Nucleic acid construct
The present invention provides a nucleic acid construct comprising: a first nucleotide sequence of interest (NOI1); a frame-slip motif or a translational readthrough motif (FSM/TRM); and a second nucleotide sequence of interest (NOI2). The invention also provides vectors and cells expressing such a construct. The invention also provides a method for modulating the relative expression of two transgenes in a nucleic acid construct which comprises the step of including a frame-slip motif or a translational readthrough motif between the two transgenes in order to reduce the expression of the downstream transgene.
1. A nucleic acid construct comprising:
a first nucleotide sequence of interest (NOI1), a translational readthrough motif (TRM), and a second nucleotide sequence of interest (NOI2), and
a nucleotide sequence encoding a cleavage site (CL), so that NOI1 and NOI2 are expressed as separate proteins,
wherein the (TRM) comprises the sequence STOP-CUAG or STOP-CAAUUA, in which “STOP” is a stop codon.
2. A nucleic acid construct according to claim 1 , wherein the translational readthrough motif comprises one or more of the follow sequences:
(SEQ ID No. 5)
UGA-CUAG
(SEQ ID No. 6)
UAG-CUAG
(SEQ ID No. 7)
UAA-CUAG
(SEQ ID No. 8)
UGA-CAAUUA
(SEQ ID No. 9)
UAG-CAAUUA
(SEQ ID No. 10)
UAA-CAAUUA.
3. A nucleic acid construct according to claim 1 which has the structure:
NOI1-TRM-CL-NOI2,
NOI1-TRM1-TRM2-CL-NOI2,
NOI1-CL1-TRM-CL2-NOI2,
NOI1-TRM-CL-SP-NOI2,
NOI1-TRM1-CL1-NOI2-TRM2-CL2-NOI3, or
NOI1-TRM1-CL1-TRM2-CL2-NOI2,
in which:
TRM1 and TRM2, which may be the same or different, are first and second translational readthrough motifs;
CL1 and CL2, which may be the same or different, are first and second nucleic acid sequences each encoding a cleavage site,
SP is an attenuated signal peptide, and
NOI3 is a third nucleotide sequence of interest.
4. A nucleic acid construct according to claim 1 wherein the cleavage site comprises a self-cleaving peptide, a furin cleavage site or a Tobacco Etch Virus cleavage site.
5. A nucleic acid construct according to claim 4 , wherein the cleavage site comprises a 2A self-cleaving peptide from an aphtho- or a cardiovirus or a 2A-like peptide.
6. A vector comprising a nucleic acid construct according to claim 1 .
7. A retroviral vector or a lentiviral vector according to claim 6 .
8. A cell comprising a nucleic acid construct according to claim 1 .
9. A method for making a cell according to claim 8 which comprises the step of introducing into a cell a nucleic acid construct comprising:
a first nucleotide sequence of interest (NOI1), a translational readthrough motif (TRM), and a second nucleotide sequence of interest (NOI2), and
a nucleotide sequence encoding a cleavage site (CL), so that NOI1 and NOI2 are expressed as separate proteins.
10. A nucleic acid construct comprising:
a first nucleotide sequence of interest (NOI1), a translational readthrough motif (TRM), and a second nucleotide sequence of interest (NOI2), and
a nucleotide sequence encoding a cleavage site (CL), so that NOI1 and NOI2 are expressed as separate proteins,
wherein NOI2 encodes a cytokine, chemokine or toxin.
11. A vector comprising a nucleic acid according to claim 10 .
12. A retroviral vector or a lentiviral vector according to claim 11 .
13. A cell comprising a nucleic acid construct according to claim 10 .
14. A method for making a cell according to claim 13 which comprises the step of introducing into a cell a nucleic acid construct comprising:
a first nucleotide sequence of interest (NOI1), a translational readthrough motif (TRM), and a second nucleotide sequence of interest (NOI2), and
a nucleotide sequence encoding a cleavage site (CL), so that NOI1 and NOI2 are expressed as separate proteins.
15. A nucleic acid construct comprising: a first nucleotide sequence of interest (NOI1), a translational readthrough motif (TRM) and a second nucleotide sequence of interest (NOI2),
wherein the nucleic acid construct is capable of producing two products when expressed in a cell:
a) a first product encoded by NOI1 alone, and
b) a second product, encoded by NOI1 and NOI2, which is produced when translational readthrough occurs, and
wherein the first and/or second product is a chimeric antigen receptor (CAR).
16. A nucleic acid construct according to claim 15 , wherein the second product is a chimeric antigen receptor (CAR) and the first product is a truncated version of the CAR, incapable of inducing CAR-mediated cell signalling.
17. A nucleic acid construct according to claim 15 , wherein the first product is a chimeric antigen receptor (CAR) comprising an intracellular signalling domain and the second product is a CAR comprising an intracellular signalling domain and one or more co-stimulatory domain(s).
18. A vector comprising a nucleic acid construct according to claim 15 .
19. A retroviral vector or a lentiviral vector according to claim 18 .
20. A cell comprising a nucleic acid construct according to claim 15 .
21. A method for making a cell according to claim 20 which comprises the step of introducing into a cell a nucleic acid construct comprising:
a first nucleotide sequence of interest (NOI1), a translational readthrough motif (TRM), and a second nucleotide sequence of interest (NOI2), and
a nucleotide sequence encoding a cleavage site (CL), so that NOI1 and NOI2 are expressed as separate proteins.