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WO1996040271A1 - Traitement des hemoglobinopathies - Google Patents

Traitement des hemoglobinopathies Download PDF

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Publication number
WO1996040271A1
WO1996040271A1 PCT/US1996/009430 US9609430W WO9640271A1 WO 1996040271 A1 WO1996040271 A1 WO 1996040271A1 US 9609430 W US9609430 W US 9609430W WO 9640271 A1 WO9640271 A1 WO 9640271A1
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nucleic acid
strand
oligonucleotide
native
donor
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PCT/US1996/009430
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Peter M. Glazer
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Yale University
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/42—Haemoglobins; Myoglobins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3511—Conjugate intercalating or cleaving agent

Definitions

  • the present invention relates to management and treatment of hemoglobinopathies, such as sickle cell anemia and ⁇ -thalassemia.
  • the invention also relates to developing research animals and cell lines for the study of hemoglobinopathies and their therapies.
  • the invention utilizes third strand oligonucleotides to target double- stranded nucleic acid sequences in or near the globin genes or in or near sequences controlling expression of those genes to cause either a desired mutation or nucleic damage to stimulate homologous recombination with a supplied donor nucleic acid.
  • Oligonucleotides (third strands) can bind to double- stranded DNA to form triple-stranded helices (triplexes) in a sequence specific manner.
  • Oligonucleotide-mediated triplex formation has been shown to prevent transcription factor binding to promoter sites and to block mRNA synthesis in vitro and in vivo (Blu e e ⁇ al., Nucleic Acids Res. 20:1777 (1992); Cooney, et al., Science 241:456 (1988); Duval-Valentin, et al., Proc. Natl. Acad. Sci. USA. 89:504 (1992); Grigoriev, et al., Proc. Natl. Acad. Sci. USA. 90:3501 (1993) ; Grigoriev, et al., J. Biol. Chem.
  • third strands are used to target or direct mutagenesis or homologous recombination to specific sites in or near selected globin genes in order to produce permanent changes in gene function and expression. Long- term blocking of the DNA target is, therefore, not necessary.
  • the fact that DNA damage and mutagenesis can be directed in this sequence specific manner by third strands is evidenced by mutagenesis "footprints" at the site resulting from unrepaired damage (Havre, et al . , Proc.
  • the third-strand binding code and binding motifs dictates the sequence specificity for binding third strands in the major groove of double-stranded DNA to form a triple-stranded helix or triplex.
  • Third-strand binding differs from the familiar Watson-Crick complementarity principle (A:T/U and G:C) for the double-stranded helix in two major respects: (1) the third-strand binding code is degenerate, and (2.) third strands bind only to double-strands which contain a sequence of adjacent (or run of) purine bases (A or G) in one of the strands, which here will be called the center or core strand.
  • the third-strand binding code is illustrated in the chart below.
  • a "+” means the bases are complementary or correspondent, and a "-" means they are not complementary or not correspondent.
  • motifs which further describe third-strand binding to purine center-strand targets.
  • the motifs describe, for example, whether the third-strand must bind parallel or antiparallel to the center target strand (polarity) ; and to some extent the motifs describe center- strand sequence and nearest neighbor effects on binding.
  • hemoglobinopathies hemoglobinopathies
  • Bunn and Forget Hemoglobin: Molecular, Genetic and Clinical Aspects
  • Hemoglobin is the blood protein which carries oxygen to tissues. It is present in large quantities in the erythrocytes (red blood cells) , which are little more than "bags" of hemoglobin. Hemoglobin messenger RNA (mRNA) is produced in pre-erythrocyte cells (erythroids) . Because they lack a nucleus, erythrocytes are not capable of directing the manufacture of hemoglobin mRNA. Young erythrocytes in the blood, called reticulocytes, carry hemoglobin mRNA and translate it into protein.
  • mRNA Hemoglobin messenger RNA
  • Hg Hg
  • Other physiological conditions present near metabolizing tissues high C0 2 , high acidity, high temperature, and high concentration of the compound 2,3- diphosphoglycerate (DPG) — cause the release of more oxygen to "the tissues at the venous Po 2 when it is demanded by high metabolism.
  • the hemoglobin protein consists of four amino-acid chains or subunits. The predominant hemoglobin in normal adults (about 92%) is called hemoglobin-A (HbA) and has two each of so-called ⁇ chains and ⁇ chains. Its four-chain structure is denoted as ⁇ ⁇ 2 .
  • a minor hemoglobin in normal adults, HbA 2/ consists of two ⁇ chains and two ⁇ chains ( ⁇ 2 5 2 ) . Other hemoglobin molecules are present in small amounts in normal adults.
  • Hemoglobin is highly-tuned by evolution to deliver oxygen as needed. For example, each stage of human development from embryo to adult has different oxygen needs and hemoglobins, which differ slightly in amino acid sequence. In early embryo, there are Gower-1 ( ⁇ 2 ) , Gower-
  • Hemoglobin Molecular, Genetic and Clinical Aspects", W.B. Saunders, Philadelphia pp. 62, 68 (1986) .
  • Sickle cell anemia ⁇ -thalassemia and ⁇ -thalassemia denote the three major disease categories.
  • Sickle cell anemia (SCA) and the much milder sickle cell trait are caused by a single amino acid change in the ⁇ chain.
  • the defective ⁇ chain is denoted by ⁇ s and hemoglobin containing ⁇ s chains is denoted as HbS.
  • the specific defect is a valine replacing the normal glutamic acid (Glu > Val) , and the underlying DNA base mutation is adenine to thymine (A)
  • ⁇ -thalassemia results from the absence of functional ⁇ chain. Over 100 different mutations have been associated with ⁇ -thalassemia (Collected in Huisman, Hemoglobin, 17:479 (1993)) .
  • ⁇ thalassemia is classified as either ⁇ + or ⁇ °, depending on whether one or both ⁇ chain DNA regions are defective. In the severe disease, ⁇ ° thalassemia, both are defective, and no ⁇ chains are produced. In the mild ⁇ + disease, some, but very little ⁇ chain is produced.
  • a single therapeutic strategy of targeting a single third-strand to a region near the locations of different ⁇ -thalassemia mutations in different patients to allow homologous recombination with a single "normal sequence" donor strand can correct the several different mutations in the different patients.
  • a single composition of matter third strand and donor DNA
  • ⁇ o-thalassemia is often much less severe if it is associated with elevation of other hemoglobins.
  • HbFH hereditary persistence of fetal hemoglobin
  • fetal hemoglobin in which fetal hemoglobin is synthesized into adult life, the presence of 20% to 30% HbF results in only mild disease (Bunn and Forget, op. ci t . , pp. 345-346; Apperley, Bailliere 's Clinical Haematolog ⁇ , 6:299 (1993)), and lesser amounts of HbF will reduce the severity of the disease (Perrine, et al . , N. Engl . J. Med. 328: 81 (1993) ) .
  • HbF The severity of SCA is reduced by the presence of HbF as well. To eliminate most disease symptoms, it is estimated that 20% HbF is needed, and as little as 10% to 12% HbF can reduce or make infrequent some disease symptoms, such as protection against stroke (Noguchi, et al . , N Engl . J. Med. 318:96 (1988); Charache, Experientia . 49:126 (1993); Jackson, et al . , J. Am. Med A ⁇ soc. Ill : 867 (1961); Davies, Blood Reviews . 7:4 (1993)) .
  • the resulting diseases are usually asymptomatic with mild anemia present in most individuals.
  • sickle cell anemia there is high risk of septicemia in infancy and early childhood; and in the more severe cases, there is high risk of childhood stroke.
  • extreme anemia due to destruction of red blood cells and painful crises due to the blocking of capillaries by sickled cells (vaso-occlusion) are major manifestations of SCA. Growth and development abnormalities, damaged organs, and a number of other complications occur (Bunn and Forget, op. cit . , pp. 510-533).
  • the disease course is variable with some patients following a severe course beginning shortly after birth with early childhood stroke, whereas other patients are infrequently ill (Powars and Hiti, AJDC, 147:1197 (1993)). About 30% of patients experience devastating disease with recurrent pain and vaso-occlusion crises that result in repeated strokes, chronic renal failure, etc. About 60% of patients have less severe disease, and 10% remain nearly symptom-free throughout life (reviewed in Apperley, Balliere 's Clinical Haematology. 6: 299 (1993)) .
  • Transfusions also carry the risk of complications and of diseases such as hepatitis and AIDS. Without adequate transfusion, morbidity and mortality occur sooner in life (Bunn and Forget, op . cit . , pp. 335-336). Since ⁇ °-thalassemia has such a severe clinical course, bone marrow transplants and other drastic therapies are justified despite the high risk of complications and even death. In the context of this invention, ABMT is less risky than current therapies. While oc-thalassemias (caused by defective or missing ⁇ chains) are known, and the methods, target sites, and third-strands directed to those sites, etc. apply to these conditions and other hemoglobinopathies as well, SCA and ⁇ ° thalassemia are more prevalent and will be used to illustrate the utility of the invention.
  • a single mutation in the ⁇ chain of hemoglobin is sufficient to cause the sickle-shaped cells characteristic of SCA.
  • the normal adenine base is mutated to a thymine, which causes an amino acid change from a negatively-charged glutamic acid to an uncharged, hydrophobic valine.
  • the normal DNA sequence surrounding and including the changed base is:
  • the altered DNA sequence surrounding and including the changed base is:
  • HbS sickle cell hemoglobin
  • HbS sickle cell hemoglobin
  • HBA HbS and HBA molecules
  • High-molecular-weight, physically- large polymers consisting of 14 double-stranded fibers of hemoglobin molecules form, which in turn distort the erythrocytes into a characteristic sickle shape.
  • the distorted erythrocytes cannot pass through the capillaries (vaso-occlusion) which is the cause of many of the severe medical problems associated with SCA, which include insufficient blood supply to tissues and organs with subsequent damage, stroke, and sickle-cell pain crises.
  • the distorted erythrocytes are selectively destroyed causing severe anemia. Prevention of aggregation, polymerization and subsequent sickling, then, is one way to manage the disease.
  • erythrocytes contain no DNA
  • hemoglobin mRNA is synthesized in precursor erythroid cells.
  • factors that influence globin gene expression in erythroid cells include:
  • trans- acting factors e. g. , proteins or other molecules
  • hemoglobin gene expression may increase or decrease hemoglobin gene expression.
  • trans- acting factors e. g. , proteins or other molecules
  • hemoglobin gene expression may increase or decrease hemoglobin gene expression.
  • the TDR method of the present invention replaces the DNA at the native site with the donor DNA, so regulation of gene expression will follow the native course, provided that the donor DNA was not purposely designed to alter the native course of gene expression.
  • ⁇ -chain gene regulation in ⁇ -thalassemia can, in principle, be caused by any ⁇ - gene-associated mutation that completely deactivates HbA. It is not surprising, then, that ⁇ °-thalassemia is caused by kinds of mutations that include nonsense and frameshift mutations and mutations that cause defective processing of the mRNA. For many specific mutations the exact base change is known. A summary and discussion of specific base mutations, as of 1988, may be found in Bunn and Forget ( op. ci t . , p. 274) . A recent summary of all known mutations may be found in Huis an (Hemoglobin. 17: 479 (1993)) . Relevant to this invention is the fact that a single or a few donor DNAs in the TDR method can be used to correct all approximately 100 known ⁇ -thalassemia mutations.
  • IVS intervening sequences
  • N stands for any nucleotide, . and the underlined AG sequence is invariant among many species, and therefore, is thought to be absolutely required for proper splicing
  • Bone marrow transplants are used to treat both sickle- cell anemia and ⁇ ° thalessemia.
  • the general procedure is as- follows:
  • Immune suppressants such as cyclophosphamide, immuran and azothiopain are administered to the patient to destroy some of the bone marrow to decrease substantially the risk of transplant immune rejection (graft vs. host disease or GVHD) .
  • immune depressants serve to decrease the numbers of abnormal embryonic stem cells in the SCA patient, which is desired to yield a high percentage of healthy, transplanted cells from the donated bone marrow. In ⁇ ° thalessemia, however, the affected pre-erythrocytes produce no functional hemoglobin, so their numbers need not be suppressed in advance of transplantation. Immune depressants leave the patient vulnerable to disease, which is a major reason for the 10% mortality rate associated with them.
  • Bone marrow from matched siblings, who are disease-free, is then injected intravenously to "home" to the bone marrow of the patient.
  • Siblings who have been matched for immune-compatibility are used; otherwise, transplant rejection and GVHD are too frequent a complication.
  • a major problem is that many SCA and ⁇ °-thalessemia patients do not have a disease-free, matched sibling to serve as bone marrow donor, which severely limits the applicability of bone marrow transplants.
  • hematopoietic stem cells that are the targets for the therapies of this invention and in general for gene therapies for these diseases in general
  • hematopoietic stem cells could be proliferated ex vivo for only a few generations.
  • Berardi, et al . (Science, 267: 104 (1995)), have just developed a stem cell isolation procedure that provides primitive hematopoietic cells in high concentrations (1 in 105 of bone marrow mononuclear cells) , so this area of stem-cell therapies is advancing rapidly.
  • the isolated cells proliferate both along lymphoid and myeloid (precursors to erythrocytres) lineages, and can be made to form sizable colonies and secondary cultures on replating with an efficiency of 40%.
  • the present invention can circumvent a number of the problems with present-day bone-marrow-transplant technology. Since the transplanted bone-marrow is treated bone marrow obtained from the patient, immune rejection and GVHD should be reduced or eliminated; thus eliminating the need for immune suppressants at least for immune-rejection reasons. For SCA, immune depressants may still be required to destroy some of the ⁇ s producing cells before transplantation. For ⁇ o-thalessemia, however, immune depressants might be eliminated entirely since the diseased cells may not need to be destroyed. The invention should then reduce or eliminate much of the morbidity and mortality associated with bone marrow transplants.
  • the present invention provides a method for effecting repair of genetic defects in the ⁇ globin gene and DNA regions involved in the expression of that gene.
  • repair is effected through homologous recombination between a native nucleic acid segment in the ⁇ -gene cluster on chromosome 11 in a human cell and a donor nucleic acid segment introduced into the cell, which comprises: a) introducing into a human cell i) an oligonucleotide third strand which comprises a base sequence capable of forming a triple helix at a binding region on one or both strands of a native nucleic acid segment, said native nucleic acid segment containing an undesired mutation in the vicinity of the human ⁇ globin gene cluster target region where the recombination is to occur, said oligonucleotide being capable of inducing homologous recombination at the target region of the native nucleic acid, and ii) a donor nucleic acid which
  • Another aspect of the present invention concerns a method for effecting homologous recombination between a native nucleic acid segment in a cell and a donor nucleic acid segment introduced into the cell, which comprises: a) contacting a donor nucleic acid segment with an oligonucleotide third strand which comprises a base sequence capable of forming a triple helix at a binding region on one or both strands of the donor nucleic acid segment in the vicinity of a target region where the recombination is to occur, said oligonucleotide being capable of inducing homologous recombination at the target region of the donor nucleic acid, and said donor having a sequence sufficiently homologous to a first nucleic acid segment in a human cell which comprises at least a portion of the human ⁇ globin gene cluster, such that the donor sequence will undergo homologous recombination with the first sequence at the target region; b) treating the nucleic acid segment by allowing the oligonucleo
  • Another aspect of the present invention concerns a method for causing a mutation at a specific DNA sequence site in a cell, which comprises: a) introducing into a cell an oligonucleotide third strand which comprises a base sequence which is capable of forming a triple helix at a binding region on one or both strands of a native nucleic acid segment which contains an undesired mutation in the vicinity of the human ⁇ globin gene cluster target region, said oligonucleotide being capable of inducing mutagenesis when bound to the binding region; b) allowing the oligonucleotide to bind to the native nucleic acid segment to form a triple stranded-nucleic acid; and c) allowing mutagenesis to occur in the target region.
  • the present invention provides a composition
  • a composition comprising: a) an oligonucleotide third strand which comprises a base sequence which is capable of forming a triple helix at a binding region on one or both strands of a native nucleic acid segment in the vicinity of the human ⁇ globin gene cluster target region, said oligonucleotide being capable of inducing homologous recombination at the target region of the native nucleic acid; and b) a donor nucleic acid which comprises a nucleic acid sequence sufficiently homologous to the native nucleic acid segment such that the donor nucleic acid will undergo homologous recombination with the native sequence at the target region when the third strand is bound to the native nucleic acid.
  • the present invention provides a composition
  • a composition comprising an oligonucleotide third strand which comprises a base sequence capable of third strand binding to a portion of one or both strands of a native nucleic acid segment in the human ⁇ globin gene-cluster of a cell, said oligonucleotide being capable of causing a mutation at a specific site of the native nucleic acid segment when bound to the native nucleic acid segment.
  • the present methods and compositions are useful in research and therapeutic applications where site specific recombination in the human ⁇ globin gene-cluster of a cell is desired.
  • the present inventions are also useful constructing cell lines and transgenic animals for the study of hemoglobinopathies.
  • FIG 1 is a schematic illustration of the targeted mutagenesis (TM) method using a psoralen-linked third strand as an example.
  • the AT base pair in boldface is the base pair to be changed to a "normal” TA base pair.
  • Long wavelength UV light (UVA) photoactivates the psoralen generating a psoralen adduct on the T in the AT base pair.
  • the damage to the AT base pair is cross-linking as indicated by "+”.
  • the damage is initiated through the psoralen reacting specifically with the T base in the AT pair, and the other base pairs in the DNA remain unchanged and are not shown in the center and right-hand parts of the Figure.
  • the cell's native DNA repair machinery attempts to repair the damage, but the machinery often makes a mistake and repairs the cross link to a TA base pair instead of the original AT base pair.
  • Figure 2 is an illustration of the targeted DNA replacement method.
  • Figure 2(A) is a schematic illustration of a mutation responsible for a genetic defect, x, and a third-strand binding site downstream from the mutation.
  • the genetic defect may be a base substitution, deletion or addition of one or more bases.
  • the third strand binding site may be located thousands of bases from the defect.
  • the mutagenic third strand binds to its targeted purine site and causes DNA damage.
  • Figure 2(C) the native cellular machinery, stimulated by the DNA damage, "aligns" the donor DNA with the defective chromosome region to allow homologous recombination to occur between the donor double strand and the chromosome defective region, which results in a repaired chromosome region, shown in Figure 2(D) .
  • Figure 3 illustrates the approximate relative locations of the ⁇ -globin and ⁇ -globin-like genes on the human chromosome 11 ⁇ -gene cluster. The cluster spans about 52 kilobases from the beginning of the embryonic ⁇ gene through the adult ⁇ gene. The ⁇ gene cluster is also shown. (Reproduced from Bunn and Forget, "Hemoglobin: Molecular, Genetic and Clinical Aspects", W.B. -Saunders, Philadelphia (1986) , p. 174) . DETAILED DESCRIPTION OF THE INVENTION
  • binding motifs For purposes of illustrating the present invention, five binding motifs are described. It is understood that practice of the invention is not limited to these motifs. Table 1 summarizes these five motifs, which are additional rules subject to the binding code. The motifs provide further instructions for defining the sequences of different third-strands that will specifically and stably bind to a single purine center-strand target. The Table also shows selected analog bases which may be substituted for the native A, G, T, and C third-strand bases.
  • the colon indicates third- strand binding of the base to the left of the colon to the center base to the immediate right of the colon.
  • the + superscript indicates that the bases are in the protonated form when they bind (the energy of binding provides the energy for protonation) .
  • DAP stands for di-aminopurine.
  • third-strands should preferably be at least about 10 bases in length, more preferably at least about 20 bases.
  • the targeted DNA replacement method may be thousands of bases from the site of the genetic defect, so there is a high probability that at least one purine run of sufficient length will be found. For example, there are on average at least 9.8x10-4 9.8 purine runs of ten bases within 10,000 bases of the site of a genetic defect.
  • analog bases for various motifs include, but are not limited to, those presented in Table 1. Other analog bases, for example, are discussed in Sun and Helene (op. cit . ) and also in co-pending U.S.
  • Table 2 shows the effect of third-strand bases in the center of a 21-mer triple helix in the pyrimidine/parallel motif as measured by melting temperature of the third-strand.
  • the test helices were composed of the single strands A 3.0 -X- 10 (Watson-Crick center strand) , T 3.0 -Y-T 10 (other Watson-Crick strand) , T ⁇ o-Z-
  • T 10 (third strand) .
  • the triple-helix bases at the mid position in the helix is denoted by Z:XY.
  • the body of the table is melting temperature in °C. Choice of the most stable base at pyrimidine center-strand sites is useful for designing third strands to purine runs interrupted by occasional pyrimidines.
  • the table shows, for example in the pyrimidine/parallel motif, that a G base in the third strand opposite a T base mismatch in the center strand leads to a more stable third strand, as its melting temperature is 16.1°C vs . -5.0°C, -0.3°C, and -3.0°c for the A, T and C bases.
  • a third-strand targeted to the site of the genetic defect is prepared with a mutagen, preferably psoralen, attached to its end. Psoralen selectively reacts with the base T. The psoralen-linked third strand is then introduced into cells in culture removed from the patient
  • the mutagen-linked third strands may be injected or delivered by intravenous infusion.
  • the third-strand binds specifically to a double- stranded chromosomal DNA sequence according to the third- strand binding code.
  • the cell culture is then bathed in long-wavelength ultraviolet light (centered at 365 nm, also known as UVA) , which causes the psoralen to damage the double-stranded DNA target by cross-linking the two strands together at the AT base-pair target site (boldface type) .
  • UVA long-wavelength ultraviolet light
  • the cell's native DNA repair mechanism recognizes the damage, and attempts to repair the damaged T base, but frequently makes the mistake of replacing the T base with an A base.
  • the targeted T to A base change is about 100-times more prevalent than background mutations caused by free psoralen (psoralen not linked to the third strand) .
  • third-strands promote damage and misrepair with high specificity.
  • This method for genetic defect repair involves targeting third strands to native DNA sequences to induce DNA damage to stimulate homologous recombination with an introduced donor nucleic acid strand.
  • the third strand modifies or damages the donor nucleic acid before it is introduced into the target cell.
  • the invention provides a method for effecting gene transfer or mutation repair at a specific sequence site on the target native nucleic acid in a cell.
  • the method utilizes two nucleic acids: (1) an oligonucleotide third strand capable of specifically binding to the binding region of a native double-stranded nucleic acid, and (2) a donor nucleic acid fragment capable of undergoing homologous recombination with the native nucleic acid targeted by the oligonucleotide.
  • the nucleic acid sequence of the donor DNA is slightly different from the native nucleic acid it is replacing by homologous recombination to, for example, repair a genetic defect.
  • the method and some of its features are illustrated in Figure 2.
  • the TDR method is more general than the TM method for two reasons: First, the genetic defect to be repaired is not required to be near a third strand binding site. In fact, the genetic defect may be thousands of bases distant from the third strand binding site. Thus, most genetic defects are potential therapeutic targets, compared to the TM method where therapeutic targets must be selected to be in or near third-strand binding sites. Secondly, the TDR method is able to correct multiple base substitutions or small or large base deletions and/or insertions, as long as the donor nucleic acid has acceptable homology with the native nucleic acid it is replacing. In targeted mutagenesis, in contrast, the genetic defect to be repaired is usually restricted to a single base substitution, or occasionally to a single base deletion or addition.
  • Targeted DNA replacement accomplishes the same therapeutic goals as viral-vector gene therapies, but with a number of advantages:
  • the repaired gene resides at its native chromosomal site, so repair and hence disease cure is permanent, and gene expression will be native.
  • viral vectors either provide only temporary cure or they integrate into chromosomes in non-native sites, so patterns of gene expression may not be compatible with the requirements of a disease cure.
  • -Donor nucleic acids smaller than whole genes may be used, which may be delivered by standard methods, IV or injection.
  • -Most human cell types are available for targeting.
  • most viral vectors are targeted to a very limited number of cell types.
  • the oligonucleotide third strand useful in either the TM or TDR methods is a synthetic or isolated oligonucleotide capable of binding with specificity to a predetermined binding region of a double-stranded native nucleic acid molecule to form a triple-stranded structure.
  • the third strand may bind solely to one strand of the native nucleic acid molecule, or may bind to both strands at different points along its length.
  • the predetermined target region of the double-stranded DNA is in or adjacent to a gene, mRNA synthesis or processing control region, or other DNA region that it is desirous to replace by homologous recombination.
  • the predetermined binding region, if adjacent to the targeted region is preferably within 10,000 nucleotides or bases from the targeted region.
  • the oligonucleotide is a single-stranded DNA molecule between about 7 and about 50, most preferably between about 10 and about 30 nucleotides in length.
  • the base composition can be homopurine, homopyrimidine, or mixtures of the two.
  • the third strand binding code and preferred conditions under which a triple-stranded helix will form are well known to those skilled in the art (Fresco U.S. Patent 5,422,251; Beal and Dervan, Science 251: 1360 (1991); Beal and Dervan, Nucleic Acids Res . , 20:2773 (1992); Broitman and Fresco, Proc. Natl . Acad. Sci .
  • the third strand need not be perfectly complementary to the duplex, but may be substantially complementary. In general, by substantially complementary is meant that one mismatch is tolerable in every about 10 base pairs.
  • the oligonucleotide may have a native phosphodiester backbone or may be comprised of other backbone chemical groups or mixtures of chemical groups which do not prevent the triple-stranded helix from forming. These alternative chemical groups include phosphorothioates, methylphospho- nates, peptide nucleic acids (PNAs) , and others known to those skilled in the art.
  • the oligonucleotide backbone is phosphodiester.
  • the oligonucleotide may also comprise one or more modified sugars, which would be well known to one of ordinary skill.
  • modified sugars include ⁇ - enantiomers.
  • the third strand may also incorporate one or more synthetic bases if such is necessary or desirable to improve third strand binding.
  • synthetic base design and the bases so designed are found in the co- pending U.S. application of Fresco, et al . entitled "Residues for Binding Third Strands to Complementary Nucleic Acid Duplexes of any Base-Pair Sequence", filed concurrently herewith.
  • the oligonucleotide may be modified with one or more protective groups.
  • the 3' and 5' ends may be capped with a number of chemical groups such as an alkyl amine group, a thiol group, cholesterol, acridine, etc.
  • the oligonucleotide third strand may be protected from exonucleases by circularization.
  • the oligonucleotide third strand should be capable of inducing either homologous recombination or targeted mutagenesis at a target region of the native nucleic acid. That may be accomplished by the binding of the third strand alone to the native nucleic acid binding region, or by a moiety attached to the oligonucleotide. In the embodiment where the binding of the third strand alone induces the recombination, the third strand should bind tightly to the binding region, i.e., it should have a low dissociation constant (K ) for the binding region.
  • K dissociation constant
  • the Ka is estimated as the concentration of oligonucleotide at which triplex formation is half-maximal.
  • the oligonucleotide has a K d less than or equal to about 10-7 M, most preferably less than or equal to about 2 X 10-8 M.
  • the K d - may be readily determined by one of ordinary skill, including estimation using a gel mobility shift assay (Durland, et al . , Biochemistry 30, 9246 (1991); see also the copending U.S. application of Glazer entitled “Triple Helix Forming Oligonucleotides for Targeted Mutagenesis” filed concurrently herewith, the content of which is incorporated by reference. ) .
  • the oligonucleotide may be chemically modified to include a mutagen at either the 5 ' end, 3 ' end, or internal portion so that the mutagen is proximal to a site where it will cause damage to the native nucleic acid.
  • the mutagen is incorporated into the oligonucleotide during nucleotide synthesis.
  • commercially available compounds such as psoralen C2 phosphoroamidite (Glen Research, Sterling VA) are inserted into a specific location within an oligonucleotide sequence in accordance with the methods of Takasugi et al . , Proc. Natl . Acad. Sci USA, 88:5602 (1991); Gia et al . , Biochemistry 31:11818 (1992); Giovannangeli, et al . , Proc. Natl . Acad. Sci . USA, 89:8631 (1992), all of which are incorporated by reference herein.
  • the mutagen may also be attached to the oligonucleotide through a linker, such as sulfo-m- maleimidonbenzoly-N-hydroxysuccinimide ester (sulfo-MBS, Pierce Chemical Company, Rockford IL) in accordance with the methods of Liu et al . , Biochem. 18:690 (1979) and Kitagawa and Ailawa, J " . Biochem. 79:233 (1976), both of which are incorporated by reference herein.
  • the mutagen is attached to the oligonucleotide by photoactivation, which causes a mutagen, such as psoralen, to bind to the oligonucleotide.
  • the mutagen can be any chemical cap_able of stimulating either mutagenesis or homologous recombination. Such stimulation can be caused by modifying the native nucleic acid in some way, such as by damaging with, for example, crosslinkers or alkylating agents.
  • the mutagen may also be a moiety which increases the binding of the third strand to the target, such as intercalators (e.g., acridine) . Such mutagens are well known to those skilled in the art.
  • the chemical mutagen can either cause the mutation spontaneously or subsequent to activation of the mutagen, such as, for example by exposure to light.
  • Preferred mutagens include psoralen and substituted psoralens such as hydroxymethyl-psoralen (HMT) that require activation by ultraviolet light; bleomycin, fullerines, mitomycin C, polycyclic aromatic carcinogens such as 1- nitrosopyrene, alkylating agents; restriction enzymes, nucleases, radionuclides such as 125 I, 5s and 2p ; and molecules that interact with radiation to become mutagenic, such as boron that interacts with neutron capture and iodine that interacts with auger electrons.
  • HMT hydroxymethyl-psoralen
  • light can be delivered to cells on the surface of the body, such as skin cells, by exposure of the area requiring treatment to a conventional light source.
  • Light can be delivered to cells within the body by fiber optics or laser by methods known to those skilled in the art.
  • Targeted flourogens that provide sufficient light to activate the mutagens can also provide a useful light source.
  • Ex-vivo exposure to light of cells such as embryonic stem cells can be carried out by procedures known to those skilled in the art of ex vivo medical treatments.
  • the donor nucleic acid used in the practice of the TDR embodiment is either a double-stranded nucleic acid, a substantially complementary pair of single stranded nucleic acids, or a single stranded nucleic acid.
  • the sequence of the donor nucleic acid at its ends is substantially homologous to the nucleic acid region which is to be replaced by homologous recombination.
  • the region of substantial homology is at least about 20 bases at each end of the donor nucleic acid.
  • substantially homology is meant that at least about 85% of the available base pairs are matching.
  • nucleic acid segments may be added according to the present invention. Such segments include a gene, a part of a gene, a gene control region, an intron, a splice junction, a transposable element, a site specific recombination sequence, and combinations thereof.
  • the donor nucleic acid strands may be gene sized, or greater or smaller. Preferably, they are at least about 40 bases in length, preferably between about 40 and about 1,000,000 bases in length. Most preferably, the lengths are between about 500 and about 3,000 bases.
  • Peripheral blood hematopoeitic precursor and stem cells can then be collected by leukapheresis and used for the present invention.
  • Techniques for isolation of hematopoeitic stem cells are well known to those skilled in the art, and may also be found in Hoffman, et al . , Hematology, Churchill-Livingstone, New York, 1995.
  • the oligonucleotide can be delivered to cells or live animals simply by exposing the cells to the oligonucleotide by including it in the medium surrounding the cells, or in live animals or humans by bolus injection or continuous infusion.
  • concentration will be readily determined by one of ordinary skill, and will depend on the specific pharmacology and pharmacokinetic situation. Typically, from about 0.1 to about 10 ⁇ M will be sufficient.
  • the nucleic acid modification or damage used to stimulate homologous recombination is targeted to the donor nucleic acid (as opposed to the native nucleic acid) either inside or outside the target cells.
  • the modified or damaged donor nucleic acid is then introduced into the target cell to stimulate homologous recombination with the native nucleic acid.
  • Modifying or damaging the donor nucleic acid outside the cell has several desirable features including: nucleic acid modification or damage can be caused with higher efficiency outside the cell; mutagens and other treatments (e. g. , psoralen-UVA) potentially toxic to the cell, animal or human can be used since the mutagen can be isolated away from the modified or damaged donor nucleic acid before the purified donor nucleic acid is introduced into the target cell; conditions (e.g., temperature, cation composition and concentration) can be controlled to maximize binding of third-strands for any binding motif; and nucleic acid modifying or damaging agents can be directly synthesized into specific sites on the donor nucleic acid by methods well known to those skilled in the art, without the use of third strands.
  • mutagens and other treatments e. g. , psoralen-UVA
  • conditions e.g., temperature, cation composition and concentration
  • nucleic acid modifying or damaging agents can be directly synthesized into specific sites on the donor nucleic acid by methods well
  • third strand sites can be engineered into the donor nucleic acid at a location where the engineered nucleic acid segment is unlikely to cause unwanted effects when the donor nucleic acid is recombined into the organism's native nucleic acid.
  • the preferred method of introducing the oligonucleotide into stem cells and erythrocyte precursors is co-incubation in the growth medium with or without the addition of cationic liposomes (Wang, et al . , Mol . and Cell Biol . , 15:1759-1768 (1995)).
  • the donor nucleic acid can be delivered to the nucleus of cells in culture or cells removed from an animal or a patient (ex vivo) by manipulations such as peptide- facilitated uptake, electroporation, calcium chloride, micro-injection, microprojectiles or other treatments well known' to those skilled in the art.
  • the donor nucleic acid can be delivered to cells or live animals or humans simply by exposing the cells to the oligonucleotide that is included in the medium surrounding the cells, or in live animals by bolus injection or continuous infusion.
  • One of the complementary single strands is delivered and the other delivered at the same time or up to 12 hours later, preferably from about 20 to about 40 minutes later.
  • the donor nucleic acid may also be introduced into the cell in the form of a packaging system which would be well known to one of ordinary skill.
  • a packaging system which would be well known to one of ordinary skill.
  • Such systems include DNA viruses, RNA viruses, and liposomes as in traditional gene therapy.
  • the preferred method of introducing the donor DNA into stem cells and erythrocyte precursors is co-incubation in the growth medium with or without the addition of cationic liposomes (Wang, et al . , Mol . and Cell Biol . , 15:1759-1768 (1995)) .
  • the patient may undergo chemotherapy to reduce or destroy the bone marrow cells in the body.
  • chemotherapy are well known to those skilled in the art of autologous bone marrow transplants, see Hoffman, et al . , Hematology, Churchill-Livingstone, New York, 1995.
  • Treated stem cells can be reintroduced into the patient by intravenous infusion, using standard methods.
  • the invention provides a method for effecting gene transfer, genetic defect repair, and targeted mutagenesis at a specific sequence site on the DNA target in the ⁇ globin gene cluster in cells such as stem cells and erythrocyte precursor cells of humans or other animals. Examples of therapeutic use are apparent. For example, if a targeted DNA region contains base changes, deletions or additions of bases which cause an inherited or somatic hemoglobinopathy such as sickle cell anemia, or ⁇ - thalassemia or ⁇ -thalassemia, then the donor nucleic acid can provide a normal gene by replacing the defective nucleic acid to correct that disorder.
  • the hemoglobinopathy may be treated by an oligonucleotide carrying a mutagen, the modification or damage from which is subsequently misrepaired to provide an active, normal or near-normal hemoglobin.
  • the preferred embodiment of the methods and compositions of the invention are in ex vivo therapies where third strands and donor DNA can be introduced into target cells outside the body.
  • the hemoglobinopathies are particularly amenable to ex vivo treatments.
  • the ⁇ -globin gene cluster ( Figure 3) is approximately 52 kilobases (kb) in length, and a large number of purine runs of sufficient length (10 bases or greater) and a number of purine runs of the preferred minimum length
  • the targeted DNA replacement method of this invention can carry out homologous recombination initiated by DNA damage at 40,000 bases or more from the site of the genetic defect to be corrected. Therefore, all genetic defects within the ⁇ gene cluster may be corrected by this method with a single third-strand binding site. It is preferred, however, that the third-strand binding site be within 10 kb of the genetic defect to be corrected.
  • the mutagen psoralen (for which mutagen- stimulated homologous recombination by the methods of this invention has been demonstrated and for which misrepair of T to A has been demonstrated) is used clinically for therapy —topically for dermatological conditions and ex vivo for bone marrow transplants to reduce the risk of graft-vs-host disease and as therapy for cutaneous T-cell lymphoma.
  • psoralen alone is relatively non-toxic in clinical use (Ortonne, Clin . Dermatol . 7:120 (1989); Taylor and Gasparro, Semin . Hematol . 29:132 (1992); Jampel, et al . , Arch . Dermatol .
  • transgenic mouse expressing only human sickle-cell hemoglobin, for example, would be extremely useful for testing therapies for this disease in advance of human trials.
  • the use of and useful and novel features of mutagen-linked third strands to either cause a desired mutation (targeted mutagenesis) or cause DNA damage to stimulate homologous recombination with a donor DNA (third-strand-directed homologous recombination or targeted DNA replacement) will be further understood in view of the following non-limiting examples.
  • Example 1 A single mutation in the ⁇ chain of hemoglobin is sufficient to cause the sickle-shaped cells characteristic of sickle cell anemia (SCA) .
  • SCA sickle cell anemia
  • the normal adenine base is mutated to a thymine, which causes an amino acid change from a negatively-charged glutamic acid to an uncharged, hydrophobic valine.
  • the normal and altered DNA sequence surrounding and including the changed base is:
  • gag 5" ATG GTG CAC CTG ACT CCT GAG GAG AAG TCT GCC GTT ACT GCC CTG 3" 3' GAC CAC GTG GAC TGA GGA CTC CTC TTC AGA CGG CAA TGA'CGG GAC 5" (SEQ ID N0:1) Altered sequence:
  • the upper strand is the coding strand where coding begins at the first triplet shown, the ATG start codon for the ⁇ globin gene.
  • the codons "gag” ' and “gug” code for the native glutamic acid and mutant valine, respectively.
  • the altered DNA sequence is imbedded in nearly uninterrupted stretches of purine bases (underlined regions designated by the circled numbers ⁇ , , ® or ®, , ⁇ ) alternating between strands that are targets for binding a third strand with strand switching; and (2) in the targeted mutagenesis method (TM method) of this invention, highly-specific damage to thymine base by psoralen-bound third strands is preferentially
  • oligonucleotides suitable for use in ' the present invention may be derived by any method known in the art, including chemical synthesis, or by cleavage of a larger nucleic acid using non-specific nucleic acid-cleaving chemicals or enzymes, or by using site specific restriction endonucleases. Psoralen and other mutagens may also be specifically bound to the ends or internal positions of the oligonucleotides by standard methods (Havre, et al . , Proc. Natl . Acad. Sci . USA 90:7879 (1993); Havre and Glazer, J. Virology 67:7324 (1993)) .
  • sequences for the 5 motifs binding solely to the ⁇ purine-rich region of the gene are:
  • the purine/antiparallel motif is the preferred purine motif for binding to the ⁇ region alone (without strand switching) since it is a slightly G-rich target.
  • the single pyrimidine base, T, in the ⁇ region may be opposite an A, G, C or T base as indicated in the third strand depicted. While there are no strongly preferred bases for the mismatch, the T base is slightly preferred. That T base in the coding strand is also the one that it is desired to change to the native A base.
  • the psoralen is preferably attached to the A, G, C or T base opposite the T base in the center strand.
  • the single pyrimidine base, T, in the ⁇ region may be opposite an A, G, C or T base as indicated in the depicted third strand. That T base is also the one that it is desired to change to the native A base.
  • the psoralen is preferably attached to the A, G, C or T base opposite the T base in the center strand.
  • the single pyrimidine base, T, in the ⁇ region may be opposite an A, G, C or T base as indicated in the depicted third strand.
  • the G base is -the preferred base opposite the T base in the center strand in this sequence (see Table 3) . That T base is also the one that it is desired to change to the native A base.
  • the psoralen is preferably attached to the A, G, C or T base opposite the T base in the center strand.
  • the G and T/antiparallel motif is the slightly preferred of the two GT motifs for binding to ⁇ region alone (without strand switching) since the target AG and GA nearest neighbor frequencies outnumber the AA and GG frequencies by 3 to 2.
  • the single pyrimidine base, T, in the ⁇ region may be opposite an A, G, C or T base as indicated in the depicted third strand. This T base is also the one that it is desired to change to the native A base.
  • the psoralen is preferably attached to the A, G, C or T base opposite the T base in the center strand.
  • the single pyrimidine base, T, in the ⁇ region may be opposite an A, G, C or T base as indicated in the depicted third strand.
  • This T base is also the one that it is desired to change to the native A base.
  • the psoralen is preferably attached to the A, G, C or T base opposite the T base in the center strand, or attached to the base immediately adjacent. Examples of a number of preferred third-strand sequences utilizing strand switching and binding to all of the ⁇ , ⁇ , ® purine-rich regions, and one example utilizing the ⁇ , ⁇ , ⁇ strand- switching scheme along with binding data, are presented below.
  • the example immediately below employs the purine/antiparallel motif, throughout. It is the preferred purine motif, since the three center strand regions are G rich. In this example the ⁇ , ⁇ , ⁇ strand-switching scheme is utilized.
  • X represents a linker (e.g., spacer phosphoroamidite 9 from Glen Research) required to both provide steric flexibility and to maintain antiparallel strand orientation after strand switching.
  • Y represents a linker required to both provide steric flexibility and to maintain antiparallel strand orientation after strand switching.
  • the double-underlined sequence was omitted (see below) . While there are no strongly preferred bases for the two mismatches, the T base is slightly preferred.
  • the example immediately below also employs the purine/antiparallel motif, throughout. It is the preferred purine motif, since the three center strand regions are G rich.
  • the ⁇ , ⁇ , ® strand-switching scheme is utilized for illustrative purposes. All the following examples could also use the ⁇ , ⁇ , ⁇ scheme.
  • X represents a linker (e.g., spacer phosphoroamidite 9 from Glen Research) required to both provide steric flexibility and to maintain antiparallel strand orientation after strand switching.
  • Y represents a linker required to both provide steric flexibility and to maintain antiparallel strand orientation after strand switching. While there are no strongly preferred bases for the two mismatches, the T base is slightly preferred.
  • the linker Z need only supply enough flexibility to make the switch.
  • Such flexible linkers include, but are not limited to, one or two natural bases (e.g., T, TT, C, CC) and others such as spacer 3, spacer 9 phosphoramidites from Glen Research (Sterling, VA) .
  • Example 2 The ⁇ -gene cluster, shown in Figure 3, is located on human chromosome 11 and contains all the ⁇ -like genes in the order they are expressed in human development, from left to right in the Figure.
  • the cluster from the beginning of the ⁇ gene to the end of the ⁇ gene spans about 52 kilobases (Kb) .
  • the targeted DNA replacement method (TDR) allows for DNA modification or damage to occur at greater than 52 Kb from the site at which a desired DNA change is to be made, so one third strand binding site may be used to repair any genetic defect or make any other alteration in the whole ⁇ -gene cluster. It is preferred, however, that the DNA damage site be within 10 kb of the site of the repair or alteration. For some clinical applications, it may be preferable that the DNA- damage site be even closer to the site at which the DNA is to be altered.
  • ⁇ globin gene sequence particularly in the introns, there are many good third-strand binding sites that may be utilized in the practice of the TDR method of the invention.
  • GenBank sequence of the chromosome-11 human-native hemoglobin-gene cluster (GenBank: LOCUS HUMHBB, 73308 bp ds-DNA) from base 60001 to base 66060 is presented below. This portion of the GenBank sequence contains the native ⁇ globin gene sequence.
  • the adenine base at position 62206 (or position 2206 as listed in SEQ ID NO:10, indicated in boldface) is mutated to a thymine.
  • the start of the gene coding sequence at position 62187-62189 is indicated by a single underline.
  • a computer search was performed on this GenBank sequence portion for third-strand binding sites, and a representative sample of sites found are indicated by double-underlines.
  • the preferred sites for the TDR method of this invention are both double-underlined and boldface.
  • third-strand binding sites are illustrative and do not constitute all the sites in the region, which are also within the scope of the invention.
  • the two preferred binding sites beginning at Gen Bank positions 62655 and 62825 (SEQ ID NO:10 positions 2655 and 2825) , are each 21 uninterrupted pyrimidines in the coding strand or 21 uninterrupted purines in the non-coding strand and are excellent third-strand binding sites. Their sequences are:
  • TTTTCTTTCC CCTTCTTTTC T SEQ ID NO:11
  • CTTTCTTTTT TTTTCTTCTC C SEQ ID NO:12
  • the purine run on the non-coding strand is 21 bases long and is not interrupted by even one pyrimidine, it exceeds the preferred minimum length of 20 bases for third strand binding.
  • the A:T base pair at the 3' end of the coding strand after the purine run are shown because it represents a good crosslinking site for psoralen attached to the end of the third strand.
  • the site is also conveniently located to cause DNA damage to stimulate homologous recombination using a donor DNA carrying desired alterations to coding regions and introns of the ⁇ -gene and to adjacent control regions of the ⁇ -gene.
  • a third-strand binding sequence located in the ⁇ 'globin gene allows for flexibility in the length of donor DNA, which may be used to optimize introduction into hematopoietic stem cells or erythrocyte precursor cells, optimize homologous recombination, or allow for donor DNA of sufficiently short length to be delivered by traditional means, injection or IV, to a patient.
  • oligonucleotides suitable for use in the present invention may be derived by any method known in the art, including chemical synthesis, or by cleavage of a larger nucleic acid using non-specific nucleic acid- cleaving chemicals or enzymes, or by using site specific restriction endonucleases. Psoralen and other mutagens may also be specifically bound to the ends or internal positions of the oligonucleotides by standard methods. Donor DNA may be prepared in the same manner. The example sequences are 21 bases and consequently bind to the entire purine run. It is understood that effective fragments thereof are included within the scope of the invention.
  • the purine motif example immediately below employs the parallel polarity, which is preferred because the target is A rich.
  • the antiparallel motif may be employed, although not preferred.
  • Psoralen crosslinking to the AT base pair at the end of the target is a preferred method of causing DNA damage, and the position where it is bound is illustrated above. It is understood that other mutagens and other positions for binding to third strands are within the scope of the invention. The examples below, therefore, will not illustrate mutagen binding. 2.
  • the T and G motif example immediately below employs the parallel polarity, which is preferred because the target has high AA and GG nearest neighbor frequencies.
  • antiparallel motif may be employed, although not preferred.
  • a third strand, in the pyrimdine/parallel motif is another example within the scope of the invention:
  • the donor DNA must contain the DNA sequence at the DNA damage site, the DNA region containing the genetic defect to be repaired or alteration to be made, and all the native codons between the two (preferably 50 or more bases of homology to the target DNA) .
  • the donor DNA may be considerably larger than the bases between and including the damage site and the repair or alteration site.
  • the donor DNA must contain both the native adenine that is to replace the mutant thymine, the third-strand binding site to be damaged, and preferably the native DNA sequence between them.
  • One example of a double- stranded donor DNA meeting these requirements is presented immediately below (only one strand of the duplex DNA is shown) .
  • the strand spans positions 62161-62760 of GenBank: LOCUS HUMHBB, or positions 2161-2760 of (SEQ ID NO:10) : TTCACTAGCA ACCTCAAACA GACACCATGG TGCACCTGAC TCCTGAGGAG 50
  • CTGTCCACTC CTGATGCTGT TATGGGCAAC CCTAAGGTGA AGGCTCATGG 350
  • donor nucleic acids include but are not limited to: longer and shorter donor DNAs that contain both the native A base at position 62206 and the third-strand binding site; donor DNAs with different codons that code for the same amino acid or a mutant amino acid that codes for a functional protein; donor DNAs with sequence variations in the introns that do not effect substantially the processing of protein.
  • Example 3 Mutations causing ⁇ ° and ⁇ + thalassemia are mostly found in the ⁇ globin gene itself or in DNA regions close to the gene. A complete list of mutations, as of 1993, may be found in Huisman (Hemoglobin, 17:479 (1993)) . Some examples of mutations causing ⁇ ° thalassemia from Huisman and for which the donor DNA of Example 2 may be used in the TDR method to repair the mutation are presented below. It is understood that all the mutations listed in Husiman and those yet to be discovered that are located at sequence positions within the region spanned by the donor DNA may be repaired using the donor DNA.
  • RNA processing mutants at splice junctions 1.
  • Example 5 The TM method may also be used to correct ⁇ thalassemia provided: (1) the mutation lies in a third strand binding site, and (2) repair of the mutation yields either a native or non-native base that results in a functional hemoglobin. Frequent base changes from the action of specific mutagens may be found in: Aguilar, et al . , Proc. Natl . Acad. Sci . USA. 90: 8586 (1993); Gupta, et al . , J. Biol . Chem. 264: 20120 (1989); Topal, Carcinogenesis . 9: 691 (1988); Moriya, Proc. Natl . Acad. Sci . USA.
  • TAG TAG
  • TGA TAG
  • example mutagens the base changes that they cause most frequently, and the amino acid resulting from that base change in a stop codon.
  • Some mutagens can change more than one base or cause more than one type of mutation, depending on the location of the mutagen on the third strand (i.e., what base in the duplex the mutagen is near upon third strand binding) , nearest neighbor bases in the duplex, and other factors.
  • a psoralen-linked third strand targeted to the T base can change this nonsense codon to AAG which codes for lysine to provide a functional ⁇ chain and hemoglobin (S. Baserga, Ph.D. Thesis, Yale University (1988)).
  • This site also fulfills the second requirement of the TM method, as it is a weak third-strand binding site using strand switching.
  • the sequence at the site spanning codons 39 through 42 in the native, ⁇ ° thalassemia, and psoralen-linked third-strand repaired is:
  • the first underlined sequence in the ⁇ o thalassemia DNA is the first purine run for third-strand binding
  • the second underlined sequence is the complement to the AAGAAA sequence to which the third-strand will bind after switching.
  • Three preferred third-strands that will bind to that portion of the ⁇ thalassemia sequence, where the psoralen is represented as pso, are:
  • Longer antiparallel third strands may be employed that utilizes additional strand switches.
  • One example is:
  • Z is a linker to provide flexibility for strand switching but does not change the polarity of the third strand.
  • Other thalassemias are caused by improper processing of introns (intervening sequences or IVS) .
  • IVS intervening sequences
  • N stands for any nucleotide
  • the underlined AG sequence is invariant among many species, and therefore, is thought to be absolutely required for proper splicing (Bunn and Forget, op. ci t . , pp. 177-178).
  • the presence of the (T or C) n sequence provides a third-strand polypurine target site on the opposite strand for repairing improper splicing by the TM method.
  • the opposite strands of both these sequences represent excellent third-strand binding sites with few mismatches and without strand switching, but in some cases switching the polarity of the third strand according to the preferred motifs is desirable.
  • the double-underlined AG bases represent the usually invariant bases necessary for proper splicing of the mRNA. For example, at the beginning of IVS-1, a G to A change at position 1 causes abberant splicing in a Mediterranean Thalassemia (Orkin, et al . , Nature 296:627, 1982). These examples illustrate the concept of converting a stop (nonsense) codon to a non-native amino acid using mutagen-linked third strands in the TM method, which yields a functional ⁇ chain.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne la prise en charge et le traitement des hémoglobinopathies telles que la drépanocytose et la β-thalassémie. L'invention concerne également la mise au point d'animaux de laboratoire et de lignées cellulaires destinés à être utilisés pour l'étude des hémoglobinopathies et de leur thérapies. Dans le traitement selon l'invention on utilise des oligonucléotides de troisième brin pour cibler des séquences nucléotidiques double brin dans ou au voisinage des gènes de globine ou dans ou au voisinage de séquences régulant l'expression desdits gènes pour provoquer soit une mutation voulue, soit une altération nucléique et stimuler la recombinaison homologue avec un acide nucléique donneur fourni.
PCT/US1996/009430 1995-06-07 1996-06-06 Traitement des hemoglobinopathies WO1996040271A1 (fr)

Priority Applications (1)

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AU63286/96A AU6328696A (en) 1995-06-07 1996-06-06 Treatment of hemoglobinopathies

Applications Claiming Priority (2)

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US47384595A 1995-06-07 1995-06-07
US08/473,845 1995-06-07

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WO1996040271A1 true WO1996040271A1 (fr) 1996-12-19

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WO (1) WO1996040271A1 (fr)

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WO1999005264A3 (fr) * 1997-07-23 1999-04-22 Roche Diagnostics Gmbh Production de proteines mutantes humaines dans des cellules humaines par recombinaison homologue
US6136601A (en) * 1991-08-21 2000-10-24 Epoch Pharmaceuticals, Inc. Targeted mutagenesis in living cells using modified oligonucleotides
WO2001083735A1 (fr) * 2000-05-03 2001-11-08 Institut Curie Methodes et compositions de realisation de recombinaison homologue
WO2001068147A3 (fr) * 2000-03-13 2001-12-13 Univ Ferrara Oligonucleotides de synthese inducteurs de la differenciation d'erythrocytes
WO2001073001A3 (fr) * 2000-03-24 2002-03-21 Us Health Mise en place de manipulation cellulaires renforçant le ciblage de genes apres oligo-mediation
WO2004012729A1 (fr) * 2002-07-31 2004-02-12 Universita' Degli Studi Di Ferrara Utilisation d'angelicine et de ses analogues structurels dans le traitement de la thalassemie
WO2004015117A3 (fr) * 2002-08-13 2004-06-03 Nl Kanker I Modification genique ciblee par des oligonucleotides a adn simple brin
EP1210123A4 (fr) * 1999-08-27 2004-08-18 Valigen Us Inc Vecteurs mutationnels d'oligodeoxynucleotides a brin simple
US8658608B2 (en) * 2005-11-23 2014-02-25 Yale University Modified triple-helix forming oligonucleotides for targeted mutagenesis
WO2017143042A2 (fr) 2016-02-16 2017-08-24 Yale University Compositions permettant d'améliorer l'édition ciblée de gènes et leurs procédés d'utilisation
WO2017143061A1 (fr) 2016-02-16 2017-08-24 Yale University Compositions et procédés pour le traitement de la mucoviscidose
WO2018187493A1 (fr) 2017-04-04 2018-10-11 Yale University Compositions et procédés d'administration in utero
WO2020033951A1 (fr) 2018-08-10 2020-02-13 Yale University Compositions et procédés d'édition de gène embryonnaire in vitro
WO2020047344A1 (fr) 2018-08-31 2020-03-05 Yale University Compositions et méthodes permettant d'améliorer l'édition génique à base d'oligonucléotides donneur
WO2020257779A1 (fr) 2019-06-21 2020-12-24 Yale University Compositions pna à gamma-hydroxyméthyle modifiée et leurs procédés d'utilisation
WO2020257776A1 (fr) 2019-06-21 2020-12-24 Yale University Compositions d'acides nucléiques peptidiques ayant des segments de liaison de type hoogsteen modifiés et leurs procédés d'utilisation
WO2021050568A1 (fr) 2019-09-09 2021-03-18 Yale University Nanoparticules pour absorption sélective de tissu ou cellulaire
US11896686B2 (en) 2014-05-09 2024-02-13 Yale University Hyperbranched polyglycerol-coated particles and methods of making and using thereof
US11918695B2 (en) 2014-05-09 2024-03-05 Yale University Topical formulation of hyperbranched polymer-coated particles

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NUCLEIC ACIDS RESEARCH, Volume 22, Number 14, issued 1994, F.P. GASPARRO et al., "Site-Specific Targeting of Psoralen Photoadducts with a Triple Helix-Forming Oligonucleotide: Characterization of Psoralen Monoadduct and Crosslink Formation", pages 2845-2852. *
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Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136601A (en) * 1991-08-21 2000-10-24 Epoch Pharmaceuticals, Inc. Targeted mutagenesis in living cells using modified oligonucleotides
WO1999005264A3 (fr) * 1997-07-23 1999-04-22 Roche Diagnostics Gmbh Production de proteines mutantes humaines dans des cellules humaines par recombinaison homologue
EP2324856A1 (fr) * 1999-08-27 2011-05-25 Valigen (US), Inc. Vecteurs mutationnels d'oligodeoxynucleotides à brin simple
EP2266627A1 (fr) * 1999-08-27 2010-12-29 Valigen (US), Inc. Vecteurs mutationnels à oligonucléotides à brin simple.
EP1210123A4 (fr) * 1999-08-27 2004-08-18 Valigen Us Inc Vecteurs mutationnels d'oligodeoxynucleotides a brin simple
US7060500B2 (en) 1999-08-27 2006-06-13 Metz Richard A Single-stranded oligodeoxynucleotide mutational vectors
WO2001068147A3 (fr) * 2000-03-13 2001-12-13 Univ Ferrara Oligonucleotides de synthese inducteurs de la differenciation d'erythrocytes
US7262175B2 (en) 2000-03-13 2007-08-28 Universita' Degli Studi Di Ferrara Synthetic oligonucleotides as inducers of erythroid differentiation
WO2001073001A3 (fr) * 2000-03-24 2002-03-21 Us Health Mise en place de manipulation cellulaires renforçant le ciblage de genes apres oligo-mediation
US7358090B2 (en) 2000-03-24 2008-04-15 The United States Of America As Represented By The Department Of Health And Human Services Establishment of cellular manipulations which enhance oligo-mediated gene targeting
WO2001083735A1 (fr) * 2000-05-03 2001-11-08 Institut Curie Methodes et compositions de realisation de recombinaison homologue
US6936418B2 (en) 2000-05-03 2005-08-30 Institut Curie Methods and compositions for effecting homologous recombination
US7572827B2 (en) 2002-07-31 2009-08-11 Universita' Degli Studi Di Ferrara Use of angelicin and of its structural analogues for the treatment of thalassemia
WO2004012729A1 (fr) * 2002-07-31 2004-02-12 Universita' Degli Studi Di Ferrara Utilisation d'angelicine et de ses analogues structurels dans le traitement de la thalassemie
WO2004015117A3 (fr) * 2002-08-13 2004-06-03 Nl Kanker I Modification genique ciblee par des oligonucleotides a adn simple brin
US8658608B2 (en) * 2005-11-23 2014-02-25 Yale University Modified triple-helix forming oligonucleotides for targeted mutagenesis
US11918695B2 (en) 2014-05-09 2024-03-05 Yale University Topical formulation of hyperbranched polymer-coated particles
US11896686B2 (en) 2014-05-09 2024-02-13 Yale University Hyperbranched polyglycerol-coated particles and methods of making and using thereof
US11136597B2 (en) 2016-02-16 2021-10-05 Yale University Compositions for enhancing targeted gene editing and methods of use thereof
WO2017143042A2 (fr) 2016-02-16 2017-08-24 Yale University Compositions permettant d'améliorer l'édition ciblée de gènes et leurs procédés d'utilisation
WO2017143061A1 (fr) 2016-02-16 2017-08-24 Yale University Compositions et procédés pour le traitement de la mucoviscidose
WO2018187493A1 (fr) 2017-04-04 2018-10-11 Yale University Compositions et procédés d'administration in utero
US12268774B2 (en) 2017-04-04 2025-04-08 Yale University Compositions and methods for in utero delivery
WO2020033951A1 (fr) 2018-08-10 2020-02-13 Yale University Compositions et procédés d'édition de gène embryonnaire in vitro
WO2020047344A1 (fr) 2018-08-31 2020-03-05 Yale University Compositions et méthodes permettant d'améliorer l'édition génique à base d'oligonucléotides donneur
WO2020257779A1 (fr) 2019-06-21 2020-12-24 Yale University Compositions pna à gamma-hydroxyméthyle modifiée et leurs procédés d'utilisation
WO2020257776A1 (fr) 2019-06-21 2020-12-24 Yale University Compositions d'acides nucléiques peptidiques ayant des segments de liaison de type hoogsteen modifiés et leurs procédés d'utilisation
WO2021050568A1 (fr) 2019-09-09 2021-03-18 Yale University Nanoparticules pour absorption sélective de tissu ou cellulaire

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