Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1999-01-20
2003-11-04
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S189000, C435S193000, C435S320100, C536S023200, C530S350000
Reexamination Certificate
active
06642043
ABSTRACT:
BACKGROUND OF THE INVENTION
This application relates to a new mutants of the enzyme dihydrofolate reductase, and to the use of these mutants as selectable markers and for gene therapy to produce drug resistant bone marrow or peripheral stem cells.
Dihydrofolate reductase (DHFR, 5,6,7,8-tetrahydrofolate:NADP+oxidoreductase, EC 1.5.1.3) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, an essential carrier of one-carbon units in the biosynthesis of thymidylate, purine nucleotides, serine and methyl compounds. DHFR is an essential enzyme in both eukaryotes and prokaryotes.
In rapidly dividing cells, the inhibition of DHFR results in the depletion of cellular tetrahydrofolates, inhibition of DNA synthesis and cell death. Because of this, folate analogs which inhibit DHFR, for example methotrexate (MTX), are used as antineoplastic agents. The utility of “antifolate” treatments of this type is limited by two factors. First, tumor tissues may rapidly develop resistance to the antifolate, rendering the treatment ineffective. Second, the treatment may be toxic to rapidly dividing normal tissues, particularly to bone marrow or peripheral stem cells.
International Patent Publication No. W094/24277, which is incorporated herein by reference, discloses mutant forms of human DHFR which have increased resistance to inhibition by antifolates used in therapy includi MTX. The specific mutants disclosed differ from wild-type human DHFR as a result of a single mutation occurring at amino acid 15, 31 or 34.
Mutations at amino acid 22 of human DHFR have also been shown to reduce the sensitivity of the enzyme to antifolate inhibition. Ercikan et al., in
Chemistry and Biology of Pteridines and Folates
, J. E. Ayling, ed., Plenum Press (1993). In these mutants, the amino acids isoleucine, methionine, phenylalanine and tyrosine are substituted for the leucine of the wild-type enzyme.
SUMMARY OF THE INVENTION
The present invention provides new mutant forms of human DHFR which have properties superior to the previously disclosed mutants. In particular, the present application is addressed to mutant forms of human DHFR which have mutations at both amino acid 22 and amino acid 31. Preferred mutant forms within the scope of the invention are Ser31Tyr22, Ser31Phe22, Gly31Tyr22, Gly31Phe22, Ala31Tyr22 and Ala31Phe22.
The mutant DHFR of the invention may be used as a selectable marker. Thus, an aspect of the present invention is a method of selecting among clones for the introduction of a non-selectable gene comprising the steps of
(a) inserting the non-selectable gene into a DNA vector comprising DNA encoding a mutant form of human dihydrofolate reductase which differs from wild-type human dihydrofolate reductase at both amino acid 22 and amino acid 31, wherein the mutant form has an amino acid with a larger volume side chain than leucine at amino acid 22 and an amino acid having a smaller volume, more hydrophilic side chain than phenylalanine at amino acid 31;
(b) introducing the vector containing the non-selectable gene into cells of a type in which the non-selectable gene and the mutant form of dihydrofolate reductase are expressed; and
(c) selecting cells which are resistant to inhibition by antifolates.
The mutant DHFR of the invention may also be used to modify the genome of human cells, particularly bone marrow cells or peripheral blood stem cells to render them resistant to chemotherapy using antifolate agents. Thus a further aspect of the invention is a method for gene therapy comprising the steps of:
(a) obtaining hematopoietic cells from a human patient;
(b) transducing into the hematopoietic cells an expressible mutant form of human dihydrofolate reductase which differs from wild-type human dihydrofolate reductase at both amino acid 22 and amino acid 31, wherein the mutant form has an amino acid with a larger volume side chain than leucine at amino acid 22 and an amino acid which having a smaller volume, more hydrophilic side chain than phenylalanine at amino acid 31; and
(c) returning the transduced cells to the human patient.
REFERENCES:
patent: WO 94/24277 (1994-10-01), None
patent: WO 97/33988 (1997-09-01), None
Rosowsky et al. J. Med. Chem. 38: 745-752, 1995.*
Schweitzer et al. JBC, 264(34) : 20786-95, 1989.*
Huang et al. Biochemistry , 33 : 11576-11585, 1994.*
Accessions XP 003991 and AAA58485.*
Dicker et al. Identification and Characterization of a Mutation in the Dihydrofolate Reductase Gene from the Methotrexate-resistant Chinese Hamster Ovary Cell Line Pro-3MtxRIII. J. Biol. Chem. May 15, 1990, vol. 265, No. 14, pp. 8317-8321, see entire document.
Schweitzer et al. Probing the Role of Two Hydrophobic Active Site Residues in the Human Dihydrofolate Reductase by Site-Directed Mutagenesis. J. Biol. Chem. Dec. 5, 1989, vol. 264, No. 34, pp. 20786-20795, see entire document.
Rosowsky et al. 2,4-diamino-5-substituted-quinazolines as Inhibitors of a Human Dihydrofolate Reductase with a Site-Directed Mutation at Position 22 and of the Dihydrofolate Reductases fromPneumocystis cariniiandToxoplasma gondii. J. Med. Chem. 1995, vol. 38, pp. 745-752, see entire document.
Banerjee et al. Transfection with a cDNA Encoding a Ser31or Ser34Mutant Human Dihydrofolate Reductase in Chinese Hamster Ovary and Mouse Marrow Progenitor Cells Confers Methotrexate Resistance. Gene. 1994, vol. 139, pp. 269-274, see entire document.
Huang et al. Nonadditivity of Mutational Effects at the Folate Binding Site ofEscherichia coliDihydrofolate Reductase. Biochemistry, 1994, vol. 33, No. 38, pp. 11576-11585, see entire document.
Schweitzer et al. Mutations in the Human Dihydrofolate Reductase. In: Chemistry and Biology of Pteridines, Pteridines and Folic Acid Derivatives, 1986, Proceedings of the Eighth International Symposium on Pteridines and Folic Acid Derivatives Chemical, Biological and Clinical Aspects, Montreal Canada. Jun. 15-20, 1986. Edited by: B.A. Cooper and V.M. Whitehead, pp. 793-797, see entire document.
Schweitzer et al. Mutations at Hydrophobic Residues in Dihydrofolate Reductase. In: Chemistry and Biology of Pteridines 1989, Pteridines and Folic Acid Derivatives, Proceedings of the Ninth International Symposium on Pteridines and Folic Acid Derivatives Chemical, Biological and Clinical Aspects, Zurich, Switzerland. Sep. 3-8, 1989. Edited by: H.-Ch. Curtis, S. Ghisla and N. Blau, pp. 760-764, see entire document.
Li et al. Development of a Retroviral Construct Containing a Human Mutated Dihydrofolate Reductase cDNA for Hematopoietic Stem Cell Transduction. Blood. Jun. 1, 1994, vol. 83, No. 11, pp. 3403-3408, see entire document.
Banerjee et al. Molecular Mechanisms of Resistance to Antifolates, A Review. Acta Biochimica Polonica, 1995, vol. 42, No. 4, pp. 457-464, see entire document.
Fan, J. et al. Demonstration of Rb-mediated drug sensitivity and growth inhibition by an inducible expression system. 1995 AACR Abstract Form.
Huang, Zheng et al. Nonadditivity of Mutational Effects at the Folate Binding Site ofEscherichia coliDihydrofolate Reductase. Biochemistry 1994, 33, 11576-11585. United States.
Li, Ming-Xia et al. Development of a Retroviral Construct Containing a Human Mutated Dihydrofolate Reductase cDNA for Hematopoietic Stem Cell Transduction. Blood, vol 83, No 11 (Jun. 1), 1994: pp 3403-3408. The American Society of Hematology.
Ercikan-Abali, Emine A. et al. Variants of Human Dihydrofolate Reductase with Substitutions at Leucine-22: Effect on Catalytic and Inhibitor Binding Properties. Molecular Pharmacology, 49:1 (1996). New York, New York.
Oefner, Christian et al. Crystal Structure of Human Dihydrofolate Reductase Complexed with Folate. Received Jan. 20, 1988/Mar. 21, 1988—EJB 880071. Central Research Units, F. Hoffman—La Roche & Co. Ltd, Basel.
Ercikan, E. et al. Effect of Codon 22 Mutations on Substrate and Inhibitor Binding for Human Dihydrofolate Reductase. Chemistry and Biology of Pteridines and Folates, Edited by J.E. Ayling et al., Plenum Press, New York, New York, 1993.
Banerjee Debabrata
Bertino Joseph R.
Ercikan-Abali Emine A.
Mineishi Shin
Sadelain Michel
Oppedahl & Larson LLP
Saidha Tekchand
Sloan-Kettering Institute for Cancer Research
LandOfFree
Double mutants of dihydrofolate reductase and methods of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Double mutants of dihydrofolate reductase and methods of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Double mutants of dihydrofolate reductase and methods of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3124016