WO1993002654A2

WO1993002654A2 - A method of inducing hemoglobin synthesis in red blood cells and uses therefor

Info

Publication number: WO1993002654A2
Application number: PCT/US1992/006685
Authority: WO
Inventors: Arthur J. Sytkowski
Original assignee: New England Deaconess Hospital
Priority date: 1991-08-09
Filing date: 1992-08-10
Publication date: 1993-02-18
Also published as: AU2449092A; WO1993002654A3

Abstract

Methods of inducing hemoglobin synthesis in red blood cell precursor cells are disclosed. Also disclosed are red blood cells with increased hemoglobin content made by the methods and their use in treating conditions of decreased oxygen-carrying capacity of the blood or any disorder or condition, which requires an increased supply of hemoglobin in a vertebrate.

Description

A METHOD OF INDUCING HEMOGLOBIN SYNTHESIS IN RED BLOOD

CELLS AND USES THEREFOR

DESCRIPTION

Background of the Invention Gene expression is accomplished by the transcription of genetic information from DNA to RNA followed by translation of mRNA to protein molecules. In transcription, mRNA molecules are synthesized by using the base sequence of one strand of DNA as a template in a polymerization reaction that is catalyzed by RNA polymerases. 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.

In 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. Thus, 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 triggers the growth and differentiation of red blood cells by binding to receptors on plasma membranes of immature erythroid precursor cells. 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. (HARRISON'S PRINCIPLES OF INTERNAL

MEDICINE, 12th Ed., p. 1517, Wilson, J.D., et al. ed. (1991)) .

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. However, transfusions carry the risk of transmitting parasitic or viral diseases, including Hepatitis B and HIV. Moreover, 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.

Summary of the Invention

In general, 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. Specifically, 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.

In particular, the artificial downregulation of the cellular gene, c-mvb, in red blood cell precursor cells leads to an increase of hemoglobin synthesis, a critical step in red blood cell (rbc) maturation and terminal differentiation. Thus, 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.

In one embodiment, 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. Alternatively, 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. For example, 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.

In another embodiment, the invention 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. For example, polyclonal or monoclonal neutralizing antibodies to p75^c"nιyb, the regulator protein encoded by c-myb. can be introduced into rbc precursor cells. 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) . In this embodiment, a substance (or agent) which decreases the amount of differentiation regulator protein is introduced into rbc precursor cells. For example, 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). In another embodiment, 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. Alternatively, 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. For example, 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.

Detailed Description of the Invention

In general, 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. That is, in either case, 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.

In particular, 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. This result is surprising because studies of the role of c-myb in hematopoiesis using antisense oligonucleotides have shown an arrest, or reduction, of hematopoietic growth accompanying decreased expression of^* the c-myb gene. (Gewirtz, AM, and B. Calabretta Science 242: 1303 (1988); Anfossi, G. et al.. Proc. Natl. Acad. Sci. USA 86: 3379 (1989) ; Cavacσiolo, D. et al.. Blood 77: 1181, (1991)). Therefore, the presence of myb protein is generally thought to be required for hematopoietic cell proliferation. Calabretta, B. et al.. Proc. Natl. Acad. Sci. USA 88: 2351-2355 (March 15, 1991).

Based on the disclosed findings, it is now possible to induce hemoglobin synthesis in rbc precursor cells, using any means which decreases the quantity, or activity, of a differentiation regulator protein present in the cells. As used herein, the term "red blood cells" (rbcs) refers to hemoglobin-containing cells, including terminally differentiated erythrocytes, as well as hemoglobin containing erythroblasts. The term "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) . (Axelrad et al..

Properties of Cells that Produce Erythrocytic Colonies in vitro In Robinson W.A. (ed.) Hemopoiesis in Culture, pp. 226-234, U.S. Government Printing Office, Washington, D.C.). 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. In particular, the erythroid cells are rbc precursor cells. The differentiation regulator protein is a gene product of a cellular gene. For example, p75^c"S^Q^_r the gene product of c-myb expression, is a differentiation regulator protein. The term "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. c-myb antisense oligonucleotides useful in the subject invention are disclosed in International Patent Application Publication Number W090/0554, by Gewitz, A.M. and B. Calabretta, which was published May 31, 1990, the teachings of which are incorporated herein by reference.

Downregulating the Expression of a Differentiation

Regulator Protein Using Antisense Oliqonucleotides One general method of decreasing the amount of a differentiation regulator protein is by downregulating its expression. For example, 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. For example, 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. Alternatively, 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. These modifications can optimize the hybridizing ability of the antisense oligonucleotide. Furthermore, 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) . The following describes the results of experiments in which erythroid precursor cells (i.e., Rauscher cells) growing in culture in the absence or presence of EPO were treated with either an antisense oligonucleotide complementary to codons 2 through 7 of, mouse c-myb (SEQ ID NO: 1), antisense c-myc (SEQ ID NO:2), antisense vesicular stomatitis virus M protein (anti-VSV) (SEQ ID NO:3) or a nonsense sequence with the same base composition as anti-myb (SEQ ID NO:4), as explained in detail in Example 1.

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.

The ability of 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 μg/mL or 40 μg/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. A high concentration of the myb protein was found in the cytosol of Rauscher erythroleukemia cells. Although 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. Importantly, treatment of Rauscher cells with antisense c-myb drastically reduced the myb protein concentration in the cells, confirming the efficacy of the treatment.

To test if c-myb oligonucleotide simultaneously arrested proliferation of cells, cells in suspension culture were treated or 18 hours and then transferred to plasma clot culture as explained in Example 3, to permit examination of Hb⁺ daughter cells arising in colonies.

Table 1. EFFECT OF ANTISENSE C-MYB OR EPO ON HEMOGLOBINIZATION OF RAUSCHER CELLS GROWN IN

PLASMA CLOT CULTURE

Inducer Hb⁺ Colonies (%)

None 2.6 ± 1.0

Anti-myb oligo (40 μg/mL) 20.0 ± 4.7

Control anti-VSV oligo (40 μg/mL) 2.6 ± l.l

EPO (10 U/mL) 21.0 ± 8.0

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. (Alper, S.L. et al. , Proc. Natl. Acad. Sci. USA 86: 5429 (1989)). EPO treatment resulted in the appearance of band 3 of 19% of the cells. However, 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 (α) and two beta like (/?- like) chains. Five genes encode .-like globin protein products, e, ^Gy, V_. ^s and β. During gestation, more than 90% of the .-like chains present in the rbcs of the fetus are gamma (y or oc_zVz) • Shortly after birth, production of the predominant /.-like chain begins to switch from y to β (α^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. (Whitney, J.B., Cell, 12.: 863-871 (1977)). Mice also produce the chain, and the α and β- like chains associate to form the hemoglobin protein as in humans. During gestation, the quantity of minor .-globin present is increased, often equal to or exceeding the quantity of β-major. However, in the adult mouse, the ratio of /?-major to /.-minor is approximately 80 to 20. (Alter, B. P. and Goff, S. C, Science 207:647-649 (1980)). In mice, the shift from Hninor to /.-major qualitatively resembles the switch from fetal (gamma or y) to adult (beta or β) globin chain in humans.

To determine what effect the down-regulation of c-myb had on the expression of /--minor and /--major globin chains, experiments were performed as described in Example 4 were performed. Results showed that addition of antisense oligonucleotide, in the absence of other agents, induced 15% of the cells to produce benzidine-reactive hemoglobin within 72 hours of culture as shown in Table 2.

TABLE 2 HEMOGLOBIN INDUCTION BY ANTISENSE c-myb

OLIGODEOXYNUCLEOTIDE

% Hemoglobinization

Untreated control 0

Antisense myb oligo 15

Control oligo 0

Sense myb oligo 0

Erythropoietin 30 Dimethyl sulfoxide 20

Approximately 30% of the cells produced hemoglobin in response to erythropoietin. 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.

To determine what effect the artificial downregulation of c-myb had on the expression of a, β- major and /Hminor globin chains, a triton-urea-acid polyacryla ide gel system was used to analyze the synthesis of the α, /?-major and /.-minor globin chains in 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 [³H]- leucine to label globin chains being actively synthesized. Artificial downregulation of c-mvb induced the synthesis of and of both forms of .-globin in Rauscher cells as shown in the Figure. Scanning densito etry revealed that antisense c-myb oligos induced a 3.51 increase in the synthesis of alpha globin, a 3.06-fold increase in the synthesis of /.-major globin and a 3.19-fold increase in /?- minor globin synthesis in Rauscher cells as shown in Table 3.

TABLE 3

GLOBIN CHAIN INDUCTION (fold-increase over control)

The relative rate of synthesis of the major and minor forms induced by antisense c-myb was 1.00:1.04

( ajor:minor) . Erythropoietin induced a 4.34-fold increase in the synthesis of α globin, a 3.23-fold increase in the synthesis of ?-major globin and a 3.30- fold increase in /.-minor globin chain synthesis. The relative induction of yff-major and /.-minor chain synthesis by erythropoietin was 1.00:1.02 (major:minor) , similar to the ratio induced by antisense c-myb treatment. It is important to note that both EPO and the downregulation of c-myb also induced a 4.34 and 3.51 increase, respectively, in α-globin synthesis. Therefore, it is likely that downregulation of c-myb expression is not only sufficient for globin chain synthesis, but that it is a critical event by which erythropoietin induces hemoglobinization. There is evidence that the ?-globin genes in Friend cells (also derived from the DBA/2 mouse, as are Rauscher cells) are independently regulated and that their relative rates of expression are dependent upon both the inducing agent and the particular cell line used (Curtis, P., et al., J. Biol. Chem. 255:8971-8974 (1980); Alter, B.P. and Goff, S.C., Science 207: 647-649 (1980)). The rates of synthesis of the /.-major and .-minor globin gene products are controlled primarily at the transcriptional level Gangluy, S., et al.. J. Biol. Chem. 263: 3216-3219 (1988). The fact that both erythropoietin and antisense c-myb oligodeoxynucleotide induced the same relative rates of synthesis of /.-major and /.-minor globin chains in Rauscher cells suggests that erythropoietin regulates transcription of the .-globin genes via the c-myb gene product. Additional Methods of Downregulating the Expression of a Differentiation Regulator Protein

Alternatively, 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. For example, 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. (Hsieh, P., et al.. Genes Dev. 4: 1951-1963 (1990); Flum, G.E. et al.. Proc. Natl. Acad. Sci. USA 87: 9436-9440 (1990); Htun, J. and J.E. Dahlberg, Science 243: 1571-1576 (1989)).

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. For example, 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. For example, antibodies (e.g., polyclonal or monoclonal), which bind to the differentiation regulator protein and block the protein's activity, can be introduced into rbc precursor cells. Various techniques exist to prepare suitable antibodies. For example, polyclonal antibodies can be isolated from the serum of animals, which have been immunized with the differentiation regulator protein. Alternatively, 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.

Utility

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) . For example, 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. These cells can then be administered to a human, using techniques known in the art. For example, 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. Likewise, 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.

Alternatively, 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. For example, 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. For example, 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. For example, in humans and in mice, regulatory switching produces a replacement of fetal chain synthesis (y in humans and β- minor in mice) by adult chain synthesis (β in humans and /?-major in mice). (Whitney, J. B., Cell 12:863-871 (1977)). 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 β- thalassemia.

For example, in sickle cell disease, hemoglobin present in the mature rbc consists of two normal α-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.

In other anemias, such as β-thalassemia, insufficient production of the /.-globin chain causes an imbalance in the α- to /?-globin chain ratio present within the rbc.

This imbalance also leads to the destruction of rbcs, and a decrease in the oxygen-carrying capacity of the blood. In such conditions, if the switch from fetal hemoglobin (gamma chain) to adult hemoglobin β chain can be prevented or reversed the oxygen-carrying capacity of the blood could be significantly increased by the presence of normal fetal hemoglobin in rbcs.

As described herein, downregulation of the c-myb cellular gene leads to an increase of the /.-minor (fetal) chain of hemoglobin, as well as an increase in the /.-major (adult) chain in the mouse. Thus, it is reasonable to predict that manipulation of the differentiation regulator protein of c-myb would result in an increase in both the fetal ( ) and adult (/?) forms of hemoglobin in humans as well. An increase in even a small proportion of fetal hemoglobin in human rbcs would significantly increase the oxygen-carrying capacity of the blood in patients with sickle cell disease, β-thalassemia and other hemoglobinopathies as well.

The present invention will now be illustrated by the following Examples, which are not to be seen as limiting in any way.

EXAMPLE 1

Cell Growth and Oligodeoxynucleotide Treatment

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 μL) 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 μg/mL were added followed by a second addition of 10 μg/mL after 24 hours. Hemoglobinized cells (Hb⁺) 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.

EXAMPLE 2

Immunocytochemical Identification of myb Protein

After incubation with oligo, cells were harvested and centrifuged onto glass slides, fixed with acetone at room temperature, and stored at -20°C. Slides were thawed and fixed in acetone for 10 minutes at 4°C, and then stained with monospecific anti-myb monoclonal antibodies (MoAbs) generated against a synthetic peptide corresponding to amino acids 94-112 of v-myb

(Microbiological Associates, Bethesda, MD) as described in Sytkowski, A.J. et al. , J. Clin. Invest. 85: 40 (1990) .

EXAMPLE 3

Plasma Clot Cultures Rauscher cells were plated in suspension culture at 1 x 10⁵ cells/mL and were treated with oligo (40 μg/mL for 18 hours) . Ten microliters of the cell suspension was added to 90 μL 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

Determination of the Expression of a . .-major and .-minor

Globin Chains

Rauscher cells (clone R404) Sytkowski, A.J. et al.. Science. 210: 74 (1980) ) were maintained in DMEM supplemented with 10% fetal bovine serum. Cells were stimulated in 24-well plates at 1 x 10⁵ cells/well (total vol. = 500 ul) in the absence or presence of recombinant human erythropoietin (40 U/ml, Elanex Pharmaceuticals) . 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 μCi/ml of ³H- 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 μl 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. After electrophoresis, the gels were fixed, soaked in ENHANCE (Dupont Chemicals) for 1 hour, washed, dried, and exposed to X-Omat x-ray film (Kodak) at -80°C. Integrated absorbance units were determined using an LKB Ultroscan LX laser densitometer.

Equivalents

Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.

Statement Regarding the Content of the Sequence Listing in Paper and Computer Readable Form The content of the Sequence Listing in paper form and of the computer readable form are the same.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Sytkowski, Arthur J.

(ii) TITLE OF INVENTION: A METHOD OF INDUCING HEMOGLOBIN SYNTHESIS IN RED BLOOD CELLS

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Hamilton, Brook, Smith & Reynolds, P.C.

(B) STREET: Two Militia Drive

(C) CITY: Lexington

(D) STATE: MA

(E) COUNTRY: USA

(F) ZIP: 02713

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US

(B) FILING DATE: (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/748,867

(B) FILING DATE: 09-AUG-1991

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Granahan, Patricia

(B) REGISTRATION NUMBER: 32,227

(C) REFERENCE/DOCKET NUMBER: NEDH91-05A (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617-861-6240

(B) TELEFAX: 617-861-9540

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDΞDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GTGTCGGGGT CTCCGGGC 18

(2) INFORMATION FOR SEQ ID NOΪ2Ϊ

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPEt nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION. SEQ ID NO:2i

AACGTTGAGG GCAT 14

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NOt3:

TTGGGATAAC ACTTA 15 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GCGCGCTTGG CTGTGGGC 18

Claims

1. A method of inducing hemoglobin synthesis in a red blood cell precursor cell, comprising decreasing the amount of a differentiation regulator protein which regulates the transcription of a gene required for hemoglobin synthesis, said differentiation regulator protein produced by a cellular gene in the red blood cell precursor cell, and for example the differentiation regulator protein is p75^c"SD^ and for example the cellular gene is c-myb.

2. A method of inducing hemoglobin synthesis in a red blood cell precursor cell comprising downregulating in a red blood cell precursor cell, the expression of a differentiation regulator protein which regulates the transcription of a gene required for hemoglobin synthesis.

3. A method of inducing hemoglobin synthesis in a red blood cell precursor cell comprising introducing into a red blood cell precursor cell, an antisense oligonucleotide, which is complementary to a cellular gene mRNA which encodes a differentiation regulator protein which regulates the transcription of a gene required for hemoglobin synthesis, in such a manner that the antisense oligonucleotide hybridizes with the cellular gene mRNA, thereby preventing translation of the mRNA into a differentiation regulator protein, and for example the cellular gene is c-myb.

4. A method of inducing hemoglobin synthesis in a red blood cell precursor cell comprising blocking or neutralizing the activity of a differentiation regulator protein in a red blood cell precursor cell, which regulates the transcription of a gene required for hemoglobin synthesis.

5. A method of inducing hemoglobin synthesis in a red blood cell precursor cell, comprising introducing into a red blood cell precursor cell, an antibody which binds to a differentiation regulator protein which regulates the transcription of a gene required for the synthesis of hemoglobin and thereby neutralizes the activity of the differentiation regulator protein, and for example the differentiation regulator protein is p75^c"m^.

6. Red blood cells produced by the method of any of Claims 1-4.

7. Use of a substance which decreases the amount or function of a differentiation regulator protein which regulates the transcription of a gene required for hemoglobin synthesis, for treating a disease or condition in an individual which requires an increase in hemoglobin synthesis, and for example the cellular gene is c-myb.

8. The use of Claim 1 , wherein the differentiation regulator protein is p75^c"!!!^.

9. Use, for treating conditions of decreased oxygen- carrying capacity of the blood in a vertebrate, of a substance, to be introduced into red blood cell precursor cells of the vertebrate, said substance being one which downregulates the expression of a differentiation regulator protein which regulates the transcription of a gene required for the synthesis of hemoglobin.

10. The use of Claim 9, wherein the substance is an antisense oligonucleotide, which is complementary to the cellular gene mRNA encoding a differentiation regulator protein which regulates the transcription of a gene required for the synthesis of hemoglobin, and for example the cellular gene is c-myb.

11. Use, for treating conditions of decreased oxygen- carrying capacity of the blood in a vertebrate, of a substance to be introduced into the red blood cell precursor cells of the vertebrate, said substance being one which blocks, or neutralizes the activity of a differentiation regulator protein which regulates the transcription of a gene required for the synthesis of hemoglobin.

12. The use of Claim 11, wherein the substance is an antibody which binds to the differentiation regulator protein, thereby blocking or neutralizing the activity of the protein, and for example the differentiation regulator protein is p75^c"SQ*.

13. Use, for the preparation of a medicament for treating conditions of decreased oxygen-carrying capacity of the blood in a vertebrate, of an agent which decreases the amount of differentiation regulator protein which is required for the synthesis of hemoglobin, for inducing hemoglobin synthesis and for increasing the hemoglobin content of the cells.

14. The use of Claim 13, wherein the differentiation regulator protein is p75^c'2Q^.

15. Use, for the preparation of a medicament for treating conditions of decreased oxygen-carrying capacity of the blood in a vertebrate, of a substance which downregulates the expression of a differentiation regulator protein which regulates the transcription of a gene required for the synthesis of hemoglobin, for inducing hemoglobin synthesis and for increasing the hemoglobin content of the cells.

16. The use of Claim 15, wherein the substance is an antisense oligonucleotide, which is complementary to a cellular gene mRNA encoding a differentiation regulator protein which regulates the transcription of a gene required for the synthesis of hemoglobin, and for example the cellular gene is c-myb.

17. Use, for the preparation of a medicament for treating conditions of decreased oxygen-carrying capacity of the blood in a vertebrate, of a substance which blocks, or neutralizes the activity of a differentiation regulator protein which regulates a gene required for the synthesis of hemoglobin, for inducing hemoglobin synthesis and for increasing the hemoglobin content of the cells.

18. The use of Claim 17, wherein the substance is an antibody which binds to the differentiation regulator protein and thereby neutralizes the activity of the differentiation regulator protein, and for example the differentiation regulator protein is p75^c"5Q!-².