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WO1997003085A1 - Intracellular action of nucleic acid ligands - Google Patents

Intracellular action of nucleic acid ligands Download PDF

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Publication number
WO1997003085A1
WO1997003085A1 PCT/US1996/011473 US9611473W WO9703085A1 WO 1997003085 A1 WO1997003085 A1 WO 1997003085A1 US 9611473 W US9611473 W US 9611473W WO 9703085 A1 WO9703085 A1 WO 9703085A1
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WIPO (PCT)
Prior art keywords
nucleic acid
fiv
ligand
rna
nucleic acids
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PCT/US1996/011473
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French (fr)
Inventor
Larry Gold
Michael Lochrie
Hang Chen
Craig Tuerk
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Nexstar Pharmaceuticals Inc
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Nexstar Pharmaceuticals Inc
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Priority to AU64867/96A priority Critical patent/AU6486796A/en
Publication of WO1997003085A1 publication Critical patent/WO1997003085A1/en
Anticipated expiration legal-status Critical
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Described herein are methods for treating intracellularly-mediated diseases or conditions with oligonucleotides. Further included herein is a method for treating intracellularly-mediated diseases or conditions with Nucleic Acid Ligands. Further included is a method for diagnosing intracellularly-mediated diseases or conditions. More particularly included herein is a method for treating diseases or conditions resulting from viral pathogens, specifically a method for treating HIV-l infection.
  • the present invention also includes methods for identifying and preparing high-affinity Nucleic Acid Ligands to FIV RT.
  • the method utilized herein for identifying such Nucleic Acid Ligands is called SELEX, an acronym for Systematic Evolution of Ligands by Exponential enrichment.
  • This invention includes high affinity Nucleic Acid Ligands of FIV RT. Further disclosed are
  • RNA ligands to FIV RT are useful in veterinary applications, diagnostic agents, or as a model for studies of RT-targeted chemotherapy for AIDS.
  • HIV-l The type-1 human immunodeficiency virus (HIV-l) is the etiological agent of acquired immunodeficiency syndrome (AIDS) (Levy (1993) Microbiol. Rev. 57:183-289).
  • AIDS acquired immunodeficiency syndrome
  • Four mononucleoside drugs (3'-azido-3'deoxythymidine (AZT) 5'-triphosphate, ddl, ddC, and d4T) have been approved for use in the United
  • FELINE IMMUNODEFICIENCY VIRUS Feline immunodeficiency virus is a lentivirus isolated from domestic cats suffering an AIDS-like disease (Pedersen et al. (1987) Science 2i5_:790-793; Pedersen et al. (1991) J. Am. Vet. Med. Assoc.122:1289-1305). FIV causes immune suppression and increases susceptibility to infections by other pathogens (Yamamoto et al. (1991) J. Am. Vet. Med. Assoc. 124:213-220; Ishida et al. (1990) Jpn. J. Vet. Sci. 52:645-648; Pederson et al.
  • FIV-infected cats develop an AIDS-related complex (ARC)-like disease which progresses to the final AIDS-like stage (Pedersen et al. (1991) J. Am. Vet. Med. Assoc. 129:1289-1305).
  • ARC AIDS-related complex
  • FIV has a wide tropic spectrum of host cell types. FIV can be isolated from the infected cat in
  • T-lymphoids CD4+ and CD8+
  • B-lymphoids B-lymphoids
  • macrophages macrophages
  • monocytes monocytes
  • RT reverse transcriptase
  • Viruses RNA Tumor Viruses vol. 2: Cold Spring Harbor Laboratory Press; Temin et al. (1970) Nature 226:211-213; Baltimore (1970) Nature 226:209-211).
  • the retroviral genome consists of three open reading frames: gag, pol, and env.
  • FIV RT encoded by the pol gene ofthe retroviral genome is a heterodimer composed of p66 and p51 subunits.
  • FIV RT uses a particular host tRNA as a primer in vivo (Bishop (1978) Annu. Rev. Biochem. 47:35-88).
  • the similarities of FIV and HIV-l RTs make FIV a useful model for studies of RT-targeted chemotherapy for AIDS.
  • FIV RT has been previously shown to be similar to the HIV-l RT in physical properties, catalytic activities, and sensitivity to the 5'-triphosphates of several nucleoside analogs that display anti-HIV activity, including AZ
  • the FIV and HIV-l RTs are also similar in sensitivity to phosphonoformate (PFA), but differ in that FIV RT is not sensitive to the other non-nucleoside inhibitors such as nevirapine (BIRG-587) and TIBO compounds, which are potent inhibitors ofthe HIV-l RT. Although very similar to one another, the FIV and HIV-l RTs are quite different from the RT of avian myeloblastosis virus in susceptibility to antiviral nucleoside analogs (Remington et al. (1994) Virol. 68:632-637).
  • the FIV system has been proven to be useful for studies of resistance to antiviral drugs (Remington et al. (1994) Virol. 68:632-637; Remington et al. (1991) J. virol. 65:308-312; Gobert et al. (1994) Antimicrob. Agents Chemother. 38:861-864). It was reported that AZT-resistant variants of FIV were isolated by in vitro selection (Remington et al. (1991) J. virol.
  • Gene therapy can be defined as the transfer of new genetic material to the cells of an individual with resulting Therapeutic benefit to the individual (Morgan and Anderson (1993) Ann Rev Biochem 62:191-217).
  • gene transfer methods include chemical techniques such as calcium phosphate coprecipitation (Graham and van der Eb (1973) Virology 52:456-467; Pellicer et al (1980) Science 202:1414-1422); mechanical techniques, such as microinjection (Anderson et al. (1980) Proc. Natl. Acad. Sci. USA 27:5399-5403) or particle-mediated delivery (“biolistics”) (Sanford et al. (1993) Meth. Enz.
  • Viral vectors are also used in gene transfer and include retroviruses and adenoviruses. Vectors derived from retroviruses have been widely used for gene expression (Gilboa (1986) BioEssay 5_:252-257).
  • Retroviral genomic RNA is replicated into double-stranded DNA (proviral DNA), which is integrated into the host chromosome where it utilizes host machinery for gene expression.
  • the amphotropic murine leukemia virus-derived vector which maintains the packaging signal, but lacks the viral structural protein genes (which are replaced by a neomycin-resistant gene as a selection marker), has been used successfully to deliver genes (Sullenger et al. (1990) Mol. Cell. Biol. 10:6512-6523).
  • SELEX Systematic Evolution of Ligands by Exponential enrichment
  • the SELEX method involves selection from a mixture of candidate oligonucleotides and step- wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity.
  • the SELEX method includes steps of contacting the mixture with the Target under conditions favorable for binding, partitioning unbound Nucleic Acids from those Nucleic Acids which have bound specifically to
  • Target molecules dissociating the Nucleic Acid-Target complexes, amplifying the Nucleic Acids dissociated from the Nucleic Acid-Target complexes to yield a ligand-enriched mixture of Nucleic Acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity Nucleic Acid Ligands to the
  • the SELEX method encompasses the identification of Wgh-affinity Nucleic Acid Ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions.
  • SELEX-identified Nucleic Acid Ligands containing modified nucleotides are described in United States Patent Application Serial No. 08/117,991, filed September 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines.
  • the SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in United States Patent Application Serial No. 08/284,063, filed August 2, 1994, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX” and United States Patent Application Serial No. 08/234,997, filed April 28, 1994, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,” respectively.
  • These applications allow the combination ofthe broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
  • Each ofthe above described patent applications which describe modifications ofthe basic SELEX procedure are specifically inco ⁇ orated by reference herein in their entirety.
  • RNA sequences are provided that are capable of binding specifically to FIV RT. Specifically included in the invention are the RNA ligand sequences shown in Figures 1 A -
  • a method of identifying Nucleic Acid Ligands and Nucleic Acid Ligand sequences to FIV RT comprising the steps of (a) preparing a Candidate Mixture of Nucleic Acids, (b) contacting the Candidate Mixture of Nucleic Acids with FIV RT, (c) partitioning between members of said Candidate Mixture on the basis of affinity to FIV RT, and (d) amplifying the selected molecules to yield a mixture of Nucleic Acids enriched for Nucleic Acid sequences with a relatively higher affinity for binding to FIV RT.
  • the present invention includes the RNA ligands to FIV RT identified according to the above-described method, including those ligands shown in Figures 1 A -IH (SEQ ID NOS:6-12). Also included are RNA ligands to FIV RT that are substantially homologous to any ofthe given ligands and that have substantially the same ability to bind FIV RT and inhibit the function of FIV RT. Further included in this invention are Nucleic Acid Ligands to FIV RT that have substantially the same structural form as the ligands presented herein and that have substantially the same ability to bind FIV RT and inhibit the function of FIV RT. The present invention also includes modified nucleotide sequences based on the RNA ligands identified herein and mixtures ofthe same.
  • Figures 1 A - IH show the sequences ofthe selected ligands to FIV RT and shows their possible secondary structures. Each subset of three classes were analyzed by an RNA folding program (Barta et al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81:3607-3611; Shelness et al. (1985) J. Biol. Chem.
  • RNA sequences of full length RNA were input into mfold program in GCG.
  • the sequences in the figure only show the randomized region; however, the ligands also include the fixed region as described in Example 1.
  • the fixed regions fold into stem-loop structures which do not interfere with the randomized region.
  • Bold uppercase letters indicate the consensus sequences.
  • Y represents pyrimidine; R represents purine; and N can be any ofthe four nucleotides; The name, Kd value and the frequency are also shown in the figure.
  • Figure 2 (SEQ ID NO: 10) summarizes the FIV RT binding boundary of ligand F5.
  • the boxed region (SEQ ID NO: 13) shows the FIV RT binding boundary.
  • the lower case letters indicate the fixed sequences.
  • the secondary structure was obtained by a computer RNA folding algorithm.
  • Figure 3 is a diagram ofthe pNEW6 retroviral expression vector, a) Structure of pNEW6 plasmid. b) Structure ofthe NEW6 provirus in infected cells. pNEW6 is a double-copy type of retroviral vector. After reverse transcription the 3' LTR is copied to the 5' end. c) Structure ofthe LTR of a NEW6 provirus. The pol II and pol III transcripts are shown as arrows and the primers used for RT-PCR are shown, d) Structure ofthe tRNA-HIV Nucleic Acid Ligand RNA chimeric gene.
  • Nucleic Acid Ligand as used herein is a non-naturally occurring Nucleic Acid having a desirable action on a Target which comprises two or more nucleotides.
  • a desirable action includes, but is not limited to, binding of the Target, catalytically changing the Target, reacting with the Target in a way which modifies/alters the Target or the functional activity ofthe Target, covalently attaching to the Target as in a suicide inhibitor, facilitating the reaction between the Target and another molecule.
  • the action is specific binding affinity for a Target molecule, such Target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the Nucleic Acid Ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein the Nucleic Acid Ligand is not a Nucleic Acid having the known physiological function of being bound by the Target molecule.
  • the Nucleic Acid Ligands ofthe invention are identified by the SELEX methodology.
  • Nucleic Acid Ligands include Nucleic Acids that are identified from a Candidate Mixture of Nucleic Acids, said Nucleic Acid Ligand being a ligand of a given Target by the method comprising: a) contacting the Candidate Mixture with the Target, wherein Nucleic Acids having an increased affinity to the Target relative to the Candidate Mixture may be partitioned from the remainder ofthe Candidate Mixture; b) partitioning the increased affinity Nucleic Acids from the remainder ofthe Candidate Mixture; and c) amplifying the increased affinity Nucleic Acids to yield a ligand-enriched mixture of Nucleic Acids.
  • Candidate Mixture is a mixture of Nucleic Acids of differing sequence from which to select a desired ligand.
  • the source of a Candidate Mixture can be from naturally-occurring Nucleic Acids or fragments thereof, chemically synthesized Nucleic Acids, enzymatically synthesized Nucleic Acids or Nucleic Acids made by a combination ofthe foregoing techniques.
  • each Nucleic Acid has fixed sequences surrounding a randomized region to facilitate the amplification process.
  • Nucleic Acid means either DNA, RNA, single-stranded or double-stranded and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that inco ⁇ orate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the Nucleic Acid Ligand bases or to the Nucleic Acid Ligand as a whole.
  • Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping. "SELEX" methodology involves the combination of selection of
  • Nucleic Acid Ligands which interact with a Target in a desirable manner, for example binding to a protein, with amplification of those selected Nucleic Acids. Iterative cycling ofthe selection amplification steps allows selection of one or a small number of Nucleic Acids which interact most strongly with the Target from a pool which contains a very large number of Nucleic Acids.
  • the SELEX methodology can be employed to obtain a Nucleic Acid Ligand to a desirable Target.
  • SELEX Target means any compound or molecule of interest for which a ligand is desired.
  • a Target can be a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc. without limitation.
  • Target means a preselected location in a biological system including tissues, organs, cells, intracellular compartments, extracellular components. The latter include hormones (endocrine paracrine, autocrine), enzymes, neurotransmitters and constituents of physiological cascade phenomena (e.g., blood coagulation, complement, etc.).
  • “Intracellular Target” means anything that is existing, occurring, or functioning within a cell or cellular compartment. This includes, but is not limited to, organelles, enzymes, proteins, viral pathogens and intracellular bacteria.
  • Gene Therapy means the transfer of new genetic material to the cells of an organism with resulting Therapeutic benefit to the organism.
  • “Therapeutic,” as used herein, includes treatment and/or prophylaxis. When used, Therapeutic refers to humans and other animals.
  • Intracellularly-mediated Disease or Condition is any disease or condition that originates from within the cell or has an adverse affect on the cell and impairs normal functioning of a cell, tissue, organ, or organism.
  • Ligand Chimeric Gene is a Nucleic Acid Ligand that has been fused to a gene which has the transcriptional regulatory elements that allow for its expression and intracellular localization.
  • the SELEX process may be defined by the following series of steps:
  • a Candidate Mixture of Nucleic Acids of differing sequence is prepared.
  • the Candidate Mixture generally includes regions of fixed sequences (i.e., each ofthe members ofthe Candidate Mixture contains the same sequences in the same location) and regions of randomized sequences.
  • the fixed sequence regions are selected either: (a) to assist in the amplification steps described below, (b) to mimic a sequence known to bind to the Target, or (c) to enhance the concentration of a given structural arrangement ofthe Nucleic Acids in the Candidate Mixture.
  • the randomized sequences can be totally randomized (i.e., the probability of finding a base at any position being one in four) or only partially randomized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).
  • the interaction between the Target and the Nucleic Acids ofthe Candidate Mixture can be considered as forming Nucleic Acid-Target pairs between the Target and those Nucleic Acids having the strongest affinity for the Target.
  • the Nucleic Acids with the highest affinity for the Target are partitioned from those Nucleic Acids with lesser affinity to the Target. Because only an extremely small number of sequences (and possibly only one molecule of Nucleic Acid) corresponding to the highest affinity Nucleic Acids exist in the Candidate Mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the Nucleic Acids in the Candidate Mixture
  • the SELEX Patent Applications also describe ligands obtained to a number of target species, including both protein Targets where the protein is and is not a Nucleic Acid binding protein.
  • the methods described herein and the Nucleic Acid Ligands identified by such methods are useful for both Therapeutic and diagnostic pu ⁇ oses.
  • Therapeutic uses include the treatment or prevention of diseases or medical conditions in felines. Diagnostic utilization may include both in vivo or in vitro diagnostic applications.
  • the SELEX method generally, and the specific adaptations of the SELEX method taught and claimed herein specifically, are particularly suited for diagnostic applications.
  • SELEX identifies Nucleic Acid Ligands that are able to bind Targets with high affinity and with su ⁇ rising specificity. These characteristics are, of course, the desired properties one skilled in the art would seek in a diagnostic ligand.
  • the Nucleic Acid Ligands ofthe present invention may be routinely adapted for diagnostic pu ⁇ oses according to any number of techniques employed by those skilled in the art. Diagnostic agents need only be able to allow the user to identify the presence of a given Target at a particular locale or concentration. Simply the ability to form binding pairs with the Target may be sufficient to trigger a positive signal for diagnostic pu ⁇ oses. Those skilled in the art would also be able to adapt any Nucleic Acid Ligand by procedures known in the art to inco ⁇ orate a labeling tag in order to track the presence of such ligand. Such a tag could be used in a number of diagnostic procedures.
  • the Nucleic Acid Ligands to FIV RT described herein may specifically be used for identification of FIV RT.
  • SELEX provides high affinity ligands of a Target molecule. This represents a singular achievement that is unprecedented in the field of nucleic acids research.
  • One embodiment ofthe present invention applies the SELEX procedure to the specific Target FIV RT.
  • the experimental parameters used to isolate and identify the Nucleic Acid Ligands to FIV RT are described.
  • the Nucleic Acid Ligand (1) binds to the Target in a manner capable of achieving the desired effect on the Target; (2) be as small as possible to obtain the desired effect; (3) be as stable as possible; and (4) be a specific ligand to the chosen Target. In most situations, it is preferred that the Nucleic Acid Ligand have the highest possible affinity to the Target.
  • the present invention includes methods of treating intracellularly-mediated diseases or conditions with oligonucleotides.
  • diseases include, but are not limited to cancer; infectious diseases, such as
  • AIDS cytomegalovirus retinitis, hepatitis, infectious mononucleosis, Leischmaniasis, candidiasis, malaria, influenza
  • dominant genetic disorders such as polycystic kidney disease, Charcot-Marie-Tooth disease, Stargardt's disease, Parkinson's disease, Alzheimer's disease, Schizophrenia, Artherosclerosis, and cancers such as those involving dominant RAS mutations.
  • a number of recessive genetic diseases result in ove ⁇ roduction or accumulation of proteins or molecules that could be bound or inhibited by oligonucleotides, induding Nucleic Acid Ligands. For example, defects in the LDL receptor leads to hypercholesterolemia.
  • Treatment comprises the introduction of oligonucleotides into cells whereby the oligonucleotide affects the activity of the Intracellular Target and thereby the disease condition is attenuated.
  • the oligonucleotide inhibits the activity ofthe Intracellular Target.
  • treatment also includes prophylaxis.
  • Introduction ofthe oligonucleotide can be a one time administration or as part of a regimen.
  • the oligonucleotide can be transiently or stably expressed depending on the type of gene or Nucleic Acid transfer procedure employed.
  • an oligonucleotide comprises two or more nucleotides.
  • the oligonucleotide comprises 3 or more nucleotides. In the preferred embodiment, the oligonucleotide comprises 7 or more nucleotides.
  • the oligonucleotide can act in any fashion to affect the activity ofthe
  • Intracellular Target except by binding to another Nucleic Acid by Watson-Crick base-pairing.
  • a method of treating intracellularly-mediated diseases or conditions with Nucleic Acid Ligands is also included in the present invention.
  • the Nucleic Acid Ligands are identified by the SELEX methodology.
  • a method of diagnosing intracellularly-mediated diseases or conditions with oligonucleotides, including Nucleic Acid Ligands are identified by the SELEX methodology.
  • the present invention further includes methods for treating or diagnosing diseases or conditions resulting from viral pathogens.
  • the methods ofthe invention can be used in the treatment or diagnosis of HIV-l infection by inserting Nucleic Acid Ligands specific for proteins involved in the replication of HIV-l into cells.
  • the Nucleic Acid Ligands inhibit the function of these proteins thereby preventing the replication of HIV-l .
  • the preferred targeted HIV-l proteins are tat, rev, and reverse transcriptase.
  • the Nucleic Acid Ligand(s) can be inserted into the cells using any gene or Nucleic Acid transfer procedure or combination of procedures.
  • the cells can be genetically engineered in vivo or in vitro. For example, cells can be removed from a patient, genetically engineered in vitro with the Nucleic Acid Ligand (either DNA or RNA) and the cells containing the Nucleic Acid
  • Ligand(s) can be readministered to a patient. This procedure is referred to herein as ex vivo treatment.
  • the engineered cells now will express the Nucleic Acid Ligand and allow it to affect the activity ofthe Intracellular Target.
  • the Nucleic Acid Ligand can be administered to a patient for delivery ofthe Nucleic Acid Ligand in vivo to the targeted cells.
  • Retroviral-mediated gene transfer is used.
  • Retroviruses are currently the most widely used transducing agent (Goff and Shenk (1993) Current Opinion in Genetics and Development 2:71-73). The life cycle ofthe retroviruses makes these agents a natural choice for the delivery of Nucleic Acid Ligands into target cells.
  • Viral vectors can be designed that retain no intact viral genes and only a minimum of viral sequences. Early in infection, the viral RNA genome is reverse transcribed into duplex DNA, and the DNA copy is efficiently integrated into the host genome.
  • RNA viruses derived from other RNA viruses and DNA viruses.
  • retroviral vectors including those derived from other RNA viruses and DNA viruses.
  • adenoviruses a family of DNA viruses, have many advantages, such as their potential to carry large segments of DNA and suitability for infecting tissues in situ (Miller (1992) Nature 252:455-459).
  • the Nucleic Acid Ligand is RNA, it is well known to one skilled in the art to produce the DNA complement for inco ⁇ oration into the adenovirus.
  • the viral vectors can be engineered to direct the expression ofthe transduced Nucleic Acid Ligands under a variety of different transcriptional regulatory sequences, as would be known to one of skill in the art.
  • the Target ofthe Nucleic Acid Ligand may be present in the nucleus or the cytoplasm.
  • other localization signals such as sequences that direct the Nucleic Acid Ligand transcript to remain in the nucleus or to go to the cytoplasm can also be inco ⁇ orated into the viral vector, as would be known to one of skill in the art.
  • the snRNAs e.g. , U6 are involved in RNA splicing and thus remain in the nucleus.
  • tRNAs are involved in translation and are thus primarily found in the cytoplasm. It would be known to one of skill in the art that fusions of Nucleic Acid Ligands to other RNA (e.g. , U6 or tRNA) can be made in a manner that would not disrupt their intracellular localization.
  • RNA e.g. , U6 or tRNA
  • Nucleic Acid Ligand is not required, and therefore stable integration into the cell's genome is not necessary.
  • other gene transfer methods can be employed (e.g., receptor-mediated, microinjection).
  • Receptor-mediated methods of gene transfer involve complexing plasmid DNA and specific polypeptide ligands that are recognized by receptors on a cell surface (Mulligan (1993) Science 260:926-932).
  • Nucleic acid ligands which are RNA can also be complexed with specific polypeptide ligands for receptor-mediated uptake.
  • the Nucleic Acid Ligands can be complexed with a lipophilic compound (e.g., cholesterol) or attached to or encapsulated in a complex comprised of lipophilic components (e.g., a liposome).
  • a lipophilic compound e.g., cholesterol
  • the complexed Nucleic Acid Ligands can enhance the cellular uptake ofthe Nucleic Acid Ligands by a cell for delivery ofthe Nucleic Acid Ligands to an Intracellular Target.
  • Nucleic Acid Ligand Complexes which is inco ⁇ orated in its entirety herein, describes a method for preparing a Therapeutic or diagnostic complex comprised of a Nucleic Acid Ligand and a lipophilic compound or a non-immunogenic, high molecular weight compound.
  • Nucleic Acid Ligands can be introduced into cells by applying intense electric fields (electroporation). High electric fields make membranes transiently permeable to large molecules, such as DNA and RNA. Direct DNA uptake can also be used to deliver Nucleic Acid Ligands to cells.
  • a plasmid is constructed that encodes the Nucleic Acid Ligand and this plasmid is injected into tissues. It is also possible to directly deliver a Nucleic Acid Ligand that is not fused to other RNAs by inco ⁇ orating it into a viral particle. This would be possible if the ligand bound to a structural component ofthe virus (such as RT) which ultimately is found intracellularly.
  • RNA and not a Ligand Chimeric Gene would be delivered in this case, the action ofthe ligand may be transient. Moreover, for treating acute intracellularly-mediated diseases there could be advantages over using stable genes to treat chronic conditions. Such ligands would be inco ⁇ orated into a murine retrovirus used for Gene Therapy by expressing the appropriate ligand and viral protein in the same packaging cell used to produce the virus.
  • One potential problem encountered in the Therapeutic and in vivo diagnostic use of Nucleic Acids is that oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is manifest. Certain chemical modifications ofthe Nucleic Acid Ligand can be made to increase the in vivo stability ofthe Nucleic Acid
  • Nucleic Acid Ligand or to enhance or to mediate the delivery ofthe Nucleic Acid Ligand.
  • Modifications ofthe Nucleic Acid Ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that inco ⁇ orate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the Nucleic Acid Ligand bases or to the Nucleic Acid Ligand as a whole.
  • modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping.
  • Nucleic Acid Ligands are derived by the SELEX method
  • the modifications can be pre- or post- SELEX modifications.
  • Pre-SELEX modifications yield Nucleic Acid Ligands with both specificity for their
  • RNA with specific high affinity for FIV RT from a degenerate library containing 40 random positions ( Figures 1 A - IH).
  • This invention includes the specific RNA ligands to FIV RT (SEQ ID NOS:6- 12), identified by the methods described in Examples 1 and 2.
  • the scope of the ligands covered by this invention extends to all Nucleic Acid Ligands of FIV RT modified and unmodified, identified according to the SELEX procedure. More specifically, this invention includes Nucleic Acid sequences that are substantially homologous to the ligands shown in Figures 1 A - IH (SEQ ID NOS: 6- 12).
  • substantially homologous it is meant a degree of primary sequence homology in excess of 70%, most preferably in excess of 80%.
  • a review ofthe sequence homologies ofthe ligands of FIV RT shown in Figures 1 A - IH (SEQ ID NOS:6-12) shows that sequences with little or no primary homology may have substantially the same ability to bind FIV RT.
  • this invention also includes Nucleic Acid Ligands that have substantially the same structure and ability to bind FIV RT as the Nucleic Acid Ligands shown in Figures 1 A - IH (SEQ ID NOS:6-12).
  • Substantially the same ability to bind FIV RT means that the affinity is within one or two orders of magnitude ofthe affinity ofthe ligands described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence — substantially homologous to those specifically described herein — has substantially the same ability to bind FIV RT.
  • This invention also includes the ligands as described above, wherein certain chemical modifications are made in order to increase the in vivo stability ofthe ligand or to enhance or mediate the delivery ofthe ligand.
  • modifications include chemical substitutions at the sugar and/ or phosphate and/or base positions of a given Nucleic Acid sequence. See, e.g., U.S. Patent Application Serial No. 08/117,991, filed September 9, 1993, entitled High Affinity Nucleic Acid Ligands Containing Modified Nucleotides which is specifically inco ⁇ orated herein by reference.
  • Other modifications are known to one of ordinary skill in the art.
  • Such modifications may be made post-SELEX (modification of previously identified unmodified ligands) or by inco ⁇ oration into the SELEX process.
  • the ligands described herein are useful in veterinary applications.
  • This invention also includes a method of inhibiting FIV RT function by administration of a Nucleic Acid Ligand capable of binding to FIV RT.
  • Example 1 describes the general procedures followed in Example 2 for the evolution of Nucleic Acid Ligands to FIV RT.
  • Example 2 describes the Nucleic Acid Ligands to FIV RT.
  • Example 3 describes the experimental procedures used in expressing a HIV-l Nucleic Acid Ligand in human cells.
  • Example 4 describes the protection of cells from HIV-l infection by a HIV-l Nucleic Acid Ligand.
  • Example 5 describes the experimental procedures used in expressing FIV RT ligands in cells.
  • Example 6 describes the inhibition of FIV replication in cells by selected inhibitory ligands.
  • Recombinant FIV RT was purified from E. coli containing the clone pRFT14 (North et al. (1989) Antimicrob. Agents Chemother. 22:915-919); virion FIV RT was purified as previously described (North et al. (1989)
  • AZR-17c FIV RT from an AZT resistant mutant
  • AZR-17c FIV RT from an AZT resistant mutant
  • the latter enzyme has decreased susceptibility to the 5 '-triphosphate of AZT.
  • AMV reverse transcriptase was purchased from Life Sciences, Inc.
  • M-MLV reverse transcriptase was purchased from GIBCO
  • HIV-l RT was generously provided by Agouron Pharmaceuticals Inc.
  • Taq DNA polymerase was purchased from Perkin Elmer Cetus, T4 polynucleotide kinase from New England Biolabs, and T7 RNA polymerase from U.S. Biochemical Co ⁇ oration. DNA and RNA oligonucleotides were synthesized on an Applied Biosystems Model 394 DNA/RNA synthesizer.
  • RNA ligands were selected from an RNA repertoire containing IO 14 unique species (Tuerk et al. (1990) Science. 249:505-510: Tuerk et al. (1992) Proc. Natl. Acad. Sci. 89:6988-6992; Chen et al. (1994)
  • the first 10 rounds of selection were performed by nitrocellulose filter partitioning in 1 mL binding buffer (50 mM Tris-HCl, pH 7.7, 200 mM potassium acetate and 10 mM dithiothreitol) containing RNA and Target protein.
  • binding buffer 50 mM Tris-HCl, pH 7.7, 200 mM potassium acetate and 10 mM dithiothreitol
  • the binding reaction was incubated at 37°C for 10 minutes and bound RNA was partitioned by nitrocellulose filtration.
  • the bound RNA was eluted into 200 ⁇ L 7 M urea and 400 ⁇ L phenol as described previously (Tuerk et al. (1992) Proc. Natl. Acad. Sci. £2:6988-6992), and recovered by ethanol precipitation.
  • cDNA was synthesized by AMV RT at 45°C.
  • the cDNA product was amplified by PCR and transcribed with T7 RNA polymerase to generate the RNA pool for the next round of selection (Beard et al. (1952) Natl. Cancer Conf. Proc. 2:1396-1411; Larder et al. (1989) Science. 242:1731-1734).
  • the polymerization inhibition assay was performed as described previously (Tuerk et al. (1992) Proc. Natl. Acad. Sci. 82:6988-6992; Chen et al. (1994) Biochemistry. 22:8746-8756).
  • the reaction contained 17.4 nM RT, 10 nM template/ 32 P 5'-end labeled primer complex, and RNA ligand.
  • the inhibition assay was performed at 37°C for 10 minutes in 20 ⁇ L polymerization buffer (50 mM Tris-HCl pH 7.7, 200 mM potassium-acetate, 6 mM MgCl 2 , 10 mM DTT, 25 ⁇ g/mL BSA, and 0.4 mM dNTPs).
  • the polymerization products were analyzed by electrophoresis on a 7 M urea, 10% polyacrylamide gel.
  • FIV RT activity was assayed as described previously (North et al. (1994) Antimicrob. Agents Chemother. 38:388-391; North et al. (1990) J. Biol. Chem. 265:5121-5128 . Reactions were carried out in a volume of 50 ⁇ L and under standard conditions containing 50 mM Tris-HCl, pH 8.5, 10 mM DTT, 0.05% Triton X-100, 250 ⁇ g /mL BSA (nuclease-free), 6 mM MgCl 2 , 0.5 OD 260 units of template/primer, the appropriate [ 3 H] dNTP (33 ⁇ Ci/mL, 20 ⁇ M) and enzyme.
  • the concentration of dNTP or of template/primer was varied as indicated.
  • the reaction mixtures were incubated at 37°C for 30 minutes, and then 40 ⁇ L samples were spotted onto Whatman No. 3 filters (2.3 cm, which were pre-soaked with 5% TCA and 1% sodium pyrophosphate). Filters were washed four times in ice-cold trichloracetic acid, 1% sodium pyrophosphate, once in 95% ethanol and the radioactivity was quantified by scintillation counting.
  • RNA sequence was randomized at 40 nucleotide positions flanked by a 25 nucleotide fixed region at the 5'-end and a 27 nucleotide fixed region at the 3'-end (Chen et al. (1994) Biochemistry. 22:8746-8756).
  • the RNA sequence was randomized at 40 nucleotide positions flanked by a 25 nucleotide fixed region at the 5'-end and a 27 nucleotide fixed region at the 3'-end (Chen et al. (1994) Biochemistry. 22:8746-8756).
  • 5'-gggaggauauuuucucagaccguaa-N 40 -uugcagcaucgugaacuaggauccggg-3' (SEQ ID NO: 1) was used as a starting pool with 5'-CCCAAGCTTAATACGACTCACTATAGGGAGGATATTTTCTCAG
  • ACCGTAA-3' (SEQ ID NO:2), which contains T7 promoter, and 5'-CCCGGA TCCTAGTTCACGATCTGCAA-3 ' (SEQ ID NO:3) as the 5' and 3' PCR primers, respectively.
  • the starting repertoire contained approximately IO 14 unique RNA species.
  • a recombinant FIV RT was used as the Target.
  • the first 10 rounds of selection were performed by nitrocellulose filtration.
  • the final 8 rounds of selection were performed using a native gel mobility retardation method (Carey et al. (1991) Methods Enzymol. 208:103-117) as the partitioning strategy. Two major shifted bands appeared on the native gel (not shown), and both complexes were collected for selection.
  • the binding affinity ofthe RNA pool could not be further improved by continued selection, and the complexity ofthe round eighteen pool was determined by RNA sequence analysis (not shown).
  • the cDNA of round- 18 pool was cloned into pUC-18 at Hind III and BamH I sites and sequenced.
  • the FIV RT binding affinity ofthe round eighteen pool was at least 10 3 -fold higher than that ofthe starting repertoire.
  • RNA sequences from round eighteen RNA molecular pool were obtained and analyzed by an RNA folding algorithm (Zuker et al. (1989) Meth. Enzymol.110:262-288; Jaeger et al. (1989) Proc. Natl. Acad. Sci. USA. £6_:7706-7710).
  • the selected RNA molecules fell into three major classes
  • Figures 1 A - IH The secondary structures presented in Figures 1 A - IH have the lowest free-energy.
  • Class I molecules containing four subsets, Fl, F2, F3, and F4 (SEQ ID NOS:6-9)) form a stem-loop or a stem-loop with internal bulge structures and contain one or two U-tract consensus sequences present in a region predicted to be single stranded.
  • Three subsets F2, F3, and F4 (SEQ ID NOS : 7-9)
  • Class II consists of three subsets of species (F5, F6, and F7 (SEQ ID NOS: 10-12)) that can form a stem-loop with internal bulge structures.
  • class II ligands have consensus AA dinucleotide in the bulge and two subsets (F6 and F7 (SEQ ID NOS: 11-12)) of this class contain an ACCA consensus in a tetra loop.
  • Class III consists of two subsets (F8 and F9).
  • F8 subset contain YAA repeats and members of F9 subset have a A-track sequence.
  • YAA'repeats and A-track sequences appear to be in unstructured regions, at least as predicted by the RNA folding program (Zuker et al. (1989) Meth. Enzymol. l£0:262-288; Jaeger et al. (1989) Proc. Natl. Acad. Sci. USA. 86:7706-7710).
  • Nine o ⁇ han sequences were also obtained among the cDNA clones (not shown).
  • the binding affinities of each subset of RNA molecules to FIV RT were measured by filter binding. As shown in Figures 1 A - IH, the dissociation constants were in the range of 1.9 nM to 24.0 nM.
  • the p66/p66 homodimeric FIV RT is not as stable as the p66/p51 heterodimer.
  • the homodimer can dissociate to form p66 monomer.
  • gel mobility retardation assays (Carey et al. (1991) Methods Enzymol. ___ . : 103-117) were performed to analyze the RNA-protein interaction of each subset of RNA molecules.
  • the RNA molecules of class I (SEQ ID NOS:6-9) bound to the FIV RT dimer and monomer almost equally well (data not shown).
  • RNA ligands bound mainly to dimeric RT, and with less affinity to monomeric RT with the exception of ligand F5 (SEQ ID NO: 10) which could bind to both forms but prefers binding to the dimeric form.
  • the RNA ligands of class III bound to both forms of RT with approximately equal affinity, however, an extra complex with an intermediate mobility was also present. This extra complex has not yet been identified. It may arise from different conformations of RNA interacting with the monomer protein, or binding of two RNA molecules with one monomer protein, or from the impurity ofthe recombinant FIV RT. It was also observed that the mobility of free RNA ligand of F8 subfamily was slower. The native gel mobility shift results indicated that the RNA ligands of the three different classes have different binding interactions with FIV RT.
  • RNA ligands may interact with FIV RT at the active site and function as inhibitors.
  • inhibitor screening experiments Tuerk et al. (1992) Proc. Natl. Acad. Sci. 82:6988-6992; Chen et al. (1994) Biochemistry. 22:8746-8756 were performed using an inhibition assay.
  • the RNA template for assaying RT activity was a fragment of plasmid pT7-l transcribed by T7 RNA polymerase (RNA sequence:
  • the selected ligand can discriminate homologues of a protein family.
  • the inhibition effect of other subsets ofthe FIV RT selected ligands (including Fla) ligands with moderate inhibitory effect on FIV RT did not inhibit AMV, M-MLV or HIV-l RT.
  • RNA ligand F5 (SEQ ID NO: 10) has the highest inhibition activity, it was further analyzed by protein binding boundary determination (Tuerk et al. (1992) Proc. Natl. Acad. Sci. 82:6988-6992; Chen et al. (1994) Biochemistry. 22:8746-8756).
  • This example provides general procedures followed and inco ⁇ orated in Example 4 describing the expression of HIV-l Nucleic Acid Ligands in human cells.
  • Recombinant HIV-l tat protein produced in E. coli, was purchased from Intracell, Inc. (Cambridge, MA).
  • the HIV-l reverse transcriptase was produced in E. coli as described (Hostomsky et al. (1991) Proc. Natl. Acad.
  • the HIV-l rev protein was produced in E. coli and provided by Maria Zapp and Michael Green (Harvard Medical School, Cambridge, MA) or purchased from Intracell, Inc.
  • the retrovirus packaging line GP+envAml2 and the retroviral expression vector pDCT-5T were obtained from Bruce Sullenger and Eli Gilboa (Duke University). Other expression vectors can be used, as would be known to one of skill in the art.
  • the reaction was vacuum-filtered through nitrocellulose filters (HAWP, Millipore, Co ⁇ ., Bedford, MA), the amount of labeled RNA retained on the filter was determined, and the apparent Kj ofthe protein for the RNA was obtained by plotting the amount of RNA bound vs. the concentration ofthe protein using Kaleidograph computer software (Synergy, Inc., Reading, PA).
  • the retroviral vector pNEW6 ( Figure 3) was constructed by replacing the polylinker of pDCT-5t (5'CCGCGGTGGATCC3') (SEQ ID NO: 14), which has Sac II and BamHI cloning sites, with one which has Hind III and BamHI cloning sites (5'CCGCGGGTCGTGTTAGAAGCTTCCCATGGATCCTTCGGGATCTG
  • Taq polymerase was obtained from Perkin-Elmer (Norwalk, CT). All molecular cloning techniques were performed essentially as described by Sambrook, et al. (1989) Molecular Cloning: A laboratory manual (2nd ed), Cold Spring Harbor Laboratories, Cold Spring Harbor, NY). The recA' E. coli strain DH5 ⁇ (Gibco, Inc., Gaithersburg, MD) was used for transformation.
  • RNA and genomic DNA were prepared from CEMss cell lines using TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer's directions.
  • PCR Polymerase chain reactions
  • RT-PCR Reverse transcription-polymerase chain reactions
  • QC-PCR was performed to accurately quantitate intracellular ligand expression. QC-PCR was performed as described by Sieber and Larrick (1993) Biotechniques 14:244-249; Piatak et al. (1993) Science 259: 1749- 1754. The plasmids used to generate competitor RNAs
  • the samples were then amplified by RT-PCR as described above for a total of 30 cycles.
  • the competitor RNAs produce a PCR product that is 42 base pairs larger than that obtained by PCR amplification ofthe SELEX ligand-containing RNAs.
  • Analysis of QC-PCR products was done on 3% NuSieve agarose (FMC, Inc., Rockland, ME) gels run in 1 x TBE buffer.
  • the concentration at which the PCR product from the competitor RNA was equal in amount to that ofthe cellular RNA (the "equivalence point") was taken as the concentration ofthe SELEX ligand-containing RNAs. In some cases three fold dilutions of competitor RNA was used to determine the SELEX ligand-containing RNA concentration more accurately.
  • the SELEX combinatorial method was used to generate RNA ligands that bind to the HIV-l tat, rev, and reverse transcriptase proteins.
  • the binding properties ofthe rev ligand, rev30A (SEQ ID NO:21) (also called rev ⁇ a), to the HIV-l rev protein have been described in detail by Jenson, et al.
  • the binding properties ofthe pseudoknot portion ofthe rtwl 7 (SEQ ID NO: 22) ligand to the HIV-l reverse transcriptase protein have been described in detail by Green, et al. (1995) J. Mol. Biol. 242:60-68.
  • the affinities ofthe selected RNAs for their Target proteins ranged from 0.1-10 nM (Table 2).
  • C ⁇ Mss cells expressing tRNAj Met alone were also infected. Prior to HIV-l infection the cells were analyzed for CD3 and CD4 expression. The level of CD 3 and CD4 expression was roughly equivalent to that of control C ⁇ Mss cells for all cell lines that were challenged with HIV-l.
  • cell lines expressing the chimeric tRNA-SELEX RNA were protected from HIV-l infection by 100 TCID 50 units of HIV-l Illb for at least 30 days, whereas cell lines that did not express a HIV Nucleic Acid
  • This example provides general procedures followed and inco ⁇ orated in Example 6 describing the expression of FIV Nucleic Acid Ligands in feline cells.
  • the murine-derived retroviral vector pNEW6 which contains the bacterial neomycin-resistant gene (neo), was modified by insertion of a polylinker sequence downstream ofthe human tRNAj Met promoter in the U3 region of LTR (Adeniyi- Jones et al. (1984) Nuc. Acids Res. 12:1101-1115).
  • F5 The cDNAs of intact FIV RT selected RNA molecule F5 (5'-GGGAGGATATTTTCTCAGACCGTAATTGCGAAGGAAAAACCGA GGTGCTTTACGCGTCAATATGCTTGCAGCATCGTGAACTAGGATC CGGG-3' (SEQ ID NO:26)) and the truncate version dF5 (5'-GTAATTGCGAAGG AAAAACCGAGGTGCTTTACG-3' (SEQ ID NO:26)) and the truncate version dF5 (5'-GTAATTGCGAAGG AAAAACCGAGGTGCTTTACG-3' (SEQ ID NO:26)) and the truncate version dF5 (5'-GTAATTGCGAAGG AAAAACCGAGGTGCTTTACG-3' (SEQ ID NO:26)) and the truncate version dF5 (5'-GTAATTGCGAAGG AAAAACCGAGGTGCTTTACG-3' (SEQ ID NO:26)
  • RNA Isolation Kit (Strategene Cloning System) according to the instruction manual. 10 ⁇ g of total RNA sample was subjected to electrophoresis on a 1% formaldehyde
  • genomic DNA was digested with restriction endonuclease Hind III or Xba I.
  • the samples were electrophoresed on a 1% agarose gel, transferred onto a Hybond-N membrane (Amersham Life Science Inc.), and UV cross-linked to the membrane with a Strategene UV Statelinker.
  • the membrane was pre-hybridized at 65°C for at least one hour in 12.5 mL of 5X Denhardts (0.1 % BSA, 0.1% Ficoll, 0.1% polyvinylpyrrolidone), IX SSPE (180 mM NaCl, 10 mM sodium phosphate pH7.7, and 1 mM EDTA pH7.7), and 2 ⁇ g/mL sonicated salmon sperm DNA.
  • the 32 P-labeled ligand-specific DNA probe or neomycin gene ( «e ⁇ )-specific probe (10 8 cpm/mL) was added to the pre-hybridization solution. The hybridization was carried out for 16 hours at 65°C.
  • the hybridized membrane was washed twice with 2X SSPE (360 mM NaCl, 21 mM sodium phosphate pH 7.7, and 2 mM EDTA pH 7.7), 0.1% SDS, at room temperature for 10 minutes, and washed with IX SSPE (180 mM NaCl, 10 mM sodium phosphate pH7.7, and 1 mM EDTA pH7.7), 0.1%
  • Genomic DNA from expression cell lines was PCR amplified with primer pFRT5- 1 (5'-TGTGAGCC GTGTGCTGCTTGGCAG-3' (SEQ ID NO: 1)
  • genomic DNA was PCR amplified in 100 ⁇ L of PCR reaction mixture containing 200 picomoles of primers pFRT5-l and pFRT3-l at 93°C for 30 seconds, 55°C for 15 seconds, and 72°C for 90 seconds, for 30 cycles.
  • primers pFRT5-l and pFRT3-l As a positive control, plasmid pNEW6-FRTl, which contained the cDNA of RNA ligand F5 (SEQ ID NO: 10) was used.
  • the PCR product (294 bp) was purified from an 1% low-melting agarose gel.
  • primer pFRT5-2 (5'-GCTTGGCAGAACA GCAGAGTGG-3' (SEQ ID NO : 30) )for forward sequencing or primer pFRT3 -2
  • the plasmid was shown to possess the desired insertion.
  • the constructed plasmids were renamed as pNEW6-FRTl (for full length) and pNEW6-FRT2 (for the truncated version), respectively ( Figure 3).
  • the insertion site is located at the 3' end of a deleted human tRNAj Met gene (Adeniyi- Jones et al.
  • the constructed plasmids were transfected into the packaging cell line GP+envAM12 (Markowitz et al. (1988) Virology 162:400-406 ) by electroporation.
  • the transfected GP+envAM12 cells were grown in the medium containing G418 for positive selection.
  • the GP+envAM12 cell line is an amphotropic cell line which expresses all the murine retroviral proteins for packaging the defective virus.
  • the defective amphotropic viruses can infect a wide range of hosts, but can only undergo a single cycle of infection (Markowitz et al. (1988) Virology 162:400-406 ).
  • the tRNAi Me, -Ligand Chimeric Gene is in the U3 region of the 3' LTR. After replication ofthe vector, the chimeric gene should be duplicated in the U3 region of both 5' and 3' LTR of proviral DNA ( Figure 3).
  • the Crandell feline kidney (CrFK) cell line was infected with the defective virus (Sullenger et.al. (1990) Mol. Cell. Biol. 10:6512-6523). The infected cells were grown in the medium containing 200 ⁇ g/mL G418 for positive selection. After two weeks, 35 G418-resistant single colonies were obtained.
  • the CrFK cell lines were named CrFK-FRTl for expression ofthe full-length FIV RT-selected RNA molecule, and CrFK-FRT2 for expression of the truncated version ofthe FIV RT-selected RNA molecule.
  • RNA polymerase III RNA polymerase III
  • tRNA promoter RNA polymerase III
  • expression ofthe selected RNA was further analyzed by Northern blot (Ausubel et al. (1992) Curr. Prot. Mol. Clon. Vol.l).
  • a double-stranded cDNA probe (155 bp) which covers the human initiation methionyl-tRNA gene, containing the ligand F5 sequences, was used for hybridization.
  • the Northern blot result suggested that the selected RNA existed as both pol II and pol III transcripts. Three pol II transcripts have been detected.
  • the 4.5kb nucleotide RNA molecule presumably is the full length transcription product ofthe murine retroviral vector, while the 2.2kb and l.Okb nucleotide RNA molecules are presumably alternatively spliced products.
  • the pol III transcription product wliich is 210 nucleotides in length, is the human initiation methionyl-tRNA-selected RNA chimeric molecule. Originally, it was expected that the selected RNA molecule would be overexpressed under the control ofthe human promoter. However, the RNA blotting analysis showed that the hybridization signal of expression level for pol III transcripts was lower than those of pol II transcripts.
  • RNA ligands The effects of expression of selected RNA ligands on cell growth were also examined by analyzing the doubling time of expression cell lines. 1X10 4 to 1X10 5 cells were seeded in the media in a T25 flask. The total number of cells was counted by tryptan blue staining every 24 to 48 hours. The doubling times of three cell lines expressing RNA ligands were in the range of 21.9 hours to 23.45 hours, while that ofthe parental cell line is 17.7 hours. It was also noticed that one cell line, CrFK-FRTl -20, had a doubling time which was slightly faster than the parental cell line. The results suggest that the expression of selected ligand have no severe effects on cell proliferation.
  • the total genomic DNA from expression CrFK cell lines were isolated.
  • the fragment of genomic DNA containing the insertion was amplified by PCR with primers pFRT5- 1 (SEQ ID NO:28) and pFRT3-l (SEQ ID NO:29) and the band was sliced from a low melting gel.
  • the plasmid pNEW6-FRTl was used as a positive control and for tracing the occurrence of PCR-introduced mutations.
  • the genomic sequencing analysis showed that no point mutations or deletions existed in the inserted cDNA of the selected RNA ligand F5 (SEQ ID NO: 10) (not shown).
  • proviral DNA was further analyzed by Southern blot.
  • the predicted duplication of LTR of proviral DNA will place the ligand template in the U3 region of both 5' and 3' LTRs.
  • the retroviral vector contains two Hind III sites and two Xba I sites in the viral LTRs (Sullenger etal. (1990) Mol. Cell. Biol. 10:6512-6523; Adeniyi-Jones et al. ( 1984) Nuc. Acids Res. 12: 1101 - 1115).
  • the restriction endonuclease Hind III will generate a 3.8-kb fragment, which contains one copy ofthe tRNAj Met -Ligand Chimeric Gene, as will Xba I digestion, while another copy ofthe chimeric gene will be associated with the host cellular chromosome.
  • the hybridization pattem with a ligand specific probe showed a 3.8-kb fragment in each ofthe four expression cell lines tested and a unique fragment in each individual cell line.
  • the DNA blotting analysis indicated the existence ofthe duplication ofthe LTR and a variation of integration sites of proviral DNA in each expression cell line.
  • the same samples were also hybridized with a neomycin gene (neo ) specific probe.
  • the 3.8-kb fragment was apparent for each cell line as predicted. DNA analyses suggested that the
  • HIV-l RT-selected RNA molecule (Tuerk et al. (1992) Proc. Natl. Acad. Sci.
  • RNA molecule dF5 CrFK cell lines expressing RNA molecule dF5 were also tested.
  • Poly(rA)-oligo(dT) was used as the template/primer complex in the experiments. All data were determined from two or more experiments with three determinants per experiment.
  • Target protein tRNAmeH'gand RNA sequence 8 dJ r SEQ target protein ID NO
  • the first three Iines are the tRNA and cloning sites (SacII and Hindlll)
  • the fourth line is the 5' fixed region used for the PCR step of SELEX
  • the fifth line is the sequence that was randomized for SELEX
  • the sixth line is the 3' fixed region used for the PCR step of SELEX
  • the seventh line is a cloning site (BamHI) and the 3' end of the transcript.
  • RNA SELEX Target lines made express ligand infected with HIV were HIV resistant 5 tRNA-tat7 HIV-l tat 35 23 6 4 tRNA-rev30A HIV-l rev 11 6 4 2 tRNA- rtwl 7 HIV-l RT 18 14 7 3
  • a HIV resistance is defined as the number of cell lines that are at least 100 fold more protected (100 fold lower p24) from HIV-l infection compared to CEMss for at least 30 days post-infection. Typically, by 30 days post-infection parental CEMss cells will produce 1,000,000 pg/ml p24 over a 4 day period.
  • Ligand dF5 is the truncated version of RNA ligand F5 according to the FIV RT binding boundary *Cell Iines are more susceptible to FIV infection than control.
  • ATTORNEY/AGENT INFORMATION (A NAME: Barry J. Swanson (B REGISTRATION NUMBER: 33,215 (C REFERENCE/DOCKET NUMBER: NEX45/PCT

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Abstract

The invention relates to methods of identifying and producing Nucleic Acid Ligands to Feline Immunodeficiency Virus (FIV) Reverse Transcriptase (RT) and homologous proteins. The Nucleic Acid Ligands are so identified and produced, one of the ligands is shown in the Figure. The invention also relates to methods for treating patients suffering from intracellularly mediated diseases or conditions by introducing oligonucleotides into the patients' cells.

Description

INTRACELLULAR ACTION OF NUCLEIC ACID LIGANDS
FIELD OF THE INVENTION
Described herein are methods for treating intracellularly-mediated diseases or conditions with oligonucleotides. Further included herein is a method for treating intracellularly-mediated diseases or conditions with Nucleic Acid Ligands. Further included is a method for diagnosing intracellularly-mediated diseases or conditions. More particularly included herein is a method for treating diseases or conditions resulting from viral pathogens, specifically a method for treating HIV-l infection.
The present invention also includes methods for identifying and preparing high-affinity Nucleic Acid Ligands to FIV RT. The method utilized herein for identifying such Nucleic Acid Ligands is called SELEX, an acronym for Systematic Evolution of Ligands by Exponential enrichment. This invention includes high affinity Nucleic Acid Ligands of FIV RT. Further disclosed are
RNA ligands to FIV RT. The oligonucleotides ofthe present invention are useful in veterinary applications, diagnostic agents, or as a model for studies of RT-targeted chemotherapy for AIDS.
BACKGROUND OF THE INVENTION
TYPE 1 HUMAN IMMUNODEFICIENCY VIRUS
The type-1 human immunodeficiency virus (HIV-l) is the etiological agent of acquired immunodeficiency syndrome (AIDS) (Levy (1993) Microbiol. Rev. 57:183-289). There are estimated to be about 1 million people infected with HIV-l in the United States and over 13 million worldwide. In some localities over 80% ofthe population is infected. The average time between HIV-l infection and death is about ten years, making the course of treatment long and expensive. There is no cure for HIV-l infection. Four mononucleoside drugs (3'-azido-3'deoxythymidine (AZT) 5'-triphosphate, ddl, ddC, and d4T) have been approved for use in the United
States for the treatment of HIV-l infection. Each of these drugs inhibits the HIV-l reverse transcriptase protein. Unfortunately, high dosages of these agents can cause undesirable side effects (Yarchoan et al. (1989) Am. J. Med. 87:191-200). In addition, the usefulness of these drugs is limited by the rapid emergence of drug resistant mutants (Larder (1989) Science 24^:1155-1158). More effective treatments or a cure for HIV-l infection would be highly desirable. As a result, other HIV-l proteins, such as HIV-l protease, are being explored as possible drug targets (Huff (1991) J. Med. Chem. 34:2305-2314; Kempf (1995) Proc. Natl. Acad. Sci. USA 22:2484-2488).
FELINE IMMUNODEFICIENCY VIRUS Feline immunodeficiency virus (FIV) is a lentivirus isolated from domestic cats suffering an AIDS-like disease (Pedersen et al. (1987) Science 2i5_:790-793; Pedersen et al. (1991) J. Am. Vet. Med. Assoc.122:1289-1305). FIV causes immune suppression and increases susceptibility to infections by other pathogens (Yamamoto et al. (1991) J. Am. Vet. Med. Assoc. 124:213-220; Ishida et al. (1990) Jpn. J. Vet. Sci. 52:645-648; Pederson et al.
(1989) Vet. Immunol. Immunopathol.21:111-129; Torten et al. (1991) J. Virol. 65:2225-2230). FIV-infected cats develop an AIDS-related complex (ARC)-like disease which progresses to the final AIDS-like stage (Pedersen et al. (1991) J. Am. Vet. Med. Assoc. 129:1289-1305). FIV has a wide tropic spectrum of host cell types. FIV can be isolated from the infected cat in
T-lymphoids (CD4+ and CD8+), B-lymphoids, macrophages, and monocytes (English et al. (1993) J. Virol. 67:5175-5186). FIV can also be found in kidney, lung, and central nervous system (Beebe et al. (1994) J. Virol. 68:). The retroviral RNA genome is replicated through a DNA intermediate by reverse transcriptase (RT) (Weiss et al. (1985) Molecular Biology of Tumor
Viruses: RNA Tumor Viruses vol. 2: Cold Spring Harbor Laboratory Press; Temin et al. (1970) Nature 226:211-213; Baltimore (1970) Nature 226:209-211). The retroviral genome consists of three open reading frames: gag, pol, and env. FIV RT encoded by the pol gene ofthe retroviral genome is a heterodimer composed of p66 and p51 subunits. FIV RT uses a particular host tRNA as a primer in vivo (Bishop (1978) Annu. Rev. Biochem. 47:35-88). The similarities of FIV and HIV-l RTs make FIV a useful model for studies of RT-targeted chemotherapy for AIDS. FIV RT has been previously shown to be similar to the HIV-l RT in physical properties, catalytic activities, and sensitivity to the 5'-triphosphates of several nucleoside analogs that display anti-HIV activity, including AZT, 2',
3'-dideoxynucleotides (ddATP, ddCTP, ddGTP and ddTTP) and 2', 3'-dideoxy-2', 3'-didehydrothymidine (d4T) 5 '-triphosphate (Cronn et al. (1992) Pharmacol 44:1375-1381; Remington et al. (1994) Virol. 6J5:632-637). The FIV and HIV-l RTs are also similar in sensitivity to phosphonoformate (PFA), but differ in that FIV RT is not sensitive to the other non-nucleoside inhibitors such as nevirapine (BIRG-587) and TIBO compounds, which are potent inhibitors ofthe HIV-l RT. Although very similar to one another, the FIV and HIV-l RTs are quite different from the RT of avian myeloblastosis virus in susceptibility to antiviral nucleoside analogs (Remington et al. (1994) Virol. 68:632-637).
The FIV system has been proven to be useful for studies of resistance to antiviral drugs (Remington et al. (1994) Virol. 68:632-637; Remington et al. (1991) J. virol. 65:308-312; Gobert et al. (1994) Antimicrob. Agents Chemother. 38:861-864). It was reported that AZT-resistant variants of FIV were isolated by in vitro selection (Remington et al. (1991) J. virol.
65:308-312). Those mutants share phenotypic similarities to the AZT-resistant mutants of HIV-l isolated from AIDS patients (Remington et al. (1994) Virol. 68:632-637) (Larder et al. (1989) Science. 243:1731-1734; Larder et al. (1989) Science 246:1155-1158; Remington et al. (1991) J. virol. 65:308-312).
GENE THERAPY
Gene therapy can be defined as the transfer of new genetic material to the cells of an individual with resulting Therapeutic benefit to the individual (Morgan and Anderson (1993) Ann Rev Biochem 62:191-217). Several gene transfer methods are known and include chemical techniques such as calcium phosphate coprecipitation (Graham and van der Eb (1973) Virology 52:456-467; Pellicer et al (1980) Science 202:1414-1422); mechanical techniques, such as microinjection (Anderson et al. (1980) Proc. Natl. Acad. Sci. USA 27:5399-5403) or particle-mediated delivery ("biolistics") (Sanford et al. (1993) Meth. Enz. 212:483-509), membrane fusion-mediated transfer via liposomes (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA £4:7413-7417), naked DNA uptake (Wolff et al (1990) Science 242:1465-1468) and receptor-mediated DNA transfer (Leamon and Low (1990) Proc. Natl. Acad. Sci. USA 88:5572-5576). Viral vectors are also used in gene transfer and include retroviruses and adenoviruses. Vectors derived from retroviruses have been widely used for gene expression (Gilboa (1986) BioEssay 5_:252-257). Retroviral genomic RNA is replicated into double-stranded DNA (proviral DNA), which is integrated into the host chromosome where it utilizes host machinery for gene expression. The amphotropic murine leukemia virus-derived vector, which maintains the packaging signal, but lacks the viral structural protein genes (which are replaced by a neomycin-resistant gene as a selection marker), has been used successfully to deliver genes (Sullenger et al. (1990) Mol. Cell. Biol. 10:6512-6523).
SELEX
A method for the in vitro evolution of Nucleic Acid molecules with highly specific binding to Target molecules has been developed. This method, Systematic Evolution of Ligands by Exponential enrichment, termed SELEX, is described in United States Patent Application Serial No. 07/536,428, entitled "Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, United States Patent Application Serial No. 07/714,131, filed June 10, 1991, entitled "Nucleic Acid Ligands," United States Patent Application Serial No. 07/931,473, filed August 17, 1992, entitled "Nucleic Acid Ligands," now United States Patent No. 5,270,163 (see also WO 91/19813) which is herein specifically incoφorated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a Nucleic Acid Ligand to any desired Target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step- wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of Nucleic Acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the Target under conditions favorable for binding, partitioning unbound Nucleic Acids from those Nucleic Acids which have bound specifically to
Target molecules, dissociating the Nucleic Acid-Target complexes, amplifying the Nucleic Acids dissociated from the Nucleic Acid-Target complexes to yield a ligand-enriched mixture of Nucleic Acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity Nucleic Acid Ligands to the
Target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, United States Patent Application Serial No. 07/960,093, filed October 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure," describes the use of SELEX in conjunction with gel electrophoresis to select Nucleic Acid molecules with specific structural characteristics, such as bent DNA. United States Patent Application Serial No. 08/123,935, filed September 17, 1993, entitled "Photoselection of Nucleic Acid Ligands" describes a SELEX based method for selecting Nucleic Acid Ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a Target molecule. United States Patent Application Serial No. 08/134,028, filed October 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine," describes a method for identifying highly specific Nucleic Acid Ligands able to discriminate between closely related molecules, termed Counter-SELEX. United States Patent Application Serial No. 08/143,564, filed October 25, 1993, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Solution SELEX," describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a Target molecule. United States Patent Application Serial No. 07/964,624, filed October 21, 1992, entitled "Methods of Producing Nucleic Acid Ligands" describes methods for obtaining improved Nucleic Acid Ligands after SELEX has been performed. United States Patent Application Serial No. 08/400,440, filed March 8, 1995, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chemi-SELEX," describes methods for covalently linking a ligand to its
Target.
The SELEX method encompasses the identification of Wgh-affinity Nucleic Acid Ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified Nucleic Acid Ligands containing modified nucleotides are described in United States Patent Application Serial No. 08/117,991, filed September 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. United States Patent Application Serial No. 08/134,028, supra, describes highly specific Nucleic Acid Ligands containing one or more nucleotides modified with 2'-amino (2'-NH2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). United States Patent Application Serial No. 08/264,029, filed June
22, 1994, entitled "Novel Method of Preparation of 2' Modified Pyrimidine Intramolecular Nucleophilic Displacement," describes oligonucleotides containing various 2'-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in United States Patent Application Serial No. 08/284,063, filed August 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX" and United States Patent Application Serial No. 08/234,997, filed April 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX," respectively. These applications allow the combination ofthe broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. Each ofthe above described patent applications which describe modifications ofthe basic SELEX procedure are specifically incoφorated by reference herein in their entirety.
SELEX LIGANDS TO HIV-l PROTEINS.
SELEX-derived Nucleic Acid Ligands to HIV-l proteins have been previously described (see U.S. Patent No. 5,270,163; U.S. Patent Application No. 07/964,624, filed October 21, 1992, entitled "Method of Producing
Nucleic Acid Ligands"; U.S. Patent Application No. 08/243,870, filed May 17, 1994, entitled "Ligands of HIV-l tat Protein"; U.S. Patent Application No. 08/238,863, filed May 6, 1994, entitled "High Affinity ssDNA Ligands of HIV-l Reverse Transcriptase"; U.S. Patent Application No. 08/361,795, filed December 21, 1994, entitled "High Affinity HIV Integrase Inhibitors" ;
U.S. Patent Application No. 08/447,172, filed May 19, 1995, entitled "High Affinity HIV-l GAG Nucleic Acid Ligands"; U.S. Patent Application No. 08/477,830, filed June 7, 1995, entitled "High Affinity HIV Nucleocapsid Nucleic Acid Ligands"). SELEX-derived Nucleic Acid Ligands that bind to the HIV- 1 tat (e.g. , ligand tat7) and rev proteins (e.g. , ligand rev30A) were derived as described in Tuerk and Waugh (1993) Gene 137:33-39: Tuerk et al. (1994) in The Polymerase Chain Reaction, eds. Ferre et al. (Birkhauser, Springer-Verlag, New York). A 30 base long SELEX-derived ligand (ligand lig2) that binds to the HIV reverse transcriptase protein has been previously described in Tuerk et al. (1992) Proc. Natl. Acad. Sci. USA 8£:6988-6992.
This "pseudoknot" ligand was extended by 30 additional bases at the 3' end and SELEX was repeated to derive an extended reverse transcriptase ligand (ligand rtwl7) as described in U.S. Patent Application Serial No. 07/964,624, filed October 21, 1992, entitled "Method of Producing Nucleic Acid Ligands." SELEX-derived Nucleic Acid Ligands that bind integrase, gag, and nucleocapsid have also been described (Allen et al. (1995) Virology
202:235; U.S. Patent Application No. 08/361,795, filed December 21, 1994, entitled "High Affinity HIV Integrase Inhibitors"; U.S. Patent Application No. 08/447,172, filed May 19, 1995, entitled "High Affinity HIV-l GAG Nucleic Acid Ligands"; U.S. Patent Application No. 08/467,362, filed June 6, 1995, entitled "High Affinity HIV Nucleocapsid Nucleic Acid Ligands")
BRIEF SUMMARY OF THE INVENTION
Described herein are methods for treating intracellularly- mediated diseases or conditions with oligonucleotides. Further included herein are methods for treating intracellularly-mediated diseases or conditions with Nucleic Acid Ligands. Also included is a method for diagnosing intracellularly-mediated diseases or conditions. Further included herein is a method for treating diseases or conditions resulting from viral pathogens. Also included in the invention is a method for treating HIV-l . In one embodiment of the present invention is a method of identifying and producing Nucleic Acid Ligands to Feline Immunodeficiency Virus (FIV) Reverse Transcriptase (RT) and homologous proteins and the Nucleic Acid Ligands so identified and produced. In particular, RNA sequences are provided that are capable of binding specifically to FIV RT. Specifically included in the invention are the RNA ligand sequences shown in Figures 1 A -
1H (SEQ ID N0S:6-12).
Further included in this invention is a method of identifying Nucleic Acid Ligands and Nucleic Acid Ligand sequences to FIV RT comprising the steps of (a) preparing a Candidate Mixture of Nucleic Acids, (b) contacting the Candidate Mixture of Nucleic Acids with FIV RT, (c) partitioning between members of said Candidate Mixture on the basis of affinity to FIV RT, and (d) amplifying the selected molecules to yield a mixture of Nucleic Acids enriched for Nucleic Acid sequences with a relatively higher affinity for binding to FIV RT.
More specifically, the present invention includes the RNA ligands to FIV RT identified according to the above-described method, including those ligands shown in Figures 1 A -IH (SEQ ID NOS:6-12). Also included are RNA ligands to FIV RT that are substantially homologous to any ofthe given ligands and that have substantially the same ability to bind FIV RT and inhibit the function of FIV RT. Further included in this invention are Nucleic Acid Ligands to FIV RT that have substantially the same structural form as the ligands presented herein and that have substantially the same ability to bind FIV RT and inhibit the function of FIV RT. The present invention also includes modified nucleotide sequences based on the RNA ligands identified herein and mixtures ofthe same.
BRIEF DESCRIPTION OF THE FIGURES
Figures 1 A - IH show the sequences ofthe selected ligands to FIV RT and shows their possible secondary structures. Each subset of three classes were analyzed by an RNA folding program (Barta et al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81:3607-3611; Shelness et al. (1985) J. Biol. Chem.
260:8637-8646 . The sequences of full length RNA were input into mfold program in GCG. The sequences in the figure only show the randomized region; however, the ligands also include the fixed region as described in Example 1. The fixed regions fold into stem-loop structures which do not interfere with the randomized region. Bold uppercase letters indicate the consensus sequences. Y represents pyrimidine; R represents purine; and N can be any ofthe four nucleotides; The name, Kd value and the frequency are also shown in the figure.
Figure 2 (SEQ ID NO: 10) summarizes the FIV RT binding boundary of ligand F5. The boxed region (SEQ ID NO: 13) shows the FIV RT binding boundary. The lower case letters indicate the fixed sequences. The secondary structure was obtained by a computer RNA folding algorithm.
Figure 3 is a diagram ofthe pNEW6 retroviral expression vector, a) Structure of pNEW6 plasmid. b) Structure ofthe NEW6 provirus in infected cells. pNEW6 is a double-copy type of retroviral vector. After reverse transcription the 3' LTR is copied to the 5' end. c) Structure ofthe LTR of a NEW6 provirus. The pol II and pol III transcripts are shown as arrows and the primers used for RT-PCR are shown, d) Structure ofthe tRNA-HIV Nucleic Acid Ligand RNA chimeric gene.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS:
"Nucleic Acid Ligand" as used herein is a non-naturally occurring Nucleic Acid having a desirable action on a Target which comprises two or more nucleotides. A desirable action includes, but is not limited to, binding of the Target, catalytically changing the Target, reacting with the Target in a way which modifies/alters the Target or the functional activity ofthe Target, covalently attaching to the Target as in a suicide inhibitor, facilitating the reaction between the Target and another molecule. In the preferred embodiment, the action is specific binding affinity for a Target molecule, such Target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the Nucleic Acid Ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein the Nucleic Acid Ligand is not a Nucleic Acid having the known physiological function of being bound by the Target molecule. In preferred embodiments ofthe invention, the Nucleic Acid Ligands ofthe invention are identified by the SELEX methodology. Nucleic Acid Ligands include Nucleic Acids that are identified from a Candidate Mixture of Nucleic Acids, said Nucleic Acid Ligand being a ligand of a given Target by the method comprising: a) contacting the Candidate Mixture with the Target, wherein Nucleic Acids having an increased affinity to the Target relative to the Candidate Mixture may be partitioned from the remainder ofthe Candidate Mixture; b) partitioning the increased affinity Nucleic Acids from the remainder ofthe Candidate Mixture; and c) amplifying the increased affinity Nucleic Acids to yield a ligand-enriched mixture of Nucleic Acids.
"Candidate Mixture" is a mixture of Nucleic Acids of differing sequence from which to select a desired ligand. The source of a Candidate Mixture can be from naturally-occurring Nucleic Acids or fragments thereof, chemically synthesized Nucleic Acids, enzymatically synthesized Nucleic Acids or Nucleic Acids made by a combination ofthe foregoing techniques.
In a preferred embodiment, each Nucleic Acid has fixed sequences surrounding a randomized region to facilitate the amplification process.
"Nucleic Acid" means either DNA, RNA, single-stranded or double-stranded and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that incoφorate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the Nucleic Acid Ligand bases or to the Nucleic Acid Ligand as a whole. Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping. "SELEX" methodology involves the combination of selection of
Nucleic Acid Ligands which interact with a Target in a desirable manner, for example binding to a protein, with amplification of those selected Nucleic Acids. Iterative cycling ofthe selection amplification steps allows selection of one or a small number of Nucleic Acids which interact most strongly with the Target from a pool which contains a very large number of Nucleic Acids.
Cycling ofthe selection/amplification procedure is continued until a selected goal is achieved. In the present invention, the SELEX methodology can be employed to obtain a Nucleic Acid Ligand to a desirable Target.
The SELEX methodology is described in the SELEX Patent Applications. "SELEX Target" means any compound or molecule of interest for which a ligand is desired. A Target can be a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc. without limitation. "Target" means a preselected location in a biological system including tissues, organs, cells, intracellular compartments, extracellular components. The latter include hormones (endocrine paracrine, autocrine), enzymes, neurotransmitters and constituents of physiological cascade phenomena (e.g., blood coagulation, complement, etc.). "Intracellular Target," means anything that is existing, occurring, or functioning within a cell or cellular compartment. This includes, but is not limited to, organelles, enzymes, proteins, viral pathogens and intracellular bacteria.
"Gene Therapy," means the transfer of new genetic material to the cells of an organism with resulting Therapeutic benefit to the organism.
"Therapeutic," as used herein, includes treatment and/or prophylaxis. When used, Therapeutic refers to humans and other animals.
"Intracellularly-mediated Disease or Condition," is any disease or condition that originates from within the cell or has an adverse affect on the cell and impairs normal functioning of a cell, tissue, organ, or organism.
"Ligand Chimeric Gene," is a Nucleic Acid Ligand that has been fused to a gene which has the transcriptional regulatory elements that allow for its expression and intracellular localization.
SELEX is described in U.S. Patent Application Serial No. 07/536,428, entitled Systematic Evolution of Ligands by Exponential Enrichment, now abandoned, U.S. Patent Application Serial No. 07/714,131, filed June 10, 1991, entitled Nucleic Acid Ligands, United States Patent Application Serial No. 07/931,473, filed August 17, 1992, entitled Nucleic Acid Ligands, now United States Patent No. 5,270,163, (see also WO 91/19813). These applications, each specifically incoφorated herein by reference, are collectively called the SELEX Patent Applications.
In its most basic form, the SELEX process may be defined by the following series of steps:
1) A Candidate Mixture of Nucleic Acids of differing sequence is prepared. The Candidate Mixture generally includes regions of fixed sequences (i.e., each ofthe members ofthe Candidate Mixture contains the same sequences in the same location) and regions of randomized sequences. The fixed sequence regions are selected either: (a) to assist in the amplification steps described below, (b) to mimic a sequence known to bind to the Target, or (c) to enhance the concentration of a given structural arrangement ofthe Nucleic Acids in the Candidate Mixture. The randomized sequences can be totally randomized (i.e., the probability of finding a base at any position being one in four) or only partially randomized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).
2) The Candidate Mixture is contacted with the selected Target under conditions favorable for binding between the Target and members ofthe
Candidate Mixture. Under these circumstances, the interaction between the Target and the Nucleic Acids ofthe Candidate Mixture can be considered as forming Nucleic Acid-Target pairs between the Target and those Nucleic Acids having the strongest affinity for the Target. 3) The Nucleic Acids with the highest affinity for the Target are partitioned from those Nucleic Acids with lesser affinity to the Target. Because only an extremely small number of sequences (and possibly only one molecule of Nucleic Acid) corresponding to the highest affinity Nucleic Acids exist in the Candidate Mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the Nucleic Acids in the Candidate Mixture
(approximately 5-50%) are retained during partitioning. 4) Those Nucleic Acids selected during partitioning as having the relatively higher affinity to the Target are then amplified to create a new Candidate Mixture that is enriched in Nucleic Acids having a relatively higher affinity for the Target. 5) By repeating the partitioning and amplifying steps above, the newly formed Candidate Mixture contains fewer and fewer weakly binding sequences, and the average degree of affinity ofthe Nucleic Acids to the Target will generally increase. Taken to its extreme, the SELEX process will yield a Candidate Mixture containing one or a small number of unique Nucleic Acids representing those Nucleic Acids from the original Candidate Mixture having the highest affinity to the Target molecule.
The SELEX Patent Applications describe and elaborate on this process in great detail. Included are Targets that can be used in the process; methods for partitioning Nucleic Acids within a Candidate Mixture; and methods for amplifying partitioned Nucleic Acids to generate enriched Candidate Mixture.
The SELEX Patent Applications also describe ligands obtained to a number of target species, including both protein Targets where the protein is and is not a Nucleic Acid binding protein.
In one embodiment ofthe present invention, the methods described herein and the Nucleic Acid Ligands identified by such methods are useful for both Therapeutic and diagnostic puφoses. Therapeutic uses include the treatment or prevention of diseases or medical conditions in felines. Diagnostic utilization may include both in vivo or in vitro diagnostic applications. The SELEX method generally, and the specific adaptations of the SELEX method taught and claimed herein specifically, are particularly suited for diagnostic applications. SELEX identifies Nucleic Acid Ligands that are able to bind Targets with high affinity and with suφrising specificity. These characteristics are, of course, the desired properties one skilled in the art would seek in a diagnostic ligand. The Nucleic Acid Ligands ofthe present invention may be routinely adapted for diagnostic puφoses according to any number of techniques employed by those skilled in the art. Diagnostic agents need only be able to allow the user to identify the presence of a given Target at a particular locale or concentration. Simply the ability to form binding pairs with the Target may be sufficient to trigger a positive signal for diagnostic puφoses. Those skilled in the art would also be able to adapt any Nucleic Acid Ligand by procedures known in the art to incoφorate a labeling tag in order to track the presence of such ligand. Such a tag could be used in a number of diagnostic procedures. The Nucleic Acid Ligands to FIV RT described herein may specifically be used for identification of FIV RT. SELEX provides high affinity ligands of a Target molecule. This represents a singular achievement that is unprecedented in the field of nucleic acids research. One embodiment ofthe present invention applies the SELEX procedure to the specific Target FIV RT. In the Example section below, the experimental parameters used to isolate and identify the Nucleic Acid Ligands to FIV RT are described.
In order to produce Nucleic Acids desirable for use as a pharmaceutical, it is preferred that the Nucleic Acid Ligand (1) binds to the Target in a manner capable of achieving the desired effect on the Target; (2) be as small as possible to obtain the desired effect; (3) be as stable as possible; and (4) be a specific ligand to the chosen Target. In most situations, it is preferred that the Nucleic Acid Ligand have the highest possible affinity to the Target.
In co-pending and commonly assigned U.S. Patent Application Serial No. 07/964,624, filed October 21, 1992 ('624), methods are described for obtaining improved Nucleic Acid Ligands after SELEX has been performed.
The '624 application, entitled Methods of Producing Nucleic Acid Ligands, is specifically incoφorated herein by reference.
The present invention includes methods of treating intracellularly-mediated diseases or conditions with oligonucleotides. Such diseases include, but are not limited to cancer; infectious diseases, such as
AIDS, cytomegalovirus retinitis, hepatitis, infectious mononucleosis, Leischmaniasis, candidiasis, malaria, influenza; dominant genetic disorders, such as polycystic kidney disease, Charcot-Marie-Tooth disease, Stargardt's disease, Parkinson's disease, Alzheimer's disease, Schizophrenia, Artherosclerosis, and cancers such as those involving dominant RAS mutations. A number of recessive genetic diseases result in oveφroduction or accumulation of proteins or molecules that could be bound or inhibited by oligonucleotides, induding Nucleic Acid Ligands. For example, defects in the LDL receptor leads to hypercholesterolemia. Treatment comprises the introduction of oligonucleotides into cells whereby the oligonucleotide affects the activity of the Intracellular Target and thereby the disease condition is attenuated. In the preferred embodiment, the oligonucleotide inhibits the activity ofthe Intracellular Target. For the puφose of this invention, treatment also includes prophylaxis. Introduction ofthe oligonucleotide can be a one time administration or as part of a regimen. In addition, the oligonucleotide can be transiently or stably expressed depending on the type of gene or Nucleic Acid transfer procedure employed. For the pmpose of this invention, an oligonucleotide comprises two or more nucleotides. More preferably, the oligonucleotide comprises 3 or more nucleotides. In the preferred embodiment, the oligonucleotide comprises 7 or more nucleotides. The oligonucleotide can act in any fashion to affect the activity ofthe
Intracellular Target except by binding to another Nucleic Acid by Watson-Crick base-pairing. Also included in the present invention is a method of treating intracellularly-mediated diseases or conditions with Nucleic Acid Ligands. In this embodiment, it is preferred that the Nucleic Acid Ligands are identified by the SELEX methodology. Further included is a method of diagnosing intracellularly-mediated diseases or conditions with oligonucleotides, including Nucleic Acid Ligands. In the preferred embodiment, the Nucleic Acid Ligands are identified by the SELEX methodology. The present invention further includes methods for treating or diagnosing diseases or conditions resulting from viral pathogens. In the preferred embodiment, the methods ofthe invention can be used in the treatment or diagnosis of HIV-l infection by inserting Nucleic Acid Ligands specific for proteins involved in the replication of HIV-l into cells. The Nucleic Acid Ligands inhibit the function of these proteins thereby preventing the replication of HIV-l . The preferred targeted HIV-l proteins are tat, rev, and reverse transcriptase.
The Nucleic Acid Ligand(s) can be inserted into the cells using any gene or Nucleic Acid transfer procedure or combination of procedures. The cells can be genetically engineered in vivo or in vitro. For example, cells can be removed from a patient, genetically engineered in vitro with the Nucleic Acid Ligand (either DNA or RNA) and the cells containing the Nucleic Acid
Ligand(s) can be readministered to a patient. This procedure is referred to herein as ex vivo treatment. The engineered cells now will express the Nucleic Acid Ligand and allow it to affect the activity ofthe Intracellular Target.
Alternatively, the Nucleic Acid Ligand can be administered to a patient for delivery ofthe Nucleic Acid Ligand in vivo to the targeted cells.
Gene transfer methods fall into three broad categories - biological (e.g., virus-derived vectors and receptor uptake), chemical (e.g., lipid-based carriers) and physical (e.g., microinjection, electroporation) (see Morgan and Anderson (1993) Ann. Rev. Biochem. 62:191-217 for review). In a preferred embodiment, retroviral-mediated gene transfer is used. Retroviruses are currently the most widely used transducing agent (Goff and Shenk (1993) Current Opinion in Genetics and Development 2:71-73). The life cycle ofthe retroviruses makes these agents a natural choice for the delivery of Nucleic Acid Ligands into target cells. Viral vectors can be designed that retain no intact viral genes and only a minimum of viral sequences. Early in infection, the viral RNA genome is reverse transcribed into duplex DNA, and the DNA copy is efficiently integrated into the host genome.
Other vectors can be used besides retroviral vectors, including those derived from other RNA viruses and DNA viruses. For example, adenoviruses, a family of DNA viruses, have many advantages, such as their potential to carry large segments of DNA and suitability for infecting tissues in situ (Miller (1992) Nature 252:455-459). In embodiments where the Nucleic Acid Ligand is RNA, it is well known to one skilled in the art to produce the DNA complement for incoφoration into the adenovirus.
The viral vectors can be engineered to direct the expression ofthe transduced Nucleic Acid Ligands under a variety of different transcriptional regulatory sequences, as would be known to one of skill in the art. In addition, the Target ofthe Nucleic Acid Ligand may be present in the nucleus or the cytoplasm. Thus, other localization signals, such as sequences that direct the Nucleic Acid Ligand transcript to remain in the nucleus or to go to the cytoplasm can also be incoφorated into the viral vector, as would be known to one of skill in the art. For example, the snRNAs (e.g. , U6) are involved in RNA splicing and thus remain in the nucleus. The tRNAs are involved in translation and are thus primarily found in the cytoplasm. It would be known to one of skill in the art that fusions of Nucleic Acid Ligands to other RNA (e.g. , U6 or tRNA) can be made in a manner that would not disrupt their intracellular localization.
Expression ofthe Nucleic Acid Ligand is not required, and therefore stable integration into the cell's genome is not necessary. Thus, other gene transfer methods can be employed (e.g., receptor-mediated, microinjection). Receptor-mediated methods of gene transfer involve complexing plasmid DNA and specific polypeptide ligands that are recognized by receptors on a cell surface (Mulligan (1993) Science 260:926-932). Nucleic acid ligands which are RNA can also be complexed with specific polypeptide ligands for receptor-mediated uptake. The Nucleic Acid Ligands can be complexed with a lipophilic compound (e.g., cholesterol) or attached to or encapsulated in a complex comprised of lipophilic components (e.g., a liposome). The complexed Nucleic Acid Ligands can enhance the cellular uptake ofthe Nucleic Acid Ligands by a cell for delivery ofthe Nucleic Acid Ligands to an Intracellular Target. U.S. Patent Application No. 08/434,465, filed May 4, 1995, entitled
"Nucleic Acid Ligand Complexes," which is incoφorated in its entirety herein, describes a method for preparing a Therapeutic or diagnostic complex comprised of a Nucleic Acid Ligand and a lipophilic compound or a non-immunogenic, high molecular weight compound.
In addition, Nucleic Acid Ligands can be introduced into cells by applying intense electric fields (electroporation). High electric fields make membranes transiently permeable to large molecules, such as DNA and RNA. Direct DNA uptake can also be used to deliver Nucleic Acid Ligands to cells. A plasmid is constructed that encodes the Nucleic Acid Ligand and this plasmid is injected into tissues. It is also possible to directly deliver a Nucleic Acid Ligand that is not fused to other RNAs by incoφorating it into a viral particle. This would be possible if the ligand bound to a structural component ofthe virus (such as RT) which ultimately is found intracellularly. Since a ligand RNA and not a Ligand Chimeric Gene would be delivered in this case, the action ofthe ligand may be transient. Moreover, for treating acute intracellularly-mediated diseases there could be advantages over using stable genes to treat chronic conditions. Such ligands would be incoφorated into a murine retrovirus used for Gene Therapy by expressing the appropriate ligand and viral protein in the same packaging cell used to produce the virus. One potential problem encountered in the Therapeutic and in vivo diagnostic use of Nucleic Acids is that oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is manifest. Certain chemical modifications ofthe Nucleic Acid Ligand can be made to increase the in vivo stability ofthe Nucleic Acid
Ligand or to enhance or to mediate the delivery ofthe Nucleic Acid Ligand. Modifications ofthe Nucleic Acid Ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that incoφorate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the Nucleic Acid Ligand bases or to the Nucleic Acid Ligand as a whole. Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping.
Where the Nucleic Acid Ligands are derived by the SELEX method, the modifications can be pre- or post- SELEX modifications. Pre-SELEX modifications yield Nucleic Acid Ligands with both specificity for their
SELEX Target and improved in vivo stability. Post-SELEX modifications made to 2'-OH Nucleic Acid Ligands can result in improved in vivo stability without adversely affecting the binding capacity ofthe Nucleic Acid Ligands. This invention includes the SELEX process for identification of Nucleic Acid Ligands of FIV RT.
In one embodiment ofthe present invention, SELEX experiments were performed in order to identify RNA with specific high affinity for FIV RT from a degenerate library containing 40 random positions (Figures 1 A - IH). This invention includes the specific RNA ligands to FIV RT (SEQ ID NOS:6- 12), identified by the methods described in Examples 1 and 2. The scope of the ligands covered by this invention extends to all Nucleic Acid Ligands of FIV RT modified and unmodified, identified according to the SELEX procedure. More specifically, this invention includes Nucleic Acid sequences that are substantially homologous to the ligands shown in Figures 1 A - IH (SEQ ID NOS: 6- 12). By substantially homologous it is meant a degree of primary sequence homology in excess of 70%, most preferably in excess of 80%. A review ofthe sequence homologies ofthe ligands of FIV RT shown in Figures 1 A - IH (SEQ ID NOS:6-12) shows that sequences with little or no primary homology may have substantially the same ability to bind FIV RT. For these reasons, this invention also includes Nucleic Acid Ligands that have substantially the same structure and ability to bind FIV RT as the Nucleic Acid Ligands shown in Figures 1 A - IH (SEQ ID NOS:6-12). Substantially the same ability to bind FIV RT means that the affinity is within one or two orders of magnitude ofthe affinity ofthe ligands described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence — substantially homologous to those specifically described herein — has substantially the same ability to bind FIV RT.
This invention also includes the ligands as described above, wherein certain chemical modifications are made in order to increase the in vivo stability ofthe ligand or to enhance or mediate the delivery ofthe ligand. Examples of such modifications include chemical substitutions at the sugar and/ or phosphate and/or base positions of a given Nucleic Acid sequence. See, e.g., U.S. Patent Application Serial No. 08/117,991, filed September 9, 1993, entitled High Affinity Nucleic Acid Ligands Containing Modified Nucleotides which is specifically incoφorated herein by reference. Other modifications are known to one of ordinary skill in the art. Such modifications may be made post-SELEX (modification of previously identified unmodified ligands) or by incoφoration into the SELEX process. As described above, because of their ability to selectively bind FIV RT, the ligands described herein are useful in veterinary applications. This invention, therefore, also includes a method of inhibiting FIV RT function by administration of a Nucleic Acid Ligand capable of binding to FIV RT.
The following Examples are provided to explain and illustrate the present invention and are not intended to be limiting ofthe invention. Example 1 describes the general procedures followed in Example 2 for the evolution of Nucleic Acid Ligands to FIV RT. Example 2 describes the Nucleic Acid Ligands to FIV RT. Example 3 describes the experimental procedures used in expressing a HIV-l Nucleic Acid Ligand in human cells. Example 4 describes the protection of cells from HIV-l infection by a HIV-l Nucleic Acid Ligand. Example 5 describes the experimental procedures used in expressing FIV RT ligands in cells. Example 6 describes the inhibition of FIV replication in cells by selected inhibitory ligands.
EXAMPLE 1. EXPERIMENTAL PROCEDURES
This example provides general procedures followed and incoφorated in Example 2 for the evolution of Nucleic Acid Ligands to FIV RT.
Materials
Recombinant FIV RT was purified from E. coli containing the clone pRFT14 (North et al. (1989) Antimicrob. Agents Chemother. 22:915-919); virion FIV RT was purified as previously described (North et al. (1989)
Antimicrob. Agents Chemother. 22:915-919); and FIV RT from an AZT resistant mutant, AZR-17c was purified as previously reported (Remington et al. (1994) Virol. 68:632-637). The latter enzyme has decreased susceptibility to the 5 '-triphosphate of AZT. AMV reverse transcriptase was purchased from Life Sciences, Inc., M-MLV reverse transcriptase was purchased from GIBCO
BRL, HIV-l RT was generously provided by Agouron Pharmaceuticals Inc., Taq DNA polymerase was purchased from Perkin Elmer Cetus, T4 polynucleotide kinase from New England Biolabs, and T7 RNA polymerase from U.S. Biochemical Coφoration. DNA and RNA oligonucleotides were synthesized on an Applied Biosystems Model 394 DNA/RNA synthesizer.
Selection Procedure
High affinity RNA ligands were selected from an RNA repertoire containing IO14 unique species (Tuerk et al. (1990) Science. 249:505-510: Tuerk et al. (1992) Proc. Natl. Acad. Sci. 89:6988-6992; Chen et al. (1994)
Biochemistry. 22:8746-8756). The first 10 rounds of selection were performed by nitrocellulose filter partitioning in 1 mL binding buffer (50 mM Tris-HCl, pH 7.7, 200 mM potassium acetate and 10 mM dithiothreitol) containing RNA and Target protein. The binding reaction was incubated at 37°C for 10 minutes and bound RNA was partitioned by nitrocellulose filtration. The bound RNA was eluted into 200 μL 7 M urea and 400 μL phenol as described previously (Tuerk et al. (1992) Proc. Natl. Acad. Sci. £2:6988-6992), and recovered by ethanol precipitation. The last 8-rounds of selections were performed by native gel shift assay (Carey et al. (1991) Methods Enzymol. 208:103-117) in 52 μL binding buffer containing 7% glycerol with appropriate RNA and protein concentrations. The binding reaction was incubated at 37°C for 10 minutes and the sample was loaded on a 8% native poly-acrylamide gel (acrylamide: bis-acrylamide is 80:1) using 25 mM Tris, 192 mM glycine, and 1 mM EDTA, pH 8.3 running buffer. Electrophoresis was carried out at 4°C for 2 hours at 200 volts. After autoradiography, RNA in the RNA-RT complexes was purified. cDNA was synthesized by AMV RT at 45°C. The cDNA product was amplified by PCR and transcribed with T7 RNA polymerase to generate the RNA pool for the next round of selection (Beard et al. (1952) Natl. Cancer Conf. Proc. 2:1396-1411; Larder et al. (1989) Science. 242:1731-1734).
Boundary Analysis
Determination ofthe 5' and 3' boundaries required for protein binding were performed as described by Tuerk et al. (Tuerk et al. (1990) J. Mol. Biol. 212:749-761; Chen et al. (1994) Biochemistry. 22:8746-8756). Briefly, RNA was partially hydrolyzed by incubation in 50 μL of 50 mM sodium-carbonate pH 9.0, 1 mM EDTA, and 0.5 mg/mL yeast tRNA at 90°C for 15 minutes. The reaction was neutralized by addition of 6 μL 3 M sodium-acetate pH 5.2 followed by ethanol precipitation. The 3' boundary was determined by incubating 30 pmoles of partially alkaline hydrolyzed 32P 5 '-end labeled RNA with protein in 1 mL binding buffer as described previously (Tuerk et al.
(1990) J. Mol. Biol. 212:749-761; Chen et al. (1994) Biochemistry. 22:8746-8756). Similarly, the 5' boundary was determined by incubating partially alkaline hydrolyzed 3' 32pCp labeled RNA with protein, as described above. Inhibition of RT Polymerization Activity
The polymerization inhibition assay was performed as described previously (Tuerk et al. (1992) Proc. Natl. Acad. Sci. 82:6988-6992; Chen et al. (1994) Biochemistry. 22:8746-8756). The reaction contained 17.4 nM RT, 10 nM template/32P 5'-end labeled primer complex, and RNA ligand. The inhibition assay was performed at 37°C for 10 minutes in 20 μL polymerization buffer (50 mM Tris-HCl pH 7.7, 200 mM potassium-acetate, 6 mM MgCl2, 10 mM DTT, 25 μg/mL BSA, and 0.4 mM dNTPs). The polymerization products were analyzed by electrophoresis on a 7 M urea, 10% polyacrylamide gel.
Kinetics
FIV RT activity was assayed as described previously (North et al. (1994) Antimicrob. Agents Chemother. 38:388-391; North et al. (1990) J. Biol. Chem. 265:5121-5128 . Reactions were carried out in a volume of 50 μL and under standard conditions containing 50 mM Tris-HCl, pH 8.5, 10 mM DTT, 0.05% Triton X-100, 250 μg /mL BSA (nuclease-free), 6 mM MgCl2, 0.5 OD260 units of template/primer, the appropriate [ 3H] dNTP (33 μCi/mL, 20 μM) and enzyme. For the experiments to determine kinetic parameters, the concentration of dNTP or of template/primer was varied as indicated. The reaction mixtures were incubated at 37°C for 30 minutes, and then 40 μL samples were spotted onto Whatman No. 3 filters (2.3 cm, which were pre-soaked with 5% TCA and 1% sodium pyrophosphate). Filters were washed four times in ice-cold trichloracetic acid, 1% sodium pyrophosphate, once in 95% ethanol and the radioactivity was quantified by scintillation counting. The template-primer used in the experiments, Poly(rA)-oligo(dT)10 was resuspended in 10 mM Tris-HCl, pH 7.5, 5.0 mM NaCl, and 0.2 mM EDTA. Kinetic parameters (Km and Ki) were determined as previously reported (North et al. (1990) J. Biol. Chem. 265:5121-5128), by using intercept values calculated from double-reciprocal plots. EXAMPLE 2. HIGH-AFFINITY OLIGONUCLEOTIDES TO FIV
RT
Selection of High-Affinity Oligonucleotides to FIV RT
The starting RNA repertoire was randomized at 40 nucleotide positions flanked by a 25 nucleotide fixed region at the 5'-end and a 27 nucleotide fixed region at the 3'-end (Chen et al. (1994) Biochemistry. 22:8746-8756). The RNA sequence
5'-gggaggauauuuucucagaccguaa-N40-uugcagcaucgugaacuaggauccggg-3' (SEQ ID NO: 1) was used as a starting pool with 5'-CCCAAGCTTAATACGACTCACTATAGGGAGGATATTTTCTCAG
ACCGTAA-3' (SEQ ID NO:2), which contains T7 promoter, and 5'-CCCGGA TCCTAGTTCACGATCTGCAA-3 ' (SEQ ID NO:3) as the 5' and 3' PCR primers, respectively. The starting repertoire contained approximately IO14 unique RNA species. In this experiment, a recombinant FIV RT was used as the Target. The first 10 rounds of selection were performed by nitrocellulose filtration. The final 8 rounds of selection were performed using a native gel mobility retardation method (Carey et al. (1991) Methods Enzymol. 208:103-117) as the partitioning strategy. Two major shifted bands appeared on the native gel (not shown), and both complexes were collected for selection. After 8 rounds of selection using the gel retardation assay, the binding affinity ofthe RNA pool could not be further improved by continued selection, and the complexity ofthe round eighteen pool was determined by RNA sequence analysis (not shown). The cDNA of round- 18 pool was cloned into pUC-18 at Hind III and BamH I sites and sequenced. The FIV RT binding affinity ofthe round eighteen pool (Kd, 7.7 nM) was at least 103-fold higher than that ofthe starting repertoire.
Eighty RNA sequences from round eighteen RNA molecular pool were obtained and analyzed by an RNA folding algorithm (Zuker et al. (1989) Meth. Enzymol.110:262-288; Jaeger et al. (1989) Proc. Natl. Acad. Sci. USA. £6_:7706-7710). The selected RNA molecules fell into three major classes
(Figures 1 A - IH). The secondary structures presented in Figures 1 A - IH have the lowest free-energy. Class I molecules (containing four subsets, Fl, F2, F3, and F4 (SEQ ID NOS:6-9)) form a stem-loop or a stem-loop with internal bulge structures and contain one or two U-tract consensus sequences present in a region predicted to be single stranded. Three subsets (F2, F3, and F4 (SEQ ID NOS : 7-9)) of class I ligands also have an ACG consensus in the loop. Class II consists of three subsets of species (F5, F6, and F7 (SEQ ID NOS: 10-12)) that can form a stem-loop with internal bulge structures. All of the class II ligands have consensus AA dinucleotide in the bulge and two subsets (F6 and F7 (SEQ ID NOS: 11-12)) of this class contain an ACCA consensus in a tetra loop. Class III consists of two subsets (F8 and F9).
Members of F8 subset contain YAA repeats and members of F9 subset have a A-track sequence. YAA'repeats and A-track sequences appear to be in unstructured regions, at least as predicted by the RNA folding program (Zuker et al. (1989) Meth. Enzymol. l£0:262-288; Jaeger et al. (1989) Proc. Natl. Acad. Sci. USA. 86:7706-7710). Nine oφhan sequences were also obtained among the cDNA clones (not shown). The binding affinities of each subset of RNA molecules to FIV RT were measured by filter binding. As shown in Figures 1 A - IH, the dissociation constants were in the range of 1.9 nM to 24.0 nM.
Native Gel Mobility Shift Analysis ofthe Interaction between Selected RNA Ligands and FIV RT.
The p66/p66 homodimeric FIV RT is not as stable as the p66/p51 heterodimer. The homodimer can dissociate to form p66 monomer. In order to determine whether the selected RNA molecules bind to the dimeric protein, monomer, or both forms, gel mobility retardation assays (Carey et al. (1991) Methods Enzymol. ___.: 103-117) were performed to analyze the RNA-protein interaction of each subset of RNA molecules. The RNA molecules of class I (SEQ ID NOS:6-9) bound to the FIV RT dimer and monomer almost equally well (data not shown). The class II (SEQ ID NOS: 10-12) RNA ligands bound mainly to dimeric RT, and with less affinity to monomeric RT with the exception of ligand F5 (SEQ ID NO: 10) which could bind to both forms but prefers binding to the dimeric form. The RNA ligands of class III bound to both forms of RT with approximately equal affinity, however, an extra complex with an intermediate mobility was also present. This extra complex has not yet been identified. It may arise from different conformations of RNA interacting with the monomer protein, or binding of two RNA molecules with one monomer protein, or from the impurity ofthe recombinant FIV RT. It was also observed that the mobility of free RNA ligand of F8 subfamily was slower. The native gel mobility shift results indicated that the RNA ligands of the three different classes have different binding interactions with FIV RT.
Inhibition of RT Polymerization Activity
RNA ligands may interact with FIV RT at the active site and function as inhibitors. To test the inhibitory activities of RNA ligands of different classes and to identify the RNA ligands with the highest inhibitory activity, inhibitor screening experiments (Tuerk et al. (1992) Proc. Natl. Acad. Sci. 82:6988-6992; Chen et al. (1994) Biochemistry. 22:8746-8756) were performed using an inhibition assay. The RNA template for assaying RT activity was a fragment of plasmid pT7-l transcribed by T7 RNA polymerase (RNA sequence:
5'-GGGAAUUCGAGCUCGGUACCCGGGAUCCUCUAGA GUCGACCUGCAGGCAUGCUAGCUUGGCACUGGGCGU CGUUUUACAACGUCGUGACGUGG-3' (SEQ ID NO:4), and DNA 3'-primer sequence: 5'-CCCACGTCACGACGTTGTAAAACGACGCCC-3' (SEQ ID NO:5)). 17.4 nM RT and 10 nM template-primer complex which was 32P labeled at the 5 '-end ofthe primer were incubated with varying amounts (0, 0.10, 0.39, 1.56, 6.26, 25.00, and 100.00 nM) of RNA ligand in the concentration range from 0.1 nM to 100 nM by successive 4-fold dilution in 20 μL of reaction buffer at 37°C for 10 minutes. Samples were analyzed by electrophoresis on a 10% polyacrylamide-7 M urea gel.
The experiments showed that the RNA ligand F5 (SEQ ID NO: 10) (Kd, 4.2 nM) of class II inhibited RNA-dependent DNA polymerase activity of homodimeric FIV RT, while other RNA ligands, including members of Fl subset, were less inhibitory than ligand F5. The starting RNA pool did not inhibit FIV RT polymerization activity, even up to a concentration of 1 μM. We also tested the inhibition activity ofthe selected RNA ligand F5
(SEQ ID NO: 10) to the wild type virion (p66/p51 heterodimeric) FIV RT, and to the RT from AZT-resistant mutant FIV (AZR-17c), in wliich the amino acid glutamate at the position 202 is changed to lysine (Remington et al. (1994) Virol. 68:632-637). The inhibition experiments indicated that the RNA ligand F5 selected against recombinant FIV RT inhibited the cDNA synthesis activity of RTs from both wild-type and an AZT-resistant mutant of FIV.
In order to test the inhibition specificity of RNA ligand F5 (SEQ ID NO: 10), other RTs (AMV RT, M-MLV RT and HIV-l RT) were used in inhibition experiments. 3.2 nM AMV RT, 16 nM HIV-l RT, and 3.2 nM M-MLV RT were used in the experiments, respectively. The experiments were performed as described above with respect to inhibition of FIV RT, except that 4-fold dilutions of inhibitor were prepared with the resulting ligand concentration from 0.98 nM to 1000 nM.
The results indicated that the selected RNA F5 (SEQ ID NO: 10) specifically inhibited FIV RT, and was much less inhibitory to the other three
RTs. Therefore, the selected ligand can discriminate homologues of a protein family. The inhibition effect of other subsets ofthe FIV RT selected ligands (including Fla) ligands with moderate inhibitory effect on FIV RT did not inhibit AMV, M-MLV or HIV-l RT.
Minimum RNA Sequences Required for Binding to FIV RT.
Because RNA ligand F5 (SEQ ID NO: 10) has the highest inhibition activity, it was further analyzed by protein binding boundary determination (Tuerk et al. (1992) Proc. Natl. Acad. Sci. 82:6988-6992; Chen et al. (1994) Biochemistry. 22:8746-8756). The experiments indicated that the mimmum sequence of RNA ligand F5 required for interaction with FIV RT consists of 28 nucleotides (position U26-C53). This 28 nucleotide sequence is predicted to form a stem-loop with an internal bulge structure (Figure 2) (SEQ ID NO: 10). According to the boundary analysis, a truncated version ofthe F5 ligand (dF5) (SEQ ID NO: 13) that contains 28 nucleotides was designed (Figure 2; as shown in the boxed region). The truncated ligand dF5 (SEQ ID
NO: 13) bound to FIV RT with a Kd of about 7.8 nM, and was able to inhibit FIV RT although its activity was slightly lower than that ofthe intact ligand F5 (SEQ ID NO: 10). As expected, the truncated ligand dF5 (SEQ ID NO: 13) could also inhibit wild type FIV RT, as well as the AZT-resistant mutant. A native gel retardation experiment indicated that the truncated ligand dF5 could bind only to the FIV RT dimer.
Kinetic Studies.
Kinetic constants were determined for the inhibition of FIV RT by selected RNA ligand F5 (SEQ ID NO: 10) using poly(rA)-oligo(dT) as the template-primer complex (summarized in Table 1). The inhibition was competitive with respect to template-primer complex and non-competitive with respect to dTTP. The ligand F5 (SEQ ID NO: 10) also inhibited FIV RT in the reaction with poly(rI)-oligo(dC), and with heteropolymeric RNA template derived from the FIV genome, with Ki values of 57 nM and 45 nM, respectively (data not shown). The RT, from an AZT-resistant mutant AZR-17c, was also inhibited by ligand F5. The enzyme from this mutant was previously shown to have a 5 -fold higher Ki than wild type FIV RT for inhibition by the active form (5 '-triphosphate) of AZT (Remington et al. (1994) Virol. 6.8:632-637). The Ki values of ligand F5 to virion and AZR-17c
RTs were approximately equal (Table 1). Interestingly, RTs from the wild type virion and from the AZT-resistant mutant were more sensitive than the recombinant FIV RT to the selected ligand F5; the Ki value for inhibition of RT from wild type virion and from AZR-17c was 31 nM and 25 nM, respectively, while the Ki value determined for inhibition of recombinant FIV
RT was 96 nM (Table 1). EXAMPLE 3. EXPERIMENTAL PROCEDURES
This example provides general procedures followed and incoφorated in Example 4 describing the expression of HIV-l Nucleic Acid Ligands in human cells.
Materials.
Recombinant HIV-l tat protein, produced in E. coli, was purchased from Intracell, Inc. (Cambridge, MA). The HIV-l reverse transcriptase was produced in E. coli as described (Hostomsky et al. (1991) Proc. Natl. Acad.
Sci. USA £8: 1148-1152) and was generously provided by Agouron, Inc. (La Jolla, CA). The HIV-l rev protein was produced in E. coli and provided by Maria Zapp and Michael Green (Harvard Medical School, Cambridge, MA) or purchased from Intracell, Inc. The retrovirus packaging line GP+envAml2 and the retroviral expression vector pDCT-5T were obtained from Bruce Sullenger and Eli Gilboa (Duke University). Other expression vectors can be used, as would be known to one of skill in the art.
Binding Affinities of Ribonucleic Acid Ligands for Target Proteins.
The binding affinities of ribonucleic acid ligands for their Target HIV-l proteins were measured by a filtration method as described (Tuerk and Gold (1990) Science 242:505-510). Briefly, radiolabeled RNA is transcribed from a PCR-generated template using T7 RNA polymerase and α-32P-CTP. The RNA (-100 frnol; 10,000 cpm) was bound to Target HIV-l proteins ranging in concentration from IO"10 to 10"6M at 37 °C. for 5 minutes in a 30 μl reaction. The buffer used for binding consisted of 200 mM potassium acetate, 10 mM dithiothreitol, 50 mM Tris-HCl, pH 7.7. The reaction was vacuum-filtered through nitrocellulose filters (HAWP, Millipore, Coφ., Bedford, MA), the amount of labeled RNA retained on the filter was determined, and the apparent Kj ofthe protein for the RNA was obtained by plotting the amount of RNA bound vs. the concentration ofthe protein using Kaleidograph computer software (Synergy, Inc., Reading, PA).
Construction of Retrovirus Vectors. The retroviral vector pDCT-5T has been described (Lee et al. (1992)
The New Biologist 4:66-74). The retroviral vector pNEW6 (Figure 3) was constructed by replacing the polylinker of pDCT-5t (5'CCGCGGTGGATCC3') (SEQ ID NO: 14), which has Sac II and BamHI cloning sites, with one which has Hind III and BamHI cloning sites (5'CCGCGGGTCGTGTTAGAAGCTTCCCATGGATCCTTCGGGATCTG
TTCCACTAGCGATCC3' (SEQ ID NO: 15)) in order to facilitate cloning of SELEX ligands. Nucleic acid ligands were amplified by polymerase chain reaction, digested with Hind III and BamHI, and then subcloned into the Hind III and BamHI sites of pNEW6. The restriction enzymes (Hind III and BamHI) and T4 DNA ligase were obtained from New England Biolabs
(Beverly, MA). Taq polymerase was obtained from Perkin-Elmer (Norwalk, CT). All molecular cloning techniques were performed essentially as described by Sambrook, et al. (1989) Molecular Cloning: A laboratory manual (2nd ed), Cold Spring Harbor Laboratories, Cold Spring Harbor, NY). The recA' E. coli strain DH5α (Gibco, Inc., Gaithersburg, MD) was used for transformation.
Cell Line Construction.
The retroviral vectors were electroporated into the amphotropic murine leukemia retrovirus packaging cell line GP+envAml2 (Markowitz et al (1988) Virology 162:400-406). The electroporation was done at 200 volts and 960 μF in a 0.4 cm cuvette using a Gene Pulser (BioRad, Richmond, CA). After electroporation, the cells were seeded in nonselective media (90% DMEM, 10%) fetal calf serum). Two days later, selection ofthe packaging cells was initiated in media containing 0.5 mg/ml (active concentration) G418
(Gibco, Inc., Gaithersburg, MD). After about two weeks of selection, the G418 resistant packaging cells were pooled and retrovirus was harvested. The retrovirus was used to infect a human T-cell line (CEMss; NIH AIDS Research and Reference Reagent Program). CEMss cells were grown in 90% RPMI, 10% fetal calf serum and CrFK cells were grown in 45% L-15, 45% DMEM, 10% fetal calf serum. Two days later, selection ofthe infected cells in media containing 0.2 (for CrFK) or 0.5 (for CEMss) mg/ml G418 (active concentration) was initiated. After three weeks of G418 selection, clonal CEMss cell lines were generated from the pool of G418 resistant cells by limiting dilution.
RNA and DNA Preparation.
Total RNA and genomic DNA were prepared from CEMss cell lines using TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer's directions.
Polymerase Chain Reactions.
Polymerase chain reactions (PCR) utilized Taq polymerase according to manfacturer's directions.
Reverse Transcription-Polymerase Chain Reactions.
Reverse transcription-polymerase chain reactions (RT-PCR) were done according to Myers and Gelfand (1991) Biochemistry 20:1661-1666 in a single reaction using rTth polymerase (Perkin Elmer) except that the magnesium chelation step was omitted. RT-PCR was performed to rapidly assess ligand expression qualitatively. Chimeric tRNAjMe,-SELEX ligand
RNAs were detected after 30 cycles of RT-PCR using primers (5'RPA: 5ΑGCAGAGTGGCGCAGCGG3' (SEQ ID NO: 16); 3'RPA2.1 : 5'CTAGTGGAACAGATCCCGAAGGATCC3' (SEQ ID NO: 17)) that hybridize to both the pol II and pol III transcripts encoded by the pNEW6 vector. These primers correspond in sequence to the extreme 5' and 3' ends ofthe chimeric tRNA-SELEX ligand RNA. The reverse transcription was carried out at 72°C. for 15 minutes and then heated to 94°C. for 1 minute to denature RNA/DNA duplexes. The PCR was carried out at 94°C. for 1 minute, then 60°C. for 30 seconds for a total 30 alternating cycles.
Deletion of Ligand Chimeric Genes was detected using primers (ML-2:
5'TAATACGACTCACTATAGGGGATCTGAACTTCTCTATTCTC3' (SEQ ID NO: 18); ML-3: 5ΑATGAAAGACCCCACCTGTA3' (SEQ ID NO: 19). These primers flank direct repeats found on each side ofthe gene which are responsible for gene deletion. The presence ofthe HIV-l RNA was detected in RNAse-free,
DNase-treated (RQ1, Promega, Madison, WI) total RNA from HIV-l infected cells after 40 cycles of RT-PCR using primers SK38 and SK39 (Perkin Elmer).
Quantitative-Competitive Reverse Transcription-Polymerase Chain
Reactions.
QC-PCR was performed to accurately quantitate intracellular ligand expression. QC-PCR was performed as described by Sieber and Larrick (1993) Biotechniques 14:244-249; Piatak et al. (1993) Science 259: 1749- 1754. The plasmids used to generate competitor RNAs
(pNEW6-tat7-QCPCR) were constructed by replacing the 13 base pair Sac II/Hind III fragment of pNEW6-tat7 with a 56 base pair fragment from pBluescript KS/+ (Stratagene, La Jolla, CA). Templates for transcription of competitor RNAs were generated by PCR amplification of pNEW6-tat7-QCPCR with the primers 5'RPA (SEQ ID NO: 16) and
3'RPA2.1 (SEQ ID NO: 17). Competitor RNAs were transcribed from these PCR products with T7 RNA polymerase gel-purified, and quantified by their absorbance at 260 nm. The competitor RNAs were diluted 10 fold from 100 attograms to 10 ng and added to 100 ng total cellular RNA that was prepared from cell lines judged to be expressing SELEX-derived ligands by RT-PCR.
The samples were then amplified by RT-PCR as described above for a total of 30 cycles. The competitor RNAs produce a PCR product that is 42 base pairs larger than that obtained by PCR amplification ofthe SELEX ligand-containing RNAs. Analysis of QC-PCR products was done on 3% NuSieve agarose (FMC, Inc., Rockland, ME) gels run in 1 x TBE buffer. The concentration at which the PCR product from the competitor RNA was equal in amount to that ofthe cellular RNA (the "equivalence point") was taken as the concentration ofthe SELEX ligand-containing RNAs. In some cases three fold dilutions of competitor RNA was used to determine the SELEX ligand-containing RNA concentration more accurately.
Affinity of Chimeric Nucleic Acid Ligands for HIV proteins.
The SELEX combinatorial method was used to generate RNA ligands that bind to the HIV-l tat, rev, and reverse transcriptase proteins. The binding properties ofthe rev ligand, rev30A (SEQ ID NO:21) (also called revόa), to the HIV-l rev protein have been described in detail by Jenson, et al. The binding properties ofthe pseudoknot portion ofthe rtwl 7 (SEQ ID NO: 22) ligand to the HIV-l reverse transcriptase protein have been described in detail by Green, et al. (1995) J. Mol. Biol. 242:60-68. The affinities ofthe selected RNAs for their Target proteins ranged from 0.1-10 nM (Table 2). Fusion ofthe SELEX ligands to the tRNAjMet used for expressing the ligands in CEMss cell lines either had little effect on their affinity (tat7 (SEQ ID NO:23)), rev30A (SEQ ID NO:24)) or reduced it significantly (rtwl 7 (SEQ ID NO:25)) (Table 3).
EXAMPLE 4. PROTECTION OF CELLS FROM HIV-l
INFECTION BY HIV-l NUCLEIC ACID LIGANDS CEMss Cell Lines.
A total of 35 (tat7 (SEQ ID NO:23)), 18 (rtwl 7 (SEQ ID NO:24)), and 11 (rev30A (SEQ ID NO:25)) clonal CEMss cell lines that contained a NEW6 provirus (encoding a tRNAjMet-SELEX Ligand Chimeric Gene) were generated. These 'cell lines were examined for expression ofthe tRNA-HIV-1 Nucleic Acid Ligand chimeric RNA by RT-PCR. The number of cell lines that express a Ligand Chimeric Gene is shown in Table 4 and ranged from 66-80%.
Some of these cell lines had a subpopulation of cells that had a deleted Ligand Chimeric Gene. This was detected by PCR analysis. Little or no deletion was found to occur in E. coli. Therefore, most ofthe gene deletion may occur during transfection ofthe packaging line or during reverse transcription in CΕMss cells. From the size ofthe deleted PCR product, it is suspected that the deletion occurs between two 21 base direct repeats that flank the Nucleic Acid Ligand gene and were generated during the construction ofthe pDCT-5T vector. If Nucleic Acid Ligand expression protects cells from HIV-l replication, then the presence of cells in the population which have deleted genes could result in cells which would replicate HIV-l . Therefore, cell lines that had evidence of deletion of HIV-l were not challenged.
The rate of cell doubling and rate of thymidine incoφoration of several cell lines was examined and found not to be significantly different from control non-transduced CΕMss cells. Therefore, the expression of chimeric tRNA-HIV SΕLΕX ligand RNAs was not noticeably toxic to CΕMss cells.
CΕMss cells expressing tRNAjMet alone, were also infected. Prior to HIV-l infection the cells were analyzed for CD3 and CD4 expression. The level of CD 3 and CD4 expression was roughly equivalent to that of control CΕMss cells for all cell lines that were challenged with HIV-l.
HIV-l Challenge.
CΕMss cell lines that express ligands or non-transduced control (CΕMss) cells (3 x IO5) were infected with HIV-l (Illb) at 100 TCID50 (moi=~0.001). After 4 hours of infection, the virus was washed out, the cells were split into three equal aliquots, and then plated at a density of 1 x lOVml in 2 ml media in 24 well plates. The cells were split and HIV-l capsid (p24) levels were assayed in triplicate every 4 days using an ELISA kit (Coulter Coφ., Hialeah, FL). The results are shown in Table 4.
In each case, cell lines expressing the chimeric tRNA-SELEX RNA were protected from HIV-l infection by 100 TCID50 units of HIV-l Illb for at least 30 days, whereas cell lines that did not express a HIV Nucleic Acid
Ligand were not protected from HIV infection. There is a 104-106 fold difference in the level of p24 produced by protected cells compared to susceptible cells. Cell lines with detectable amounts of deleted Ligand Chimeric Genes were not included in the data shown in Table 4. Such cell lines were always susceptible to HIV-l infection adding further support to the notion that Nucleic Acid Ligands protect cells from HIV-l infection. The protection observed can be overcome in the cell lines by infection with 500 TCID50 units of HI V-1 Illb.
EXAMPLE 5. EXPERIMENTAL PROCEDURES
This example provides general procedures followed and incoφorated in Example 6 describing the expression of FIV Nucleic Acid Ligands in feline cells.
Materials
The Crandell feline kidney (CrFK) cell line and the Petaluma strain of FIV were gifts generously provided by Dr. Tom North at University of Montana. CrFK is available from the ATTC and the Petaluma strain of FIV is available from the NIH AIDS Research and Reference Reagent Program.
Generation of Expression Retroviral Vector and Derivation of Expression Cell Lines.
The murine-derived retroviral vector pNEW6, which contains the bacterial neomycin-resistant gene (neo), was modified by insertion of a polylinker sequence downstream ofthe human tRNAjMet promoter in the U3 region of LTR (Adeniyi- Jones et al. (1984) Nuc. Acids Res. 12:1101-1115). The cDNAs of intact FIV RT selected RNA molecule F5 (5'-GGGAGGATATTTTCTCAGACCGTAATTGCGAAGGAAAAACCGA GGTGCTTTACGCGTCAATATGCTTGCAGCATCGTGAACTAGGATC CGGG-3' (SEQ ID NO:26)) and the truncate version dF5 (5'-GTAATTGCGAAGG AAAAACCGAGGTGCTTTACG-3' (SEQ ID
NO:27)) were cloned into murine vector pNEW6 with restriction enzymes Hind III and BamHI, respectively. Plasmid DNA (lμg) was electroporated into the amphotropic packaging cell line GP+envAM12 (Markowitz et al. (1988) Virology 162:400-406) with a Bio-Rad gene pulser, and transduced cells were selected with G418 (700μg/mL). G418-resistant colonies were pooled, and the virus-containing supernatant (Remington et al. (1994) Virol. 68:632-637) was used to infect the Crandell feline kidney cell line (CrFK) (North et al. (1989) Antimicrob. Agents Chemother. 22:915-919) in the presence of 8 μg/mL of Polybrene, to create an expression cell line. The infected CrFK cells were grown in the 45% Leibowitz-IS, 45% DEM, 10% fetal calf serum (Gibco BRL) in the presence of 200 μg/mL G418 (Gibco BRL). The G418-resistant colonies were isolated by using cloning cylinders, expanded to cell lines, and used in subsequent experiments (Freshney (1994) Culture of Animal Cells: A Manual of Basic Technique. 3rd Ed. Wiley-Liss Press.
RNA Analyses
Total cellular RNA was isolated with the RNA Isolation Kit (Strategene Cloning System) according to the instruction manual. 10 μg of total RNA sample was subjected to electrophoresis on a 1% formaldehyde
(pH>4) agarose gel with IX MOPS running buffer (20 mM 3-[N-Mθφhlino] propane-sulphonic acid, 50 mM sodium acetate pH 7.0, and 1 mM EDTA pH 7.0) (Ausubel et al. (1992) Curr. Prot. Mol. Clon. Vol.1 The samples were transferred to Hybond-N membrane (Amersham Life Science, Inc.), and hybridization was carried out in 12.5 mL of hybridization buffer (0.1 % BSA,
0.1% Ficoll, 0.1% polyvinylpyrolidone, 180 mM NaCl, 10 mM sodium phosphate pH 7.7, 1 mM EDTA pH 7.7, 50% v/v formamide, and 20 μg/mL sonicated salmon sperm DNA) at 42°C overnight, as described by Ausubel et al. (Ausubel et al. (1992) Curr. Prot. Mol. Clon. Vol.1). The probe was the 32P-internal labeled PCR product of RNA ligand F5 (SEQ ID NO:10) (108 cpm/mL). The hybridized membrane was washed twice at room temperature for 10 minutes with 2X SSPE (360 mM NaCl, 21 mM sodium phosphate pH 7.7, and 2 mM EDTA pH 7.7), 1% SDS, and auto-radiographed. After exposure, the membrane was stained with methylene blue to visualize ribosome RNA as an internal control. The dried membrane was soaked in 5% acetic acid for 15 minutes at room temperature, and stained in 0.004% methylene blue and 0.5 M sodium acetate for 10 minutes, and destained with water for one hour.
DNA Analyses Chromosomal DNA was isolated by the Genomic DNA Isolation
System (Gibco BRL), as indicated by the instruction manual. 5 μg genomic DNA was digested with restriction endonuclease Hind III or Xba I. The samples were electrophoresed on a 1% agarose gel, transferred onto a Hybond-N membrane (Amersham Life Science Inc.), and UV cross-linked to the membrane with a Strategene UV Statelinker. The membrane was pre-hybridized at 65°C for at least one hour in 12.5 mL of 5X Denhardts (0.1 % BSA, 0.1% Ficoll, 0.1% polyvinylpyrrolidone), IX SSPE (180 mM NaCl, 10 mM sodium phosphate pH7.7, and 1 mM EDTA pH7.7), and 2 μg/mL sonicated salmon sperm DNA. The 32P-labeled ligand-specific DNA probe or neomycin gene («eø)-specific probe (108 cpm/mL) was added to the pre-hybridization solution. The hybridization was carried out for 16 hours at 65°C. The hybridized membrane was washed twice with 2X SSPE (360 mM NaCl, 21 mM sodium phosphate pH 7.7, and 2 mM EDTA pH 7.7), 0.1% SDS, at room temperature for 10 minutes, and washed with IX SSPE (180 mM NaCl, 10 mM sodium phosphate pH7.7, and 1 mM EDTA pH7.7), 0.1%
SDS, at 65°C for 15 minutes. The membrane was analyzed by autoradiography.
Genomic DNA Sequencing
Genomic DNA from expression cell lines was PCR amplified with primer pFRT5- 1 (5'-TGTGAGCC GTGTGCTGCTTGGCAG-3' (SEQ ID
NO:28)) and primer pFRT3-l (5'-CCATGCCTT GCAAAATGGCGA-3' (SEQ ID NO:29)). 50 ng genomic DNA was PCR amplified in 100 μL of PCR reaction mixture containing 200 picomoles of primers pFRT5-l and pFRT3-l at 93°C for 30 seconds, 55°C for 15 seconds, and 72°C for 90 seconds, for 30 cycles. As a positive control, plasmid pNEW6-FRTl, which contained the cDNA of RNA ligand F5 (SEQ ID NO: 10) was used. The PCR product (294 bp) was purified from an 1% low-melting agarose gel. The sample in the low-melting gel was directly subjected to sequence analysis with either primer pFRT5-2 (5'-GCTTGGCAGAACA GCAGAGTGG-3' (SEQ ID NO : 30) )for forward sequencing or primer pFRT3 -2
(5'-GGCGTTACTTAAGCTAGCACGC-3' (SEQ ID NO:31)) for reverse sequencing, using the Sequenase Version 2.0 DNA Sequencing Kit (USB) according to the instruction manual.
FIV Infection
The Petaluma strain of FIV (Pedersen et al. (1987) Science 235:790-793) and a virus derived from a molecular clone of FIV, 34TF10 (Remington et al. (1994) Virol. 68:632-637) were used. Virus was grown and maintained in Crandell feline kidney (CrFK) cells as described supra (North et al. (1989) Antimicrob. Agents Chemother. 22:915-919). FIV infectivity was determined by the focal formation assay. Uninfected cells were infected with 20-60 focus-forming units of FIV per well (IO4 cells/well). After 4 days, the medium was removed and the cells were fixed with methanol. Infectious foci were detected by reacting the cells with a polyclonal antiserum obtained from FIV-infected specific-pathogen-free cats (Remington et al. (1994) Virol.
68:632-637), followed by reacting with peroxidase-conjugated anti-cat immunoglobulin (Organon Teknika) (Remington et al. (1994) Virol. 68:632-637). The peroxidase stain was developed with H2O2 and amino-ethyl-carbazole. The infectious foci are present as areas of red cells against a background of unstained cells.
EXAMPLE 6. INHIBITION OF FIV REPLICATION IN CELL
CULTURE BY SELECTED INHIBITORY RNA LIGANDS Construction ofthe Expression Vector pNEW6-FRT and Expression Cell Lines CrFK.
The murine derived retroviral vector consists ofthe genomic structure of Moloney murine leukemia virus, with the retroviral open reading frames replaced by a neomycin-resistant gene (neo) and pBR322 sequence (Sullenge ;r: et al. (1990) Mol. Cell. Biol. 10:6512-6523). A fragment of human tRNAiMet gene, which contains a poly-linker, was engineered into the U3 region ofthe long terminal repeat (LTR). The cDNAs of FIV RT-selected RNA ligand F5 (SEQ ID NO: 10), and the truncated version dF5 (SEQ ID NO: 13), determined from the FIV RT binding boundary, were cloned into a murine derived vector pNEW6 between restriction sites Hind III and Bam HI. The plasmid was shown to possess the desired insertion. The constructed plasmids were renamed as pNEW6-FRTl (for full length) and pNEW6-FRT2 (for the truncated version), respectively (Figure 3). The insertion site is located at the 3' end of a deleted human tRNAjMet gene (Adeniyi- Jones et al. (1984) Nuc. Acids Res. 12:1101-1115) in the U3 region ofthe 3' LTR ofthe murine retroviral vector. The FIV RT-selected RNA ligands can be transcribed by either RNA polymerase II, RNA polymerase III, or both.
The constructed plasmids were transfected into the packaging cell line GP+envAM12 (Markowitz et al. (1988) Virology 162:400-406 ) by electroporation. The transfected GP+envAM12 cells were grown in the medium containing G418 for positive selection. The GP+envAM12 cell line is an amphotropic cell line which expresses all the murine retroviral proteins for packaging the defective virus. The defective amphotropic viruses can infect a wide range of hosts, but can only undergo a single cycle of infection (Markowitz et al. (1988) Virology 162:400-406 ). As discussed supra, the tRNAiMe,-Ligand Chimeric Gene is in the U3 region of the 3' LTR. After replication ofthe vector, the chimeric gene should be duplicated in the U3 region of both 5' and 3' LTR of proviral DNA (Figure 3).
The Crandell feline kidney (CrFK) cell line was infected with the defective virus (Sullenger et.al. (1990) Mol. Cell. Biol. 10:6512-6523). The infected cells were grown in the medium containing 200 μg/mL G418 for positive selection. After two weeks, 35 G418-resistant single colonies were obtained. The CrFK cell lines were named CrFK-FRTl for expression ofthe full-length FIV RT-selected RNA molecule, and CrFK-FRT2 for expression of the truncated version ofthe FIV RT-selected RNA molecule.
Expression of the Selected RNA Molecule in CrFK Cell Lines
Total cellular RNA was isolated from CrFK cell lines transduced with selected RNA ligand vectors. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated the expression of selected RNA ligands F5 (SEQ ID NO: 10) or dF5 (SEQ ID NO: 13) (data not shown) in all 34 cell lines. However, RT-PCR analysis could not distinguish whether the selected RNA transcription was driven by RNA polymerase II or RNA polymerase III. The tRNAiMet-Ligand Chimeric Gene should be present in the U3 regions of 5' and 3' LTR of proviral DNA. The presence ofthe chimeric gene in the 5' LTR is outside the viral transcription unit (pol II), possibly resulting in the additional transcription ofthe chimeric gene by RNA polymerase III, utilizing the tRNA promoter. Expression ofthe selected RNA was further analyzed by Northern blot (Ausubel et al. (1992) Curr. Prot. Mol. Clon. Vol.l). A double-stranded cDNA probe (155 bp) which covers the human initiation methionyl-tRNA gene, containing the ligand F5 sequences, was used for hybridization. The Northern blot result suggested that the selected RNA existed as both pol II and pol III transcripts. Three pol II transcripts have been detected. The 4.5kb nucleotide RNA molecule presumably is the full length transcription product ofthe murine retroviral vector, while the 2.2kb and l.Okb nucleotide RNA molecules are presumably alternatively spliced products. The pol III transcription product, wliich is 210 nucleotides in length, is the human initiation methionyl-tRNA-selected RNA chimeric molecule. Originally, it was expected that the selected RNA molecule would be overexpressed under the control ofthe human
Figure imgf000044_0001
promoter. However, the RNA blotting analysis showed that the hybridization signal of expression level for pol III transcripts was lower than those of pol II transcripts. The effects of expression of selected RNA ligands on cell growth were also examined by analyzing the doubling time of expression cell lines. 1X104 to 1X105 cells were seeded in the media in a T25 flask. The total number of cells was counted by tryptan blue staining every 24 to 48 hours. The doubling times of three cell lines expressing RNA ligands were in the range of 21.9 hours to 23.45 hours, while that ofthe parental cell line is 17.7 hours. It was also noticed that one cell line, CrFK-FRTl -20, had a doubling time which was slightly faster than the parental cell line. The results suggest that the expression of selected ligand have no severe effects on cell proliferation.
DNA Analyses of Expression Cell Line CrFK
The total genomic DNA from expression CrFK cell lines were isolated. The genomic DNA from four cell lines, CrFK-FRTl-3, CrFK-FRTl-7, CrFK 1-8, and CrFK-FRTl -20, were further analyzed. In order to determine if any mutations had occurred, the fragment of genomic DNA containing the insertion was amplified by PCR with primers pFRT5- 1 (SEQ ID NO:28) and pFRT3-l (SEQ ID NO:29) and the band was sliced from a low melting gel. The plasmid pNEW6-FRTl was used as a positive control and for tracing the occurrence of PCR-introduced mutations. The genomic sequencing analysis showed that no point mutations or deletions existed in the inserted cDNA of the selected RNA ligand F5 (SEQ ID NO: 10) (not shown).
The integration and structure of proviral DNA was further analyzed by Southern blot. The predicted duplication of LTR of proviral DNA will place the ligand template in the U3 region of both 5' and 3' LTRs. The retroviral vector contains two Hind III sites and two Xba I sites in the viral LTRs (Sullenger etal. (1990) Mol. Cell. Biol. 10:6512-6523; Adeniyi-Jones et al. ( 1984) Nuc. Acids Res. 12: 1101 - 1115). Therefore, the restriction endonuclease Hind III will generate a 3.8-kb fragment, which contains one copy ofthe tRNAjMet-Ligand Chimeric Gene, as will Xba I digestion, while another copy ofthe chimeric gene will be associated with the host cellular chromosome. The hybridization pattem with a ligand specific probe showed a 3.8-kb fragment in each ofthe four expression cell lines tested and a unique fragment in each individual cell line. The DNA blotting analysis indicated the existence ofthe duplication ofthe LTR and a variation of integration sites of proviral DNA in each expression cell line. The same samples were also hybridized with a neomycin gene (neo ) specific probe. The 3.8-kb fragment was apparent for each cell line as predicted. DNA analyses suggested that the
LTR was duplicated, and proviral DNA was integrated into the cellular chromosome DNA with a different integration site in each different cell line. The genomic sequencing analysis indicated that the cDNA of selected RNA ligand F5 (SEQ ID NO: 10) was the same as the original insert.
Inhibition of FIV Infectivity in Cells Expressmg Selected Inhibitory RNA Molecules
To determine the effect of selected RNA expression on a single cycle of FIV replication, CrFK cells were infected with the Petaluma strain of FIV (as described supra) (Remington et al. (1994) Virol. 68:632-637). Infectivity and the generation of progeny virus were monitored by focus formation four days post-infection. Six cell lines tested, expressing full length RNA ligand F5 (SEQ ID NO: 10) , showed that the retroviral infectivity was inhibited greater than 50% as compared to the parental cells (Table 5). In two cell lines tested (CrFK-FRTl -7 and CrFK-FRTl -8), the viral infectivity was reduced as much as 99%. As control experiments, CrFK cells harboring the vector without FIV RT selected ligand expression and CrFK cells expressing an
HIV-l RT-selected RNA molecule (Tuerk et al. (1992) Proc. Natl. Acad. Sci.
82:6988-6992) were examined and showed no inhibition of FIV infectivity.
This is consistent with the in vitro experiments which show that HIV-l RT-selected pseudoknot RNA does not inhibit the polymerization activity of
FIV RT.
CrFK cell lines expressing RNA molecule dF5 were also tested. In vitro experiments indicated that the truncated RNA dF5 (SEQ ID NO: 13) also inhibits FIV RT polymerization activity. The results show that five cell lines inhibit FIV infectivity in the range of 86% to 99% (Table 6).
While the invention has been described with respect to certain specific embodiments, it will be appreciated that many modifications and changes may be made by those skilled in the art without departing from the spirit ofthe invention.
Table 1. Ki and Km Values for FIV RT
Enzyme Variable Km + SEM (μM) Km ± SEM (nM) Mode of Inhibition
Recombinant FIV RT dTTP 3.3 +0.2 89 + 8.4 non-competitive
Poly (rA) - oligo (dT) 0.9 +0.01 96 + 7.2 competitive
Viron FIV RT dTTP 3.3 + 0.2 37 + 3.6 non-competitive
4
Poly (rA) - oligo (dT) 0.5 +0.01 31 + 2.1 competitive
AZR - 17c RT dTTP 6.6 +0.2 45 +3.8 non-competitive
Poly (rA) - oligo (dT) 0.7 +0.01 25 +4.2 competitive
Poly(rA)-oligo(dT) was used as the template/primer complex in the experiments. All data were determined from two or more experiments with three determinants per experiment.
Table 2. Sequences of HIV-l Nucleic Acid Ligand RNAs.
Ligand Name HIV target protein HIV SELEX ligand RNA sequence3 K^ of ligand SEQ ID NO for target protein tat7 HIV-l tat 5' GGGAGCUCAGAAUAAACGCUCAA 10 nM 20 cacgaaggaaacggagggaaucuugaagaacccggaccac UUCGACAUGAGGCCCGGAUCUAUUGAAAC rev30A HIV-l rev 5' GGGAGCCA ACACCAC A AUUCCAAUCAAG I nM 21 ggugcauugagaaacacguuuguggacucuguau CUAUGAAAGAAUUUUAUAUCUCUAUUGAAAC rtwl 7 HIV- l RT 5' GGUCCGAAGUGCAACGGGAAAAUGCACU 0.1 nM 22 caugggagcccaucgauucugguguugc CUAUGAAAGAAUUUUAUAUCUCUAUUGAAAC O *s a The first line is the sequence of 5' fixed region used for the PCR step of SELEX, the second line is the sequence of the region that was randomized for the SELEX process, and the third line is the sequence of the 3' fixed region used for the PCR step of SELEX.
Figure imgf000049_0001
Table 3. Sequences of tRNA-Nucleic Acid Ligand chimeric RNAs.
Ligand Name Target protein tRNAmeH'gand RNA sequence8 dJ r SEQ target protein ID NO
tat7 HIV-l tat 5' AGCAGAGUGGCGCAGCGGAAGCGUGCUGGGCCCAU 0.6 nM 23
AACCCAGAGGUCGAUGGAUCGAAACCCCGGAUCGU ACCGCGGGUCGUGUUAGAAGCUU
CUAAUACGACUCACUAUAGGGAGCUCAGAAUAAACGCUCAA cacgaaggaaacggagggaaucuugaagaacccggaccac UUCGACAUGAGGCCCGGAUCUAUUGAAAC GGAUCCUUCGGGAUCUGUUCCACUAGCGAUCCGUUU 3' rev30A HIV-l rev 5' AGCAGAGUGGCGCAGCGGAAGCGUGCUGGGCCCAU 2 nM 24
AACCCAGAGGUCGAUGGAUCGAAACCCCGGAUCGU ACCGCGGGUCGUGUUAGAAGCUU
CUAAUACGACUAUAGGGAGCCAACACCACAAUUCCAAUCAAG ggugcauugagaaacacguuuguggacucuguau CUAUGAAAGAAUUUUAUAUCUCUAUUGAAAC GGAUCCUUCGGGAUCUGUUCCACUAGCGAUCCGUUU 3' rtwl 7 HIV- l RT 51 AGCAGAGUGGCGCAGCGGAAGCGUGCUGGGCCCAU 200 nM 25
AACCCAGAGGUCGAUGGAUCGAAACCCCGGAUCGU ACCGCGGGUCGUGUUAGAAGCUU
CUAAUACGACUCACUAUAGGUCCGAAGUGCAACGGGAAAAUGCACU caugggagcccaucgauucugguguugc CUAUGAAAGAAUUUUAUAUCUCUAUUGAAAC GGAUCCUUCGGGAUCUGUUCCACUAGCGAUCCGUUU 3'
Figure imgf000049_0002
a For each RNA sequence shown, the first three Iines are the tRNA and cloning sites (SacII and Hindlll), the fourth line is the 5' fixed region used for the PCR step of SELEX, the fifth line is the sequence that was randomized for SELEX, the sixth line is the 3' fixed region used for the PCR step of SELEX, and the seventh line is a cloning site (BamHI) and the 3' end of the transcript.
Table 4. Protection of CEMss cell lines from HIV-l infection by tRNA-Nucleic Acid Ligand chimeric RNAs.
Number of Number of expressing Number of expressing
Chimeric Number of cell lines that cell lines that were cell lines that
RNA SELEX Target lines made express ligand infected with HIV were HIV resistant5 tRNA-tat7 HIV-l tat 35 23 6 4 tRNA-rev30A HIV-l rev 11 6 4 2 tRNA- rtwl 7 HIV-l RT 18 14 7 3
TOTAL : 64 43 17
CONTROLS : tRNA alone none 5 3 3 0 various*5 various 31 0 31 0
TOTAL : 36 3 34
4 co
a HIV resistance is defined as the number of cell lines that are at least 100 fold more protected (100 fold lower p24) from HIV-l infection compared to CEMss for at least 30 days post-infection. Typically, by 30 days post-infection parental CEMss cells will produce 1,000,000 pg/ml p24 over a 4 day period.
b These include cells that had a tRNA-SELEX ligand gene but did not express the gene.
Figure imgf000050_0001
Table 5. Inhibition of FIV Infection by Expression of Ligand F5
Cell Line % Relative Infectivity % Inhibition of infectivity
CrFK p185 100%
CrFK-FRTM 17% 83%
CrFK-FRT1-2 43% 57%
CrFK-FRT1-3 1166%
CrFK-FRT1-5 19% 81%
CrFK-FRT1-7 0.90% 99%
CrFK-FRT1-8 0.98% 99%
CrFK-FRT1-11 26% 74%
Figure imgf000051_0001
virus infection killed cells
a All data were obtained by at least triplets. *Cell Iines are more susceptible to FIV infection than control.
Table 6. Inhibition of FIV Infection by Expression of Ligand dF5
Cell Line % Relative Infectivity % Inhibition of infectivity
CrFK pi 85 100%
QFK-FRT2-3 1010% *
CrFK-FRT2-7 14% 86%
CrFK-FRT2-16 12% 88% Ul o
CrFK-FRT2-19 1.1% 99%
CrFK-FRT2-22 8.5% 92%
CrFK-FRT2-23 1.4% 99%
aAII data were obtained by at least triplets. Ligand dF5 is the truncated version of RNA ligand F5 according to the FIV RT binding boundary *Cell Iines are more susceptible to FIV infection than control.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: LARRY GOLD
MICHAEL LOCHRIE HANG CHEN CRAIG TUERK (ii) TITLE OF THE INVENTION: INTRACELLULAR ACTION OF
NUCLEIC ACID LIGANDS (iii) NUMBER OF SEQUENCES: 31 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Swanson and Bratschun, L.L.C.
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( i) CURRENT APPLICATION DATA:
(A APPLICATION NUMBER: PCT/US96/_ (B FILING DATE: (C CLASSIFICATION:
( ii) PRIOR APPLICATION DATA:
(A APPLICATION NUMBER: 08/521,515 (B FILING DATE: 30-AUGUST-1995
( ii) PRIOR APPLICATION DATA: (A APPLICATION NUMBER: 60/000,872 (B FILING DATE: ll-JULY-1995
(viii) ATTORNEY/AGENT INFORMATION: (A NAME: Barry J. Swanson (B REGISTRATION NUMBER: 33,215 (C REFERENCE/DOCKET NUMBER: NEX45/PCT
(ix) TELECOMMUNICATION INFORMATION: (A TELEPHONE: (303) 793-3333 (B TELEFAX: (303) 793-3433 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 92 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GGGAGGAUAU UUUCUCAGAC CGUAANNNNN NNNNNNNNNN NNNNNNNNNN 50 NNNNNNNNNN NNNNNUUGCA GCAUCGUGAA CUAGGAUCCG GG 92
(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CCCAAGCTTA ATACGACTCA CTATAGGGAG GATATTTTCT CAGACCGTAA 50
(2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CCCGGATCCT AGTTCACGAT CTGCAA 26
(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GGGAAUUCGA GCUCGGUACC CGGGAUCCUC UAGAGUCGAC CUGCAGGCAU 50 GCUAGCUUGG CACUGGGCGU CGUUUUACAA CGUCGUGACG UGG 93 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CCCACGTCAC GACGTTGTAA AACGACGCCC 30
(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GGGAGGAUAU UUUCUCAGAC CGUAAGUACC GAAUGUGCUU UUYGGCCCGA 50 UUUUUGGCCC CUGCAGUUGC AGCAUCGUGA ACUAGGAUCC GGG 93
(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 92 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GGGAGGAUAU UUUCUCAGAC CGUAAUUUUU RGUGCURUCA CGUGCACAUU 50 UUUUAGCUGC UYAAUUUGCA GCAUCGUGAA CUAGGAUCCG GG 92
(2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 92 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GGGAGGAUAU UUUCUCAGAC CGUAAAACUU UUGUGGCUUU NACGACCACC 50 AUUUUGUGUU GUAACUUGCA GCAUCGUGAA CUAGGAUCCG GG 92 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 92 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 : GGGAGGAUAU UUUCUCAGAC CGUAAYRGGA GGUUUGUCAU ACACGAAUGA 50 CUUUUUUAGC UACAUUUGCA GCAUCGUGAA CUAGGAUCCG GG 92
(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 92 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GGGAGGAUAU UUUCUCAGAC CGUAAUUGCG AAGGAAAAAC CGAGGUGCUU 50 UACGCGUCAA UAUGCUUGCA GCAUCGUGAA CUAGGAUCCG GG 92
(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GGGAGGAUAU UUUCUCAGAC CGUAACCUAG UUWAAUCRCA CCAGUCGGAC 50 UCUUCAGCCG AAAACAUUGC AGCAUCGUGA ACUAGGAUCC GGG 93
(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 92 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GGGAGGAUAU UUUCUCAGAC CGUAACUAGU UCAACCACCA GGGAGCACAU 50 GACUCCAUCA GCAUAUUGCA GCAUCGUGAA CUAGGAUCCG GG 92
(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RΝA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: UUGCGAAGGA AAAACCGAGG UGCUUUAC 28
(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CCGCGGTGGA TCC 13
(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CCGCGGGTCG TGTTAGAAGC TTCCCATGGA TCCTTCGGGA TCTGTTCCAC 50 TAGCGATCC 59
(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: AGCAGAGTGG CGCAGCGG 18 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CTAGTGGAAC AGATCCCGAA GGATCC 26
(2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DΝA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: TAATACGACT CACTATAGGG GATCTGAACT TCTCTATTCT C 41
(2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: AATGAAAGAC CCCACCTGTA 20
(2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 92 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GGGAGCUCAG AAUAAACGCU CAACACGAAG GAAACGGAGG GAAUCUUGAA 50 GAACCCGGAC CACUUCGACA UGAGGCCCGG AUCUAUUGAA AC 92
(2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GGGAGCCAAC ACCACAAUUC CAAUCAAGGG UGCAUUGAGA AACACGUUUG 50 UGGACUCUGU AUCUAUGAAA GAAUUUUAUA UCUCUAUUGA AAC 93
(2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GGUCCGAAGU GCAACGGGAA AAUGCACUCA UGGGAGCCCA UCGAUUCUGG 50 UGUUGCCUAU GAAAGAAUUU UAUAUCUCUA UUGAAAC 87
(2) INFORMATION FOR SEQ ID NO: 23 (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 239 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RΝA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: AGCAGAGUGG CGCAGCGGAA GCGUGCUGGG CCCAUAACCC AGAGGUCGAU 50 GGAUCGAAAC CCCGGAUCGU ACCGCGGGUC GUGUUAGAAG CUUCUAAUAC 100 GACUCACUAU AGGGAGCUCA GAAUAAACGC UCAACACGAA GGAAACGGAG 150 GGAAUCUUGA AGAACCCGGA CCACUUCGAC AUGAGGCCCG GAUCUAUUGA 200 AACGGAUCCU UCGGGAUCUG UUCCACUAGC GAUCCGUUU 239
(2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 236 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: AGCAGAGUGG CGCAGCGGAA GCGUGCUGGG CCCAUAACCC AGAGGUCGAU 50 GGAUCGAAAC CCCGGAUCGU ACCGCGGGUC GUGUUAGAAG CUUCUAAUAC 100 GACUAUAGGG AGCCAACACC ACAAUUCCAA UCAAGGGUGC AUUGAGAAAC 150 ACGUUUGUGG ACUCUGUAUC UAUGAAAGAA UUUUAUAUCU CUAUUGAAAC 200 GGAUCCUUCG GGAUCUGUUC CACUAGCGAU CCGUUU 236
(2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 234 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: AGCAGAGUGG CGCAGCGGAA GCGUGCUGGG CCCAUAACCC AGAGGUCGAU 50 GGAUCGAAAC CCCGGAUCGU ACCGCGGGUC GUGUUAGAAG CUUCUAAUAC 100 GACUCACUAU AGGUCCGAAG UGCAACGGGA AAAUGCACUC AUGGGAGCCC 150 AUCGAUUCUG GUGUUGCCUA UGAAAGAAUU UUAUAUCUCU AUUGAAACGG 200 AUCCUUCGGG AUCUGUUCCA CUAGCGAUCC GUUU 234
(2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 92 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GGGAGGATAT TTTCTCAGAC CGTAATTGCG AAGGAAAAAC CGAGGTGCTT 50 TACGCGTCAA TATGCTTGCA GCATCGTGAA CTAGGATCCG GG 92
(2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GTAATTGCGA AGGAAAAACC GAGGTGCTTT ACG 33
(2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: TGTGAGCCGT GTGCTGCTTG GCAG 24
(2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERIZATION: .
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: CCATGCCTTG CAAAATGGCG A 21
(2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DΝA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: GCTTGGCAGA ACAGCAGAGT GG 22
(2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: GGCGTTACTT AAGCTAGCAC GC 22

Claims

CLAIMS:
1. A method of identifying nucleic acid ligands to FIV RT, comprising: a) preparing a candidate mixture of nucleic acids; b) contacting the candidate mixture of nucleic acids with FIV RT, wherein nucleic acids having an increased affinity to FIV RT relative to the candidate mixture may be partitioned from the remainder ofthe candidate mixture; c) partitioning the increased affinity nucleic acids from the remainder ofthe candidate mixture; and d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding FIV RT, wherein nucleic acid ligands of FIV RT may be identified.
2. The method of claim 1 further comprising: e) repeating steps b), c), and d.
3. The method of claim 1 wherein said candidate mixture of nucleic acids is comprised of single stranded nucleic acids.
4. The method of claim 1 wherein said candidate mixture of nucleic acids is comprised of single stranded ribonucleic acids.
5. The method of claim 1 wherein said candidate mixture of nucleic acids is comprised of single stranded deoxyribonucleic acids.
6. The method of claim 1 wherein said candidate mixture of nucleic acids is comprised of double stranded nucleic acids.
7. A purified and isolated non-naturally occurring nucleic acid ligand to FIV RT.
8. The nucleic acid ligand to FIV RT of claim 7 identified according to the method of claim 1.
9. The nucleic acid ligand to FIV RT of claim 7 identified according to the method of claim 2.
10. The nucleic acid ligand of claim 7, wherein said ligand is an RNA selected from the group consisting ofthe sequences set forth in Figures 1 A - IH (SEQ ID NOS:6-12), or the corresponding complementary sequences thereof.
11. The nucleic acid ligand of claim 7, wherein said ligand is an RNA substantially homologous to and has substantially the same ability to bind FIV RT as a ligand selected from the group consisting ofthe sequences set forth in Figures 1 A - IH (SEQ ID NOS:6-12), or the corresponding complementary sequences thereof.
12. The nucleic acid ligand of claim 7, wherein said ligand has substantially the same structure and substantially the same ability to bind FIV RT as a ligand selected from the group consisting ofthe sequences set forth in Figures 1 A - IH (SEQ ID NOS:6-12), or the corresponding complementary sequences thereof.
13. A method for the treatment of intracellularly mediated diseases or conditions in patients suffering therefrom comprising: administering to said patient an oligonucleotide that enters into cells and thereby attenuates said disease or condition.
14. The method of claim 13 wherein said intracellularly mediated disease or condition includes an Intracellular target involved in the mediation of said intracellularly mediated disease or condition.
15. The method of claim 13 wherein said intracellularly mediated disease or condition is selected from the group consisting of cancer, infectious diseases and dominant genetic disorders.
16. The method of claim 15 wherein said infectious disease is selected from the group consisting of AIDS, cytomegalovirus retinitis, hepatitis, infectious mononucleosis, Leischmaniasis, candidiasis, malaria and influenza.
17. The method of claim 15 wherein said domrnant genetic disorder is selected from the group consisting of polycystic kidney disease, Charcot-Marie-Tooth disease, Stargardt's disease, Parkinson's disease, Alzheimer's disease, schizophrenia and atherosclerosis.
18. The method of claim 13 wherein said oligonucleotide comprises three or more nucleic acid residues.
19. The method of claim 14 wherein said oligonucleotide binds said intracellular target.
20. The method of claim 19 wherein said oligonucleotide is an inhibitor ofthe biological activity said intracellular target.
21. The method of claim 14 wherein said oligonucleotide is a nucleic acid ligand.
22. The method of claim 14 wherein said oligonucleotide affects the activity of said intracellular target by means other than by Watson-Crick base pairing with said intracellular target.
23. The method of claim 21 wherein said nucleic acid ligand is identified according to the method: a) preparing a candidate mixture of nucleic acids; b) contacting the candidate mixture of nucleic acids with said intracellular target, wherein nucleic acids having an increased affinity to said intracellular target relative to the candidate mixture may be partitioned from the remainder ofthe candidate mixture; c) partitioning the increased affinity nucleic acids from the remainder ofthe candidate mixture; and d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding said intracellular target, whereby nucleic acid ligands of said intracellular target may be identified.
24. The method of claim 14 wherein said intracellularly mediated disease or condition is AIDS.
25. The method of claim 24 wherein said intracellular target is selected from the group consisting of HIV-rev protein, HIV-l reverse transcriptase protein, HIV-l tat protein, HIV Integrase protein, HIV-l GAG protein, HIV Nucleocapsid protein.
26. The method of claim 24 wherein said oligonucleotide is introduced into the cells of said patient by gene therapy techniques.
27. The method of claim 14 wherein said oligonucleotide is intracellularly expressed in the cells of said patient.
PCT/US1996/011473 1995-07-11 1996-07-10 Intracellular action of nucleic acid ligands Ceased WO1997003085A1 (en)

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US52151595A 1995-08-30 1995-08-30
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Cited By (2)

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Publication number Priority date Publication date Assignee Title
DE10116829A1 (en) * 2001-04-04 2002-11-14 Nascacell Gmbh Neuropeptide binding nucleic acids
JP2006525796A (en) * 2003-02-27 2006-11-16 イェダ リサーチ アンド デベロップメント カンパニー リミテッド Nucleic acid molecules, polypeptides, antibodies, and compositions containing them useful for treating and detecting influenza virus infection

Citations (1)

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US5275813A (en) * 1987-08-26 1994-01-04 The Regents Of The University Of California Methods and compositions for vaccinating against feline immunodeficiency virus

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US5275813A (en) * 1987-08-26 1994-01-04 The Regents Of The University Of California Methods and compositions for vaccinating against feline immunodeficiency virus

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10116829A1 (en) * 2001-04-04 2002-11-14 Nascacell Gmbh Neuropeptide binding nucleic acids
JP2006525796A (en) * 2003-02-27 2006-11-16 イェダ リサーチ アンド デベロップメント カンパニー リミテッド Nucleic acid molecules, polypeptides, antibodies, and compositions containing them useful for treating and detecting influenza virus infection
JP2010279385A (en) * 2003-02-27 2010-12-16 Yeda Res & Dev Co Ltd Nucleic acid molecule, polypeptide, antibody, and composition including the same, each useful for treating and detecting influenza virus infection
US8357789B2 (en) 2003-02-27 2013-01-22 Yeda Research And Development Co. Ltd. Nucleic acid molecules, polypeptides, antibodies and compositions for treating and detecting influenza virus infection
US9029526B2 (en) 2003-02-27 2015-05-12 Yeda Research And Development Co. Ltd. Nucleic acid molecules, polypeptides, antibodies and compositions for treating and detecting influenza virus infection

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