 9A is a schematic map of the plasmid pssDNA-Express-C constructed in accordance with the present invention.
  • Figure 9B is a schematic representation of the cloning sites, inverted repeats, and PBS region (arrow) for plasmid pssDNA-Express-C.
  • Figure 9C is a sequence map of the cloning sites, inverted repeats, and PBS region (arrow) for plasmid pssDNA-Express-C.
  • Figure 10A shows the partial sequence of the 23 rd codon of h-ras antisense binding sequence with the 10-23 DNA enzyme sequence inserted between the 5' and 3' complimentary sequences.
  • Figure 10B shows the partial sequence of c-raf kinase antisense binding sequence with the 10-23 DNA enzyme sequence inserted between the 5' and 3' complimentary sequences.
  • Figure 10C shows the partial sequence of pleiotrophin antisense binding sequence with the 10-23 DNA enzyme sequence inserted between the 5' and 3' complimentary sequences.
  • Figure 10D shows the partial sequence of tat antisense binding region of the SIV sequence with the 10-23 DNA enzyme sequence inserted between the 5' and 3' complimentary sequences.
  • Figures 11A and 1 IB are gels showing the results of PCR reverse transcriptase assays (J. Silver, et al, 21 Nucleic Acids Res. 3593-3594 (1993)) on RNA/ssDNA extracts from HeLa cell lines transformed with the plasmid pTest constructed in accordance with the present invention.
  • Lanes, Fig. 11 A (1) size markers, (2) untransformed HeLa cells, (3) HeLa cells transformed with the vector pcDNA3JZeo + , (4) HeLa cells transformed with pssXA, (5) positive control containing 2 milliunits of MoMuLV reverse transcriptase, and (6) negative control containing no protein extract.
  • the arrow indicates the 150 bp amplification product.
  • Lanes, Fig. 11B (1) clone A12, (2) B8, (3) B12, (4) D7, (5) positive control containing 2 milliunits of MoMuLV reverse transcriptase, and (6) negative control containing no protein extract.
  • Figure 12 are gels showing PCR amplification of single-stranded DNA from extracts of HeLa cell lines transformed with the plasmid pTest constructed in accordance with the present invention. RNA ssDNA preparations were used as templates in PCR reactions using primers specific to the expected ssDNA product.
  • RNA/ssDNA PCR template isolated from stably transformed colony A12 (HeLa cell line stable transfected with plasmid pssDNA-Express- A) (1) used with no prior treatment, (2) treated with SI nuclease, (3) treated with RNAse A. Lane (4) total RNA/ssDNA from a colony stably transformed with the pcDNA3JZeo + vector alone.
  • RNA/ssDNA PCR template isolated from stably transformed colony B12 (HeLa cell line stably transfected with plasmid pssDNA-Express- A) (1) used with no prior treatment, (2) treated with SI nuclease, (3) treated with PJSfAse A. Lane (4) total RNA/ssDNA from a colony stably transformed with the pcDNA3JZeo + vector alone. Lane (5), positive control template, plasmid pTest.
  • Figure 13 is a gel showing PCR amplification of single-stranded DNA from extracts of cells transformed with the plasmid pTelo constructed in accordance with the present invention.
  • RNA/ssDNA was harvested from transfected cell cultures at 36 hrs post transfection and used as templates for PCR reactions using primers specific to the expected ssDNA product. 25 bp lane marker used. Lanes 1-5 with isolation from HeLa cell line A12, (1) total RNA/ssDNA fraction, (2) total RNA treated with SI nuclease, (3) total RNA treated with RNAse A, (4) negative control, (5) positive control telomere DNA plasmid.
  • Figure 14 is a gel showing premature truncation of ss-cDNA transcription and read through products by in vitro reverse transcriptase reaction.
  • Lanes 1-5 represent plasmid constructs with stem-loop structures constructed in accordance with the present invention and various sequences of interest cloned within the loop region.
  • RNA template was produced by in vitro transcription with T7 RNA polymerase under standard conditions, then treated with DNAse to remove plasmid template. Phenol/chloroform extracted RNA pools were then reverse transcribed with mouse Moloney reverse transcriptase, treated with RNAse A for 15 mins. and resolved on a 6% acrylamide gel at 45 V for 30 mins. 25 bp marker at borders.
  • FIG. 15 shows a Northern blot of an antisense producing vector constructed in accordance with the present invention producing an antisense sequence against c-raf kinase (Fig. 10B) and including the "10-23 DNA enzyme" after transfection of plasmid pssDNA-Express-C (Fig. 9) including that sequence into a lung cancer cell line in vitro.
  • ss-cDNA single-stranded deoxyribonucleic acid
  • yeast prokaryotic cells
  • eukaryotic cells with or without flanking nucleotide sequences.
  • Methods and constructs are described which use biological rather than the in vitro, or artificial, synthesis of ss-cDNA of desired nucleotide base composition. Because biological, i.e., enzymatic reactions, are used in these methods they are applicable to any in vivo system.
  • a vector (as used herein, the term “vector” refers to a plasmid or modified viral construct used to deliver and manipulate DNA segments of interest) system was designed to produce any DNA sequence as a ss-cDNA molecule, free of most contiguous vector sequences, within yeast, prokaryotic, and/or eukaryotic cells.
  • the vector system contains all the necessary enzymatic functions and signaling instructions to enable the host cell to which the vector is delivered to produce ss-cDNA.
  • the cell to which the vector of the present invention is delivered produces an RNA transcript (Fig.
  • the vector comprises two plasmids that are co-transfected into a suitable host cell, which can be yeast or any prokaryotic or eukaryotic cell, to produce a ssDNA including the sequence of interest in the cell.
  • a second system is described in which the vector system comprises a single plasmid containing the sequence of interest that is transfected into a suitable host cell for production of an inhibitory nucleic acid, such as the antisense sequence, from the sequence of interest.
  • 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. 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
  • biomolecules that vary widely in size, structure and composition
  • examples of such molecules include (but are not limited to): (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.
  • thrombin a multifunctional regulatory protein that converts fibrinogen to fibrin in the process of clot formation
  • bradykinin a nonapeptide kinin involved in blood pressure regulation and implicated in hypotension
  • PGF2 alpha a prostaglandin or fatty acid derivative that exhibits hormonal activity.
  • inhibitory nucleic acids 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. These different types of inhibitory nucleic acids are described in Helene, C. and J. Toulme, 1049 Biochim. Biophys. Acta. 99-125 (1990).
  • 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 such as the ssDNA enzymes, including the so-called "10-23 enzyme” as described herein.
  • the first approach O 00/22114 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.
  • 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.
  • 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 is used to selectively suppress certain cellular functions by inhibition of translation of mRNA encoding critical proteins. Inhibitory nucleic acids targeting mRNA have been shown to work by several different mechanisms to inhibit translation of the encoded protein(s).
  • an inhibitory nucleic acid is the sequence that is complementary to regions of c-myc mRNA, which inhibits c-myc protein expression in a human promyelocytic leukemia cell line, HL60, which overexpresses the c-myc proto-oncogene. Wickstrom E. L., et al, 85 Proc. Natl. Acad. Sci. USA 1028-1032 (1988); Harel-Bellan, A., et al, 168 Exp. Med. 2309-2318 (1988).
  • inhibitory nucleic acids produced in the cell can also utilize the third approach of designing the "sense" strand of the gene or mRNA to trap or compete for enzymes or binding proteins involved in mRNA translation.
  • inhibitory nucleic acids are used to induce chemical inactivation or cleavage of the target genes or mRNA. Chemical inactivation occurs, for instance, by induction of crosslinks between the inhibitory nucleic acid and the target nucleic acid within the cell and by the method contemplated herein, namely, the cleavage of the target mRNA by the sequence having enzymatic activity against mRNA that is incorporated into the cassette of the present invention.
  • the present invention comprises a set of genetic elements adapted for delivery into a cell to produce ss-DNA in vitro or in vivo.
  • the set of genetic elements is incorporated into at least one vector for delivery into the cell and includes (a) a RNA dependent DNA polymerase (reverse transcriptase) gene, and O 00/22114
  • a cassette including (1) an inverted tandem repeat (IR), (2) a sequence of interest located either between the inverted repeat (IR) or 3 Jo the inverted repeat, and (3) a primer binding site (PBS) for the reverse transcriptase that is located 3' to the inverted repeat.
  • the vector system also preferably includes the functions and signaling instructions for transcription of these elements in vivo and the functions and signaling instructions for translation of the reverse transcriptase (RT) gene.
  • Additional elements that may be included in the set of genetic elements of the present invention include a restriction endonuclease (RE) gene, which may be included for a purpose described below, and a sequence having enzymatic activity when the linearized ss-DNA folds into the secondary configuration which confers enzymatic activity upon the sequence.
  • RE restriction endonuclease
  • the enzymatic sequence is preferably located within a sequence of interest, regardless of whether the sequence of interest is located between the inverted tandem repeats or between the 3' aspect of the inverted repeat and the PBS.
  • the vector comprises a two plasmid system, and the first plasmid includes the RNA-dependent DNA polymerase (reverse transcriptase) gene and an RNAse H gene, linked with a histidine-proline linker to a restriction endonuclease gene.
  • the plasmid into which these genes were inserted contains the necessary transcriptional and translational control elements for these genes along with polyadenylation tailing sequences.
  • a plasmid pssDNA-Express-A as shown in Fig. 4 in one of the preferred embodiments described herein.
  • a second, “B” plasmid was constructed which, in the embodiment described herein, includes the cassette comprised of three of the above-listed elements, namely, a primer binding sequence (PBS) matched to the reverse transcriptase, a sequence of interest, and an inverted tandem repeat.
  • PBS primer binding sequence
  • pMN-new-link and pssDNA-Express-4B the latter being shown in Fig.
  • the sequence of interest is positioned either between the inverted tandem repeats or in a 5' position (with respect to the mRNA transcript) to the primer binding sequence (PBS), the PBS being located at the most 3' aspect of the mRNA transcript.
  • the sequence of interest is located (1) inbetween the inverted repeats, (2) between the inverted repeats and the PBS, and/or (3) both between the inverted repeats and inbetween the inverted repeats and the PBS.
  • plasmid B also includes a combination of transcriptional control elements. However, in at least one preferred embodiment described herein, the B plasmid does not include (or require) translational control elements since no protein product is produced from this construct.
  • the vector system of the present invention comprises a single plasmid, designated as plasmid C, in which all of the elements of the set of genetic elements listed above were incorporated with the exception of the RE gene, which was not included for reasons to be made clear in the following discussion.
  • the components of the cassette included in the B plasmid of the two plasmid system described above e.g., the primer binding sequence (PBS), sequence of interest, and inverted tandem repeat, reside in the untranslated 3' portion of the reverse transcriptase polyprotein in the C plasmid.
  • the resulting mRNA transcript contains the coding region for the reverse transcriptase-RNAse H polyprotein and, at the end of translation at the stop signals, the additional mRNA transcript contains (3' to this translated protein) the elements from the B plasmid with further 3' downstream signaling events for polyadenylation signals, which remain intact from the RT-RNAse H component.
  • an appropriate promoter in the preferred embodiments described herein, the RSV promoter was utilized
  • sequence of interest (including the DNA enzyme) is inserted into the C plasmid only in a 3' position to the inverted repeats and unwanted vector sequences are removed by premature truncation of the ss-cDNA product as the transcript encounters the relatively stable stem and is unable to continue transcribing the ss-cDNA from the mRNA transcript. More specifically, as will be made apparent in the following description, each of the sequences cf interest was inserted only within the Bam H I-Pac I restriction sites of the pssDNA-Express-C plasmid.
  • the plasmids include cloning sites for insertion of the sequence of interest. Both Not 1 sites (located between the inverted repeats) and Pac I/BamH I (3' to the inverted repeats, e.g., between the inverted repeats and the PBS) sites are provided in the preferred embodiment of the B plasmid described herein.
  • the preferred C plasmid described herein includes only the Pac I/BamH I sites for this purpose. However, those skilled in the art who have the benefit of this disclosure will recognize that these particular cloning sites were chosen for the particular systems described herein and that other cloning sites may be equally useful for this same purpose.
  • a plasmid comprising the two plasmid vector system described herein was not intended to include the sequence of interest, but those skilled in the art having the benefit of this disclosure will recognize that, if a two plasmid vector system is to be used, the elements of the set of genetic elements of the present invention, and particularly the sequence of interest, may be inserted into either plasmid as may be convenient.
  • the nucleic acid sequence that is referred to herein as a cassette provides the template for synthesis of ss-cDNA in target cells. It is this element which includes the sequence of interest, inverted tandem repeat, and primer binding site. As is the case for the other elements of the set of genetic elements of the present invention, this genetic element is preferably regulated by an appropriate wide spectrum or tissue-specific promoter/enhancer, such as the CMV promoter, or combination of promoters/enhancers, located upstream of the genetic element. Also as is the case for the other genetic elements, the promoter/enhancer can either be constitutive or inducible promoter.
  • the cassette also includes a downstream polyadenylation signal sequence so that the mRNA produced by the sequence of interest has a poly(A) tail.
  • the primer-binding site (PBS) for initiation of priming for cDNA synthesis is located between the 3' inverted tandem repeat and the polyadenylation signal.
  • the PBS is a sequence which is complementary to a transfer RNA (tRNA) which is resident within the eukaryotic target cell.
  • tRNA transfer RNA
  • the PBS takes advantage of the proline tRNA.
  • the PBS utilized in connection with the presently preferred embodiment of the invention that is described herein was taken from the actual 18 nucleotide sequence region of mouse Moloney virus. See 293 Nature 81.
  • the PBS used was taken from the nucleotide sequence of HIV. Y. Li, et al, 66 J, Virology 6587-6600 (1992). In short, any PBS that is matched to the reverse transcriptase which is utilized in connection with the method of the present invention is utilized for this purpose.
  • the cassette of the present invention also comprises a pair of inverted tandem repeats. After digestion of the mRNA from the mRNA-cDNA heteroduplex by RNAse H and the release of the ss-cDNA, the inverted repeats cause the ss-cDNA to fold back upon itself to form the stem of a stem-loop structure, the stem structure being comprised of double stranded, anti-parallel DNA.
  • the ss-cDNA which is produced is transcribed with the encoded 5' and 3' regions flanking the stem (made up of the inverted repeat) and a loop containing the sequence of interest.
  • One or more functional genetic elements may be designed into the double stranded portion, i.e., the inverted repeat, that forms the stem of the stem-loop intermediate.
  • the functional genetic element is one or more restriction endonuclease site(s) which is cut by the restriction endonuclease produced from the above-described restriction endonuclease gene (in the case of those plasmids which include an RE gene), but those skilled in the art will recognize that the inverted repeat can be designed to include other functional genetic elements such as a specific enzyme recognition sequence (other than a restriction endonuclease site), eukaryotic and/or prokaryotic receptor recognition sites, promoter or promoter/enhancer sites for driving expression of a target sequence (that may or may not be likewise designed into the stem) in isolated cw-oriented fashion.
  • the stem is then cut (also termed digested or cleaved) by any of the many restriction endonuclease enzymes which recognize the cut site designed into the stem (note that the endonuclease recognition site may be designed into the stem even though the RE gene is not included in the vector system of the present invention) to release the ss-cDNA loop.
  • the loop portion of the ss-cDNA which does not form any apparent duplex DNA, is immune to restriction endonuclease activity since restriction endonucleases recognize only double stranded DNA as a target substrate.
  • restriction endonuclease site(s) need not be designed into the inverted repeats which form the stem of the ste - loop intermediate if the second aspect of the present invention is being utilized.
  • the inverted repeats may contain eukaryotic, prokaryotic, or viral protein DNA binding sites, which can act to competitively titer out selected cellular proteins.
  • Combinations of restriction sites or other sequence specific elements may be included in the inverted tandem repeats depending on the base pair composition chosen for the construction of inverted repeats such that linear or precisely cut stem-loop intermediate forms of ss-DNA are produced. It is generally preferred to use synthetically constructed functional genetic elements in the inverted tandem repeat since (if the functional genetic element is a restriction endonuclease site) it is unlikely that a naturally occurring inverted repeat would have the properly aligned restriction sites.
  • the cassette which comprises one of the elements of the set of genetic elements of the present invention optionally also includes a DNA sequence with catalytic activity.
  • DNA enzyme a DNA sequence with catalytic activity.
  • the present invention is used to particular advantage when the sequence of interest serves as the template for synthesis of an inhibitory nucleic acid that is an antisense sequence. For that reason, the examples set out herein describe production of four antisense sequences of interest as set out in Fig.
  • SIV simian immunodeficiency virus
  • the nucleic acid sequence having enzymatic activity which comprises the cassette of the present invention can be any such enzymatic sequence.
  • the sequence having the desired catalytic activity is inserted into the cassette in either or both of the two locations, e.g., (a) between the inverted repeats and inside the sequence of interest or (b) inside the second sequence of interest that is located 3' to the inverted repeats and 5' to the PBS.
  • the resulting aptamer is specific for the target of the sequence of interest and is therefore used to target other DNA sequences, mRNA sequences, and any other suitable substrate, to inhibit or change DNA or mRNA splicing mechanisms, or even to directly alter the cellular genome in a specific manner.
  • Nucleic acid sequences with enzymatic activity that are appropriate for use in connection with the present invention include, but are not limited to, sequences having RNAse activity such as the so-called “10-23" and “8-17 enzymes" (Santoro, S.W. and G.F. Joyce, supra (1997)) and other metal- dependent RNAses (Breaker, R.R. and G.F. Joyce, 1 Biol. Chem. 223-229 (1994); Breaker, R.R. and G.F. Joyce, 2 Biol. Chem. 655-660 (1995)) and histidine- dependent RNAse (Roth, A. and R.R. Breaker, 95 Proc. Natl Acad. Sci. USA 6027-6031 (1998)); sequences having DNAse activity such as copper-dependent DNAse
  • RNA kinase acitivity such as calcium-dependent DNA kinase
  • nucleic acid sequence having enzymatic activity utilized in the examples described herein is the "10-23 enzyme" (Santoro, S.W. and G.F. Joyce, supra (1997)), and it is this enzymatic sequence which is represented schematically in Figs. 3A and 3B.
  • any of the sequences reported in the above-listed literature will function for the intended purpose when inserted into the cassette of the present invention.
  • RNA-dependent DNA polymerase or reverse transcriptase (RT) gene which also comprises one element of the set of genetic elements of the present invention
  • the reverse transcriptase/RNAse H gene from Moloney murine leukemia virus was used to advantage in the examples described herein.
  • the reverse transcriptase/RNAse H gene from the human immunodeficiency virus (HIV) has also been tested.
  • retro viral reverse transcriptase/RNAse H genes may be used to advantage in connection with the present invention including those from retroviruses such as SIV, strains of hepatitis B, hepatitis C, bacterial retron elements, and retrons isolated from various yeast and bacterial species, it being preferred that the reverse transcriptase/RNase H gene be a reverse transcriptase/RNase H gene that is regulated by an appropriate upstream promoter/enhancer such as the cytomegalovirus (CMV) or Rouse sarcoma virus (RSV) promoter for expression in eukaryotic cells.
  • CMV cytomegalovirus
  • RSV Rouse sarcoma virus
  • RNA-dependent DNA polymerases usually have an associated RNase H component enzyme within the same coding transcript.
  • the present invention does not require the naturally-occurring RNase H gene for a particular reverse transcriptase.
  • RNase H gene for a particular reverse transcriptase.
  • the target cell may itself have sufficient endogenous RNase H to fulfill this function.
  • the target cell may itself have sufficient endogenous reverse transcriptase activity from, for instance, prior retroviral infection, to fulfill this function.
  • the reverse transcriptase/RNase H gene also preferably includes a downstream polyadenylation signal sequence so that the mRNA produced from the reverse transcriptase/RNase H gene includes a 3' ⁇ oly(A) tail for mRNA stability.
  • a downstream polyadenylation signal sequence so that the mRNA produced from the reverse transcriptase/RNase H gene includes a 3' ⁇ oly(A) tail for mRNA stability.
  • multiple poly(A) tails are available and are routinely used for production of expressed eukaryotic genes.
  • the reverse transcriptase produced in the cell synthesizes a complementary DNA
  • cDNA using as the template the genetic element including the sequence of interest described below.
  • the gene encoding the restriction endonuclease (used only in the two plasmid system, and not even a required component of that system) may be any of several genes which encode for restriction endonucleases, and preferably those that are controlled by one or more constitutive or inducible wide spectrum and/or tissue-specific promoters/enhancers as discussed below.
  • the particular restriction endonucleases utilized herein were Mbo II and Fok I, but those skilled in the art who have the benefit of this disclosure will recognize that any restriction endonuclease (type I, II, IIS, or III) site may be included in the inverted tandem repeat. These enzymes "clip,” or digest, the stem of the stem-loop intermediate to linearize the sequence of interest as single-stranded DNA.
  • RE gene may be regulated by an appropriate constitutive or inducible promoter/enhancer located upstream from the restriction endonuclease gene such as the CMV or RSV promoter for expression in human cells, in plasmid pssDNA-Express-A described herein, the RE gene (Mbo II) is linked to the RT-
  • the RE gene also preferably includes a downstream polyadenylation signal sequence so that the mRNA transcript from the restriction endonuclease gene will have a 3' poly(A) tail.
  • tissue-specific or wide spectrum promoters/enhancers may also be used to advantage to regulate the reverse transcriptase/RNAse H gene, the RE gene (if utilized), and the 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 and many other viral or mammalian promoters.
  • promoters/enhancers which are appropriate for use in connection with the elements of the present invention may include, but are not limited to, HSVtk (S.L. McKnight, et al, 217 Science 316 (1982)), human ⁇ -globulin promoter (R. Breathnach, et al, 50 Ann. Rev. of Biochem. 349 (1981)), ⁇ -actin (T. Kawamoto, et al, 8 Mol. Cell Biol. 267 (1988)), rat growth hormone (P.R. Larsen, et al, 83 Proc. Natl. Acad. Sci. U.S.A. 8283 (1986)), MMTV (A.L.
  • the vector When the elements comprising the set of genetic elements of the present invention are incorporated into a vector, it is preferred that the vector contain other specialized genetic elements to facilitate the identification of cells that carry the vector and/or to increase the level of expression of the cassette.
  • the specialized genetic elements include selectable marker genes so that the vector can be transformed and amplified in a prokaryotic system.
  • the most commonly used 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 may be used that confer to the cell resistance to an antibiotic or other drug or alter the phenotype of the cell such as morphological changes, loss of contact inhibition, or increased growth rate.
  • 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. Incorporation of these components into an appropriate vector allows two convenient methods for removing predetermined vector sequences after the production of ssDNA.
  • the loop portion of the ssDNA stem-loop intermediate that is produced is comprised of the nucleotide sequence of interest and, after digestion with the restriction endonuclease, the loop is released as linearized, single-stranded cDNA without flanking sequences.
  • reverse transcription is terminated at the stem of the stem-loop structure such that the resulting ssDNA is produced without flanking sequences.
  • the cassette is designed with an inverted repeat which forms a stem that is more stable than the stem of the stem-loop intermediate from which the ssDNA is produced by digestion of the stem in accordance with the first aspect of the present invention.
  • factors such as the length of the inverted repeat e.g., the number of nucleotides comprising the repeat
  • the identity of the bases comprising the repeat can be manipulated (using, for instance, the //; vitro system described herein) to obtain a stem having as little or as much stability as may be desired to obtain production of the first or second sequence of interest or a mix of both sequences.
  • the intact stem-loop ss-cDNA structure can function similarly in many applications as the linearized ss-cDNA form. Consequently, the cassette is also used to advantage without the restriction endonuclease gene and associated regulatory elements and/or with a sequence of interest which lacks the corresponding restriction endonuclease site.
  • a cassette can be made which encodes a ss-cDNA that has a "trimmed" stem-loop structure.
  • the restriction endonuclease sites encoded in the inverted repeats flanking the sequence of interest are designed such that the stem portion (after duplex formation) is digested with the corresponding restriction endonuclease so as to cut the dsDNA comprising the stem in a way that removes a portion of the stem and the associated flanking sequences yet leaves sufficient duplex DNA that the transcript retains the above-described stem-loop structure.
  • Such a ss-cDNA 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 predetermined sequence i.e., aptamers
  • stem structure is used in the cell as a competitor to titer out a selected protein(s) that regulates specific gene function.
  • a ss-cDNA stem-loop is produced in accordance with the present invention in a cell that contains a binding site for a selected positive transcription factor such as adeno virus El a.
  • Adeno virus El a modulates expression of several adenoviral and cellular genes by affecting the activity of cell-encoded transcription factors, resulting in the changing of normal cells to transformed cells. Jones, et al, 2 Genes Dev. 267-281 (1988).
  • the duplex stem of the stem-loop intermediate produced in accordance with the present invention is therefore designed to function to "bind up" this transcription factor, preventing the protein from binding a promoter, and thus inhibiting expression of the 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 Ela 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 rav, mos, src, and trk oncogenes.
  • TPA 12-O-tetradecanolyphorbol 13 -acetate
  • the mechanism involves inhibition of the function of the transcription factor family AP-1.
  • nucleotide sequence can be inserted into the genetic element which encodes the "loop" portion of the stem-loop intermediate to ultimately carry out a desired inhibitory function, e.g., antisense binding, down regulation of a gene, and so on as herein described.
  • the present invention is used to construct complex secondary ssDNA structures which confer biologic reactions on the cDNA transcript based on conformational secondary structure folding.
  • Such secondary structure can be engineered to serve any of several functions.
  • the sequence of interest may include (but is not limited to) a sequence which is incorporated into the loop portion of the single-stranded cDNA transcript which forms so- called “clover leaf or "crucible” like structures such as those found in the long terminal repeats of adeno-associated virus or in retrotransposons. Under correct circumstances, such structure is integrated in site-specific manner into the host genome.
  • 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 the infected 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, 80 Proc. Natl.
  • the vector including the set of genetic elements of the present invention is advantageously employed to deliver anti-sense, triplex, or any other inhibitory nucleic acid or single-stranded nucleotide sequence of interest, using known digestion and ligation techniques to splice the particular sequence of interest into the cassette (between inverted tandem repeat or between PBS and inverted tandem repeat).
  • digestion and ligation techniques to splice the particular sequence of interest into the cassette (between inverted tandem repeat or between PBS and inverted tandem repeat).
  • 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. For instance, a likely change is to change the promoter so as to confer advantageous expression characteristics on the cassette in the system in which it is desired to express the sequence of interest.
  • the present invention takes the form of a kit comprised of a plasmid having the above-described RNA-dependent DNA polymerase and restriction endonuclease genes cloned therein as well as a multiple cloning site (MCS) into which the user of the kit inserts a particular sequence(s) of interest.
  • MCS multiple cloning site
  • the cloning site into which the sequence(s) of interest is inserted is located between the above-described inverted tandem repeats, e.g., 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, and sent to the user.
  • the kit preferably also includes the restriction endonuclease for the cloning site into which the sequence of interest is to be cloned, the ligases and other enzymes, along with suitable buffers, for ligating the sequence of interest into the plasmid, and a map of the plasmid and/or other instruction for the user.
  • the sequences of interest are delivered to a host cell either by co-transfection of the cells with two plasmids, designated A and B, each plasmid being designed and constructed to include one or more of the genetic elements listed above, or a single "C" plasmid.
  • the B plasmid encodes the cassette including the sequence of interest, nested within flanking sequences that include the inverted repeat, and the primary binding site that provides the post-transcriptional processing signals that mediate the conversion of the mRNA into single-stranded DNA.
  • the B plasmid also includes the second sequence of interest when this second aspect of the invention is utilized (as set out above, when the RE gene is omitted from the construct of the present invention, for instance in the single "C" plasmid described herein, it is this second sequence of interest that encodes the particular inhibitory nucleic acid having the desired activity). Activities required for processing the primary gene product of the B plasmid into single-stranded DNA, with the removal of vector sequences and processing signals, specifically the reverse transcriptase/RNAse H, and restriction endonuclease, are expressed from the A plasmid. The single-stranded DNA sequence that is released by interaction of the transcriptional products of these components in vivo is free to bind intracellular targets such as mRNA species and DNA promoters in antisense and triplex strategies.
  • the B plasmid includes cloning sites (Not I sites were utilized in one embodiment of the B plasmid described herein) between which any DNA sequence of interest is placed (as noted above, in the examples described herein, the sequences include a "stuffer," or test, sequence, telomeric repeats, h-ras, c-raf kinase, a region encoding the angiogenic growth factor pleiotrophin, and the region encoding tat from SIV).
  • sequences include a "stuffer," or test, sequence, telomeric repeats, h-ras, c-raf kinase, a region encoding the angiogenic growth factor pleiotrophin, and the region encoding tat from SIV).
  • Flanking the cloning sites are signals directing the processing of the primary mRNA transcript, produced from a promoter (a CMV promoter was utilized in the preferred B plasmid described herein), into the desired single-stranded inhibitory nucleic acid.
  • a CMV promoter was utilized in the preferred B plasmid described herein
  • the A and B plasmids are co-transfected into a cell line of choice for constitutive expression of ssDNA.
  • the sequence of interest is cloned into that plasmid and transfected into the cell line for further processing.
  • this processing proceeds in three steps following transcription of the single-stranded DNA region (i.e., sequence of interest, inverted repeats, and PBS):
  • reverse transcriptase which in the preferred embodiment described herein is reverse transcriptase expressed by the A or C plasmid (in the preferred embodiment described herein, the reverse transcriptase is Moloney mouse leukemia virus (MoMuLV) reverse transcriptase), proceeding from the primer binding site lying 3' to the sequence of interest (including the sequence with enzymatic activity), inverted repeats, and primer binding site as shown in Fig. 1;
  • MoMuLV Moloney mouse leukemia virus
  • RNAse H digestion of the resulting heteroduplex either by the RNAse H activity of the reverse transcriptase polyprotein or by endogenous RNAse H activity, to release the single- stranded DNA precursor from its RNA complement; and (3) Removal of flanking sequences by either restriction endonuclease digestion of the stem of a stem-loop intermediate formed upon Watson-Crick base pairing of the bases comprising the inverted repeat or by premature termination of the cDNA transcript by formation of the stem-loop secondary structure by the self- complementary inverted tandem repeats.
  • ODN Oligodeoxynucleotides
  • PCR Polymerase chain reactions
  • oligonucleotides and the designed restriction endonuclease cut sites for Not I and Mbo II were the oligonucleotides of length 129 (Seq. ID 4) and 121 (Seq. ID 5) bases.
  • a tRNA primer binding sequence (PBS) for recognition by primer for reverse transcriptase was designed into the oligonucleotides. The PBS was located 3' downstream from the inverted tandem repeat.
  • Standard restriction endonuclease digests were then carried out with Not I, Fok I, Mbo II (appropriate for each ohgonucleotide for which a restriction endonuclease cut site was present) and EcoR I (as a negative control) with 10 units of enzyme in a total reaction volume of 15 ⁇ l and appropriate reaction buffers.
  • the digests were confirmed by electrophoretic gel analysis.
  • the results of this experiment showed that a synthetic ss-cDNA with inverted repeats formed duplex DNA.
  • the duplex DNA presumably the stem of a stem-loop structure, formed specific and recognizable restriction endonuclease sites from the sequences in the inverted tandem repeats, which were designed so as to form Not I and Fok I restriction endonuclease sites by Watson-Crick base pairing of the bases comprising the inverted tandem repeats.
  • EXAMPLE 2 fn vitro Formation of ss-DNA From Cassette Transcripts Two test plasmids were constructed to conduct this experiment. pcDNA3J/Zeo(+) was digested with Nhe I and Apa l under standard conditions. Two sets (A and B) of 5' phosphorylated oligonucleotides, which were designed to be complementary with each other (see attached sequence listings) were allowed to hybridize.
  • Hybridization was performed by heating the complementary oligonucleotides at 95°C and then allowing them to cool to room temperature for 15 minutes.
  • the resultant duplex ohgonucleotide linkers with appropriate cohesive ends were ligated under standard conditions to the previously prepared pcDNA3J/Zeo(+) vector.
  • Positive circular test plasmids were linearized by digestion with Pme I, and standard reverse transcription reactions were carried out as follows. To each tube was added 0J ⁇ g of linearized plasmid DNA, 25 units of T7 polymerase (i.e., a DNA-dependent RNA polymerase), 2.5 mM rNTP's (ribonucleotide triphosphates rUTP, rCTP, rGTP, rATP) and appropriate buffer. The reaction tube was incubated at 37°C for 90 minutes. After incubation, 10 units of DNAase was added and incubated for 15 minutes at 37°C. The reaction was terminated by incubation at 70°C for 10 minutes. Standard cDNA synthesis reactions were carried out using the above reactions.
  • T7 polymerase i.e., a DNA- dependent RNA polymerase
  • 2.5 rNTP's ribonucleotide triphosphates rUTP, rCTP, rGTP, rATP
  • reaction tubes were incubated at 70°C and allowed to anneal by cooling to room temperature over 15 minutes.
  • the mixture was divided equally into four tubes, in which either 10 units of restriction enzyme Not I, Fok I, Mbo II, or EcoR I was added along with the appropriate reaction buffer.
  • the tubes were incubated at 37°C for 1 hour. Replicate reactions were treated with SI nuclease (specific for ssDNA). The DNA from the above reactions was resolved by gel electrophoresis.
  • tissue culture cells used in these experiments encoded and endogenously expressed eukaryotic DNA-dependent RNA polymerase II, which utilizes the RSV promoter located upstream of the cloned genetic element that is the linker/stuffer segment described in Example 1, above. It will be recognized by those skilled in the art who have the benefit of this disclosure that any eukaryotic promoter can be used for this purpose. It is further recognized by such persons that a vector-encoded DNA-dependent RNA polymerase will also function in this capacity.
  • Plasmid constructs The ODNs were allowed to hybridize in 1 ⁇ l (5 ⁇ g/ ⁇ l in water) in four separate tubes which were incubated at 70°C for 5 min and allowed to hybridize for 15 min at room temperature. Standard restriction endonuclease digests were carried out (EcoR I used as a negative control) with 10 units of enzyme in a total reaction volume of 15 ⁇ l and appropriate reaction buffers. DNA fragments were resolved in and isolated from agarose gels. The selection of positive clones on ampicillin plates was performed after transformation into competent XL 1 -Blue MRF cells (Stratagene) as described by Maniatis, et al (1989).
  • the first "B" plasmid was derived from pcDNA3J/Zeo(+) (Fig. 7A) by digesting with restriction endonucleases Nhe I and Apa I, which are located in the multiple cloning site (MCS).
  • Nhe I and Apa I restriction endonucleases
  • MCS multiple cloning site
  • pssD ⁇ A-Express-B The structure of pssD ⁇ A-Express-B is shown in Fig. 5. Mammalian cells containing the plasmid are selectable with the antibiotic zeocin. The position and general arrangement of key regions of the insert are shown in Fig. 5 A and the specific arrangement of the sequences of the insert are shown in Fig. 5B. The exact positions of the structural features are shown in Fig. 5C. Transcription of the insert region is driven by the cytomegalovirus promoter and terminated in the BGH polyA region. The R ⁇ A transcript contains the MoMuLV core promoter along with some flanking regions of this promoter, the positions of which are indicated in Fig. 5B.
  • Reverse transcriptase synthesizes a copy of the (+) (top) strand, using the tR ⁇ A pro as a primer, beginning at the position of the core promoter.
  • Digestion of the R ⁇ A strand by R ⁇ Ase H releases a single-stranded D ⁇ A sequence containing the complementary inverted repeats IR-L and IR-R (Fig. 1).
  • Duplex formation by these repeats, shown in Fig. 2 creates a stem-loop with the sequence of interest in the loop.
  • the stem contains a recognition site for Mbo II, GAAGA, positioned so that the enzyme, which cleaves 8/7 bases 3' to the GAAGA recognition site, releases the sequence of interest from the flanking vector sequences.
  • the B plasmid expressing the telomeric repeat sequence pTelo as the sequence of interest was obtained in similar fashion, inserting the annealed, Not I compatible oligodeoxynucleotides Seq.
  • pMN-new-link A third B plasmid, termed pMN-new-link, in which the stem was comprised of 29 base pairs (rather than the 27 base pair stem site of pMNV) was constructed because the 29 base pair stem gives a more stable stem-loop structure in the stem-loop intermediate than the 27 base pair stem site of pMNV.
  • pNM-new-link was formed by ligating two self-complementary ODN's, ODN-NM-new-link(+) and ODN- NM-new-link(-), between the two Not I sites within pM ⁇ V.
  • the A plasmid, pss-D ⁇ A-Express-A (Fig. 4), was prepared from pBK-RSV (Stratagene) using XL-1 Blue MRF' as the host cell.
  • a mouse cell line expressing Moloney murine leukemia virus was obtained from the American Type Culture Collection (#CRL-1858).
  • the virus R ⁇ A was isolated and prepared for reverse transcriptase-PCR (RT-PCR).
  • RT-PCR reverse transcriptase-PCR
  • a 2.4 kb fragment containing the coding sequence of MoMuLV-RT was PCR-amplified using primers as set out in Seq. ID l/OD ⁇ -RT(-) (primer position at nucleotide #2545) and Seq.
  • 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 J.M Coffin (Ed.), Retroviruses, 757, Appendix 2, New York: Cold Spring Harbor Press (1997)), but peptides truncated at the amino terminus retain full activity (N.
  • the peptide encoded by this construct includes a part of the integrase gene, which follows the reverse transcriptase in the MoMuLV polyprotein, but is not relevant here such that the length of the construct was selected because of the availability of a convenient restriction site for cloning.
  • the bacterium Moraxella bovis which encodes the restriction endonuclease MboII (Bocklage, H Chapter et al, 19 Nucleic Acids Res. 1007-1013 (1991)), was obtained from the American Type Culture Collection (ATCC#10900). Genomic DNA was isolated from M bovis and used as the template DNA in the PCR. A 1.2 kb fragment containing the Mbo II gene was amplified by PCR using as primers Seq. ID 3/ODN-Mbo(+) (primer position at nucleotide #887) and Seq. ID 8/ODN-Mbo(-) (primer position at nucleotide #2206).
  • primers contain mismatches designed to introduce a Hind III site into the 5' primer and an Xba I site into the 3' downstream primer.
  • the 1.2 kb DNA amplification product copying the M. bovis genome between positions 888 and 2206, therefore contains the coding region for the Mbo II protein.
  • the amplification product was digested with Hind III and Xba I.
  • pBK-RSV was digested with Xba I and Nhe I to remove the promoter region.
  • the Nhe I end was converted to a Sac I end using the linker formed by annealed oligodeoxynucleotides Seq. ID 6/ODN-N>S(+) and Seq. ID 7/ODN-N>S(-).
  • the reverse transcriptase and Mbo II 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 produce pBK-RSV-RT/Mbo.
  • the double- stranded sequence formed by annealing the oligodeoxynucleotides Seq. ID 9/ODN- HisPro(+) and Seq. ID 10/ODN-HisPro(-), encoding alternate histidine and proline amino acids was inserted into pBK-RSV-RT/Mbo by digesting with Hind III.
  • the his-pro linker, with compatible Hind III ends, was inserted at the Hind III site to produce plasmid pBK- RSV-RT/Mbo-L and the orientation was confirmed by sequencing.
  • sequencing pBK-RSV-RT/Mbo-L revealed a frame shift mutation at the 5'-end of the Mbo II domain. This mutation was corrected, and the extraneous part of the integrase gene of MoMuLV was simultaneously removed, by excising the fragment of the plasmid lying between the Ase I and Bgl II sites, which encodes the 5'-end of the Mbo II gene, the his-pro linker region, and the integrase gene fragment, and replacing with an insert containing a modified his-pro linker and 5'-Mbo II gene fragment.
  • the modified his-pro linker increased the number of histidines by one, to six, and included at the 5'-end a number of unique restriction sites.
  • the 5 '-end of the Mbo II gene was modified to replace the leucine at the N-terminus that was introduced by the mismatch in the PCR primer to the original methionine and to optimize codon usage for expression of this segment of the gene in mammalian cells.
  • the repair construct was obtained by mutually-primed DNA synthesis from two templates, ODN-Rep(+) and ODN-Rep(-), that have complementary sequences of 16 bases at the 3 '-ends. These oligodeoxynucleotides were annealed and extended with the modified SEQUENASETM DNA polymerase enzyme (United States Biochemical Corp.).
  • pssDNA-Express-A (plasmid A).
  • Fig. 8A The structure of pssDNA-Express-A is shown in Fig. 8A.
  • sequences encoding an active fragment of the MoMuLV reverse transcriptase and the M. bovis Mbo II restriction enzyme were cloned between the Nhe I and Xma I sites of the eukaryotic expression vector pBK-RSV. Transcription of the cloned region is driven by the RSV promoter and selection for transformed cells is carried out in the presence of the antibiotic G418 (neomycin).
  • Reverse transcriptase and Mbo II are expressed as a single, bifunctional protein chain with the two functional domains separated by a short, histidine and proline rich linker.
  • ss-cDNA was isolated from cells transfected 48-72-hr earlier. As described above, the ss-cDNA, which co-localizes with RNA, was carried out using trizol reagent (Gibco Life Technologies, Gaithersburg, MD). Assays for specific ss-cDNA species were carried out by both PCR based assays (Fig. 12 for pTest and Fig. 13 for pTelo) for internal fragment and by denatured single stranded gel electrophoresis with subsequent nylon blotting and probing with an internal biotin-labeled probe.
  • the vector pNM-New-Link (which after single stranded conversion contains the more stable 29 base pair stem structure) was used for in vitro experiments to demonstrate premature termination of the reverse transcriptase cDNA transcript at the 3' aspect of the stem structure. As shown in Fig. 14, there was also some "read through” of this transcript through the stem structure as well (see the larger bands in Fig. 14). The sequence of interest produced from this premature termination is the second sequence of interest referred to herein.
  • ODNs were prepared and plasmids constructed in the same manner as described in Example 3, above.
  • a second embodiment of the first of the two plasmids comprising a two plasmid embodiment of the vector system of the present invention is the "4B" plasmid.
  • the 4B plasmid like plasmid pssDNA-Express-B described in Example 3, was derived from pcDNA3J/Zeo(+) (Invitrogen Corp.), shown in Fig. 7A.
  • pssDNA-Express-4B was constructed by digesting with restriction endonucleases Hind III and Not I at positions 911 and 978, respectively.
  • the double- stranded linker region having compatible Hind III and Not I ends which is formed by annealing the synthetic, single stranded oligodeoxynucleotides ODN-5'-N/M(link)2-H/N and ODN-3'-N/M(link)2-H/N was ligated under standard conditions into the digested pcDNA3J/Zeo(+) transformed into Sure II cells (Stratgene, Inc.).
  • the ODNs were allowed to hybridize in 1 ⁇ l (5 ⁇ g/ ⁇ l in water) in Ependorf tubes that were incubated at 70°C for 5 minutes and allowed to hybridize for 15 minutes at room temperature. Appropriate clones were selected and sequenced to assure proper insertion of the linker region.
  • pcDNA3J/Zeo(+)/NM-link2-gag The resulting plasmid was termed pcDNA3J/Zeo(+)/NM-link2-gag and re-named pssDNA-Express-4B.
  • pssDNA-Express-4B is shown in Fig. 7B and is the plasmid into which the sequence of interest is cloned.
  • the two Not I sites as positions 935 and 978, respectively (see Fig. 7B), were used. These two sites are contained within the inverted tandem repeats.
  • the second plasmid of the two plasmid system which comprises this second embodiment of the vector of the present invention is the A plasmid, pssDNA-Express- A, shown in Fig. 8A, prepared as described in Example 3.
  • the vector system of the present invention may also take the form of a single plasmid.
  • plasmid pssDNA-Express-A was digested with Sac I Xma I to remove the Mbo II gene (Fig. 8B).
  • a linker region comprised of oligonucleotides 5'-(link)2-Hind/Xba and 3'-(link)2-Hind/Xba (Table 1), which were allowed to anneal at 70°C for 15 minutes and slowly cooled to room temperature, was ligated into the plasmid after digestion under standard conditions.
  • Positive clones were harvested and sequenced to verify linker placement and this plasmid was then digested with Xba and Hind III.
  • the plasmid pssDNA-Express-B was then digested with Hind III and Xba and the corresponding 300 base pair DNA fragment containing the previously described inverted tandem repeats, multiple cloning site, and PBS was cloned into the digested plasmid to give pssDNA-Express-C (Fig. 9A). Standard ligation reactions were performed and transformed into Sure II cells (Stratagene, Inc.). Transformed positive colonies were harvested and positive clones were identified by restriction analysis.
  • sequences of interest were cloned into the multiple cloning site of pssDNA- Express-C by using the Bam H I and Pac I sites in the multiple cloning site (Fig. 9B).
  • Table 1 The sequences of interest as listed in Table 1, each including the "10-23 DNA enzyme" inserted between the 5' and 3' aspects of the antisense sequence (Fig. 9C) and shown in Figs. 10A-10D, were synthesized for these constructs, and similar procedures were utilized for inserting each of the four sequences of interest.
  • Each construct was prepared by allowing the paired oligonucleotides to anneal at 70°C for 15 minutes and cooling to room temperature, followed by ligation into the plasmid under standard conditions.
  • ssDNA constructs were transfected into HeLa cell lines. Assays for ssDNA were performed by PCR and by dot-blot analyses 24-48 hours after transfection. Reverse transcriptase activity was assayed using the RT-PCR assay developed by Silver, J., et al, supra, after transfection with pssDNA-Express-A plasmid. Individual colony isolates of stably substituted HeLa cell lines (A12 and B12) were additionally assayed for RT activity. The ss-cDNA was isolated from cells transfected 48-72-hr earlier using trizol reagent.
  • Assays for specific ss-cDNA species were 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.
  • the ssDNA sequence of interest produced in accordance with the method of the present invention is produced from either the position between the inverted repeats after digestion of the stem of the stem-loop intermediate or from the position between the inverted repeats and the primer binding site by premature termination of the reverse transcriptase cDNA transcript at the 3' aspect of the stem structure.
  • the sequence of interest produced from this premature termination is the second sequence of interest referred to herein. Fig.
  • sequence of interest is cloned (or synthesized) between the designed inverted tandem repeats and represents the sequence in the "loop" after ssDNA production and subsequent stem-loop formation.
  • the sequence of interest to be produced can be of any base (i.e., A,T,G,C) composition as long as the sequence does not interfere with the formation of the stem of the stable stem-loop intermediate, which may optionally include functional genetic element such as a specific enzyme recognition sequence, for instance, a site which serves as a substrate for a particular restriction endonuclease.
  • any restriction endonuclease may also be used to digest (or cleave) the stem portion of the stem-loop intermediate as long as the recognition site for that particular restriction endonuclease has been designed into the inverted tandem repeat.
  • the cassette described herein is comprised of three genetic elements, a sequence of interest, a tandem inverted repeat, and a primer binding site.
  • Other genetic elements include an optional restriction endonuclease gene and a reverse transcriptase gene, each of these genes being provided with appropriate promoters as described herein.
  • the mouse Moloney leukemia virus reverse transcriptase gene described for use as the reverse transcriptase gene of the cassette can be replaced with other reverse transcriptase genes (the reverse transcriptase gene from Human immunodeficiency virus was one such gene which was noted above) and that promoters other than the CMV promoter may be used to advantage.
  • promoters other than the CMV promoter may be used to advantage.
  • restriction endonuclease genes are listed above, but those skilled in the art will recognize from this description that the list set out above is not exhaustive and that many other restriction endonuclease genes will function to advantage in connection with the present invention.

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Abstract

L'invention concerne des méthodes et compositions de production d'ADN complémentaire simple brin dans des cellules eucaryotes et plus particulièrement une cassette d'ADN produisant un ADN complémentaire simple brin in vivo. La cassette contient le gène codant une transcriptase inverse du virus de leucémie murine de Moloney/ribonucléase H, un gène endonucléase de restriction bactérienne et une séquence intéressante produisant une matrice d'ARN à partir de laquelle la transcriptase inverse synthétise un ADN complémentaire simple brin d'une séquence particulière. On modifie ensuite l'ADN complémentaire simple brin pour retirer toutes les séquences de vecteurs flanquantes en profitant de la structure « souche-boucle» de l'ADN complémentaire simple brin qui se forme suite à l'inclusion d'une répétition en tandem inversée qui permet à l'ADN complémentaire simple brin de se replier pour former une souche d'ADN double brin dans la séquence intéressante. La souche double brin contient un ou plusieurs éléments génétiques fonctionnels tels qu'un site de reconnaissance d'endonucléase de restriction et la boucle, qui reste comme un ADN complémentaire simple brin, est composée de n'importe quelle séquence nucléotidique voulue. Cette configuration permet à la souche double brin de la souche-boucle intermédiaire d'être clivée par l'endonucléase ou les endonucléases de restriction correspondants spécifiques du site dans la souche, et la boucle, ou séquence intéressante, est ensuite libérée comme une partie d'ADN linéaire, simple brin. Cette partie d'ADN simple brin libérée (ou clivée) contient des informations de séquences minimales, s'il en est, en amont à 5' ou en aval à 3' à partir de la souche double brin antérieure qui contient le site de coupure d'endonucléase de restriction. L'utilisation de ce système pour produire de l'ADN simple brin dans des cellules hôtes est illustrée par les transfections in vivo réalisées grâce au système vectoriel d'ADN selon l'invention.
PCT/US1999/023936 1994-04-29 1999-10-12 PRODUCTION D'ADN SIMPLE BRIN $i(IN VIVO) WO2000022114A1 (fr)

Priority Applications (10)

Application Number Priority Date Filing Date Title
CA002345936A CA2345936A1 (fr) 1998-10-09 1999-10-12 Production d'adn complementaire monocatenaire dans une cellule
EP99950296A EP1119615B1 (fr) 1998-10-09 1999-10-12 Production d'adn simple brin a l'interieur d'une cellule
DK99950296T DK1119615T3 (da) 1998-10-09 1999-10-12 Fremstilling af ssDNA in vivo
MXPA01003643A MXPA01003643A (es) 1998-10-09 1999-10-12 Produccion de adnss in vivo.
BR9914773-4A BR9914773A (pt) 1998-10-09 1999-10-12 Conjunto de elementos genéricos, método para a produção de dna de cordão único, transcrição de mrna, construção de ácido nucléico, transcrição de ssdna, vetor, sistema vetor, célula hospedeira, conjunto para a produção de uma sequência de ácido nucléico de cordão único, método para a produção in vivo ou in vitro de uma sequência de ácido nucléico de cordão único, transcrição de cdna de cordão único, ácido nucléico inibidor, molécula heteroduplex, e composição farmacêutica
AU62988/99A AU6298899A (en) 1998-10-09 1999-10-12 Production of ssdna (in vivo)
JP2000576005A JP2004503203A (ja) 1998-10-09 1999-10-12 ＩＮＶＩＶＯでのｓｓＤＮＡの産生
DE69933382T DE69933382T2 (de) 1998-10-09 1999-10-12 Herstellung von ssdna innerhalb der zelle
IL142492A IL142492A (en) 1998-10-09 2001-04-05 Genetic structures for producing ssDNA transcripts are regulated within a cell, compositions, and methods of use
US11/586,797 US20070160581A1 (en) 1994-04-29 2006-10-26 Production of ssDNA in vivo

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