Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-12-23
2001-07-31
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S069100, C435S325000, C435S355000, C435S357000, C435S372000, C435S366000, C435S348000, C435S252300, C435S183000, C435S189000, C536S023100, C536S023200, C536S023500, C536S024300, C536S024310
Reexamination Certificate
active
06268138
ABSTRACT:
Throughout this application various publications are referenced by the names of the authors and the year of the publication within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF INVENTION
Recent advances in autologous bone marrow transplant strategies indicate that normal hematopoiesis can be promptly restored in patients treated with myelotoxic agents (drugs or radiation) by re-injection of autologous peripheral blood (CD34
+
) “stem cells” (Gianni, et al., Lancet 2:580, 1989). In addition, it has very recently been reported that CD34
+
cells can be transduced in vitro at high efficiency with retroviral vectors expressing specific genes (Bregni, et al., Blood 80:1418, 1992). These technology open the way to approaches in which the in vitro transduction of specific genes into autologous CD34
+
cells followed by reinoculation into patients can be used to transduce genes of therapeutic significance. This gene therapy approach includes the reconstitution of drug-resistant hematopoietic cells allowing for subsequent treatment with higher dose myelotoxic chemotherapy in cancer patients.
SUMMARY OF INVENTION
This invention provides a vector which comprises a nucleic acid molecule encoding a human cytosolic aldehyde dehydrogenase.
In addition, this invention provides a vector which comprises a nucleic acid molecule encoding a human glutamylcysteine synthetase.
In addition, this invention provides a retroviral vector, which comprises a nucleic acid molecule encoding a human cytosolic aldehyde dehydrogenase.
In addition, this invention provides a retroviral vector, which comprises a nucleic acid molecule encoding a human glutamylcysteine synthetase.
In addition, this invention provides a vector, which comprises a nucleic acid molecule encoding a human cytosolic aldehyde dehydrogenase and a glutamylcysteine synthetase.
In addition, this invention provides a method for reducing the toxic effects of a cyclophosphamide in a subject which comprises replacing the subject's hematopoietic cells with hematopoietic cells of having the retroviral vector which comprises a nucleic acid molecule encoding a human cytosolic aldehyde dehydrogenase so as to reduce the toxic effects of the cyclophosphamide in the subject.
In addition, this invention provides a method for introducing a selectable marker into a mammalian cell which comprises transfecting the cell with a nucleic acid molecule encoding a human cytosolic aldehyde dehydrogenase.
In addition, this invention provides a method for selecting mammalian cells expressing protein of interest which comprises: a). introducing into the cells a nucleic acid molecule comprising a nucleic acid molecule encoding the protein of interest and the nucleic acid molecule encoding human cytosolic aldehyde dehydrogenase; b.) culturing the resulting transfected cells; and c.) selecting cells which express human cytosolic aldehyde dehydrogenase, so as to obtain cells which express the protein of interest.
In addition, this invention provides a method for reducing the toxic effects of a cyclophosphamide in a subject which comprises replacing the subject's hematopoietic cells with hematopoietic cells of having the retroviral vector which comprises a nucleic acid molecule encoding a human glutamylcysteine synthetase so as to reduce the toxic effects of the cyclophosphamide in the subject.
In addition, this invention provides a method for introducing a selectable marker into a mammalian cell which comprises transfecting the cell with a nucleic acid molecule encoding human glutamylcysteine synthetase.
In addition, this invention provides a method for selecting mammalian cells expressing protein of interest which comprises: a). introducing into the cells a nucleic acid molecule comprising a nucleic acid molecule encoding the protein of interest and the nucleic acid molecule encoding human glutamylcysteine synthetase; b.) culturing the resulting transfected cells; and c.) selecting cells which express human glutamylcysteine synthetase, so as to obtain cells which express the protein of interest.
In addition, this invention provides an isolated mammalian nucleic acid molecule encoding a cytosolic aldehyde dehydrogenase. The isolated mammalian nucleic acid molecule may have substantially the same sequence shown in
FIG. 4
(SEQ ID NO: 1).
In addition, this invention provides an isolated mammalian nucleic acid molecule encoding a glutamylcysteine synthetase. The isolated mammalian nucleic acid molecule may have substantially the same sequence shown in
FIG. 6
(SEQ ID NO: 3).
In addition, this invention provides a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence of the nucleic acid molecule of the isolated mammalian nucleic acid molecule encoding an cytosolic aldehyde dehydrogenase or a glutamylcysteine synthetase.
In addition, this invention provides a method of detecting expression of an aldehyde dehydrogenase in a cell which comprises obtaining total mRNA from the cell, contacting the mRNA so obtained with a labelled nucleic acid molecule of the isolated mammalian nucleic acid molecule encoding an cytosolic aldehyde dehydrogenase under hybridizing conditions, determining the presence of mRNA hybridized to the molecule, and thereby detecting the expression of the cytosolic aldehyde dehydrogenase in the cell.
In addition, this invention provides a method of producing a polypeptide having the biological activity of a mammalian cytosolic aldehyde dehydrogenase which comprises growing the host cells of the host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.
In addition, this invention provides a method of detecting expression of a glutamylcysteine synthetase in a cell which comprises obtaining total mRNA from the cell, contacting the mRNA so obtained with a labelled nucleic acid molecule of the isolated mammalian nucleic acid molecule encoding an cytosolic aldehyde dehydrogenase under hybridizing conditions, determining the presence of mRNA hybridized to the molecule, and thereby detecting the expression of the cytosolic aldehyde dehydrogenase in the cell.
In addition, this invention provides a method of producing a polypeptide having the biological activity of a mammalian glutamylcysteine synthetase which comprises growing the host cells of the host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.
In addition, this invention provides an antibody directed against an amino acid molecule a cytosolic aldehyde dehydrogenase.
In addition, this invention provides an antibody directed against an amino acid molecule a glutamylcysteine synthetase.
In addition, this invention provides an immunoassay for measuring the amount of a mammalian cytosolic aldehyde dehydrogenase in a biological sample comprising steps of: a) contacting the biological sample with at least one antibody, either monoclonal or ployclonal, to form a complex with said antibody and the cytosolic aldehyde dehydrogenase, and b) measuring the amount of the cytosolic aldehyde dehydrogenase in said biological sample by measuring the amount of said complex.
In addition, this invention provides a transgenic nonhuman mammal which comprises the isolated mammalian nucleic acid molecule encoding an cytosolic aldehyde dehydrogenase or a glutamylcysteine synthetase.
In addition, this invention provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a cytosolic aldehyde dehydrogenase so placed as to be transcribed into antisense mRNA complementary to mRNA encoding the cytosolic aldehyde dehydrogenase and which hybridizes to mRNA encoding the mammalian cytosolic a
Dalla-Favera Riccardo
Gianni Alessandro Massimo
Cooper & Dunham LLP
Guzo David
The Trustees of Columbia University in the City of New York
White John P.
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