Double mutants of dihydrofolate reductase and methods of using same
AssigneeSloan-Kettering Institute for Cancer Research
InventorsJoseph R. Bertino, Emine A. Ercikan-Abali, Debabrata Banerjee, Shin Mineishi, Michel Sadelain
Life of this patent
Estimating the term…
Abstract
New mutant forms of human dihydrofolate reductase (DHFR) which have properties superior to the previously disclosed mutants have mutations at both amino acid 22 and amino acid 31. Specific mutant forms are Ser31Tyr22, Ser31Phe22, Gly31Tyr22, Gly31Phe22, Ala31Tyr22 and Ala31Phe22. The mutant DHFR of the invention maybe used as a selectable marker, and 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.
Claims12
A mutant form of human dihydrofolate reductase having activity as a dihydrofolate reductase, said mutant form differing from wild-type human DHFR as defined by Seq. ID No. 7 as a result of a set of mutations comprising a first mutation wherein an uncharged amino acid with a larger volume side chain than leucine is introduced at the amino acid corresponding to amino acid 22 in the wild-type sequence and a second mutation wherein an amino acid which has a smaller volume, more hydrophilic side chain than phenylalanine is introduced at the amino acid corresponding to amino acid 31 in the wild type sequence.
cDNA encoding a mutant form of human dihydrofolate reductase having activity as a dihydrofolate reductase, said mutant form differing from wild-type human DHFR as defined by Seq. ID No. 7 as a result of a set of mutations comprising a first mutation wherein an uncharged amino acid with a larger volume side chain than leucine is introduced at the amino acid corresponding to amino acid 22 in the wild-type sequence and a second mutation wherein an amino acid which has a smaller volume, more hydrophilic side chain than phenylalanine is introduced at the amino acid corresponding to amino acid 31 in the wild type sequence.
A DNA vector comprising DNA encoding a mutant form of human dihydrofolate reductase having activity as a dihydrofolate reductase, said mutant form differing from wild-type human DHIFR as defined by Seq. ID No. 7 as a result of a set of mutations comprising a first mutation wherein an uncharged amino acid with a larger volume side chain than leucine is introduced at the amino acid corresponding to amino acid 22 in the wild-type sequence and a second mutation wherein an amino acid which has a smaller volume, more hydrophilic side chain than phenylalanine is introduced at the amino acid corresponding to amino acid 31 in the wild type sequence.
A mammalian cell which produces a mutant form of human dihydrofolate reductase having activity as a dihydrofolate reductase, said mutant form differing from wild-type human DHFR as defined by Seq. ID No. 7 as a result of a set of mutations comprising a first mutation wherein an uncharged amino acid with a larger volume side chain than leucine is introduced at the amino acid corresponding to amino acid 22 in the wild-type sequence and a second mutation wherein an amino acid which has a smaller volume, more hydrophilic side chain than phenylalanine is introduced at the amino acid corresponding to amino acid 31 in the wild type sequence.
Dependencies are inferred from the claim text.