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WO1997003085A1 - Action intracellulaire de ligands d'acide nucleique - Google Patents

Action intracellulaire de ligands d'acide nucleique 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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Prior art keywords
nucleic acid
fiv
ligand
rna
nucleic acids
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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
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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

L'invention se rapporte à des procédés d'identification et de production de ligands d'acide nucléique pour la transcriptase inverse (RT) du virus immunodéficient félin (FIV) et pour des protéines homologues. Les ligands d'acide nucléique sont ainsi identifiés et produits, l'un des ligands étant décrit par la figure. L'invention se rapporte également aux méthodes de traitement des patients souffrant de maladies ou pathologies induites intracellulairement en introduisant des oligonucléotides dans les cellules du patient.
PCT/US1996/011473 1995-07-11 1996-07-10 Action intracellulaire de ligands d'acide nucleique Ceased WO1997003085A1 (fr)

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DE10116829A1 (de) * 2001-04-04 2002-11-14 Nascacell Gmbh Neuropeptid bindende Nukleinsäuren
JP2006525796A (ja) * 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 インフルエンザウイルス感染を処置および検出するために有用な核酸分子、ポリペプチド、抗体、およびそれらを含有する組成物

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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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GENE, 1993, Vol. 137, TUERK et al., "In Vitro Evolution of Functional Nucleic Acids: High-Affinity RNA Ligands of HIV-1 Proteins", pages 33-39. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10116829A1 (de) * 2001-04-04 2002-11-14 Nascacell Gmbh Neuropeptid bindende Nukleinsäuren
JP2006525796A (ja) * 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 インフルエンザウイルス感染を処置および検出するために有用な核酸分子、ポリペプチド、抗体、およびそれらを含有する組成物
JP2010279385A (ja) * 2003-02-27 2010-12-16 Yeda Res & Dev Co Ltd インフルエンザウイルス感染を処置および検出するために有用な核酸分子、ポリペプチド、抗体、およびそれらを含有する組成物
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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