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WO1999004800A1 - Compositions d'acides nucleiques et procedes d'introduction d'acides nucleiques dans des cellules - Google Patents

Compositions d'acides nucleiques et procedes d'introduction d'acides nucleiques dans des cellules Download PDF

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Publication number
WO1999004800A1
WO1999004800A1 PCT/US1998/015130 US9815130W WO9904800A1 WO 1999004800 A1 WO1999004800 A1 WO 1999004800A1 US 9815130 W US9815130 W US 9815130W WO 9904800 A1 WO9904800 A1 WO 9904800A1
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nucleic acid
molecule
cells
aptamer
sequence
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PCT/US1998/015130
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English (en)
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Andrew H. Segal
Jeffrey Wilson
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Genitrix, Llc
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Priority to AU85058/98A priority Critical patent/AU8505898A/en
Publication of WO1999004800A1 publication Critical patent/WO1999004800A1/fr
Priority to US09/834,109 priority patent/US20020106647A1/en
Priority to US10/960,128 priority patent/US20050153312A1/en
Priority to US12/069,441 priority patent/US20090191170A1/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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—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 against growth factors, growth regulators, cytokines, lymphokines or hormones
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—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 against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2310/13—Decoys
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • the invention relates to the introduction of exogenous nucleic acid molecules into cells.
  • nucleic acids into cells are of great utility for both physicians and experimental biologists.
  • genes encoding proteins can be transferred to treat diseases or to facilitate laboratory experiments.
  • antisense nucleic acid molecules which inhibit synthesis of a given polypeptide or polynucleotide by binding to the corresponding template, can be similarly applied, as can polynucleotide enzymes ("ribozymes").
  • ribozymes polynucleotide enzymes
  • the entire complex, including the nucleic acid, can then be internalized by the cells via receptor-mediated endocytosis.
  • ligands used have included monoclonal antibodies (see, e.g., Ferkol et al (1993) J Clin Invest 92:2394-400; Buschle et al (1995) Hum Gene Ther 6;753-61), microbe-derived polypeptides (see, e.g., Ding et al
  • nucleic acid molecules have traditionally been conceived of as linear molecules that encode information, it has recently been appreciated that some nucleic acid sequences adopt conformations that allow them to bind to other molecules, such as polypeptides, specifically and with high affinity via non- Watson-Crick interactions. Nucleic acid molecules with this property are known as "nucleic acid ligands”.
  • Nucleic acid ligands consisting of RNA, single-stranded DNA, and double-stranded DNA have been described. Little is known about the structural features that determine the binding capacity and specificity of such ligands. Nevertheless, it is clear that a proper secondary structure is indispensable. "Secondary structure” refers to stable structural features formed by intrastrand Watson-Crick base pairing. Specific types of secondary structural motifs, defined by their internal juxtapositions of paired and unpaired segments, include, e.g., motifs conventionally known as hairpin loops, symmetric hairpin loops, asymmetric bulged hairpin loops, bulges, and pseudoknots (Schimmel (1989) Cell 58:9-12).
  • Nucleic acid ligands have been used to bind soluble molecules and inhibit their biological activities (see, for example, Jellinek et al (1994) Biochemistry 33:10450-56; Bock et al (1992)
  • Nucleic acid ligands that bind to cell surface molecules are also possible (Gold and Tuerk, U.S. Pat. 5, 270,163; Gold and Tuerk, U.S. Pat. 5, 475, 096), and for the purposes of the invention are termed "aptamers".
  • the invention pertains to bifunctional nucleic acid molecules that comprise an aptamer and another useful nucleic acid sequence that is not an aptamer.
  • nucleic acid molecules herein referred to as
  • bifunctional nucleic acid molecules which can bind to a cell surface with at least micromolar affinity and which comprise: a first nucleic acid which comprises an aptamer bonded to a second nucleic acid that possesses a biological activity (herein referred to as a "biological effector sequence") and which is not an aptamer.
  • the invention thus relates to a nucleic acid molecule comprising a first nucleic acid sequence comprising an aptamer covalently linked to a second nucleic acid sequence comprising a biological effector sequence.
  • the invention also relates to a nucleic acid molecule comprising a first nucleic acid sequence comprising an aptamer linked via Watson-Crick base pairing to a second nucleic acid sequence comprising a biological effector sequence.
  • a molecule of the invention may also further comprise a third nucleic acid sequence which is an aptamer that is covalently linked to the nucleic acid molecule.
  • a molecule of the invention may also further comprise a third nucleic acid sequence which is an aptamer that is linked via Watson-Crick base pairing to the nucleic acid molecule.
  • the third nucleic acid sequence comprises an aptamer that is different from the first nucleic acid sequence comprising an aptamer.
  • a molecule of the invention comprises DNA or RNA.
  • nucleic acid molecule as used herein is intended to include structures that comprise nucleotides covalently bound to each other to form polymers that can, for example, be linear, cyclic, and/or branched.
  • the bonds between sequential nucleotides in the polymer are referred to herein as a "backbone" of the molecule and can be, for example, phosphodiester, phosphorothioate, phosphoramidate, thioformacetal, carboxamide, methylphosphonate, or peptide bonds.
  • the molecules can be single-stranded and/or multi-stranded, but in a preferred embodiment are single- or double-stranded.
  • a nucleic acid molecule can include naturally occurring and non-naturally occurring nucleotides, and can include non- nucleotide structures, e.g. amino acids, carbohydrates, and metal ions.
  • the nucleotides may be chemically substituted at the ribose and/or phosphate and/or base positions.
  • the nucleotides can incorporate 5-pyr, 2'-amino, 2'-fluoro, or 2'-O-methyl groups.
  • a nucleic acid molecule can include structures that are linked to each other by means other than covalent bonds, e.g. by Watson-Crick base pairing between overlapping complementary sequences.
  • an “aptamer” is a nucleic acid molecule that is capable, by virtue of secondary and/or tertiary structure, of binding to a cell surface molecule such as a carbohydrate or a protein in a selective fashion, preferably with an affinity as strong as in the micromolar range (1-100 uM) and more preferably with an affinity even stronger, e.g., in the nanomolar to picomolar range (1- 100 nM affinity and 1-lOOpM affinity). That is, the aptamer will selectively bind to the target molecule or cell with an affinity that is at least 10-fold greater affinity than the affinity with which the aptamer binds to a non-target molecule.
  • an aptamer will comprise about 10-400 nucleotides, more preferably 20-100, and most preferably 25-50.
  • Selective binding refers to specific binding to a target cell surface molecule but not to most other molecules on the cell surface or on other cells that do not contain the targeted cell surface molecule.
  • the aptamer and the biological effector sequence are linked via a covalent bond that is a phosphodiester bond.
  • a nucleotide sequence of the bifunctional nucleic acid molecule can contribute to the biologic 90 function of both.
  • the aptamer may be 3' to the biological effector sequence or 5' to the biological effector sequence.
  • the aptamer and the biological effector sequence can be linked in a 3'-3' manner, e.g. via an acetal group.
  • a bifunctional nucleic acid molecule can comprise multiple aptamers and/or biological effector sequences.
  • the 95 aptamer and biological effector sequence are admixed with a polycation, such as poly-L-lysine.
  • the aptamer and the biological effector sequence are linked via a biotin-streptavidin interaction.
  • the aptamer of a bifunctional nucleic acid molecule be able to bind to a cell-surface polypeptide present on a cell, preferably with nanomolar to picomolar (lpM - 100 100 nM), preferably 10pM-50 nM, most preferably 50pM- 10 nM) affinity.
  • the biological effector sequence of a bifunctional nucleic acid molecule comprises a sequence that encodes a biologically or therapeutically useful polypeptide or polynucleotide.
  • the sequence is double-stranded DNA and comprises a promoter that is operably linked to the coding sequence.
  • the biological effector sequence is an mRNA, that is an RNA that translates to a polypeptide, which preferably comprises a 5' cap and a 3' poly-A sequence.
  • the biological effector sequence comprises a sequence that is antisense to a nucleic acid that is present in the target cell.
  • the antisense sequence can either 1) inhibit transcription of the target, or 2) inhibit reverse 110 transcription of the target, or 3) inhibit translation of the target, or 4) inhibit replication of the target, or 5) inhibit the target from assuming a functional conformation.
  • the antisense sequence is complementary to at least five contiguous nucleotides of the target, or is complementary to at least seven contiguous nucleotides of the target, or is complementary to at least ten contiguous nucleotides of the target,
  • the antisense sequence may form a Watson-Crick base-paired hybrid with a target RNA or DNA and inhibit expression (translation or transcription or regulatory binding) to the target molecule.
  • the biological effector sequence comprises a nucleic 120 acid enzyme, e.g. a ribozyme.
  • a bifunctional nucleic acid molecule can bind more than one cell surface molecule at once via its aptamers.
  • the aptamer can concatimerize and comprises first and second unpaired nucleic acids, each of which is complementary to itself; however, neither of which is complementary to the other.
  • This 125 structure allows concatemers of the aptamer to form and incorporate into the bifunctional nucleic acid molecule.
  • a concatemer is at least two aptamers, and up to 1000 or even more (10,000) aptamers, but is preferably in the range of two- 500 aptamers, and most preferably in the range of two - 50 aptamers.
  • the invention also pertains to vectors which include the bifunctional nucleic acid molecules of the invention, cells which into which have been introduced such vectors, and cells into which have been introduced the molecules of the invention.
  • the invention pertains to nucleic acid molecules that can be used in the production of bifunctional nucleic acid molecules.
  • these molecules 135 are DNA or RNA molecules that can serve as templates for the assembly of bifunctional nucleic acid molecules, e.g. by possessing Watson-Crick complementarity to a bifunctional nucleic acid molecule and by directing the assembly of a complementary nucleic acid, e.g. by the action of a nucleotide polymerase.
  • the invention pertains to compositions that include the nucleic acid 140 molecules of the invention and that include substances that can facilitate the use of these nucleic acid molecules in vivo and/or in vitro.
  • the composition includes a pharmaceutically acceptable carrier.
  • the composition includes a substance that improves the uptake of exogenous polynucleotides by cells, e.g. by inducing endocytosis and/or 145 phagocytosis and/or macropinocytosis and/or micropinocytosis and/or potocytosis.
  • such compositions can include substances that disrupt intracellular vesicles containing the nucleic acid molecules of the invention.
  • composition of the invention comprise a bifunctional nucleic acid molecule and a substance that facilitates the intracellular trafficking of the biological effector 150 sequence to its site of action.
  • substances can include, for example, those that facilitate release from an intracellular vesicle or those that facilitate translocation to the nucleus.
  • the invention pertains to methods of introducing biological effector sequences into cells.
  • the complexes of the invention are admixed in vitro with cells 155 bearing the receptor for the aptamer. It is further preferred that the cells are subjected to transfection with the complexes utilizing, e.g., calcium phosphate precipitation or electroporation. In another preferred embodiment, the cells are treated with an endosomal disruption agent. In another embodiment, the complexes of the invention are administered to an organism, e.g. an animal, a mammal, e.g. a primate, e.g. a human. 160 In another aspect, the invention features a method of introducing a biological effector sequence into an organism, comprising introducing a biological effector sequence into a cell by contacting the bifunctional nucleic acid with a host cell, and administering the cell to an organism.
  • the invention is based on the recognition that a bifunctional nucleic acid, that is, a first nucleic acid sequence which is an aptamer specific for a cell surface molecule such as a protein or carbohydrate that is bound to a second nucleic acid sequence that is a biological effector
  • the bifunctional nucleic acid molecule is composed of two nucleic acid sequences or fragments that are bound together via covalently bonding or via Watson-Crick base-pairing along a portion of the two sequences, the portion being long enough to provide sufficient stability for the two molecules to form a hybrid and to facilitate entry of the second sequence into the target
  • An aptamer specific for a given target molecule is isolated from a heterogeneous library of polynucleotides by affinity purification against the target molecules. For example, in the 80 "SELEX" procedure described in Gold and Tuerk, U.S. Pat. 5, 270, 163, the following steps are performed:
  • the library is prepared as follows. For example, a random collection of oligonucleotides can be generated by programming an automated synthesizer to select a solution of multiple bases for incorporation at one or more steps. Ideally, this library contains at least 85 about 10 9 nucleic acids, and no more than about 10 18 . If an RNA library is desired, it may be convenient to first obtain a corresponding DNA library and then, after cloning into a suitable plasmid, obtain the RNA by in vitro transcription.
  • the library is contacted with the target molecule under physiological chemical conditions. Contacting can occur either with ligands and target free in solution, or with the target 90 bound to a support.
  • supports include agarose or sepharose matrices.
  • the target can be coupled to such matrices either covalently or noncovalently.
  • the candidate aptamers with the highest affinities for the target are then partitioned from those with lower affinities.
  • the target is a polypeptide and the candidate/target mixture is passed through a 95 nitrocellulose membrane and the membrane washed with, e.g., 200mM potassium acetate, 50mM
  • Tris-HCl pH 7.7 This often elutes free candidates ("losers") while leaving aptamer/target complexes bound.
  • the "winners” are then released from the target by washing the membrane with, e.g., 200ul 7M urea, 20mM Na citrate (pH 5.0), ImM EDTA with 500 ul phenol (equilibrated with 0.1M Na acetate pH 5.2).
  • free candidates are eluted from a support-bound target using a suitable buffer. The target is then uncoupled from the support and the aptamer/target complexes collected.
  • the nucleic acid molecules are isolated by standard denaturation techniques, such as phenol extraction.
  • cells are obtained that do not express the desired target.
  • the library is admixed with the cells, and the unbound candidates are collected. These candidates are then admixed with cells that are identical to those above, 10 except that they do express the target. For example, they may be transfected with a gene encoding a polypeptide target, or enzymatically modified to bear a carbohydrate target.
  • the cells are spun down and the unbound candidates in the supernatant discarded. The cells are then treated with phenol to release the aptamers.
  • the selected polynucleotides are amplified, typically by PCR. If the candidates are 15 RNA, they are reverse transcribed, the DNA is amplified, and RNA is again generated by reverse transcription as above.
  • the product is a library enriched for capacity to bind the target.
  • Steps 2-5 are repeated. In each round, binding and/or partitioning conditions can be made more stringent. At least ten rounds are usually required for optimal results.
  • Cells that can bear targets for aptamers include bacterial, fungal, plant, yeast and 20 mammalian cells, e.g., malignant tumor cells, fibroblasts, endothelial cells, epithelial cells including respiratory epithelial cells, pluripotent stem cells, leukocytes, endocrine cells such as islet cells, hepatocytes, keratinocytes, melanocytes, pericytes, germ cells, neurons, myocytes including cardiac myocytes, osteocytes, osteoblasts and chondrocytes.
  • mammalian cells e.g., malignant tumor cells, fibroblasts, endothelial cells, epithelial cells including respiratory epithelial cells, pluripotent stem cells, leukocytes, endocrine cells such as islet cells, hepatocytes, keratinocytes, melanocytes, pericytes, germ cells, neurons, myocytes including cardiac myocytes, osteocytes, osteoblasts and
  • Cell surface molecules that are candidate targets for aptamers include, e.g., the transferrin 25 receptor, the asialoglycoprotein receptor, the TSH receptor, FGF receptors, CD3, the IL-2 receptor, the growth hormone receptor, the insulin receptor, the acetylcholine receptor, adrenergic receptors, VEGF receptors, and receptors for viruses such as the adenovirus receptor.
  • Aptamers useful according to the invention include but are not limited to aptamers specific for the human MDR1 and MRP genes, including SEQ ID Nos: 67-117, SEQ ID Nos: 30 129-179 and 185-196, SEQ ID Nos: 199-235, SEQ ID Nos: 251-290, SEQ ID Nos: 293-384 of
  • WO 96/40715 aptamers specific for insulin receptor antibody, including SEQ ID Nos.: 4-15 of WO 95/30775; aptamers specific for peripheral blood mononuclear cells, including SEQ ID Nos: 7-39 of WO 96/34874; aptamers specific for multidrug resistance (MDR) phenotype, including SEQ ID NosL 88091 of WO 96/40715; aptamers specific for TNF ⁇ , including SEQ ID Nos: 209- 35 255 of WO 96/40717; and aptamers specific for cytokines, including those disclosed in WO 96/40717.
  • MDR multidrug resistance
  • the invention pertains to nucleic acid molecules that can be used in the production of bifunctional nucleic acid molecules.
  • these molecules are preferred embodiments, these molecules
  • RNA molecules that can serve as templates for the assembly of bifunctional nucleic acid molecules, e.g. by possessing Watson-Crick complementarity to a bifunctional nucleic acid molecule and by directing the assembly of a complementary nucleic acid, e.g. by the action of a nucleotide polymerase.
  • bifunctional nucleic acid molecule-encoding sequences Such molecules are referred to herein as "bifunctional nucleic acid molecule-encoding sequences", and are useful for producing bifunctional nucleic acid molecules
  • Bifunctional nucleic acid molecule-encoding sequences can include sequences that are not Watson-Crick complementary to a bifunctional nucleic acid molecule, e.g. sequences that regulate and/or direct the activity of nucleotide polymerases, e.g. promoter sequences.
  • Another aspect of the invention pertains to cloning vectors containing bifunctional
  • cloning vector refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
  • plasmid refers to a circular, double-stranded nucleic acid loop into which additional nucleic acid segments can be ligated.
  • viral vector Another type of vector, wherein additional nucleic acid
  • Cloning vectors can include one or more nucleic acid sequences that can regulate biochemical processes by cis-acting mechanisms, e.g. promoters and/or binding sites for template-directed nucleotide polymerases.
  • Examples of cloning vectors include pBR322 (Watson (1988) Gene 70:399-403), pUC19 (Yanisch-Perron et al (1985) Gene 33:103-19), and pBK-CMV and pBK-RSV (both available from Stratagene, La Jolla, CA).
  • the recombinant cloning vectors of the invention comprise a nucleic acid of the invention in a form useful for producing larger quantities of a molecule of the invention than are available prior to operations on the vector in vivo or in vitro.
  • a bacterial cloning plasmid containing a bifunctional nucleic acid molecule sequence or a bifunctional nucleic acid molecule- encoding sequence can be used to amplify the number of copies of the sequence by introduction 65 of the plasmid into a suitable bacterial host.
  • cloning vector is a plasmid which comprises: 1) a template for a bifunctional nucleic acid molecule, which bifunctional nucleic acid molecule comprises an RNA aptamer and an RNA biological effector sequence linked via a phosphodiester bond; and 2) cis-acting sequences sufficient to allow in vitro transcription by a suitable RNA polymerase, e.g. T7 polymerase.
  • Recombinant 70 cloning vectors of the invention can also include vectors that lack a biological effector sequence and a biological effector-sequence encoding sequence but include: 1) an aptamer or an aptamer- encoding sequence and 2) a site, e.g.
  • a multiple cloning site which can facilitate the insertion of a biological effector sequence or a biological effector-sequence encoding sequence in such a manner that the resulting sequence is, or can serve as a template for, a bifunctional nucleic acid 75 molecule in which the aptamer and the biological effector sequence are joined by a phosphodiester bond.
  • RNA molecules can be produced by in vitro transcription. 80
  • a gene encoding the desired RNA is cloned into a plasmid so that the gene is operably linked to a promoter for a DNA-dependent RNA polymerase, e.g., T7 RNA polymerase.
  • the plasmid is then admixed, in a suitable buffer, with the RNA polymerase under conditions such that transcription occurs.
  • the polymerase chain reaction can be used to synthesize a specific DNA sequence 85 (Mullis, U.S. Pat. 4, 683, 202).
  • a double-stranded template sequence is admixed with a thermophilic DNA-dependent DNA polymerase, excess 5' and 3' primers, and excess free nucleotides in a suitable buffer.
  • the mixture is subjected to repeated thermal cycling in a manner that allows melting and reannealing of complementary sequences and coordinated activation and deactivation of the polymerase.
  • Nucleic acid molecules can also be synthesized by machines that execute chemical polymerization of nucleotides.
  • One such machine is produced by Applied Biosystems of Foster City, CA.
  • nucleic acid enzymes e.g. ribozymes
  • nucleic acid sequences that can regulate biochemical processes by cis-acting mechanisms e.g -9-promoters or ribosome binding sites
  • a sequence is “antisense” if it is Watson-Crick complementary to and can inhibit the
  • coding sequences that are useful according to the invention include, but are not limited to, sequences encoding the cystic fibrosis transmembrane regulator (Genbank Accession No. M28668), which is useful for treating cystic fibrosis; Factor VIII (Genbank Ace. No. E00527), which is useful for treating hemophilia; hemoglobin beta chain (Genbank Ace. No.
  • 310 is useful for treating malignant tumors; and antisense, ribozyme, and nucleic acid ligand molecules.
  • nucleic acid sequences that possess enzymatic activity and are useful according to the invention include, but are not limited to, ribozymes directed at mRNAs for proteins such as N-ras (Scherr et al (1997) J Biol Chem 272: 14304-13) and c-fos (Scanlon et al
  • nucleic acid sequences that possess enzymatic activity and are useful according to the invention include, but are not limited to, hairpin ribozymes; hammerhead ribozymes; Group I introns; Group II introns; ribozymes directed at molecules involved in the life cycle of viruses such as hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, foot and mouth 25 disease virus, mouse hepatitis virus, Moloney murine leukemia virus, bovine leukemia virus, influenza B virus, herpes viruses, lymphocytic choriomeningitis virus, potato leafroll virus, arenaviruses, potato virus YN, respiratory syncytial virus, retroviruses, rhabdoviruses, hemorrhagic fever agents, coronaviruses, rhinoviruses, myxoviruses, para
  • ribozymes directed at beta amyloid protein precursor mRNA ribozymes directed at alpha actin;
  • Tetrahymena ribozymes Tetrahymena ribozymes; ribozymes directed at phosphorothionate bonds; ribozymes directed at oncogenes such as H-ras, c-fos, c-myc, PML RAR alpha bcr/abl, p53, AML1/MTG8, lck, fyn; ribozymes directed at thymidylate synthetase mRNA; ribozymes directed at mdr-1 mRNA; ribozymes directed at mdr-2 mRNA; ribozymes directed at components of telomerase; ribozymes
  • ribozymes derived from chicory yellow mottle virus; ribozymes derived from arabis mosaic virus; ribozymes directed at amelogenin mRNAs; ribozymes directed at serum amyloid A mRNAs; ribozymes directed at osteopontin mRNA; polynucleotide kinase ribozymes; ribozymes directed at lipoxygenase mRNAs; ribozymes directed at cytokine mRNAs; at ribozymes directed at NF-IL6 mRNAs; RNA ligase ribozymes; DNA ligase ribozymes;
  • ribozymes directed at alkaline phosphatase mRNAs; Neurospora VS ribozymes; DNA enzymes such as DNA metalloenzyme DNA ligases; ribozymes directed at beta-glucuronidase mRNAs; ribozymes directed at plasminogen activator inhibitor mRNAs; self-alkylating ribozymes; ribozymes directed at glucose-regulated protein mRNAs; ribozymes directed at yeast ADEl gene mRNA; ribozymes directed at insulin receptor substrate mRNAs; RNA cyclase ribozymes;
  • ribozymes directed at tumor necrosis factor alpha mRNAs ribozymes directed at urokinase receptor mRNAs; ribozymes directed at atrial natriuretic factor mRNAs; ribozymes directed at pleiotrophin mRNAs; ribozymes directed at beta-2 microglobulin mRNAs; DNA-cleaving ribozymes; ribozymes directed at coliphages; Didydium ribozymes; chloroplast ribozymes; RNA polymerase ribozymes; DNA polymerase ribozymes; ribozymes directed at Drosophila white
  • RNAs 355 mRNAs; ribozymes that hydrolyze phosphoric acid; ribozymes directed at calretinin mRNAs;
  • 360 tobacco ringspot virus ribozymes barley yellow dwarf virus ribozymes; Chlamydomonas ribozymes; ribozymes that can specifically cleave single-stranded DNA; ribozymes directed at histone mRNAs, and ribozymes directed at glucan branching enzyme mRNAs.
  • antisense nucleic acids examples include, but are not limited to, molecules that are antisense to genes for K-ras (Alemany et al (1996) Cancer
  • antisense molecules useful according to the invention include those that have as their targets nucleic acids encoding gastrin, urokinase, Ha-ras, reverse transcriptase, HIV TAR, HIV gag, HIV rev, HIV nef, mycobacterial proteins,
  • Tobacco Mosaic Virus proteins hepatitis B virus proteins, hepatitis C virus proteins, hepatitis D virus proteins, hepatitis E virus proteins, Hepatitis G virus proteins, foot and mouth disease virus proteins, mouse hepatitis virus proteins, Moloney murine leukemia virus proteins, bovine leukemia virus proteins, influenza A virus proteins, influenza B virus proteins, herpes virus proteins, lymphocytic choriomeningitis virus proteins, potato leafroll virus proteins, arenavirus
  • Aptamers and biological effector sequences can be linked using many methods. For example, if an aptamer and a biological effector sequence comprise mutually complementary
  • each of the aptamer and biological effector sequence will be sufficiently long to permit the two molecules to form a hybrid that will not dissociate under the following conditions: at least 15'C, pH 7.4, 125mM NaCl in physiological compatible buffer. This length typically is in the range of 8-50 base pairs, preferably 10-20 base pairs, most preferably, 10-15
  • a functional assay can be performed in which the hybrid molecule is used to transfect a target cell for which the aptamer-portion of the bifunctional nucleic acid is specific (in terms of its selectively recognizing a cell surface molecule on the target cell); if the biological effector sequence is detected in the cell (either its direct presence or detection of a product or activity
  • the aptamer and biological effector fragments are admixed in a suitable buffer and heated to approximately 90°C. The mixture is then allowed to cool to a temperature that allows Watson-Crick base pairing between the complementary sequences. The temperature below which this occurs can be estimated by adding together the temperature values of each base of one of the complementary sequences, viz. 2°C for A or T and 4°C for G or C. If it is desired to
  • the ratio of each component in the bifunctional nucleic acid molecule can be manipulated by adjusting the molar ratios of the nucleic acid molecules in the annealing reaction. Furthermore, the average length of the concatemers can be manipulated by adding, in appropriate molar ratio, a "termination sequence".
  • a termination sequence comprises a nucleotide sequence that allows incorporation into a concatemerized bifunctional nucleic acid molecule via Watson-Crick base pairing, but does not comprise a second sequence that would allow further concatemerization.
  • Another method of assembling a bifunctional nucleic acid molecule involves admixing the aptamer and the biological effector sequence with a polycation, e.g. a polyamine, e.g.
  • polylysine This is particularly useful for assembling noncovalently linked DNA sequences, since the polyanionic charge of the DNA allows assembly via electrostatic attraction.
  • ligase enzymes to covalently link an aptamer and a biological effector sequence.
  • Ligases catalyze the formation of a phosphodiester bond between two nucleic acid molecules. For example, T4 DNA
  • T4 RNA ligase uses single stranded DNA or RNA.
  • E. coli DNA ligase acts on duplex DNA molecules which have compatible cohesive ends.
  • the bifunctional nucleic acid is assayed for its ability to transfer genes into a target cell.
  • the biological effector sequence of the bifunctional nucleic acid contains a marker gene for firefly luciferase.
  • the bifunctional nucleic acid contains a gene whose expression will have a beneficial therapeutic effect.
  • the bifunctional nucleic acid is incubated with thecells. After incubation, the cells are lysed and are assayed for gene expression.
  • the luciferase reporter luciferin and
  • 445 ATP are added to lysed cells and the light emitted is measured with a luminometer.
  • Cells are harvested on the day of assay by centrifugation at 1200 rpm for 5 min at room temperature.
  • the cell pellet is resuspended in phosphate buffered saline and re-centrifuged. This operation is performed twice.
  • the cell pellet is then suspended in RPMI 1640 (Gibco Ltd.) to make up a suspension of 2.7 X 10 6 cells per ml.
  • the cells are then aliquoted into tubes and 0.75 ml of
  • the cells are electroporated at 300 V and 250 ⁇ F using
  • Each 0.5 ml of transfected cell suspension is transferred to a well of a 12 well plastic culture plate containing 1.5 ml of RPMI 10% FBS.
  • the original transfection tube is rinsed with a further 1 ml of medium and the wash transferred to the culture dish making a final volume of 3 ml.
  • the culture plate is then incubated at 37°C for 24-72 h in an atmosphere of 5% CO,.
  • each well in the culture dish are transferred to centrifuge tubes and the cells collected by centrifugation at 13,000 rpm.
  • the pellet is resuspended in 0.12 ml of Lysis Buffer (100 mM sodium phosphate, pH 7.8; 8 MM MgCl 2 , 1 mM EDTA; 1% Triton X-100 and 15% glycerol) and agitated with a pipette.
  • the lysate is centrifuged at 13,000 rpm for 1 minute and the supernatant collected. 80 ⁇ l of the supernatant are transferred to a luminometer tube. The luciferase activity is then assayed
  • the assay buffer used is Lysis buffer containing 10 mM Luciferin and 100 mM ATP. Light produced by the luciferase is integrated over 4 sec and is described as relative light units (RLU) The data are converted to RLU/ml of lysate, RLU/cell or RLU/mg protein (protein concentration of the lysate having been determined in this case by the BioRAD Lowry assay).
  • a patient that is subject to a viral or genetic disease may be treated in
  • a bifunctional nucleic acid of the invention can be administered to the patient, preferably in a biologically compatible solution or a pharmaceutically acceptable vehicle, by ingestion, injection, inhalation or any number of other methods.
  • the dosages administered will vary from patient to patient; a "therapeutically effective dose" will be determined by the level of enhancement of function
  • a composition including a bifunctional nucleic acid will be administered in a single dose in the range of 10 ng - 100 ug/kg body weight, preferably in the range of 100 ng - 10 ug/kg body weight, such that at least one
  • the therapeutic gene will, of course, be associated with appropriate regulatory sequences for expression of the gene in the target cell.
  • ExjyivQ treatment is also contemplated within the present invention.
  • Cell populations can be removed from the patient or otherwise provided, transduced with a therapeutic gene in accordance with the invention, then reintroduced into the patient.
  • ex_ ⁇ vo cell dosages will be
  • Such dosages will usually be in the range of 10 5 -10 8 cells per patient, daily weekly, or intermittently; preferably 10 6 - 10 7 cells per patient.
  • a bifunctional nucleic acid according to the invention may be used to treat X-linked ⁇ -globulinemia.
  • the bifunctional nucleic acid will contain the Bruton's
  • the therapeutic gene may also encode a splice site and poly A tail, which may include portions of the human ⁇ globin locus splice and poly A signals; i.e., a BamHI Xbal 2.8 kb 3' splice/poly A flanking sequence containing exon 2 IVSII - exon 3 - - polyA sequences.
  • a bifunctional nucleic acid containing the Bruton's tyrosine kinase gene is assembled as described herein and used to treat X-linked ⁇ -globulinemia by introducing the bifunctional nucleic acid directly into a patient for in vivo gene therapy or contact the bifunctional nucleic acid with pre-B cells for ex vivo therapy, as described in Martensson et al.; Eur. Jour. Immunol. 1987, 17:1499; Okabe et al., Eur. Jour. Immunol. 1992, 22:37; and Banerji et al., Cell 33:729, 1983, and
  • a bifunctional nucleic acid for treatment of X-linked ⁇ -globulinemia will include a ligand for targeting of a preB cell.
  • ligands are well-known in the art and will be specific for and capable of targeting one or more of the following cell surface markers: CD9, CD 10, CD 19, CD20, CD22, CD24, CD38, CD40, CD72, and CD74.
  • a bifunctional nucleic acid described herein also may be used for treatment of Gaucher's disease.
  • Gaucher's disease stems from one of two different genetic mutations.
  • Gaucher's type 1 is a CGG — > CAG mutation, which results in an Arg — > Gin substitution at position 119 of the : ⁇ -glucocerebrosidase polypeptide (Graves, DNA 7:521, 1988) .
  • Gaucher's type 2 is a CTG -> CCG mutation, which results in a Leu --> Pro substitution at position 444 of the Z-glucocerebrosidase
  • a therapeutic bifunctional nucleic acid useful according to the invention includes the ⁇ -glucocerebrosidase gene, as described in Horowitz et al, 1989, Genomics 4:87-96, which is carried, as disclosed in Horowitz et al., on a 9722 base pair fragment extending from a BamHI site
  • a bifunctional nucleic acid containing the ⁇ -glucocerebrosidase gene is assembled as described herein and used to treat Gaucher's disease by introducing the bifunctional nucleic acid directly into the host for in vivo treatment, or into isolated macrophages for ex vivo therapy, as described in Immunology and Cell Biology, 1993, Vol. 71, pages 75-78 and introducing the transfected macrophages into a patient afflicted with Gaucher's disease. Expression of the wild type
  • the bifunctional nucleic acid will contain an aptamer that specifically targets a cell surface antigen on a macrophage.
  • aptamers may be easily prepared from procedures well-known in the art and described hereinabove, for example, aptamers having specificity for and capable of targeting one or more of the following cell surface markers: CD14, CD16, CD26, CD31, CDw32, CD36, 35 CD45RO, CD45RB, CD63, CD71, CD74, CD23, CD25 and CD69.
  • the cells targeted for in vivo or ex vivo gene transfer in accordance with the invention include any cells to which the delivery of the therapeutic gene is desired. Such cells will bear a cell surface marker for which a corresponding specific ligand is available or can be prepared to allow for cell-specific targeting according to the invention.
  • cells of the immune system such as 40 T-cells, B-cells, and macrophages, hematopoietic cells, and dendritic cells, each cell of which bears one or more well-known cell surface receptors having corresponding aptamers which may be selected for use as a targeting ligand in the bifunctional nucleic acid of the invention, depending upon the selected cell.
  • stem cells may be used for gene transfer after enrichment procedures (see, for example, European Patent Applications 0 455 482 and 0 451 611, 45 which disclose methods for separating stem cells from a population of hematopoietic cells).
  • unseparated hematopoietic cells and stem cell populations may be used as a target population for DNA transfer as described herein.
  • compositions including the molecules of the 50 invention and another substance (or substances) that can facilitate the use of bifunctional nucleic acid molecules to influence a cell's biologic function.
  • Such compositions can, for example, include substances that facilitate the uptake of exogenous nucleic acids by enhancing endocytosis, phagocytosis, potocytosis, micropinocytosis, and/or macropinocytosis (uptake enhancing agents).
  • Uptake enhancing agents can include, for example, cytokines, e.g. M-CSF, PDGF, EGF, and HGF, 55 as well as components of bacteria such as Shigella and Salmonella species.
  • Other examples of uptake enhancing agents can include substances used in conventional transfection methods, such as transfection mediated by calcium phosphate.
  • substances that can facilitate the use of a bifunctional nucleic acid molecule to introduce a biological effector sequence into a cell include substances that allow the 60 internalized nucleic acid to escape from a subcellular vesicle ("escape agents").
  • Nucleic acid molecules labeled for visualization e.g. fluorescently labeled, appear in a punctate pattern if they are contained in subcellular vesicles.
  • a substance is an escape agent if, when admixed with a cells and labeled nucleic acid molecules, the molecules can form a less punctate and more diffuse pattern than if the substance is omitted from the mixture.
  • a substance can
  • 565 also be an escape agent according to the invention if, when admixed with cells and a nucleic acid template for a polypeptide or a polynucleotide, expression of the encoded molecule by the cell is at least 20%) greater than if the substance is omitted.
  • Escape agents can include, e.g., adenovirus particles (Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Christen et al. (1993) Proc. Natl. Acad. Sci USA 90:2122-2126), portions of
  • diphtheria toxin including the transmembrane portion (Fisher and Wilson ( 1997) Biochem J 321 :49-
  • perfringolysin O (Gottschalk et al (1995) Gene Ther 2:498-503), peptides comprising sequences derived from influenza hemagglutinin (e.g., Plank et al (1994) J Biol Chem 269:12918-24), chloroquine (Guy et al (1995) Mol Biotechnol 3:237-48), and glycerol (Zauner et al (1997) Exp Cell Res 232:137-45).
  • Another type of escape agent is an activator of protein kinase C, such as
  • escape agents include listeriolysin O, various phospholipases, and nuclease inhibitors.
  • pharmaceutically/pharmacologically acceptable i.e., applicable to mixing with pharmaceutical agents for in vitro testing such as for example for ex vivo uses which may require further testing or processing for use in mammals or humans
  • physiologically compatible i.e., applicable for injection into an animal, particularly a human
  • biologically compatible i.e., acceptable for in vitro use on live cells but not necessarily acceptable, although not excluding acceptability, for in vivo use
  • Bifunctional nucleic acids according to the invention may be delivered to cells using additional cell transfection enhancing agents, such as lipids (e.g., cationic lipids), peptides (e.g., cationic peptides) or synthetic polymers, for example, soluble DNA/polylysine complexes can be generated (Li et al., Biochem. J. 12, 1763 (1973)). Polylysine complexes tagged with
  • asialoglycoprotein have been used to target DNA to hepatocytes in vitro (Wu and Wu, J. Biol. Chem. 262, 4429 (1987); U.S. Patent 5,166,320). Lactosylated polylysine (Midoux et al. (1993) Nuc. Acids Res. 21, 871-878) and galactosylated histones (Chen et al. (1994) Human Gene Therapy 5, 429-435) have been used to target plasmid DNA to cells bearing lectin receptors, and insulin conjugated to polylysine (Rosenkrantz et al. (1992) Exp. Cell Res. 155, 323-329) to cells bearing insulin receptors.
  • Peptides derived from the amino acid sequences of viral envelope proteins have been used in gene transfer when coadministered with polylysine DNA complexes (Plank et al. (1994) J. Biol.
  • WO 95/02698 used viral components to attempt to increase the efficiency of cationic lipid gene transfer.
  • the invention pertains to methods of introducing nucleic acid molecules into cells. Such methods can apply, for example, to prokaryotic or eukaryotic cells in vitro or in vivo.
  • in vitro refers to circumstances in which cells are harbored and/or cultivated under artificial conditions.
  • in vivo refers to circumstances in which cells are located on or within an organism.
  • in vitro methods of the invention can include admixture of compositions of the
  • Such methods can include the use of substances that can facilitate the use of bifunctional nucleic acid molecules to introduce a biological effector sequence into cells, e.g. substances that can be included in compositions of the invention.
  • Methods of the invention can include uses of such substances other than inclusion in a composition of the invention, for example application of the substance to
  • the bifunctional nucleic acid molecule can be admixed with a suitable buffer, e.g. Tris- EDTA or phosphate-buffered saline. Typically, 0. lug- 100 ug of bifunctional nucleic acid molecule in buffer is admixed with 10 3 -10 7 cells that bear the target of the bifunctional nucleic acid molecule's aptamer. 620
  • bifunctional nucleic acid molecules are delivered to a cell via receptor-mediated transfer in combination with conventional transfection methods to achieve a synergistic effect. Examples of such methods follow:
  • Naked nucleic acid can be introduced into cells by forming a mixture of the nucleic acid and DEAE-dextran and incubating the mixture with the
  • DEAE-dextran transfection is only applicable to in vitro modification of cells and can be used to introduce nucleic acid transiently into cells but is not preferred for creating stably transfected cells. Thus, this method can be used for short term production of a gene product but is not a method of choice for long-term production of a gene product. Protocols for DEAE-dextran-
  • Electroporation Naked nucleic acid can also be introduced into cells by incubating the 635 cells and the nucleic acid together in an appropriate buffer and subjecting the cells to a high- voltage electric pulse.
  • the efficiency with which nucleic acid is introduced into cells by electroporation is influenced by the strength of the applied field, the length of the electric pulse, the temperature, the conformation and concentration of the nucleic acid and the ionic composition of the media. Electroporation can be used to stably (or transiently) transfect a wide
  • Liposome-mediated transfection Naked nucleic acid can be introduced into cells by mixing the nucleic acid with a liposome suspension containing cationic lipids. The nucleic acid/liposome complex is then incubated with cells. Liposome mediated transfection can be used to stably (or transiently) transfect cells in culture in vitro. Protocols can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (e's.) Greene Publishing Associates, (1989), Section 9.4 650 and other standard laboratory manuals. Additionally, gene delivery in vivo has been accomplished using liposomes. See for example Nicolau et al. (1987) Meth. Enz.
  • a precipitate containing the nucleic acid and calcium phosphate For example, a HEPES- buffered saline solution can be mixed with a solution containing calcium chloride and nucleic acid to form a precipitate and the precipitate is then incubated with cells. A glycerol or dimethyl sulfoxide shock step can be added to increase the amount of nucleic acid taken up by certain cells. CaPO4- mediated transfection can be used to stably (or transiently) transfect cells and is only applicable to
  • naked nucleic acid is introduced into cells in culture (e.g., by one of the methods described hereinafter described hereinafter.
  • 670 drugs such as G418, hygromycin and methotrexate.
  • Selectable markers may be introduced on the same nucleic acid molecule as the gene(s) of interest or may be introduced on a separate molecule.
  • In vivo methods of the invention can include methods of administration of a composition of the invention to an organism that harbors cells into which introduction of a biological effector sequence is desired.
  • the organism can be any plant or animal, but in a preferred embodiment is
  • the targeted cells can be integral to the recipient organism or can, for example, be commensal microbes harbored by that organism.
  • Routes of administration can include, e.g., intravenous, intraarterial, intrathecal, intraperitoneal, subcutaneous, intradermal, epicutaneous, oral, ocular, mucosal, or aerosol.
  • nucleic acid internalization of a nucleic acid by a cell can be detected in 680 several ways.
  • nucleic acid encodes a polypeptide or a polynucleotide
  • intemalization can be indicated by an increase in synthesis of the encoded molecule by the cell.
  • nucleic acid encodes or comprises an antisense sequence, a ribozyme, or a nucleic acid ligand
  • intemalization can be indicated by a decrease in net synthesis of the target molecule by the cell or a decrease in activity of the target molecule.
  • the nucleic acid is conjugated to a detectable marker, e.g. a radioactive marker, an enzyme such as alkaline phosphatase or horseradish peroxidase, or a fluorochrome such as fluorescein isothiocyanate or phycoerythrin.
  • a detectable marker e.g. a radioactive marker, an enzyme such as alkaline phosphatase or horseradish peroxidase, or a fluorochrome such as fluorescein isothiocyanate or phycoerythrin.
  • a detectable marker e.g. a radioactive marker, an enzyme such as alkaline phosphatase or horseradish peroxidase, or a fluorochrome such as fluorescein isothiocyanate or phycoerythrin.
  • the nucleic acid can be botinylated and admixed with a streptavidin-linked marker.
  • the conjugates are then admixed with the cells according to a method of the invention, extracellular nucleic acid removed, e.g. by denaturation or exonuclease treatment and washing, and the marker detected. If the nucleic acid is internalized, marker uptake will be at least 20% greater than when marker alone is used in the assay.
  • the aptamer and biological effector of a bifunctional nucleic acid molecule can be detected separately in such an assay if each is conjugated to a different marker, e.g.
  • composition of the invention is administered in vivo or applied in vitro in such a manner and amount and on such a schedule that a biological effector sequence is introduced into an appropriate number and type of target cells such that the desired effect
  • the specific conditions of a method can depend, for example, on the aptamer and the biological effector sequence, the nature of the target cells, and the effect desired.
  • Bifunctional nucleic acid molecules described herein may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in, liquid prior to infection can also be prepared.
  • the preparation can also be emulsified, or the bifunctional nucleic 705 acid molecules encapsulated in liposomes.
  • the active ingredients are often mixed with carriers which are pharmaceutically acceptable and compatible with the active ingredient.
  • pharmaceutically acceptable carrier refers to a carrier that does not cause an allergic reaction or other untoward effect in subjects to whom it is administered.
  • a “biologically compatible carrier” is one which is compatible with in vitro cellular transfection. Suitable pharmaceutically acceptable
  • the 710 carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the formulation can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, and/or pH buffering agents.
  • Bifunctional nucleic acid molecules of the invention can be administered parenterally, by
  • suppositories and in some cases, oral formulations or formulations suitable for distribution as aerosols.
  • traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%.
  • the bifunctional nucleic acid molecules of the invention can be formulated into the compositions as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups can also be derived from
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the bifunctional nucleic acid molecules are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the subject's body mass. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and may be peculiar to each subject. It will be apparent to those of skill in the art that the therapeutically effective amount of bifunctional nucleic acid molecules of this invention will depend, inter alia, upon the administration schedule, the unit dose of aptamer and biological 740 effectors sequence administered, whether the bifunctional nucleic acid molecules are administered in combination with other agents, the health of the recipient, and the therapeutic activity of the particular bifunctional nucleic acid molecule. In general, though, the dosage required for therapeutic efficacy will range from about 0.1 ug to 20 mg nucleic acid/kg body mass/dose.
  • Cells transfected with the bifunctional nucleic acid molecules of the invention can 745 subsequently be administered to a subject to achieve a therapeutic effect, e.g. to provide a polypeptide which is encoded by the biological effector sequence and in which the subject is deficient.
  • the cells will be formulated as a suspension in a pharmaceutically acceptable carrier, e.g. a physiologically compatible buffer. While the number of cells required for a therapeutic effect will vary according to both subject and cell characteristics, it will generally be between about 750 10 3 and 10 9 cells/dose.
  • a cloning vector comprising a bifunctional nucleic acid molecule- encoding sequence is prepared in the following manner: The following oligodeoxynucleotides
  • nucleotide sequence including the E. coli beta galactosidase gene is prepared by digesting the pUC19 plasmid (Genbank accession number X02514) with Nar I and Hind III, and isolating the resulting 212 base pair fragment by agarose gel electrophoresis and elution. The annealed oligonucleotide and the pUC19-derived fragment are ligated using T4 DNA ligase. The resulting molecule is ligated to pSP70 plasmid (Promega, Madison, WI) that has been digested with Cla I
  • RNA bifunctional nucleic acid molecule is obtained by subjecting the plasmid to standard in vitro transcription procedures using SP6 RNA polymerase.
  • a nucleotide sequence including the E. coli beta galactosidase gene is prepared by digesting the pUC19 plasmid (Genbank accession number X02514) with Hae I and Hind III, and isolating the resulting 212 base pair fragment by agarose gel electrophoresis and elution. The annealed oligonucleotide and the pUC19-derived fragment are ligated using T4 DNA ligase. The resulting molecule is ligated to pSP70 plasmid (Promega)
  • RNA bifunctional nucleic acid molecule is obtained by subjecting the plasmid to standard in vitro transcription procedures using SP6 RNA polymerase.
  • Example 1 or Example 2 is introduced into CMS5 mouse fibrosarcoma cells that have been engineered to express a recombinant fusion polypeptide consisting of the intracellular and transmembrane portions of a fibroblast growth factor receptor (Genbank Ace. No. M34185) fused in- frame to the human VEGF 165 protein (see Swiss-Prot PI 5692), with the VEGF portion
  • bifunctional nucleic acid molecule is admixed in a suitable buffer with about 10 3 - 10 7 of these cells in vitro. Subsequent expression of beta galactosidase is determined by X-gal staining.
  • Example_4 A library of RNA's, each having 32 randomized bases between two fixed sequences of 795 30-45 bases, is constructed.
  • An aptamer for the transferrin receptor (Genbank Ace. No. Ml 1507) is isolated by SELEX, using the receptor's extracellular domain as a target.
  • An antisense oligodeoxynucleotide which binds to the template region of the RNA component of human telomerase (5TAGGGTTAG3') (Feng et al (1995) Science 269:1236-41) is synthesized and treated with calf alkaline phosphatase.
  • the aptamer and the antisense biological effector 800 sequence are ligated using T4 RNA ligase.
  • the resulting bifunctional nucleic acid is useful for treating malignant tumors.
  • the bifunctional nucleic acid binds to transferrin receptor-bearing cells and delivers to the cell antisense oligonucleotide that binds to the RNA component of human telomerase , thus inhibiting function of telomerase enzyme.
  • the aptamer of Example 4 is synthesized with an additional 5'GGGGGGGCCCCCCC3' at the 5' end and an additional 5'AAAAAAAUUUUUU3' at the 3' end.
  • the antisense sequence of Example 4 is synthesized with an additional 5'AAAAAAAUUUUUUU3' at the 3' end.
  • the molecules are admixed in a molar ratio of 8 aptamers: 1 antisense for annealing into 810 concatemers.
  • the resulting bifunctional nucleic acid is useful for treating malignant tumors.
  • a phosphorothioate antisense oligodeoxynucleotide designed to inhibit expression of the reporter protein Enhanced Green Fluorescent Protein (EGFP) was Watson-Crick hybridized to an
  • AAACGTAACGTTGCTTACTCTATGTAGTTCC3' (purchased from Midland Certified Reagent Co. Midland, TX, where it was purified by trityl-selective perfusion HPLC).
  • the antisense and aptamer molecules were simultaneously added in a 1 :1 molar ratio to 2 x 10 6 EGFP-expressing Jurkat cells in 500 ⁇ L of a buffer consisting of ImM CaCl 2 , ImM
  • the remaining 750 ⁇ L of cells and nucleic acid molecules were then incubated for 42 hours at 37°C with 95%>O 2 , 5% CO 2 . Then another 250 ⁇ L sample was drawn from each sample. These cells were then prepared and analyzed for fluorescence as at time zero.
  • Jurkat cells showed that, at 42 h, mean cell fluorescence of cells incubated in a 0.5 ⁇ M concentration of antisense alone was 13.4% below that of appropriate control cells incubated with neither aptamer nor antisense. The mean fluorescence of cells incubated with 0.5 ⁇ M of the aptamer-antisense molecule was 25.7% below
  • This bifunctional molecule is a 98 base single stranded oligodeoxynucleotide which has a sequence of: 860 5'CTACCTACGATCTGACTAGCCGGACATGAGCGTTACAAGGTGCT
  • a bifunctional nucleic acid is constructed using the aptamer of Example 6 and an antisense molecule consisting of the 14 3' bases of the antisense molecule of Example 6 865 appended 3' to the phosphorothioate antisense molecule GEM 91, which inhibits the expression of HIV gag and is described in Veal et al, 1998, Antiviral Research 38:63-73. Aptamer and antisense are annealed in PBS at room temperature.
  • the bifunctional nucleic acid molecule can target the antisense to T cells and is useful for treating HIV infection.
  • the reporter gene pEGFP-n2 from Clontech Laboratories is linearized by digestion with the restriction enzyme ApaLI in a non-coding region leaving a 5 'sense overhang with the sequence ACGT on one end and an identical overhang on the 5 'antisense strand.
  • the following oligodeoxynucleotides are synthesized: 1) 5' TGCAGGGGGGGGGGAACTACATGAGAG 3', which has the first 4 bases at 5' end
  • AAACGTAACGTTGCTTACTCTATGTAGTTCC3 ' which is a high affinity aptamer for human
  • L-selectin (SEQ ID#134, Parma et al, PCT Publication WO 96/40703)
  • the first two oligodeoxynucleotides are annealed together by room temperature incubation and then are ligated to the linearized pEGFP-n2 plasmid with T4 DNA ligase.
  • the linearized pEGFP-n2 plasmid now has two linkers ligated to both of its ends which are complementary to a 15 base region of
  • the pEGFP-n2 plasmid is incubated with the aptamer at a 1 :2 molar ratio in a buffer consisting of ImM CaCl 2 , ImM MgCl 2 , 125mM NaCl, 5mM KC1, and 20mM HEPES, pH 7.4 for 30 minutes.
  • Wild type Jurkat cells at a concentration of 5 x 10 5 which endogenously express human L-selectin are then added to the plasmid and aptamer mix, and incubation continues for another 30 minutes. The mix is then equally divided
  • a control group consists of cells which receive only linkers ligated to plasmid in a 2:1 895 molar ratio. For each group of cells a range of final pEGFP-n2 concentrations of 0 through 500 pM up to 1 ⁇ M is used. The aptamer-plasmid molecule will yield a transfection efficiency at least 20%) higher than that of plasmid alone, as measured by number of selected clones.
  • example 6 would entail using the same human L-selectin aptamer
  • Antisense 1 has the sequence: 5 GGTACCACTCGTTCCCGGATGGATGCTAGAC3' which is identical to the antisense used in example 6 and consists of an EGFP hybridization region in the first 18 bases of the 5' end and an aptamer hybridization region at the 14 bases of the 3' end.
  • A2 has the sequence
  • the A3 molecule contains aptamer hybridization regions at both the 5' and 3' region and maintains the EGFP antisense sequence in the internal 18 phosphorothioate base pairs: 5'
  • aptamer, A 1,A2, and A3 can be admixed in a molar ratio 3 : 1 : 1 :2, so that the nucleic acid molecules thus formed will on average comprise three aptamer units to four antisense units. These molecules can then be used in the method otherwise described in Example 6.

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Abstract

L'invention concerne des molécules bifonctionnelles d'acides nucléiques lesquelles peuvent se fixer à une surface cellulaire et comprennent un premier acide nucléique comprenant un aptamère fixé à un second acide nucléique, la séquence d'effecteur biologique, possédant une activité biologique. L'invention concerne également des matrices, des vecteurs ainsi que des cellules hôtes comprenant les molécules bifonctionnelles d'acides nucléiques ainsi que des procédés d'introduction de la séquence d'effecteur biologique dans un organisme par administration d'une cellule hôte transfectée avec la séquence d'effecteur biologique.
PCT/US1998/015130 1996-07-24 1998-07-22 Compositions d'acides nucleiques et procedes d'introduction d'acides nucleiques dans des cellules WO1999004800A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU85058/98A AU8505898A (en) 1997-07-22 1998-07-22 Nucleic acid compositions and methods of introducing nucleic acids into cells
US09/834,109 US20020106647A1 (en) 1996-07-24 2001-04-12 Nucleic acid compositions and methods of introducing nucleic acids into cells
US10/960,128 US20050153312A1 (en) 1996-07-24 2004-10-07 Nucleic acid compositions and methods of introducing nucleic acids into cells
US12/069,441 US20090191170A1 (en) 1996-07-24 2008-02-08 Nucleic acid compositions and methods of introducing nucleic acids into cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US89809497A 1997-07-22 1997-07-22
US08/898,094 1997-07-22

Publications (1)

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WO1999004800A1 true WO1999004800A1 (fr) 1999-02-04

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Country Link
AU (1) AU8505898A (fr)
WO (1) WO1999004800A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19916417A1 (de) * 1999-04-01 2000-10-19 Schering Ag Amyloidspezifisches Aptamer
WO2002062949A3 (fr) * 2000-10-20 2002-10-31 Canji Inc Regulation de l'expression genique au moyen d'aptameres
WO2003102142A2 (fr) * 2002-05-29 2003-12-11 Henry M. Jackson Foundation For The Advancement Of Military Medicine Inc. Methodes d'identification de biomarqueurs genomiques et proteomiques pour la mucoviscidose, jeux ordonnes d'echantillons comprenant ces biomarqueurs et methodes d'utilisation de ces jeux ordonnes d'echantillons
WO2005007859A2 (fr) * 2003-07-11 2005-01-27 Sirna Therapeutics, Inc. Inhibition par interference d'arn de l'expression genique de la carboxylase d'acetyl-coa au moyen d'un acide nucleique interferant court
EP1555874A2 (fr) * 2002-10-10 2005-07-27 Oxford Biomedica (UK) Limited Regulation des genes au moyen d'apatameres et de complexes modulateurs a des fins de therapie genique
WO2006042112A3 (fr) * 2004-10-05 2006-12-21 California Inst Of Techn Acides nucleiques a regulation d'aptameres et leurs utilisations
WO2012016188A2 (fr) 2010-07-30 2012-02-02 Alnylam Pharmaceuticals, Inc. Procédés et compositions pour l'administration d'agents actifs
US8865667B2 (en) 2007-09-12 2014-10-21 California Institute Of Technology Higher-order cellular information processing devices
US9029524B2 (en) 2007-12-10 2015-05-12 California Institute Of Technology Signal activated RNA interference
US9040495B2 (en) 2007-08-28 2015-05-26 California Institute Of Technology General composition framework for ligand-controlled RNA regulatory systems
US9145555B2 (en) 2009-04-02 2015-09-29 California Institute Of Technology Integrated—ligand-responsive microRNAs

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992014843A1 (fr) * 1991-02-21 1992-09-03 Gilead Sciences, Inc. Aptamere specifique de biomolecules et procede de production
US5270163A (en) * 1990-06-11 1993-12-14 University Research Corporation Methods for identifying nucleic acid ligands
US5399346A (en) * 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
US5442049A (en) * 1992-11-19 1995-08-15 Isis Pharmaceuticals, Inc. Oligonucleotides for modulating the effects of cytomegalovirus infections

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5399346A (en) * 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
US5270163A (en) * 1990-06-11 1993-12-14 University Research Corporation Methods for identifying nucleic acid ligands
WO1992014843A1 (fr) * 1991-02-21 1992-09-03 Gilead Sciences, Inc. Aptamere specifique de biomolecules et procede de production
US5442049A (en) * 1992-11-19 1995-08-15 Isis Pharmaceuticals, Inc. Oligonucleotides for modulating the effects of cytomegalovirus infections

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19916417A1 (de) * 1999-04-01 2000-10-19 Schering Ag Amyloidspezifisches Aptamer
EP1410021A4 (fr) * 2000-10-20 2005-02-16 Canji Inc Regulation de l'expression genique au moyen d'aptameres
WO2002062949A3 (fr) * 2000-10-20 2002-10-31 Canji Inc Regulation de l'expression genique au moyen d'aptameres
US6949379B2 (en) 2000-10-20 2005-09-27 Canji, Inc. Aptamer-mediated regulation of gene expression
EP1410021A2 (fr) * 2000-10-20 2004-04-21 Canji, Inc. Regulation de l'expression genique au moyen d'aptameres
WO2003102142A3 (fr) * 2002-05-29 2004-11-25 Jackson H M Found Military Med Methodes d'identification de biomarqueurs genomiques et proteomiques pour la mucoviscidose, jeux ordonnes d'echantillons comprenant ces biomarqueurs et methodes d'utilisation de ces jeux ordonnes d'echantillons
WO2003102142A2 (fr) * 2002-05-29 2003-12-11 Henry M. Jackson Foundation For The Advancement Of Military Medicine Inc. Methodes d'identification de biomarqueurs genomiques et proteomiques pour la mucoviscidose, jeux ordonnes d'echantillons comprenant ces biomarqueurs et methodes d'utilisation de ces jeux ordonnes d'echantillons
EP1555874A2 (fr) * 2002-10-10 2005-07-27 Oxford Biomedica (UK) Limited Regulation des genes au moyen d'apatameres et de complexes modulateurs a des fins de therapie genique
EP1555874A4 (fr) * 2002-10-10 2006-10-04 Oxford Biomedica Ltd Regulation des genes au moyen d'apatameres et de complexes modulateurs a des fins de therapie genique
WO2005007859A2 (fr) * 2003-07-11 2005-01-27 Sirna Therapeutics, Inc. Inhibition par interference d'arn de l'expression genique de la carboxylase d'acetyl-coa au moyen d'un acide nucleique interferant court
WO2005007859A3 (fr) * 2003-07-11 2005-12-01 Sirna Therapeutics Inc Inhibition par interference d'arn de l'expression genique de la carboxylase d'acetyl-coa au moyen d'un acide nucleique interferant court
WO2006042112A3 (fr) * 2004-10-05 2006-12-21 California Inst Of Techn Acides nucleiques a regulation d'aptameres et leurs utilisations
US9315862B2 (en) 2004-10-05 2016-04-19 California Institute Of Technology Aptamer regulated nucleic acids and uses thereof
US9309568B2 (en) 2004-10-05 2016-04-12 California Institute Of Technology Aptamer regulated nucleic acids and uses thereof
US9040495B2 (en) 2007-08-28 2015-05-26 California Institute Of Technology General composition framework for ligand-controlled RNA regulatory systems
US8865667B2 (en) 2007-09-12 2014-10-21 California Institute Of Technology Higher-order cellular information processing devices
US9029524B2 (en) 2007-12-10 2015-05-12 California Institute Of Technology Signal activated RNA interference
US9145555B2 (en) 2009-04-02 2015-09-29 California Institute Of Technology Integrated—ligand-responsive microRNAs
WO2012016188A2 (fr) 2010-07-30 2012-02-02 Alnylam Pharmaceuticals, Inc. Procédés et compositions pour l'administration d'agents actifs

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