WO1993002654A2 - Procede pour induire la synthese de l'hemoglobine dans les globules rouges du sang et utilisations de ce procede - Google Patents

Procede pour induire la synthese de l'hemoglobine dans les globules rouges du sang et utilisations de ce procede Download PDF

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WO1993002654A2
WO1993002654A2 PCT/US1992/006685 US9206685W WO9302654A2 WO 1993002654 A2 WO1993002654 A2 WO 1993002654A2 US 9206685 W US9206685 W US 9206685W WO 9302654 A2 WO9302654 A2 WO 9302654A2
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hemoglobin
synthesis
regulator protein
cells
differentiation regulator
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WO1993002654A3 (fr
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Arthur J. Sytkowski
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New England Deaconess Hospital
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    • 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
    • C12N15/1135—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 oncogenes or tumor suppressor genes
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • RNA polymerases bind to a DNA strand at particular sites called promoters.
  • Transcriptional regulation is one mechanism of controlling gene expression. Some promoters are competent to support initiation by RNA polymerase, although extraneous proteins (e.g., negative regulator factors) may act to prevent the initiation process. In other cases the polymerase itself is not adequate and ancillary proteins (e.g., transcriptional activators) are necessary for initiation to occur.
  • extraneous proteins e.g., negative regulator factors
  • extraneous proteins e.g., negative regulator factors
  • ancillary proteins e.g., transcriptional activators
  • hematopoiesis i.e., blood cell development
  • pluripotent stem cells in the bone marrow divide to form committed precursor cells, which mature along distinct pathways.
  • the different types of blood cells are produced in different numbers, and the production of each must be regulated individually to meet changing needs.
  • hematopoiesis necessarily involves some complex transcriptional and translational controls.
  • the development of red blood cells from undifferentiated precursor cells in bone marrow requires the regulatory influence of the glycoprotein hormone erythropoietin (EPO) .
  • EPO erythropoietin
  • EPO erythropoietin
  • the immature erythroid precursor cells undergo subsequent maturation, ending as terminally differentiated erythrocytes, which circulate in the blood for approximately 120 days.
  • a key event in the maturation of red blood cell precursor cells is the production of hemoglobin, an iron-containing protein.
  • the primary role of the erythrocyte is to transport oxygen from the lungs to tissue. Oxygen is picked up in the lungs from inspired air, bound to hemoglobin, and transported to the tissue blood capillaries. In the tissue, at the proper oxygen pressure, the oxygen is released from the hemoglobin, leaves the capillary and diffuses through the tissue.
  • Oxygenation of tissue depends on three main factors: blood flow, the affinity of the hemoglobin for oxygen and the oxygen-carrying capacity of blood.
  • the oxygen- carrying capacity of blood is a function of hemoglobin concentration.
  • the oxygen-carrying capacity of blood can be decreased as a result of a reduction in the total number of red blood cells present, as in various anemias, resulting from radiation or chemotherapeutic treatment of malignancies, or chronic renal disease. Decreased oxygen- carrying capacity of blood is also seen in a number of hemoglobinopathies, such as sickle cell anemia or thalassemia which affects the ability of hemoglobin to carry the oxygen molecule.
  • EPO has proven useful in treating patients suffering from some of these conditions. However, in certain cases ad inistration of EPO is unproductive. For example, individuals suffering from sickle cell anemia already have elevated levels of EPO in their blood, due to the significant destruction of their abnormal red blood cells. In these cases, the administration of additional EPO has no effect on increasing the oxygen-carrying capacity of the blood.
  • Blood transfusions provide an alternative to EPO treatment.
  • transfusions carry the risk of transmitting parasitic or viral diseases, including Hepatitis B and HIV.
  • repeated blood transfusions can also result in hemochromatosis, a disease resulting from excess deposits of iron (from hemoglobin) in tissue. Therefore, a method of regulating hemoglobin synthesis to increase the oxygen-carrying capacity of the blood would be very useful.
  • the present invention relates to a method of inducing hemoglobin synthesis in red blood cell precursor cells thereby increasing the hemoglobin content of red blood cells and, thus, increasing the oxygen- carrying capacity of blood.
  • the hemoglobin content of red blood cells is relatively increased over the amount of hemoglobin normally present in red blood cell precursor cells and red blood cells.
  • Hemoglobin synthesis is induced by regulating the quantity, or activity, of a differentiation regulator protein in red blood cell precursor cells.
  • This differentiation regulator protein regulates transcription of a gene (or genes) required for differentiation of red blood cell precursor cells.
  • the differentiation regulator protein is a product of a cellular gene, such as c-myb.
  • red blood cell precursor cells leads to an increase of hemoglobin synthesis, a critical step in red blood cell (rbc) maturation and terminal differentiation.
  • rbc red blood cell
  • an increase in hemoglobin synthesis in precursor cells leads to an increase in hemoglobin content in red blood cells (rbcs) , and consequently, an increase in the oxygen-carrying capacity of blood.
  • the invention relates to a method of inducing hemoglobin synthesis in rbc precursor cells by downregulation of the expression of a cellular gene by introducing into precursor cells antisense oligonucleotides, which hybridize with the mRNA transcribed by the cellular gene, thereby regulating translation of the mRNA to produce the encoded differentiation regulator protein.
  • the expression of differentiation regulator protein can be downregulated by any method which regulates the binding of a transcriptional activator to a gene encoding the differentiation regulator protein.
  • sense or antisense DNA complementary to a portion of the cellular gene can be introduced into rbc precursor cells, in which it hybridizes with a portion of the cellular gene, forming a "third strand" and thereby inhibiting the transcription of DNA to mRNA.
  • the invention in another embodiment, relates to a method of inducing hemoglobin synthesis in rbc precursor cells by neutralizing or, blocking, the activity of the differentiation regulator protein in the rbc precursor cell.
  • This can be accomplished by introducing into precursor cells antibodies which bind to the differentiation regulator protein and, thus, neutralize, or block, its activity.
  • antibodies which bind to the differentiation regulator protein and, thus, neutralize, or block, its activity.
  • the antibodies will bind to the regulator protein, blocking its ability to regulate transcription, thus, inducing hemoglobin synthesis in rbc precursor cells.
  • the invention also relates to red blood cells with increased hemoglobin content produced by the disclosed methods and their use in treating anemias and other conditions which result from a decreased oxygen-carrying capacity of blood in vertebrates (particularly a human or other mammal) .
  • a substance (or agent) which decreases the amount of differentiation regulator protein is introduced into rbc precursor cells.
  • the substance can be introduced into precursor cells ex vivo to induce hemoglobin synthesis, resulting in red blood cells with increased content of hemoglobin relative to the amount of hemoglobin normally present in the cells.
  • These red blood cells, with increased hemoglobin content can be introduced into a vertebrate (e.g., via a bone marrow transplant or transfusion).
  • the red blood cells introduced into the vertebrate can be rbc precursor cells, introduced into the vertebrate under conditions such that the precursor cells differentiate into red blood cells with increased hemoglobin content.
  • the red blood cells introduced into the vertebrate can be mature, terminally differentiated erythrocytes with increased hemoglobin content.
  • Therapy can also be accomplished in vivo, by administering the substance to a human, in such a manner that the substance is targeted to the individual's rbc precursor cells.
  • the substance can be contained within a biocompatible encapsulant, such as a liposome, which contains antibodies specific to rbc precursor cell surface antigens (e.g., ERY-1) .
  • the encapsulant containing the substance can then be administered to a vertebrate, in such a manner that the encapsulant contacts and fuses with a rbc precursor cell, and the substance is introduced into the precursor cell.
  • the methods of inducing hemoglobin synthesis of the subject invention can be used on an individuals own precursor cells, as well as on cells from another individual.
  • An important advantage of using the methods of the subject invention is that the individual's own cells can be used as the source of precursor cells and, therefore, the problems associated with transfusions (e.g., infections) are obviated.
  • the present invention relates to a method of inducing hemoglobin synthesis in red blood cell (rbc) precursor cells by regulating the quantity, or activity, of a differentiation regulator protein, in the precursor cell, which is a gene product of a cellular gene, such as c-myb.
  • the differentiation regulator protein is a transcription factor which regulates the transcription of a gene, or genes, required for synthesis of hemoglobin in erythroid cells.
  • the regulation effect of the differentiation regulator protein can increase or decrease transcription of a gene required for hemoglobin synthesis. In either case, the outcome is the same.
  • hemoglobin synthesis is induced and the hemoglobin content of red blood cell precursor cells and red blood cells is increased to an amount greater than would occur in the absence of the differentiation regulator protein and, consequently, the oxygen-carrying capacity of the blood is also increased.
  • the downregulation of the cellular gene, c-myb. in rbc precursor cells leads to an increase of hemoglobin synthesis, which, in turn, leads to an increased hemoglobin content in red blood cells and thus, increases the oxygen-carrying capacity of blood.
  • the present invention is based on the finding that treatment of red blood cell (rbc) precursor cells with an antisense oligonucleotide to the c-myb gene downregulates myb protein and induces hemoglobinization of the precursor cells, but does not adversely affect proliferation of red blood cells.
  • rbc red blood cell
  • antisense oligonucleotide to the c-myb gene downregulates myb protein and induces hemoglobinization of the precursor cells, but does not adversely affect proliferation of red blood cells.
  • red blood cells refers to hemoglobin-containing cells, including terminally differentiated erythrocytes, as well as hemoglobin containing erythroblasts.
  • red blood cell precursor cells refers to precursor cells of the erythroid lineage, and includes colony forming unit- erythroid (CFU-E) and burst forming unit-erythroid (BFU- E) .
  • CFU-E is a cell that give rise to a small colony of 8 to 50 mature, hemoglobin containing erythroblasts in 2 days (mouse) to 7 days (human) .
  • Burst forming unit-erythroid (BFU-E) is a less mature erythroid precursor cell that gives rise to a large cluster of colonies of mature erythroblasts (several hundred to several thousand cells) in 8 days (mouse) to 14 days (human) . (Gregory and Eaves, Blood. 49: 855-864 (1977)) .
  • the term "differentiation regulator protein”, as used herein, refers to a transcription factor which regulates the transcription of genes required for the synthesis of hemoglobin in erythroid cells.
  • the erythroid cells are rbc precursor cells.
  • the differentiation regulator protein is a gene product of a cellular gene.
  • p75 c" S Q ⁇ r the gene product of c-myb expression is a differentiation regulator protein.
  • cellular gene refers to a gene, which controls cellular growth and differentiation. Examples of cellular genes include c-myb. c-myc. c-fos and c- un.
  • antisense oligonucleotides which are complementary to a cellular gene mRNA, which encodes a differentiation regulator protein, can be introduced into rbc precursor cells, in such a manner that the antisense oligonucleotides hybridize with the mRNA transcribed by the cellular gene and inhibit translation of the mRNA into the encoded differentiation regulator protein.
  • the antisense oligonucleotide can be introduced using methods known in the art.
  • the oligonucleotide can be introduced, as disclosed herein, by incubating rbc precursors in the presence of antisense oligonucleotides and under conditions which allow entry of oligonucleotide into the cell.
  • other methods such as direct intracellular microinjection, infection of cells with modified vectors (e.g., plasmids or retroviral vectors) or fusion of cells with other cells, sperm or liposomes which contain the oligonucleotide.
  • Antisense oligonucleotides can be introduced into rbc precursor cells .in vivo, to induce the synthesis of hemoglobin.
  • Antisense oligonucleotides can also be introduced into rbc precursor cells in vitro to induce hemoglobin synthesis in rbc precursor cells. The resulting rbc precursor cells can then be introduced into an individual in which increased hemoglobin is described (e.g. to treat anemia) .
  • Antisense oligonucleotides of the subject invention can include unmodified nucleic acids or modified nucleic acids. Modifications can occur at any of a number of locations along the length of the oligonucleotide. For example, a modified nucleic acid can be added to the 5' end of the oligonucleotide, the 3' end or both, as well as to an internal phosphate group or a base.
  • antisense oligonucleotides can be oligodeoxynucleotides or oligoribonucleotides of a length optimized for hybridizing with the cellular gene mRNA.
  • the appropriate length of the oligonucleotides, as well as modifications, can be optimized to facilitate uptake into erythroid precursor cells, increase resistance to degradation of the oligonucleotides in the cell and to strengthen binding between the oligonucleotide and the cellular gene mRNA.
  • Preferred antisense oligonucleotides for use in the subject invention are oligonucleotides which hybridize to at least a portion of the c-myb gene. Particularly antisense oligonucleotides directed to (which hybridize at) a site at or near the initiation codon for protein synthesis. As described herein, oligonucleotides were synthesized on an automated DNA synthesizer. It is possible, however, to produce the desired sequences by other methods, such as by using genetically engineered organisms (e.g., genetically modified bacteria or viruses) .
  • erythroid precursor cells i.e., Rauscher cells
  • erythroid precursor cells i.e., Rauscher cells
  • EPO erythroid precursor cells
  • mouse c-myb SEQ ID NO: 1
  • antisense c-myc SEQ ID NO:2
  • anti-VSV antisense vesicular stomatitis virus M protein
  • SEQ ID NO:4 a nonsense sequence with the same base composition as anti-myb
  • Hb + cells After 48 hours of incubation, hemoglobinized cells were scored. 17% of the cells treated with antisense c- myb were hemoglobinized (Hb + ) . Treatment of replicate cultures with EPO instead of the antisense oligonucleotide also yielded 17% hemoglobinization. Treatment of replicate cultures with antisense c-myb and EPO simultaneously yielded 18% Hb + cells. The absence of an additive effect of antisense c-myb oligonucleotide and EPO strongly suggests that both agents are operating on the same responsive population of cells. Importantly, addition of antisense c-myc f anti-VSV or a nonsense sequence with the same base composition as anti-myb which were used as controls did not induce Hb + cells. Addition of anti-VSV oligo plus EPO resulted in 10% Hb + cells.
  • antisense c-myb to induce hemoglobin synthesis in these cells was concentration dependent.
  • Modified oligonucleotides were also shown to induce hemoglobin synthesis in a concentration dependent manner. Specifically, phosphorothioate oligonucleotide (S-oligo) was shown to induce synthesis in a concentration dependent manner. Addition of 20 ⁇ g/mL or 40 ⁇ g/mL of antisense c- vb S-oligo for 48 hours yielded 5% and 12% Hb + cells, respectively. That antisense c-myb decreases myb protein was demonstrated using an antipeptide monoclonal antibody as described in Example 2.
  • myb protein binds DNA and has been found concentrated in the nucleus of some cells, other cells, including normal T lymphocytes, exhibit higher cytosolic concentration very similar to that shown here.
  • treatment of Rauscher cells with antisense c-myb drastically reduced the myb protein concentration in the cells, confirming the efficacy of the treatment.
  • Anti-myb oligo (40 ⁇ g/mL) 20.0 ⁇ 4.7
  • Control anti-VSV oligo (40 ⁇ g/mL) 2.6 ⁇ l.l
  • Table 1 shows that in the absence of inducer, or in the presence of control oligonucleotides, only 2.6% of the colonies were hemoglobinized (Hb + ) . Colonies containing greater than 4 Hb + cells were scored as Hb + colonies. Most contained no Hb + cells. However, antisense c-myb or EPO resulted in 20% and 21% Hb + colonies, respectively. Importantly, the plating efficiencies observed under all conditions were identical (15% ⁇ 2%) , as were the sizes of the colonies. No evidence for an expected growth inhibiting action of the antisense c-myb on erythroid precursor cells was found.
  • EPO and c-myb treated erythroid precursor cells were stained for the membrane anion transport protein, band 3, which is expressed by mature erythrocytes, but not by immature erythroid precursors.
  • band 3 which is expressed by mature erythrocytes, but not by immature erythroid precursors.
  • EPO treatment resulted in the appearance of band 3 of 19% of the cells.
  • the band was not detected on cells treated with the antisense c-myb oligonucleotide. Therefore, although antisense c-myb treatment clearly results in hemoglobin synthesis, it does not induce expression of all mature erythrocyte characteristics. Hemoglobin is composed of two moieties.
  • Globin is the protein moiety of hemoglobin and heme is the iron- containing moiety. Normally, four globin chains associate to form the intact hemoglobin protein. In humans, hemoglobin consists of two alpha ( ⁇ ) and two beta like (/?- like) chains. Five genes encode .-like globin protein products, e, G y, V . s and ⁇ . During gestation, more than 90% of the .-like chains present in the rbcs of the fetus are gamma (y or oc z Vz) • Shortly after birth, production of the predominant /.-like chain begins to switch from y to ⁇ ( ⁇ t? 2 ) • This switching process continues throughout the first year of life.
  • Rauscher cells derived from DBA/2 mice, produce two forms of .-globin upon differentiation, a /.-minor form and a /.-major form.
  • Mice also produce the chain, and the ⁇ and ⁇ - like chains associate to form the hemoglobin protein as in humans.
  • the quantity of minor .-globin present is increased, often equal to or exceeding the quantity of ⁇ -major.
  • the ratio of /?-major to /.-minor is approximately 80 to 20. (Alter, B. P. and Goff, S. C, Science 207:647-649 (1980)).
  • the shift from Hninor to /.-major qualitatively resembles the switch from fetal (gamma or y) to adult (beta or ⁇ ) globin chain in humans.
  • DMSO a chemical inducer of differentiation of these cells, induced hemoglobinization of 20% of the cells. Hemoglobin synthesis in cells treated with control, non-specific oligodeoxynucleotide was not detected.
  • a triton-urea-acid polyacryla ide gel system was used to analyze the synthesis of the ⁇ , /?-major and /.-minor globin chains in Rauscher cell treated with c-mvb antisense oligodeoxynucleotides.
  • Rauscher cell treated with c-mvb antisense oligodeoxynucleotides As described in Example 4, cells were treated with antisense c-myb oligodeoxynucleotide nucleotide for three days, and then incubated with [ 3 H]- leucine to label globin chains being actively synthesized.
  • the expression of a differentiation regulator protein can be downregulated by any method which prevents the binding of a gene transcriptional activator to a gene encoding the differentiation regulator protein.
  • sense, or antisense DNA which is complementary to a portion of the cellular gene can be introduced into rbc precursor cells. In the cells, the DNA hybridizes with a portion of the cellular gene, forming a "third strand", thereby inhibiting the transcription of DNA to mRNA.
  • a sense, or antisense, oligodeoxynucleotide which is complementary to a portion of the cellular gene can be introduced into rbc precursor cells in vitro or in vivo by methods known in the art.
  • any of the above disclosed methods for introducing antisense oligonucleotides which are complementary to a cellular gene mRNA can be used.
  • Other methods which can be used include electroporation, calcium phosphate precipitation and DEAE dextran.
  • Another general method of decreasing the amount of a differentiation regulator protein is by neutralizing, or blocking, the activity of a differentiation regulator protein in the rbc precursor cell.
  • antibodies e.g., polyclonal or monoclonal
  • bind to the differentiation regulator protein and block the protein's activity can be introduced into rbc precursor cells.
  • suitable antibodies e.g., polyclonal antibodies can be isolated from the serum of animals, which have been immunized with the differentiation regulator protein.
  • monoclonal antibodies can be isolated from the supernatant of hybridomas or from serum or ascites fluid of animals inoculated with the hybridoma.
  • Monoclonal antibodies can also be obtained by recombinant DNA technology.
  • the antibodies can be introduced into rbc precursor cells in vitro or in vivo by methods known to those skilled in the art.
  • Red blood cells with increased hemoglobin content produced by methods described herein are useful for treating anemia or any disorder or condition, which results from decreased oxygen-carrying capacity of vertebrate blood (particularly in a human or other mammal) .
  • the red blood cells can be used to treat individuals with anemia, such as anemia caused by chemotherapy, patients with chronic renal failure or patients suffering from sickle cell disease.
  • An important advantage of the methods described herein is that hemoglobin synthesis can be induced even when rbc precursor cells are refractory to EPO administration.
  • a substance (or agent) which decreases the amount of differentiation regulator protein can be introduced into rbc precursor cells ex vivo to induce hemoglobin synthesis in rbc precursor cells.
  • the hemoglobinized rbc precursor cells can be transplanted into the bone marrow of an individual and allowed to subsequently differentiate into mature rbcs with an increased hemoglobin content.
  • differentiated rbcs with increased hemoglobin content can be transfused (e.g., via parenteral injection) into the vertebrate's bloodstream.
  • a distinct advantage of the present invention is that either of the above-described procedures can be done with the individual's own precursor red blood cells.
  • a substance which decreases the amount of a differentiation regulator protein can be introduced into rbc precursor cells in vitro, by methods that ensure that the substance specifically enters red blood cell precursors and not other cells of the vertebrate.
  • the substance can be contained within a biocompatible encapsulant, such as a liposome, which contains antibodies specific to rbc precursor cell surface antigens (e.g., ERY-1) . (Bacon, E. R. and Sytkowski, A. J., Blood. 69: 103-108 (1987); Yokoch, T., et al.. Blood. 63: 1376 (1984)).
  • the encapsulant containing the substance can then be administered to a vertebrate, so that the encapsulant contacts and fuses with rbc precursor cells, thereby introducing the substance into the precursor cells.
  • the substance can also be encoded in a vector which specifically recognizes red blood cell precursors.
  • parvoviruses specifically infect rbc precursor cells (Ozawa, K. et al.. J. Virol.. 62: 2508-2511 (1988) ; Ozawa, K. and N. Young, J. Virol.. 6_1: 2627-2630 (1987)). Therefore, a modified parvovirus vector, which includes the genes for targeting rbc precursors, but not the genes involved in inhibiting red blood cell proliferation, can be engineered to include genes encoding a substance which decreases the amount of a differentiation regulator protein. Administration of the modified parvovirus vector into a vertebrate would therefore introduce the substance specifically into rbc precursor cells, but not into other cells of the vertebrate.
  • the genes for mammalian /.-globin and .-like globins are closely linked on the same chromosome and the expression of the genes for many of these various chains are regulated in a sequential fashion.
  • regulatory switching produces a replacement of fetal chain synthesis (y in humans and ⁇ - minor in mice) by adult chain synthesis ( ⁇ in humans and /?-major in mice).
  • the ability to regulate the switch from fetal to adult chain synthesis could be beneficial in a number of hemoglobinopathies, such sickle cell anemia and ⁇ - thalassemia.
  • hemoglobin present in the mature rbc consists of two normal ⁇ -globin chains and two mutant /.-globin chains.
  • This /.-globin mutation causes a change in the solubility of clathrate present in the rbc.
  • the formation of insoluble clathrates disrupts the oxygen-carrying capacity of the rbc and eventually, leads to the destruction of the abnormal cell.
  • the replacement of even a small proportion of the abnormal /.-globin present with a normal globin chain, such as fetal globin ( ⁇ globin) would block clathrate formation and significantly increase the oxygen-carrying capacity of the blood.
  • Rauscher cells (clone EMS111) (Spangler, R. et al. f J. Biol. Chem. 266: 681 (1991) were cultured in 96-well plates, 1,500 cells/well (100 ⁇ L) in the absence of presence of 10 U recombinant human EPO (rhuEPO)mL (Elanex Pharmaceuticals, Bothell, WA) .
  • Oligonucleotides (O-oligos or phosphorothioate modified oligodeoxynucleotides (S- oligos) ) , 40 ⁇ g/mL were added followed by a second addition of 10 ⁇ g/mL after 24 hours.
  • Hemoglobinized cells were scored by benzidine staining after 48 hours (200 cells scored) (Rifkind, R.A. et al.. "Assay for the Commitment of Murine Erythroleukemia Cells to Differentiate", In Vitro Aspects of Erythropoiesis (M Murphy Ed.) New York, NY Springer-Verlag p. 266 (1978)). Oligos were synthesized using an Applied Biosystems DNA Synthesizer 380A (Foster City, CA) and were purified by ethanol precipitation.
  • Plasma Clot Cultures Rauscher cells were plated in suspension culture at 1 x 10 5 cells/mL and were treated with oligo (40 ⁇ g/mL for 18 hours) . Ten microliters of the cell suspension was added to 90 ⁇ L of plasma clot medium in 96-well plates and incubated for 3 days at 37°C (4 wells/determination). The clots were removed onto glass slides, fixed, and stained as described in Sytkowski, A.J. et al.. Science 210:74-76 (1980) . EXAMPLE 4
  • Antisense oligodeoxynucleotides complementary to codons 2- 7 of c-myb (SEQ ID N0:1) or control oligodeoxynucleotides with the same base composition as the antisense c-myb oligo (SEQ ID NO: 4) were added at 40 ug/ml followed by another addition of 10 ug/ml after 24 hours. After 72 hours of incubation, aliquots (50 ul) were removed to determine hemoglobinization by benzidine staining.
  • Globin chain synthesis was determined by pulse- labelling of the remaining cells with 25 ⁇ Ci/ml of 3 H- leucine (Amersham International pic) as described in Alter, B.P., et al. , Brit. J. Haematol. 44: 527-534 (1980) .
  • the labeled cells were incubated at 37°C for four hours and lysed in sample buffer (5 ml 8 M Urea, 0.5 ml acetic acid, 0.5 ml 2-mercaptoethanol, and 2 mg pyronin Y) .
  • Globin chains were separated by electrophoresis under reverse polarity on 12% polyacryla ide gels (60:0,4 acrylamide:bis-acrylamide) containing 6 M freshly deionized urea, 5% acetic acid, and 2% Triton X-100 (Sigma Chemicals) using 5% acetic acid as the electrophoresis buffer. Gels were preelectrophoresed for 60 minutes at 200 V. The anode buffer was removed, and the wells were overlaid with 20 ⁇ l 1M cysteamine (Aldrich Chemicals) . Next, fresh anode buffer was added, and the gels were run for 1 hour at 150 V. The samples then were loaded and electrophoresed at 8.5 mA for 17 hours.

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Abstract

L'invention se rapporte à des procédés pour induire la synthèse de l'hémoglobine dans des cellules précurseurs des globules rouges du sang. L'invention décrit également des globules rouges du sang ayant une teneur en hémoglobine accrue, qui sont produits par les procédés décrits, ainsi que leur utilisation dans le traitement d'états où la capacité du sang à contenir de l'oxygène est réduite, ou de tout autre trouble ou état qui nécessite un apport accru d'hémoglobine chez un vertébré.
PCT/US1992/006685 1991-08-09 1992-08-10 Procede pour induire la synthese de l'hemoglobine dans les globules rouges du sang et utilisations de ce procede WO1993002654A2 (fr)

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US74286791A 1991-08-09 1991-08-09
US742,867 1991-08-09

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WO1993002654A2 true WO1993002654A2 (fr) 1993-02-18
WO1993002654A3 WO1993002654A3 (fr) 1993-03-18

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AU (1) AU2449092A (fr)
WO (1) WO1993002654A2 (fr)

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Publication number Priority date Publication date Assignee Title
US5646042A (en) * 1992-08-26 1997-07-08 Ribozyme Pharmaceuticals, Inc. C-myb targeted ribozymes
US5658780A (en) * 1992-12-07 1997-08-19 Ribozyme Pharmaceuticals, Inc. Rel a targeted ribozymes
EP0710721A3 (fr) * 1994-11-02 1999-09-15 Takeda Chemical Industries, Ltd. Procédé pour élucider la fonction d'une protéine
WO2002072116A1 (fr) * 2001-03-08 2002-09-19 Max-Delbrück-Centrum für Molekulare Medizin Cellules sanguines transformees a l'aide de myb et leur utilisation pour le criblage de substances actives
KR100436176B1 (ko) * 1995-08-03 2004-11-06 꼼미사리아 아 레네르지 아토미끄 회전식융해로

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WO1990005445A1 (fr) * 1988-11-07 1990-05-31 Temple University Of The Commonwealth System Of Higher Education Oligonucleotides non codants du proto-oncogene c-myb et leurs emplois

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CLINICAL RESEARCH vol. 38, no. 2, April 1990, THOROFARE, N.J.,US; page 289A Y. CHERN ET AL. 'A c-myb antisense oligonucleotide induces erythropoiesis in-vitro in the absence of erythropoietin' *
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5646042A (en) * 1992-08-26 1997-07-08 Ribozyme Pharmaceuticals, Inc. C-myb targeted ribozymes
US5817796A (en) * 1992-08-26 1998-10-06 Stinchcomb; Dan T. C-myb ribozymes having 2'-5'-linked adenylate residues
US5658780A (en) * 1992-12-07 1997-08-19 Ribozyme Pharmaceuticals, Inc. Rel a targeted ribozymes
US6410224B1 (en) 1992-12-07 2002-06-25 Ribozyme Pharmaceuticals, Inc. Ribozyme treatment of diseases or conditions related to levels of NF-κB
EP0710721A3 (fr) * 1994-11-02 1999-09-15 Takeda Chemical Industries, Ltd. Procédé pour élucider la fonction d'une protéine
KR100436176B1 (ko) * 1995-08-03 2004-11-06 꼼미사리아 아 레네르지 아토미끄 회전식융해로
WO2002072116A1 (fr) * 2001-03-08 2002-09-19 Max-Delbrück-Centrum für Molekulare Medizin Cellules sanguines transformees a l'aide de myb et leur utilisation pour le criblage de substances actives

Also Published As

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AU2449092A (en) 1993-03-02
WO1993002654A3 (fr) 1993-03-18

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