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WO1998034945A1 - Modification ciblee du gene ccr-5 - Google Patents

Modification ciblee du gene ccr-5 Download PDF

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Publication number
WO1998034945A1
WO1998034945A1 PCT/US1998/002314 US9802314W WO9834945A1 WO 1998034945 A1 WO1998034945 A1 WO 1998034945A1 US 9802314 W US9802314 W US 9802314W WO 9834945 A1 WO9834945 A1 WO 9834945A1
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Prior art keywords
gene
ccr
odns
modified
sequence
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PCT/US1998/002314
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English (en)
Inventor
Rich B. Meyer, Jr.
Igor V. Kutyavin
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Epoch Pharmaceuticals, Inc.
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Priority to AU62715/98A priority Critical patent/AU6271598A/en
Publication of WO1998034945A1 publication Critical patent/WO1998034945A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs

Definitions

  • This invention is in the field of agents useful in the targeting and modification of host genes involved in Human Immunodeficiency Virus (HIV) infection. More particularly, it is in the field of triplex-fo ⁇ ning oligonucleotides capable of inducing modification of a target sequence at a specific site.
  • HIV Human Immunodeficiency Virus
  • HIV is known to be responsible for causing acquired immune deficiency syndrome (AIDS). HIV infects various cells of the immune system, in particular, macrophages and T-cells. Infection of macrophages is typical of early stages of infection while, at later stages, infection of T-cells predominates. It has been known for some time that, although the T-cell-specific cell-surface CD4 molecule serves as a receptor for HIV, CD4 alone is not sufficient for viral entry. Hence, it was believed that cofactors existed that, along with CD4, participated in the entry of HIV into the cell. Recently, such cofactors have been identified and partially characterized. For T-cell tropic HIV isolates the fusin protein acts as a co-receptor. Feng et al.
  • ⁇ 32 a naturally-occurring mutation in the CCR-5 gene, denoted ⁇ 32, results in protection from HIV infection. Liu et al. (1996) Cell 86:367-377; Samson et al. (1996) Nature 382:722-725; and Dean et al. (1996) Science 273:1856-1862.
  • the ⁇ 32 mutation is a 32-nucleotide deletion, causing a shift in the reading frame of the CCR-5 mRNA which leads to premature translation termination to form a truncated protein (see Figure 1).
  • the present invention provides compositions and methods for targeted alteration of the CCR-5 (CKR-5) gene in living cells, using triplex-forming oligonucleotides with attached crosslinking agents. Such alterations can cause heritable nucleotide sequence changes, some of which may result in altered cells that are resistant to infection by HIV.
  • the compositions and methods of the invention may be used prophylactically, to prevent HIV infection by blocking entry of the virus into macrophages, or therapeutically, to prevent further viral spread in an already-infected individual.
  • the portion shown begins at nucleotide 745.
  • the 12-nucleotide polypurine stretch which serves as a target for the modified ODNs of the invention is boxed. Enclosed within a dashed box and labeled " ⁇ 32" is the sequence deleted in the ⁇ 32 mutation, which is characteristic of certain individuals that are resistant to HIV infection.
  • the primer for first strand synthesis for LM-PCR (see Examples E and F) is shown by a horizontal arrow.
  • EcoRI site used as an internal standard for LM-PCR analysis is indicated.
  • a portion of the complementary strand, written in lower case, shows the sequence surrounding the targeted reaction site and indicates the principal site of reaction (circled G residue).
  • the lower portion of the figure shows the sequence and structure of some of the TFOs used for modification of the CCR-5 gene.
  • Figure 2 Efficiency and specificity of triplex-directed alkylation of a 60 base-pair model duplex (corresponding to nucleotides 891-950 of the CCR-5 sequence shown in Figure 1) by TFO 1 and TFO 2.
  • the noncoding (polypyrimidine-containing) strand was 2 P-labeled.
  • Figure 2 A shows alklylation products resulting from reaction of the model duplex with TFO 1 (containing normal G residues);
  • Figure 2B shows alklylation products resulting from reaction of the model duplex with TFO 2 (all G residues substituted by ppG). Results in 2A and 2B are shown as a function of coralyne concentration.
  • Figure 2C shows the location of alkylated bases resulting from reaction with TFO 1 or TFO 2 at 8 ⁇ M coralyne, determined by heat/piperidine treatment of the reactions, compared to an A+G sequencing ladder of the target sequence.
  • FIG. 3 Targeted modification of HT-29 cell genomic DNA by a modified triplex-forming oligonucleotide.
  • TFO 2 SEQ ID NO: 6
  • the leftmost lane is a control reaction lacking TFO, showing the product of LM-PCR defined by the upstream EcoRI site.
  • the center lane shows the results of modification at 20°C and the right lane shows results of modification at 37°C.
  • the arrow indicates bands resulting from modification of the target DNA.
  • Figure 4 Targeted modification of the CCR-5 gene in permeabilized HT-29 cells.
  • Lane 1 shows analysis of DNA from cells to which no TFO was added; lane 2 shows analysis of DNA from cells to which were added a non- targeted TFO conjugated to a phenylacetate mustard, lane 3 shows analysis of DNA from cells to which 5 ⁇ M modified TFO was added, and lane 4 shows analysis of DNA from cells to which 20 ⁇ M modified TFO was added.
  • the present invention encompasses modified oligonucleotides (ODNs) capable of forming a triple-stranded structure with a region of the CCR-5 gene.
  • ODNs are modified by attachment of a cross-linking agent.
  • the attached crosslinking agent reacts with targeted nucleotides in the DNA of the CCR-5 gene. As a result, a stable change is introduced into the CCR-5 gene.
  • anti- gene Triplex forming anti-gene oligonucleotides
  • anti- gene A variation of the "antisense” approach to rational drug design is termed "anti- gene.”
  • antisense ODNs target single stranded mRNA
  • anti-gene ODNs hybridize with and are capable of inhibiting the function of double-stranded DNA. More specifically, anti-gene ODNs form sequence-specific triple-stranded complexes with a double stranded DNA target and thus interfere with the replication or transcription of selected target genes.
  • RNA viruses, nucleic acid-free viroids and prions are examples of the antigene ODNs.
  • DNA is the repository for all genetic information, including regulatory control sequences and non-expressed genes, such as dormant proviral DNA genomes.
  • target for antisense ODNs which is mRNA
  • anti-gene ODNs have broader applicability and are potentially more powerful than antisense ODNs that merely inhibit mRNA processing and translation.
  • Anti-gene ODNs in the nuclei of living cells can form sequence-specific complexes with chromosomal DNA.
  • the resultant triplexes can inhibit replication and/or transcription of the target double stranded DNA.
  • the DNA binding properties of the ODNs of the invention are strengthened and enhanced by virtue of the crosslinking agents attached to the ODNs, providing a greater likelihood that covalent linkages between the anti-gene ODN and the target DNA sequence will be generated.
  • covalent linkages may often result in the generation of one or more mutations at or near the target site, endowing the anti-gene ODNs of the invention with the ability to cause longer lasting effects than those achieved by corresponding antisense inhibition of mRNA function.
  • Mammalian cell DNA does not undergo turnover; in fact, cells possess sophisticated pathways capable of repairing lesions in DNA that may arise from environmental insults or from spontaneous rearrangements.
  • mRNA is transient and may exist only for minutes within a cell.
  • anti-sense ODNs will provide relatively short term effects.
  • anti-gene ODNs have the potential to generate more lasting changes and, once within the cell, the ODNs naturally concentrate in the nucleus, their site of action.
  • ODNs may be enhanced by attachment of an ODN to carrier, including but not limited to a lipophilic or lysosomotrophic carrier, with or without an intervening cleavable peptide linker, as disclosed in U.S. Patent 5,574,142.
  • carrier including but not limited to a lipophilic or lysosomotrophic carrier, with or without an intervening cleavable peptide linker, as disclosed in U.S. Patent 5,574,142.
  • Anti-gene therapy is based on the observation that certain DNA sequences are capable of forming triple-stranded complexes.
  • the third strand resides in the major groove of the Watson Crick base-paired double helix, where it hydrogen bonds to one of the two parental strands.
  • the triplets indicated below demonstrate the binding code which governs the recognition of base pairs by a third base. In each case, the third strand base is presented first and is followed by the base pair; hydrogen bonding between the first two bases maintains the third base interaction.
  • Cytosine/thymidine-, guanine/adenine- and guanine/thymidine-containing ODNs can bind, in a sequence-specific fashion, to homopurine runs in double-stranded DNA.
  • These recognition motifs are based on Hoogsteen or reverse Hoogsteen base pairing.
  • the ODN In the C/T recognition motif, the ODN is parallel to the homopurine strand of the duplex; in the G/A recognition motif, the ODN is anti-parallel to the homopurine strand; in the G/T recognition motif, the ODN may bind parallel or anti-parallel to the homopurine strand of the duplex, depending on the G content of the third strand.
  • These recognition motifs may be sequence-dependent.
  • the sequence specificity of anti-gene ODNs using the C/T recognition motif permits hybridization of such ODNs to homopurine runs in plasmid DNA and in yeast chromosomes. Since ODN binding is restricted to homopurine runs, it would be advantageous to identify additional heterocyclic bases or base analogues that can recognize the remaining two base pairs, i.e., C-G and T-A. While guanosine can be used in the third strand to recognize T-A base pairs, this interaction involves only one hydrogen bond and is relatively unstable.
  • anti-gene ODNs can be modified with a variety of pendant groups designed to augment their activity.
  • the present invention encompasses ODNs modified with intercalating groups, cleaving agents, reporter groups, reactive groups and/or crosslinking moieties appended within or at the termini of anti-gene
  • ODNs Upon triplex formation, these groups may interact with the adjacent duplex. Further, in the C/T recognition motif, substitution of 5-methyl cytosine for cytosine in the third strand ODN significantly stabilizes triplexes formed with guanosine-rich homopurine runs.
  • ODNs with modified backbones such as oligonucleoside methyl- phosphonates and phosphorothioates, are capable of forming triple-stranded complexes and are encompassed in the present invention.
  • ODNs comprise a chain of nucleotides which are linked to one another by phosphate ester linkages.
  • Each nucleotide typically comprises a heterocyclic base, a sugar moiety attached to the heterocyclic base, and a phosphate moiety which esterifies a hydroxyl function of the sugar moiety.
  • the principal naturally-occurring nucleotides include uracil, thymine, cytosine, guanine or adenine as the heterocyclic base, and ribose or 2'-deoxyribose as the sugar moiety.
  • the ODNs of the present invention may comprise ribonucleotides, deoxyribonucleotides, or modified sugars or sugar analogues such as are known to those of skill in the art.
  • pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose and lyxose are non-limiting examples of sugar moieties which are encompassed by the present invention.
  • the sugar moiety may be in a pyranosyl or a furanosyl form and may be attached to the heterocyclic base in either the ⁇ or ⁇ anomeric configuration.
  • Preferred sugar moieties of the present invention include the furanosides of ribose, deoxyribose, arabinose and 2'-O-methyl ribose.
  • the phosphorus derivative or modified phosphate group which may be attached to the sugar or sugar analogue moiety may be a monophosphate, diphosphate, triphosphate, alkylphosphate, alkanephosphate, phosphorothioate, phosphorodithioate or the like.
  • the internucleotide linkages will be those normally found in polynucleotides or those with similar steric properties including, but not limited to, phosphodiester, phosphotriester, phosphorothioate and methyl phosphonate.
  • the phosphate group in the ODNs of the present invention will form a phosphodiester.
  • Alternative backbones, such as peptide nucleic acids, are also contemplated by the present invention.
  • heterocyclic bases of the modified ODNs of the invention may be those normally found in nucleic acids, as well as those naturally-occurring and synthetic modified bases that are known to those of skill in the art. Examples include, but are not limited to, uracil, thymine, cytosine, 5-methyl cytosine, guanine, adenine, hypoxanthine, pyrrolo [2, 3-d] pyrimidines and pyrazolo [3, 4-d] pyrimidines.
  • Particularly preferred in the present invention are the guanine analogue 6-aminopyrazolo[3,4-- ]pyrimidin-4(3H)- one (ppG) and the adenine analogue 4-amino-lH-pyrazolo[3,4-d]pyrimidine, both of which facilitate triplex formation by oligonucleotides into which they are incorporated. See co-owned U.S. Patent Application Serial No. 08/848,373, filed April 30, 1997.
  • modified ODNs of the present invention may include one or more modified base and/or sugar moieties, as long as other criteria of the invention (such as ability to form a triple helix) are satisfied.
  • modified nucleotides might even enhance the properties of the ODNs, with respect to certain criteria.
  • ODNs can be prepared by automated chemical synthesis, using any one of a number of commercial DNA synthesizers, such as those provided by Applied Biosystems.
  • reactive groups such as amino groups can be attached at either end of, or internally to, the ODN, using commercially available reagents, such as are available from Applied Biosystems, Clontech and Glen Research, among others.
  • Such reactive groups serve as a point of attachment for the linker arm A of the crosslinker, to which the leaving group L is attached (see below). Attachment of crosslinking agents to
  • ODNs is accomplished according to the procedures outlined in PCT publication WO 94/17092.
  • the length and sequence of the ODNs of the invention are such that triplex formation with a target sequence will proceed under normal experimental or physiological conditions.
  • the ODNs of the invention are modified by the attachment of a cross-linking agent.
  • the cross-linking agent can be attached at either end of the ODN, or at internal positions.
  • the attached cross-linking agent reacts with nearby nucleotides in the target sequence, leading to modification of the target sequence at a specific site, the site of modification depending on the nature of the cross-linking agent and its position in the ODN.
  • any suitable cross-linking agent known in the art can be incorporated into the ODNs of the present invention, provided they meet certain requirements.
  • each cross- linking agent must be covalently bonded to a site on the ODN.
  • the length and steric orientation of the cross-linking agent must be such that it can reach a suitable reaction site in the target DNA sequence after the ODN is hybridized with the target.
  • the cross-linking agent must have a reactive group which will react with a reactive group of the target DNA sequence.
  • the cross-linking agents can be covalently attached to the heterocyclic bases or base analogues, to the sugar or modified sugar residues, or to the phosphate or modified phosphate functions of the ODNs by any method known in the art.
  • a cross-linking agent comprises two groups or moieties, namely the reactive group, which is typically and preferably an electrophilic leaving group (L), and an "arm" (A) which attaches the leaving group L to the respective site on the ODN.
  • the leaving group L may be chosen from, for example, such groups as chloro, bromo, iodo, SO 2 R'", or S + R'"R"", where each of R'" and R"" is independently alkyl or aryl or R'" and R"" together form a C,. 6 alkylene bridge. Chloro, bromo and iodo are preferred.
  • haloacetyl groups such as -COCH 2 I, and bifunctional "nitrogen mustards", such as -N-[(CH 2 ) 2 -C1] 2 are preferred.
  • the leaving group will be altered by its leaving ability. Depending on the nature and reactivity of the particular leaving group, the group to be used is chosen in each case to give the desired specificity.
  • linker arm A can be regarded as a single entity which covalently bonds the ODN to the leaving group L, and maintains the leaving group L at a desired distance and steric position relative to the ODN
  • A can be constructed in a synthetic scheme where a bifunctional molecule is covalently linked to the ODN (for example by a phosphate ester bond to the 3' or 5'' terminus, or by a carbon-to-carbon bond to a heterocyclic base) through its first functionality, and is also covalently linked through its second functionality (for example an amine) to a hydrocarbyl bridge (alkyl bridge, alkylaryl bridge or aryl bridge, or the like) which, in turn, carries the leaving group.
  • a bifunctional molecule is covalently linked to the ODN (for example by a phosphate ester bond to the 3' or 5'' terminus, or by a carbon-to-carbon bond to a heterocyclic base) through its first functionality, and is also covalently linked through its second functionality
  • a general formula of the cross linking function is thus -A-L, or -A-L 2 where L is the above defined leaving group and A is a moiety that is covalently linked to the ODN.
  • the A moiety itself should be unreactive (other than through L) under the conditions of hybridization of the ODN with the target DNA sequence, and should maintain L in a desired steric position and distance from the desired site of reactions such as an N-7 position of a guanosine residue in the target DNA sequence.
  • the length of the A group should be equivalent to the length of a normal alkyl chain of approximately 2 to 50 carbons.
  • An exemplary more specific formula for a class of preferred embodiments of the cross-linking function is -(CH 2 ) q - Y - (CH 2 ) ra - L, where L is the leaving group, defined above, each of m and q is independently 0 to 8, inclusive, and where Y is defined as a "functional linking group".
  • a "functional linking group” is a group that has two functionalities, for example -NH 2 and -OH, or -COOH and - OH, or -COOH and -NH 2 , which are capable of linking the (CH 2 ) q and (CH 2 ) m bridges.
  • An acetylenic terminus (HC ⁇ C-) is also a suitable functionality for Y, because it can be coupled to certain heterocycles, as described, for example, in PCT Publications WO 94/17092 and WO 96/40711.
  • N(R,) - (CH 2 ) p -L describes a "nitrogen mustard", which is a class of potent alkylating agents.
  • Particularly preferred within the scope of the present invention are those modified ODNs wherein the cross-linking agent includes the functionality - N(R,) - (CH 2 ) p -L where L is halogen, preferably chlorine; even more preferred are those modified ODNs where the cross linking agent includes the grouping - N-[(CH 2 ) 2 -L] 2 (a "bifunctional" N-mustard).
  • Particularly relevant to the present invention are those mustards having the structures 4-(p- (bis-(2-chloroethyl)amino)phenyl)butyryl (chlorambucil mustards) and 2-(p-(bis-(2- chloroethyl)amino)phenyl)acetyl (phenylacetate mustards).
  • a bifunctional N-mustard (or other cross linking function having two reactive groups) is included in the cross-linking agent.
  • One such cross-linking agent attached to the ODN is sufficient, as there is evidence in accordance with PCT Publication WO 94/17092 that, after hybridization, the modified ODN attaches to both strands of the target double stranded DNA sequence.
  • crosslinking agents described above may be found in PCT Publication WO 94/17092; PCT Publication WO 96/40711 and Kutyavin et al. (1994) J. Amer. Chem. Soc. 115:9303-9304.
  • the modified ODNs of the invention are administered to cells by any method of nucleic acid transfer known in the art, including, but not limited to, transformation, co- precipitation, electroporation, neutral or cationic liposome-mediated transfer, microinjection or gene gun.
  • the modified ODNs may be attached to carriers and/or connected to carriers by cleavable linkers, such carriers and linkers including, but not limited to, those disclosed in co-owned U.S. Patent 5,574,142.
  • the modified ODNs of the invention are suitable for in vitro, in vivo and ex vivo therapy and may be administered parenterally, intravenously, subcutaneously, orally or by any other method known in the art.
  • the modified ODNs of the invention can be combined with a pharmaceutically acceptable excipient for administration to a mammalian subject.
  • a pharmaceutically acceptable excipient is usually nontoxic and nontherapeutic. Examples of such excipients are water, saline, Ringer's solution, dextrose solution, and Hank's solution.
  • Nonaqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.
  • Parenteral vehicles may also take the form of suspensions containing viscosity-enhancing agents, such as carboxymethylcellulose, sorbitol or dextran.
  • the excipient will also usually contain minor amounts of substances that enhance isotonicity and chemical stability.
  • buffers include, but are not limited to phosphate buffer, bicarbonate buffer and tris buffer; while examples of preservatives include, but are not limited to thimerosal, m- or o-cresol, formalin and benzyl alcohol.
  • Standard formulations will be either liquids or solids which can be dissolved in a suitable liquid medium as a suspension or a solution.
  • the vehicle may comprise dextrose, human serum albumin, preservatives, etc., to which sterile water, saline, buffer or other solvent can be added prior to administration.
  • modified triplex-forming ODNs might be capable of directing a crosslinking reaction to a specific site in a target sequence within a living cell.
  • ODNs capable of triplex formation at the polypurine stretch in the CCR-5 gene. These were modified by the attachment of a crosslinking group (or groups) and the modified ODNs were tested for their ability to generate targeted modification at or near the CCR-5 gene polypurine stretch.
  • the sequences of these ODNs are as follows:
  • ODN 1 consists of SEQ ID NO. 2 modified by the attachment of the following crosslinking group at the 5' terminus:
  • n 0 or 1.
  • compositions of the invention thus encompass modified ODNs of the sequence depicted in SEQ ID NO: 3 or portions thereof sufficient to effect triple-strand binding to the CCR-5 gene.
  • the OD s have the sequence depicted in SEQ ID NO: 2.
  • the modified ODNs were tested in three systems: 1) in vitro with a double- stranded ODN target; 2) in vitro with naked genomic DNA as target and 3) in living cells with the cell genome as target. Experimental procedures and results of these tests are presented in the Examples below.
  • ClAmb is chlorambucil, 4-(p-(bis-(2-chloroethyl)amino)phenyl)butyryl, attached to either the 5'- or 3'- end of the oligo via an aminohexyl linker.
  • PhAc mustard is 2-(p-(bis-(2-chloroethyl)amino)phenyl)acetyl, described in PCT Publications WO 94/17092 and WO 96/40711.
  • ODNs were prepared from 1 ⁇ mol of the appropriate CPG support on an ABI 394 (Perkin-Elmer) using the protocol provided by the manufacturer.
  • Protected ⁇ - cyanoethyl phosphoramidites of 2'-deoxynucleosides, CPG supports, deblocking solutions, capping reagents, oxidizing solutions and tetrazole solutions were obtained from Glen Research.
  • the guanine residues in certain ODNs were replaced by 6-aminopyrazolo[3,4- ]pyrimidine-4(3H)-one, (or ppG), using a phosphoramidite prepared as described by Seela et al. (1988) Helv. Chim. Acta 71: 1191-1199.
  • ODNs containing a 5' aminohexyl tail were conjugated with the 2,3,5,6- tetrafluorophenyl esters of either chlorambucil or phenylacetate mustard as described by Reed et al. (1998) Bioconjugate Chem 9:64-71, and isolated from reaction mixtures by HPLC with 50-70% yield. All manipulations with collected HPLC fractions were performed in ice-cold solutions. Conjugated ODNs were dissolved in water and stored at - 70°C. The integrity of the conjugated nitrogen mustard was assessed as described by Reed et al, supra.
  • the two strands of the target sequence were synthesized separately and one of the strands was 5'- 32 P-labeled and mixed in buffer (140 mM KC1, 10 raM MgCl 2 , 1 mM spermine, 20 mM HEPES pH 7.2) with a two-fold excess of unlabeled complementary strand.
  • the mixture was heated for 1 min at 95°C and then incubated at 37°C for 30 min. Then the modified triplex-forming ODN (Table 1) was added.
  • the final concentration of 32 P-labeled strand in the mixture was 2 x 10 "8 M and of the unlabeled strand was 4 x 10 "8 M.
  • the concentration of triplex-forming ODN added to the mixture was 2 x 10 "6 M.
  • coralyne was added to the mixture to a final concentration of 10 x 10 "6 M.
  • the mixture was incubated at 37°C for 15 hr, then analyzed by denaturing gel electrophoresis. After electrophoresis, the gel was transferred onto paper, dried down and imaged using a Bio-Rad molecular imager (with its associated computer software) to measure the ratio between crosslinked product and overall radioactivity in each lane of the gel. Results are shown in Table 1. Table 1
  • a synthetic 60 base-pair duplex target was used.
  • the sequence of this target extended from nucleotides 891-950 as shown in Figure 1.
  • the targeting reaction mixture contained 20 nM labeled duplex 60-mer, 2 ⁇ M conjugated ODN, 8 ⁇ M coralyne, 20 mM HEPES, pH 7.2, 140 mM KCl, 10 mM MgCl 2 , and 1 mM spermine, incubated at 37°C for 4 hours.
  • TFOs 12-mer triplex-forming oligonucleotides
  • TFO 1 contained normal guanines
  • TFO 2 all guanines were replaced with 6- aminopyrazolo[3,4--flpyrimidin-4(3H)-one (ppG) (SEQ ID NO: 6).
  • ODNs containing the guanine analogue ppG have increased rates of triplex formation and greater triplex stability. See co-owned U.S. Patent Application Serial No. 08/848,373, filed April 30, 1997.
  • TFO 1 and TFO 2 were designed to alkylate the N7 position of the guanine residue on the noncoding strand opposite C-930.
  • TFO 2 results obtained using TFO 2 show that substitution of guanine residues with ppG had a significant positive effect on reaction efficiency (Figure 2B).
  • TFO 2 provided 74% alkylation at 8 ⁇ M coralyne and 2% target alkylation in the absence of coralyne.
  • the effects of ppG on efficiency of alkylation of the CCR-5 gene are consistent with its ability to facilitate triplex formation, as disclosed in co-owned U.S. Patent Application Serial No. 08/848,373, filed April 30, 1997.
  • Genomic DNA from HT29 adenocarcinoma cells was prepared with a Wizard Genomic DNA Purification Kit (Promega), using the protocol provided by the manufacturer.
  • LM-PCR Quantitative Ligation-Mediated PCR
  • the first modification was to generate an internal control site by restriction digestion of the DNA after treatment with the reactive TFO, to allow quantitation of the amount of site-specific alkylation.
  • Selection of a restriction endonuclease was based on the enzyme having a recognition site upstream (5') of the alkylated base, and no recognition sites between that base and the downstream (3') sequence complementary to the first strand primer.
  • An EcoRI site upstream of (on the 5' side of) the targeted G residue on the noncoding strand was used.
  • the procedure for LM-PCR was as follows. TFO-treated DNA samples were digested to completion with EcoRI by incubation for 3 hr under optimal conditions (according to the manufacturer) with a three-fold excess of restriction enzyme. The volume was adjusted to 100 ⁇ L with water and the DNA was precipitated with ethanol.
  • the pellet was resuspended in 10 m Tris-HCl, pH 7.5, 1 m EDTA, to give a DNA concentration of about 0.5 ⁇ g/ ⁇ l.
  • To 5 ⁇ l of this chilled solution in a PCR tube was added 25 ⁇ l of the first strand synthesis solution (Mueller et al, supra).
  • First strand synthesis was primed with the following oligonucleotide: 5'-TCCATACAGTCAGTATCAATTCTGG-3' (SEQ ID NO: 7)
  • the third primer was labeled with 32 P at its 5' end and had the sequence:
  • An image of an electrophoretic gel shows LM-PCR products of unmodified (leftmost lane) and TFO 2-modified target DNA (center lane, modification at 20°C; rightmost lane, modification at 37°C). Since one of the CCR-5 alleles of the HT-29 cells used in this study has the ⁇ 32 mutation, and the primer overlaps the site of the ⁇ 32 deletion, only a single EcoRI band is obtained, corresponding to the wild-type CCR-5 allele.
  • the sequences of the two strands of the target are shown in Table 1 (SEQ ID NOs: 4 and 5) above the sequence of the modified triplex-forming ODN
  • the modified strand corresponds to the upper of the two target strands in Table 1(SEQ ID NO: 4) and is modified primarily at the G residue five nucleotides from the 5' end of the sequence of that strand shown in Table 1. Almost quantitative modification of the targeted genomic DNA was obtained with 2 ⁇ M TFO 2 in the presence of 8 ⁇ M coralyne. Only trace target alkylation was detected by LM-PCR when modification was conducted in the absence of coralyne.
  • HT-29 cells were plated into 6-well 35 mm plates at 4.0 x 10 5 cells per well and were allowed to adhere for 4 hr at 37°C in complete medium. The cells were then washed with phosphate-buffered saline (PBS) and treated for 5 min at 37°C with 350 ⁇ l of permeabilization buffer (137 mM NaCl, 100 mM PIPES, pH 7.4, 5.6 mM glucose, 2.7 mM KCl, 2.7 mM EGTA, 1 mM ATP, 0.1% bovine serum albumin) containing 500 Units/ml Streptolysin O (preactivated for 15 min at room temperature in the presence of 2.5 ⁇ M DTT), 8 ⁇ M coralyne and 5 ⁇ M or 20 ⁇ M of TFO 2 (SEQ ID NO: 6) with a phenylacetate mustard moiety conjugated to the 5' terminus through a hexylamine linker.
  • PBS phosphate-buffered
  • the phenylacetate mustard has a slightly longer half-life in cells than does the chlorambucil mustard.
  • This TFO was also modified with a 3'-hydroxyhexyl phosphate (as described in Example B) to slow the rate of nuclease digestion within cells. Streptolysin O was used to render cells permeable to ODNs. Spiller et ⁇ /. (1995) Antisense Res. Dev. 5:13-21. After treatment, 5 ml complete medium was added to each well and cells were incubated for another 6 hr at 37°C. After incubation, cells were washed with PBS, trypsinized and DNA was isolated as described in Example E. DNA modification was determined by LM-PCR, as described in Example E.
  • the results obtained in this experiment are depicted in Figure 4.
  • the image of an electrophoretic gel shows LM-PCR products of target DNA from cells that were untreated (lane 1), treated with a nontargeting TFO with a conjugated phenylcetate mustard (lane 2), or treated with 5 ⁇ M (lane 3) or 20 ⁇ M (lane 4) phenylacetate-modified TFO.
  • the efficiency of site-directed DNA modification was quantitated by comparison of the PCR product obtained from modification at the targeted G residue to the PCR product defined by the EcoRI site. Analysis of these and similar results indicate that the efficiency of site- directed DNA modification, obtained using the procedure described above, was as follows:

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Abstract

La présente invention concerne des oligonucléotides modifiés, capables de former des complexes à trois brins avec le gène CCR-5 récepteur de la chémokine. Les oligonucléotides portent des groupes réticulants capables d'induire la modification ciblée du gène CCR-5. De telles modifications peuvent altérer la capacité du produit du gène CCR-5 à servir comme corécepteur pour les virus de l'immunodéficience humaine.
PCT/US1998/002314 1997-02-06 1998-02-06 Modification ciblee du gene ccr-5 WO1998034945A1 (fr)

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WO2000012523A1 (fr) * 1998-08-26 2000-03-09 Epoch Pharmaceuticals, Inc. Conjugues oligonucleotidiques de diaziridinyl-aryle et de bis-[di(chloroethyl)amino]-aryle, et reactifs pour leur preparation
WO2001021832A1 (fr) * 1999-09-23 2001-03-29 Isis Innovation Limited Gene de susceptibilite pour des affections intestinales inflammatoires
US6511826B2 (en) 1995-06-06 2003-01-28 Human Genome Sciences, Inc. Polynucleotides encoding human G-protein chemokine receptor (CCR5) HDGNR10
US6743594B1 (en) 1995-06-06 2004-06-01 Human Genome Sciences, Inc. Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5)
US6916653B2 (en) 1998-01-15 2005-07-12 King's College London Ribozymal nucleic acid
US7175988B2 (en) 2001-02-09 2007-02-13 Human Genome Sciences, Inc. Human G-protein Chemokine Receptor (CCR5) HDGNR10
US7393934B2 (en) 2001-12-21 2008-07-01 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
WO2008086529A2 (fr) * 2007-01-11 2008-07-17 Yale University Compositions et méthodes destinées à l'inactivation ciblée de récepteurs de surface cellulaire pour le vih
US7501123B2 (en) 2004-03-12 2009-03-10 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
WO2011133803A1 (fr) * 2010-04-21 2011-10-27 Helix Therapeutics, Inc. Compositions et méthodes d'inactivation ciblée de récepteurs de surface cellulaire de vih
US8658608B2 (en) 2005-11-23 2014-02-25 Yale University Modified triple-helix forming oligonucleotides for targeted mutagenesis
EP2759596A1 (fr) * 2006-05-04 2014-07-30 Novartis AG Acide ribonucléique interférent court (siRNA)
US11136597B2 (en) 2016-02-16 2021-10-05 Yale University Compositions for enhancing targeted gene editing and methods of use thereof
US12268774B2 (en) 2017-04-04 2025-04-08 Yale University Compositions and methods for in utero delivery

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7160546B2 (en) 1995-06-06 2007-01-09 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US6511826B2 (en) 1995-06-06 2003-01-28 Human Genome Sciences, Inc. Polynucleotides encoding human G-protein chemokine receptor (CCR5) HDGNR10
US6743594B1 (en) 1995-06-06 2004-06-01 Human Genome Sciences, Inc. Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5)
US6759519B2 (en) 1995-06-06 2004-07-06 Human Genome Sciences, Inc. Antibodies to human G-protein chemokine receptor HDGNR10 (CCR5receptor)
US6800729B2 (en) 1995-06-06 2004-10-05 Human Genome Sciences, Inc. Human G-Protein chemokine receptor HDGNR10 (CCR5 receptor)
US6916653B2 (en) 1998-01-15 2005-07-12 King's College London Ribozymal nucleic acid
WO2000012523A1 (fr) * 1998-08-26 2000-03-09 Epoch Pharmaceuticals, Inc. Conjugues oligonucleotidiques de diaziridinyl-aryle et de bis-[di(chloroethyl)amino]-aryle, et reactifs pour leur preparation
GB2371622A (en) * 1999-09-23 2002-07-31 Isis Innovation Susceptibility gene for inflammatory bowel disease
WO2001021832A1 (fr) * 1999-09-23 2001-03-29 Isis Innovation Limited Gene de susceptibilite pour des affections intestinales inflammatoires
US7175988B2 (en) 2001-02-09 2007-02-13 Human Genome Sciences, Inc. Human G-protein Chemokine Receptor (CCR5) HDGNR10
US7393934B2 (en) 2001-12-21 2008-07-01 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US7501123B2 (en) 2004-03-12 2009-03-10 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US8658608B2 (en) 2005-11-23 2014-02-25 Yale University Modified triple-helix forming oligonucleotides for targeted mutagenesis
EP2759596A1 (fr) * 2006-05-04 2014-07-30 Novartis AG Acide ribonucléique interférent court (siRNA)
WO2008086529A2 (fr) * 2007-01-11 2008-07-17 Yale University Compositions et méthodes destinées à l'inactivation ciblée de récepteurs de surface cellulaire pour le vih
WO2008086529A3 (fr) * 2007-01-11 2009-02-19 Univ Yale Compositions et méthodes destinées à l'inactivation ciblée de récepteurs de surface cellulaire pour le vih
WO2011133803A1 (fr) * 2010-04-21 2011-10-27 Helix Therapeutics, Inc. Compositions et méthodes d'inactivation ciblée de récepteurs de surface cellulaire de vih
US11136597B2 (en) 2016-02-16 2021-10-05 Yale University Compositions for enhancing targeted gene editing and methods of use thereof
US12268774B2 (en) 2017-04-04 2025-04-08 Yale University Compositions and methods for in utero delivery

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