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 - 21 . (canceled)
22 . A nucleic acid construct comprising:
a nucleotide sequence encoding a chimeric antigen receptor, a translational readthrough motif (TRM), and a nucleotide sequence encoding IL-12 or Flexi-IL12, and a nucleotide sequence encoding a cleavage site (CL), so that the CAR and the IL-12 or Flexii-IL12 are expressed as separate proteins.
23 . The nucleic acid construct according to claim 22 , wherein the translational readthrough motif comprises one of the following 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.
24 . The nucleic acid construct according to claim 22 which has the structure:
CAR-TRM-CL-IL12 or
CAR-TRM-CL-Flexi-IL 12.
25 . The nucleic acid construct according to claim 22 , wherein the cleavage site comprises a self-cleaving peptide, a furin cleavage site or a Tobacco Etch Virus cleavage site.
26 . The nucleic acid construct according to claim 25 , wherein the cleavage site comprises a 2A self-cleaving peptide from an aphtho-or a cardiovirus or a 2A-like peptide.
27 . A vector comprising a nucleic acid construct according to claim 22 .
28 . A retroviral vector or a lentiviral vector according to claim 27 .
29 . A cell comprising a nucleic acid construct according to claim 22 .
30 . A method for making a cell according to claim 29 which comprises the step of introducing into a cell a nucleic acid construct comprising:
a nucleotide sequence encoding a chimeric antigen receptor (CAR), a translational readthrough motif (TRM), and a nucleotide sequence encoding IL-12 or Flexi-IL12, and a nucleotide sequence encoding a cleavage site (CL), so that the CAR and the IL-12 or Flexi-IL12 are expressed as separate proteins.
31 . A method of blocking immune-suppressive signalling in a tumor microenvironment in a subject comprising administering to the subject a cell according to claim 29 , wherein the CAR recognizes a tumor-specific antigen on the tumor.
32 . A method of increasing T cell survival in a tumor microenvironment in a subject comprising administering to the subject a cell according to claim 29 , wherein the CAR recognizes a tumor-specific antigen on the tumor.
33 . A method of treating a solid tumor in a subject comprising administering to the subject a cell according to claim 29 , wherein the CAR recognizes a tumor-specific antigen on the tumor.