Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-09-01
2003-09-02
Wilson, Michael (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S021000, C800S022000, C800S025000, C800S004000, C800S005000
Reexamination Certificate
active
06613957
ABSTRACT:
1. INTRODUCTION
The present invention relates to the synthesis of functional human hemoglobin and other proteins in erythroid tissues of transgenic non-human animals or erythroid cell lines. In addition, the present invention provides for novel expression vectors which may be used to produce &agr;-globin as well as other proteins of interest in quantity in the red blood cells of transgenic animals or erythroid cell lines; these proteins may subsequently be purified and utilized for a multitude of purposes. The human hemoglobin produced in transgenic animals according to the invention can be used as an effective, nonimmunogenic red blood cell substitute for transfusion in humans which is free of hepatitis virus and human retrovirus contamination.
2. BACKGROUND OF THE INVENTION
2.1. Globin Genes and Hemoglobin
Native hemoglobin exists as a tetrameric protein consisting of two &agr; chains and two &bgr; chains. Each &agr; and &bgr; chain binds a heme residue in noncovalent linkage. The &agr; and &bgr; chains are also held together by noncovalent bonds resulting from hydrogen bonding and Van der Waals forces. Hemoglobin constitutes about 90% of the total protein in red blood cells, 100 ml of whole blood is capable of absorbing approximately 21 ml of gaseous oxygen.
2.1.1. &agr; and &bgr; Globin Genes
Different molecular species of hemoglobin are produced during the embryonic, fetal, and adult life of an animal. The genes encoding the globin molecules expressed during the various developmental stages are arranged in clusters. In humans and most other mammals the &agr; and &bgr;-like gene clusters are arranged in order of their expression during development, with the embryonic genes followed by the fetal and adult globin genes (Watson et al., 1987, in “Molecular Biology of the Gene”, Fourth Edition, The Benjamin/Cummings Publishing Co., Inc., Menlo Park, Calif., p. 650). This developmental order is not obligatory, however; for example, in the chicken, the adult &bgr;-globin genes are flanked by embryonic genes.
In humans, the &agr; globin gene cluster is located on chromosome 16 and the &bgr; globin gene cluster is located on chromosome 11. The human &bgr; globin gene cluster comprises one embryonic (&egr;), two fetal (
G
&ggr; and
A
&ggr;) and two adult &dgr; and &bgr;) globin genes, which reside within approximately 50 kb of chromosomal DNA in the order 5′-&egr;-
G
&ggr;-
A
&ggr;-&dgr;-&bgr;-3′ (Fritsch et al., 1980, Cell 19:959-972).
Expression of the human &bgr;-like globin genes is precisely regulated in three important ways; they are expressed only in erythroid tissue, only during defined stages of development, and are produced at very high levels so as to rapidly establish the developmentally appropriate hemoglobin as the dominant protein in the red blood cell. The process by which the red blood cell ceases to transcribe one particular globin gene and begins to express another is referred to as “hemoglobin switching”. A great deal of study has been directed toward the regulatory mechanisms responsible for the switching process.
Research has indicated that DNA sequences involved in the regulation of human &bgr;-globin gene expression are located both 5′ and 3′ to the translation initiation site (Wright et al., 1984, Cell 38:251-263). Analysis of constructs with &bgr;-globin gene fragments inserted upstream of a reporter gene have demonstrated that sequences located immediately upstream, within, and downstream of the gene contribute to the correct temporal and tissue specific expression (Behringer et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:7056-7060; Kollias et al., 1987, Nucl. Acids Res. 15:5739-5747). Using murine erythroleukemia (MEL) and K562 cells, at least four separate regulatory elements required for appropriate expression of the human &bgr;-globin gene have been identified: (i) a globin specific promoter element; (ii) a putative negative regulatory element, and (iii and iv) two downstream regulatory sequences with enhancer-like activity, one of which is located in the second intron of the &bgr;-globin gene and the other located approximately 800 basepairs (bp) downstream of the gene (Behringer et al., 1987, Proc. Nat. Acad. Sci. U.S.A. 84:7056-7060). Hesse et al. (1986, Proc. Natl. Acad. Sci. U.S.A. 83:4312-4316) identified a similar enhancer sequence downstream of the chicken &bgr;-globin gene in cultured chicken erythroid cells (see also Choi and Engel, 1986, Nature 323:731-734).
2.1.2. DNase I Hypersensitivity Sites
Active chromatin domains have been associated with overall sensitivity to DNase I digestion relative to unexpressed genes or DNA outside the active chromatin domain. Hypersensitivity sites are superimposed on the increased sensitivity of active chromatin; these DNase I hypersensitivity (HS) sites comprise approximately 100 to 200 bp of DNA which are highly susceptible to cleavage by the nuclease action of DNase I. DNase I hypersensitive sites are mapped by (i) treating nucleic acid with DNase I; (ii) isolated DNA from the nuclei; (iii) digesting the isolated DNA with a restriction enzyme; (iv) fractionating the restriction enzyme-cut DNA (i.e. by gel electrophoresis); (v) blotting the fractionated DNA on nitrocellulose; and (vi) hybridizing the nitrocellulose with a labeled probe corresponding to a subfragment of nucleic acid sequence located near the gene of interest. In addition to the full length fragment generated by the restriction enzyme, a multitude of shorter bands generated by DNase I will appear if the probe represents an area of the DNA contained in a DNase I hypersensitive site (Watson et al., 1987, in “Molecular Biology of the Gene,” Fourth Edition, The Benjamin/Cummings Publishing Co., Menlo Park, Calif., pp. 692-693).
Several years ago, Tuan et al. (1985, Proc. Natl. Acad. Sci. U.S.A. 82:6384-6388) and Forrester et al. (1986, Proc. Natl. Acad. Sci. U.S.A. 83:1359-1363) mapped sites that were super-hypersensitive to DNase I digestion 6-22 kilobases (kb) upstream of the &egr;-globin gene and 19 kb downstream of the &bgr;-globin gene. The sites were found specifically in erythroid tissue at all stages of development.
FIG. 5
depicts the location of these sites in the human &bgr;-globin locus. Tuan et al. (1985, Proc. Natl. Acad. Sci. U.S.A. 82:6384-6388) observed that the major DNase I hypersensitive sites, HS I, HS II, and HS IV, situated upstream of the &bgr;-globin gene, appeared to be strongly associated with &bgr;-like globin gene expression since they were found to be present in K562 cells, human erythroleukemia cells, and adult human nucleated bone marrow cells (which express &bgr;-like globin genes) but to be absent in HL60 cells, which do not express the &bgr;-like globin genes. These experiments suggest that the super-hypersensitive sites define locus activation regions which open a large chromosomal domain for expression specifically in erythroid cells and thereby dramatically enhance globin gene expression. Furthermore, the structure of mutant loci from patients with several hemoglobinopathies suggests that the upstream hypersensitivity sites are required for efficient &bgr;-globin gene expression in humans. English and Dutch &ggr;&dgr;&bgr;-thalassemia appears to result from deletions that remove all of the upstream hypersensitivity sites (FIG.
5
); although the &bgr;-globin gene is intact in these patients, no &bgr;-globin mRNA is produced from the mutant alleles.
2.2. Transgenic Animals
The term “transgenic animals” refers to non-human animals which have incorporated a foreign gene into their genome; because this gene is present in germline tissues, it is passed from parent to offspring. Exogenous genes are introduced into single-celled embryos (Brinster et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:4438-4442). Transgenic mice have been shown to express globin (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6376-6380), transferrin (McKnight et al., 1983, Cell 34:335-341), immunoglobulin (Brinster et al., 1983, Nature 306:332-336; Ritchie, et al., 1984, Nature 312:517-520; Goodhardt et al.
Behringer Richard R.
Brinster Ralph L.
Palmiter Richard D.
Ryan Thomas M.
Townes Tim M.
Needle & Rosenberg P.C.
Paras, Jr. Peter
The UAB Research Foundation
Wilson Michael
LandOfFree
Synthesis of functional human hemoglobin and other proteins... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Synthesis of functional human hemoglobin and other proteins..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Synthesis of functional human hemoglobin and other proteins... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3110135