Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-09-18
2002-11-05
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06475740
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to gene therapy methods for treating hemoglobinopathies.
The major form of adult human hemoglobin, HbA, consists of a tetramer of two &agr;-globin chains and two &bgr;-globin chains ((&agr;
2
&bgr;
2
). Hemoglobinopathies, such as sickle cell anemia and &bgr;
O
-thalassemia, are caused by a failure to produce normal levels of &bgr;-globin. Hemoglobin A
2
(HbA
2
), which consists of a tetramer of two &agr;-globin chains and two &dgr;-globin chains (&agr;
2
&dgr;
2
), is produced in low amounts in most sickle cell patients and in normal adults (2-3% of total hemoglobin) (Steinberg et al., Blood 78:2165, 1991). HbA
2
is a potent inhibitor of the sickle hemoglobin (HbS; &agr;
2
&bgr;
S
2
) polymerization characteristic of sickle cell anemia (Nagel et al., Proc. Natl. Acad. Sci. USA 76:670, 1979), and shares some functional activity with HbA.
SUMMARY OF THE INVENTION
We have shown that modification of the &dgr;-globin gene promoter to include a binding site for the erythroid krüppel-like factor (EKLF) polypeptide that binds to the &bgr;-globin gene promoter (hereinafter “&bgr;-EKLF”) results in increased expression from the &dgr;-globin promoter. A modified &bgr;-EKLF (hereinafter “&dgr;-EKLF”) that binds to the wild type &dgr;-globin gene promoter can thus be used to induce &dgr;-globin expression.
Accordingly, in one aspect, the invention features a method of inducing &dgr;-globin gene expression in a cell, such as an erythrocyte precursor cell (e.g., an erythrocyte burst-forming cell (BFC-E) or an erythrocyte colony-forming cell (CFC-E)) or an erythrocyte. In this method, a nucleic acid encoding a &dgr;-EKLF polypeptide is introduced into the cell, or a precursor of the cell. Cells into which the nucleic acid can be introduced include erythrocyte precursors, such as BFC-E and CFC-E. Preferably, the nucleic acid is introduced into a pluripotent hematopoietic stem cell, which is capable of self renewal, and thus, in the context of gene therapy methods (see below), minimizes the number of treatments required.
The cell into which the nucleic acid is introduced can be in a mammal or can be a cell that has been removed from a mammal for introduction of the nucleic acid, after which the cell, or progeny thereof, are introduced into a mammal. Typically, this method is carried out for a human patient. In one particular example, the nucleic acid can be introduced into a hematopoietic stem cell that has been obtained from a patient. Induction of &dgr;-globin gene expression by this method can be used in gene therapy methods for the treatment of hemoglobinopathies, such as sickle cell anemia and &bgr;
0
-thalassemia.
The invention also features &dgr;-EKLF polypeptides, which are identical to &bgr;-EKLF, except that they contain one or more modifications that permit them to bind to double stranded DNA containing the sequence 5′-TGA AAC CCT-3′ or the sequence 5′-CTA ATG AAA-3′. The modifications that generate &dgr;-EKLF polypeptides are generally in a DNA-binding amino acid of a zinc finger of the &bgr;-EKLF polypeptide. For example, modifications can be made in any of amino acids -1 and/or 2-6 in any of the three &bgr;-EKLF zinc fingers (see below and FIG.
2
).
Preferably, the &dgr;-EKLF polypeptide of the invention is in a substantially pure preparation. By “substantially pure” is meant a preparation that is at least 60% by weight (dry weight) a &dgr;-EKLF polypeptide. Preferably the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight a &dgr;-EKLF polypeptide. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Also featured in the invention is a nucleic acid, such as a nucleic acid containing deoxyribonucleotides (DNA), ribonucleotides (RNA), or combinations or modifications thereof, encoding &dgr;-EKLF polypeptides. Preferably, the nucleic acid is in the form of purified DNA. By “purified DNA” is meant DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally occurring genome of the organism from which it is derived. The term thus includes, for example, a recombinant DNA molecule that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA molecule that is part of a hybrid gene encoding additional polypeptide sequence.
The invention also includes cells, such as hematopoietic stem cells or erythrocyte precursor cells, e.g., BFC-E or CFC-E, that contain nucleic acids encoding &dgr;-EKLF polypeptides.
Vectors containing nucleic acids encoding &dgr;-EKLF polypeptides are also included in the invention. The vectors of the invention include those that can be used in gene therapy methods for treating hemoglobinopathies, such as sickle cell anemia and &bgr;
0
-thalassemia. For example, adeno-associated viral (AAV) vectors and retroviral vectors (e.g., moloney murine leukemia viral vectors) can be used in the invention. Vectors that can be used for amplifying nucleic acids encoding &dgr;-EKLF polypeptides in bacteria are also included in the invention. Preferably, the nucleic acids encoding &dgr;-EKLF polypeptides are operably linked to a promoter, for example, the &bgr;-globin promoter, or a non-tissue specific promoter, such as the Cytomegalovirus promoter. By “operably linked” is meant that a gene and a regulatory sequence(s), such as a promoter, are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins or proteins which include transcriptional activation domains) are bound to the regulatory sequence(s).
The invention also features methods for identifying &dgr;-EKLF polypeptides. In these methods, a nucleic acid containing either the sequence 5′-TGA AAC CCT-3′ or the sequence 5′-CTA ATG AAA-3′ is contacted with a candidate polypeptide that has been modified such that it differs from wild type &bgr;-EKLF by at least one amino acid, for example, an amino acid in a &bgr;-EKLF zinc finger that binds DNA. &dgr;-EKLF polypeptides are then identified by their ability to bind to this nucleotide sequence.
Expression of therapeutic levels of gene products in gene therapy methods is often difficult to achieve. Transcription factors are generally required in lower quantities than other types of therapeutic gene products. Thus, an advantage of the present invention is that it provides a gene therapy method involving expression of a transcription factor, &dgr;-EKLF.
Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.
REFERENCES:
patent: 5789538 (1998-08-01), Rebar et al.
Ngo et al, The Protein Folding Problem and Tertiary Stucture Prediction, Merts et al (eds.), Birkhauser, Boston, pp. 433 and 492-495, 1994.*
Cowie et al., “DNA Sequences Involved in Transcriptional Regulation of the Mouse &bgr;-Globin Promotor in Murine Erythroleukemia Cells,” Mol. Cell. Biol. 8:3122 (1988).
Dierks et al., “Three Regions Upstream from the Cap Site Are Required for Efficient and Accurate Transcription of the Rabbit &bgr;-Globin Gene in Mouse 3T6 Cells,” Cell 32:695 (1983).
Myers et al., “Fine Structure Genetic Analysis of a &bgr;-Globin Promoter,” Science 232:613 (1986).
Nagel et al., “Structural Bases of the Inhibitory Effects of Hemoglobin F and Hemoglobin A2on the Polymerization of Hemoglobin S,” Proc. Natl. Acad. Sci USA 76:670 (1979).
Steinberg et al., “Hemoglobin A2: Origin, Evolution,and Aftermath,” Blood 78:2165 (1991).
Bieker, “Isolation, Genomic Structure, and Expression of Human Erythroid Kruppel-Like Factor (EKLF),” DNA and Cell B
Donze David
Townes Tim M.
McKelvey Terry
Needle & Rosenberg P.C.
The UAB Research Foundation
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