Nucleic acid construct

A nucleic acid construct comprising the following structure:

• A-X-B-Y-C
• in which
• (a) each of A B, and C is a nucleic acid sequence encoding a polypeptide of interest (POI), wherein said POI are different proteins, and
• (b) X and Y are nucleic acid sequences which may be the same or different, each of which encodes a cleavage site,
• wherein at least two of the POIs are cell surface transmembrane proteins (CSTMP) that each comprise an extracellular domain, a transmembrane domain, and an endodomain, and wherein the endodomain of each of said CSTMP further comprises an intracellular retention signal which directs said POI to an intracellular compartment, such that, when said nucleic acid construct is expressed in a cell, the CSTMP are expressed at the cell surface, with the proviso that the cell surface expression of each CSTMP with the intracellular retention signal is reduced compared to expression from an equivalent construct which does not include the intracellular retention signal;
• said intracellular retention signals comprising an endocytosis signal;
• a Golgi retention signal;
• a trans-Golgi network (TGN) recycling signal;
• an endoplasmic reticulum (ER) retention signal;
• or a lysosomal sorting signal.

A method for modulating the relative cell surface expression of a first cell surface transmembrane protein (CSTMP) expressed with a second CSTMP that is different from the first CSTMP from a single nucleic acid construct, the method comprising:

• (a) providing the single nucleic acid expression construct containing a nucleotide sequence that encodes the first CSTMP and a nucleotide sequence that encodes the second CSTMP,
• wherein the first CSTMP and the second CSTMP each comprises an extracellular domain, a transmembrane domain, an endodomain, and
• wherein the endodomain of one of the first CSTMP and the second CSTMP further comprises an intracellular retention signal, wherein the intracellular retention signal directs said CSTMP to an intracellular compartment, such that, when said nucleic acid expression construct is expressed in a cell, the CSTMP with the intracellular retention signal is expressed at the cell surface, with the proviso that the cell surface expression of the CSTMP with the intracellular retention signal is reduced compared to expression from an equivalent construct which does not include the intracellular retention signal,
• said intracellular retention signal comprising an endocytosis signal;
• a Golgi retention signal;
• a trans-Golgi network (TGN) recycling signal;
• an endoplasmic reticulum (ER) retention signal;
• or a lysosomal sorting signal;
• (b) transfecting a cell with the nucleic acid expression construct;
• and
• (c) culturing the cell under conditions in which the cell expresses the first cell surface transmembrane protein and the second cell surface transmembrane protein.