In vitro production of red blood cells with proteins comprising sortase recognition motifs

A genetically engineered enucleated red blood cell, which comprises on the surface a first sortaggable surface fusion protein comprising (a) a first type I red blood cell transmembrane protein or a first type II red blood cell transmembrane protein;

• (b) a first peptide;
• and (c) a first therapeutic protein;
• wherein:
• (a) when the N-terminus of the first sortaggable surface fusion protein is exposed to the extracellular space, the first peptide is either N-terminal oligoglycine or N-terminal oligoalanine fused to the first type I red blood cell transmembrane protein and is exposed to the extracellular space, and the first peptide is fused to the first therapeutic protein via a first sortase recognition motif, or
• (b) when the C-terminus of the first sortaggable surface fusion protein is exposed to the extracellular space, the first peptide is a first sortase recognition motif which is fused to the C-terminus of the first type II red blood cell transmembrane protein and is exposed to the extracellular space, and the first peptide is fused to the first therapeutic protein via an oligoglycine or oligoalanine;
• and
• the first sortase recognition motif is the amino acid sequence LPXTG (SEQ ID NO:
• 1), LPXT (SEQ ID NO:
• 50), or LPXTA (SEQ ID NO:
• 2), wherein X is any amino acid residue;
• and
• wherein the genetically engineered enucleated red blood cell has a circulatory survival rate of greater than seven days.