A method is described for regulating gene expression related to iron metabolism to ameliorate diseases that include sickle cell disease, cancers, neurodegenerative diseases, Friedreich's ataxia and other neuromuscular disorders, and atherosclerosis. This approach is illustrated by recent findings that show that ferritin-H, an iron-binding protein that is present in cell nuclei, can repress the human .beta.-globin gene, the gene that is mutated in sickle cell disease. Increased expression of ferritin-H or a related ferritin-family peptide, given to effected cells either as the peptide itself (or a part thereof), as an expression clone of the ferritin-H-subfamily gene, or via a gene regulator that increases expression of the ferritin-H-subfamily gene itself, prevents or ameliorates expression of the disease state in disorders where increased availability of iron is implicated in the etiology of the disease, including those named above.

Description of the Invention

SUMMARY OF THE INVENTION

The inventors have discovered that a nuclear ferritin-H subfamily of iron-sequestering proteins is a gene regulatory protein in human cells. Specifically, we have found that nuclear ferritin binds to a specific DNA sequence that is centrally placed in the promoter of the human .beta.-globin gene and that the effect of the ferritin-H binding is to repress transcription of this gene in transfected cells. Thus, a ferritin-H gene or peptide targeted to the correct cells offers a cure for sickle cell disease in which the .beta.-globin gene is mutated, as well as other genetic diseases where there is mismanagement of iron. Over expression of ferritin-H up to 500.times. in human cells is not harmful, does decrease the labile iron pool, decreases proliferation in cancer cell lines, and promotes apoptosis in cancer cells.

The present invention relates to a method and/or composition of altering the phenotype of a cell from the inside via producing a change in gene expression, i.e., gene expression therapy. Methods are described for transferring a gene for ferritin-H or other ferritin-H family peptides into a cell so that the ferritin-H gene is expressed therein and, as a result of this ferritin-H is produced. This alters the phenotype of the cell either through the ferritin itself regulating the expression of another gene associated with the disease phenotype (as in the inventors' well studied example of sickle cell disease) or through the ferritin changing the iron balance within the cell which, in turn, results in a change in gene expression that alters the phenotype. The phenotype of the effected cell can also be altered by delivering the expressed peptide itself (i.e., ferritin), or a part thereof, directly into the diseased cell or to the cell before it exhibits the disease phenotype. Induction of expression of the endogenous ferritin-H gene in the appropriate cells by stimulating its transcription is a third approach to gene regulation therapy; this will be done by applying to the cells exogenous cytokines or other agents. H-ferritin is known to increase in response to TNF.alpha. or IL-1.beta., whereas L-ferritin selectively increases in response to exogenously added iron. And a fourth approach is to alter the cell phenotype by gene regulation therapy by delivering an antisense oligonucleotide that would prevent expression of a specific ferritin family peptide by inhibiting translation and/or transcription of its mRNA.

Which of these approaches to gene regulation therapy will be applicable will depend on the etiology of each of the specific diseases described herein, all of which involve mismanagement of iron. Ferritin gene expression may either ameliorate or exacerbate depending on the type of ferritin expressed, on the specific disease, or the stage of disease at which intervention is initiated. The approach of choice may be to increase ferritin-H (e.g., by giving an expression vector of the ferritin-H gene) or to decrease a specific ferritin type (e.g., by an antisense oligonucleotide). Either of these approaches will achieve the desired effect. It is the balance between expression of ferritin-H and other ferritins that results in the cellular change in phenotype. Increasing the amount of ferritin-H in relation to the concentrations of the other ferritin proteins achieves the desired genetic regulation that ameliorates the effects of genetic diseases that cause the mismanagement of iron.

Delivering ferritin-H, a ferritin-H gene or derivatives thereof to erythroid precursor or stem cells represses expression of the mutated adult .beta.-globin gene in sickle cell disease and concomitantly to stimulate .gamma.(fetal)-globin gene expression thereby effecting a phenotypic cure.

In neurodegenerative diseases and neuromuscular disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and Friedreich's ataxia, excess iron (or mismanagement of iron) is an etiologic or exacerbating agent. H-ferritin is selectively expressed in neurons and is also located in neuronal nuclei. H-ferritin declines in the brain with age and is low in particular brain regions in AD, PD, and HD. H-ferritin alone among the known ferritins possesses ferroxidase activity, and the presence of ferritin-H in the brain is protective against excess or free iron. Increasing H-ferritin in specifically localized neurons of this class of patients ameliorates the symptoms and progression of these diseases. Alternatively, the delivery of specific ferritin antisense oligonucleotides to glia and other associated CNS cell types in specific brain regions may be the preferred route of gene regulation therapy, as an approach to lowering levels of iron stores in such cells. Those skilled in the art will understand that the best method of increasing intracellular ferritin-H or a derivative thereof iron will depend on a variety of factors including, but not limited to, the type of tissue being targeted, the desired level of intracellular ferritin-H or derivative, the disease being treated and the current level of intracellular ferritin in the targeted cells.

In cancers, it is also clear that excess iron is involved as an etiologic or exacerbating agent. H-ferritin is known to be increased intracellularly in a number of cancers (e.g., breast cancer), whereas L-ferritin is increased in the serum of many cancer patients (e.g., neuroblastoma). The increase in H-ferritin expression seen in a number of cancers is the cell's attempt to protect itself against free/excess iron; and in such a case, very early delivery of H-ferritin, an H-ferritin gene, or stimuli to increase endogenous ferritin-H gene expression may be the best therapeutic choice. In skin cancer, where either UV or infrared light induce endogenous ferritin-H, the ferritin is protective against some routes of oxidative damage.

Excess iron in atherosclerotic plaques is an etiologic or exacerbating agent; and gene regulation therapy to increase ferritin-H in the cells of these plaques slows or halts the disease process.

An important aspect of the invention is the finding that ferritin-H represses the human adult .beta.-globin gene by specifically binding the DNA sequence SEQ ID NO: 1, CAGTGC, in the .beta.-globin promoter. Many other genes have this sequence in their promoters, including the human .epsilon.- and .gamma.-globin genes that are stimulated by ferritin. Thus, the context of the CAGTGC motif, including the surrounding DNA as well as the distance of the motif from the start site of transcription, affects whether ferritin represses or activates. Some of the genes that have this promoter motif are expressed in apoptosis, and others are genes that are involved in iron metabolism. Using cytokines to push cancer cells into apoptosis is a route to a cure, and stimulating ferritin-H expression by this means is a powerful therapy.

The CAGTGC motif that we have discovered shares homology with important, previously discovered elements including the ARE (antioxidant response element) that has the sequence RTGACnnnGC (where R is a pyrimidine and n is any of the four standard nucleotides) and a sequence RTGR that is preferentially subject to cleavage by Fe++ mediated Fenton reactions. There are numerous other genes involved in health and disease that can be regulated through ferritin binding to these elements.

The above considerations have meaning as treatments because of the important discovery that nuclear ferritin (i.e., ferritin-H and derivatives thereof) represses gene expression from the human .beta.-globin gene promoter by binding to the CAGTGC motif located in the region of -150 base pairs from the transcription start site, as shown by our in vitro DNA binding experiments and by our gene co-transfection experiments in CV-1 cells.

DETAILED DESCRIPTION OF THE INVENTION

It has been found that ferritin is a repressor of the human .beta.-globin gene, the same gene that is mutated in sickle cell disease and in some forms of .beta.-thalassemia. The repressor is a nuclear form of ferritin (Broyles et al., "A Ferritin-Like Protein Binds to a Highly Conserved CAGTGC Sequence in the .beta.-globin promoter, In Sickle Cell Disease and Thalassaemias: New Trends in Therapy, of the ferritin H subfamily of ferritin peptides. Briefly, the inventors have found the following:

1) A ferritin-family protein from human K562 erythroleukemia cell nuclear extracts (as well as pure human ferritin-H) binds to the promoter of the human .beta.-globin gene (the promoter that drives the mutated form of the gene in sickle cell) at -150 bp from the transcription start site, in vitro. The binding is very specific to that DNA sequence.

2). An expression clone of ferritin-H represses this .beta.-globin promoter in transient co-transfection experiments. This is very reproducible in multiple experiments with two different reporter genes, with no repression seen by control/null plasmids.

3) Ferritin-H no longer represses if the promoter contains a mutated binding site. The inventors have a perfect control plasmid--a .beta.-globin promoter mutated only in the ferritin-H binding site and hooked to the same reporter gene (CAT, in this case). This is not only the perfect control for the transfections, but it also connects the in vitro DNA binding with in vivo function quite nicely.

Since a decrease in .beta.-globin expression is compensated by an increase in gamma (fetal)-globin expression in human erythroids cells, and since a modest amount of this switching is known to totally ameliorate sickle cell and wholly or partially ameliorate .beta.-thalassemias, this new finding makes ferritin useful for curing the phenotype of these classic genetic diseases.

Reports in the scientific literature indicate that H ferritin (heavy chain ferritin) is decreased by 50% in aged rat brains and in other neurodegenerative diseases such as Alzheimer's and show that ferritin-H is found in the neurodegenerative diseases where iron-mediated oxidative damage has been demonstrated, as in Parkinson's disease and possibly Huntington's disease. There are also studies that indicate a protective role of ferritin against cancers, such as liver and skin cancers. It has been reported that UV light induces ferritin production in skin cells and that ferritin is protective against UV damage. Indeed, ferritin H can be used to treat any diseases in which cellular injury is caused by iron-mediated oxidative damage.

Delivering the ferritin-H peptide or a truncated form of it to erythroid precursor cells is a more effective, more natural form of therapy than the partial measures currently in use to treat sickle cell disease and .beta.-thalassemias. Similar delivery of ferritin-derived peptides provides effective treatments and protection in Alzheimer's and other neurodegenerative diseases and cancers. The peptide can also be delivered as a fusion protein, with parts or all of the ferritin-H peptide fused to another protein such as transferrin or other ligand for which specific receptors exist on the surface of erythroid precursor cells, neurons, or other cell types for which protection is desired. The making of fusion proteins targeted to specific tissues is well know to those skilled in the art. Alternatively, an expression clone that encodes ferritin-H or a part of it, delivered to erythroid precursor cells, to hematopoietic stem cells, to neurons or to other tissue cells in an appropriate vector, either ex vivo or in vivo; and the protein expressed from such a vector also cures and protects against disease.

The ferritin-H described here is distinct from other known trans-acting proteins in its physical properties and its proposed function as a repressor that binds primarily to the .beta.-globin promoter. The H -ferritin subfamily is represented by a larger number of genes than the L-ferritin subfamily and includes a cluster of genes/pseudogenes on the X chromosome. One of these, ferritin-X, appears to encode a peptide identical in size and very similar in predicted three-dimensional structure to ferritin-H.

The possibility remains that the actual DNA-binding of the b-globin promoter -150 CAGTGC motif is mediated in vivo by a ferritin-associated protein that would be protected from proteinase K and heat treatments and react with anti-ferritin antisera because of its strong association with ferritin. However, if this is the case, it is a protein that is ubiquitous in human nuclear extract and there would be no need to upregulate it and it is ineffective in the absence of ferritin-H. Upregulation of ferritin-H is enough.

From the inventors' transient expression assays, it is clear that ferritin-H can repress the human b-globin gene and that this repression is mediated by binding of ferritin-H and/or a co-repressor to the -150 region of the promoter containing a highly conserved CAGTGC motif (FIGS. 1 and 6 (see Original Patent)). The binding site of this ferritin-H is within an important ARE required for activation of transcription of the .beta.-globin gene. Thus, the binding of this protein and displacement of other factors could be important in the repression of the human .beta.-globin gene as apparently the mouse BB1 protein (which recognizes the same sequence) is in the repression of the mouse .beta.-major globin gene in uninduced MEL cells. Subsequent interaction of this binding site with upstream negative regulatory regions creates a tightly-bound complex that prevents binding of other positive factors such as GATA-1 as well as sterically hinder the formation of an active transcription complex on the proximal promoter, by DNA looping.

FIG. 7 (see Original Patent) shows a schematic representation of the Ferritin-H protein having a bound iron ion. The active site responsible for ferroxidase activity has been elucidated. However, the active site or sites the protein responsible for transcription repression has not been identified. As with all other gene regulation proteins, derivatives of Ferritin-H will repress DNA transcription as well as or better than Ferritin-H itself. These derivatives include fragments of Ferritin proteins and any fusion proteins into which the active site or sites of Ferritin H responsible for transcription repression has been spliced. Ferritin H derivatives may also include larger transcription or translation products of a Ferritin family protein. Ferritin-H derivatives further include any mimetic proteins that represses DNA transcription by means of an active site that is substantially the same as the Ferritin-H active site responsible for DNA binding and transcription repression. Those skilled in the art will appreciate that the ferritin active site or sites responsible for repression of DNA transcription may include both DNA binding and protein binding sites. Ferritin-H derivatives may be found in fragments of any of the ferritin family proteins.

Ferritin H is only one member of the family of Ferritin proteins. Ferritin H and Ferritin L are the most studied. There are likely to be Ferritin family proteins that have not yet been identified. Ferritin family proteins are generally involved in iron metabolism. Now that the inventors have elucidated the gene regulatory activity of Ferritin H and its derivatives, it is likely that other Ferritin family proteins will also have gene regulatory functions.

The ability of Ferritin family proteins to bind to the 5' promoter region of the beta globin gene was ascertained only after lengthy and rigorous experimentation as described below. The first example shows that Ferritin-H binds to the CAGTGC ferritin binding site, SEQ ID NO: 1, found at bases -148 to -153 of the 5' promoter region of the human beta-globin gene. Example 2 (see Original Patent) shows that in addition to binding to the ferritin binding site, ferritin-H binds to another nuclear protein that binds to the beta-globin 5' promoter region further upstream of the ferritin binding site. FIGS. 8 through 12 (see Original Patent) show the experiments directed toward elucidating the mechanism by which ferritin-H represses the human beta[Greek symbol]-gene. These results represent work-in-progress and show that human K562 cell nuclear ferritin interacts with other DNA-binding proteins to repress this promoter, especially upstream silencer-binding proteins via DNA-looping."
 

Claim 1 of 5 Claims

1. A method for repressing production of beta-globin proteins and increasing production of gamma-globin proteins in a human cell, the method comprising: providing at least one human beta-globin producing cell; providing a vector encoding ferritin-H; and inserting, in vitro, the vector encoding ferritin-H into the at least one beta-globin producing cell, whereby ferritin-H is produced in the cell, and the ferritin-H produced represses production of beta-globin proteins in the cell, and activates production of gamma-globin proteins in the cell.

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