Method for expression of specific gene
View Patent ↗Disclosed is a cell which can express a non-natural oligomeric protein, which has, introduced therein, a gene encoding an exogenous polypeptide corresponding to at least one endogenous polypeptide constituting a natural oligomeric protein, and in which the expression of the endogenous polypeptide is inhibited.
1. A cell expressing a non-natural T cell receptor (TCR), comprising:
(i) recombinant nucleic acid sequences which encode exogenous α and β polypeptide chains that constitute the non-natural TCR and that correspond to the cell's endogenous α and β polypeptide chains that constitute the cell's natural TCR having a variable region and a constant region; and
(ii) two siRNAs, one which inhibits the expression of the endogenous α polypeptide chain of the natural TCR and the other which inhibits the expression of the endogenous β polypeptide chain of the natural TCR by RNA interference,
wherein:
the expression of the endogenous α and β polypeptide chains of the natural TCR are inhibited by the siRNAs,
the expression of the exogenous α and β polypeptide chains of the non-natural TCR, which partially differ in amino acid sequence from the endogenous α and β polypeptide chains of the natural TCR, are not inhibited by the siRNAs; and
each of the nucleotide sequences encoding the exogenous α and β polypeptide chains is modified without changing the encoded amino acid sequence in a region of the polypeptide chain on which one of the siRNAs acts so that the region of the polypeptide chain encodes the same amino acid sequence in the exogenous polypeptide chain as in the corresponding endogenous polypeptide chain, and
wherein the expression of each of the endogenous α and β polypeptide chains constituting the natural TCR is inhibited by RNA interference where the siRNAs target a sequence on mRNAs that encodes the constant region of each of the endogenous α and β polypeptide chains.
2. A process for producing a cell expressing a non-natural TCR, which comprises carrying out the following steps:
(a) introducing into a cell capable of expressing the natural TCR (i) recombinant nucleic acid sequences which encode exogenous α and β polypeptide chains that constitute the non-natural TCR and that correspond to the cell's endogenous α and β polypeptide chains that constitute the cell's natural TCR having a variable region and a constant region and (ii) two siRNAs, one of which is capable of inhibiting the expression of the endogenous α polypeptide chain of the natural TCR and the other of which is capable of inhibiting the expression of the endogenous β polypeptide chain of the natural TCR by RNA interference; and
(b) inhibiting the expression of the endogenous α and β polypeptide chains of the natural TCR by RNA interference with the siRNAs,
wherein the expression of the exogenous α and β polypeptide chains of the non-natural TCR, which partially differ in amino acid sequence from the endogenous α and β polypeptide chains of the natural TCR, are not inhibited by the siRNAs, and each of the nucleotide sequences encoding the exogenous α and β polypeptide chains is modified without changing the encoded amino acid sequence in a region of the polypeptide chain on which one of the siRNAs acts so that the region of the polypeptide chain encodes the same amino acid sequence in the exogenous polypeptide chain as in the corresponding endogenous polypeptide chain,
wherein the expression of each of the endogenous α and β polypeptide chains constituting the natural TCR is inhibited by RNA interference where the siRNAs target a sequence on mRNAs that encodes the constant region of each of the endogenous α and β polypeptide chains.
3. A method for forming a non-natural TCR, which comprises carrying out the following steps:
(a) introducing into a cell capable of expressing the natural TCR (i) recombinant nucleic acid sequences which encode exogenous α and β polypeptide chains that constitute the non-natural TCR and that correspond to the cell's endogenous α and β polypeptide chains that constitute the cell's natural TCR having a variable region and a constant region and (ii) two siRNAs, one of which is capable of inhibiting the expression of the endogenous α polypeptide chain of the natural TCR and the other of which is capable of inhibiting the expression of the endogenous β polypeptide chain of the natural TCR by RNA interference; and
(b) inhibiting the expression of the endogenous α and β polypeptide chains of the natural TCR by RNA interference with the siRNAs,
wherein the expression of the exogenous α and β polypeptide chains of the non-natural TCR, which partially differ in amino acid sequence from the endogenous α and β polypeptide chains of the natural TCR, are not inhibited by the siRNAs, and each of the nucleotide sequences encoding the exogenous α and β polypeptide chains is modified without changing the encoded amino acid sequence in a region of the polypeptide chain on which one of the siRNAs acts so that the region of the polypeptide chain encodes the same amino acid sequence in the exogenous polypeptide chain as in the corresponding endogenous polypeptide chain,
wherein the expression of each of the endogenous α and β polypeptide chains constituting the natural TCR is inhibited by RNA interference where the siRNAs target a sequence on mRNAs that encodes the constant region of each of the endogenous α and β polypeptide chains.
4. The cell according to claim 1 , wherein each of the siRNAs is encoded by a nucleic acid construct in the cell and transcribed from a promoter on the nucleic acid construct.
5. The cell according to claim 4 , wherein the nucleic acid construct and the recombinant nucleic acid sequences encoding the exogenous α and β polypeptide chains of the non-natural TCR are introduced into the cell with a single vector.