By Alexandra Grba
Engineered human red blood cells (RBCs) have become an attractive means of delivering drugs, immunomodulatory agents and diagnostic imaging probes into the body. They confer several advantages for therapeutic delivery over other methods, such as stem cell-based therapies. Namely, the use of modified RBCs is safer, longer-lasting and can target both wide or specific areas in the delivery of therapeutic payloads (Shi et al., 2014). Recent developments involve sortase tagging, which can be used to conjugate RBC membrane proteins to a wide variety of functional probes, including small molecules, peptides, and proteins, which could allow for the systemic delivery of therapeutic agents (Shi et al., 2014). Shi and colleagues conducted a series of experiments using engineered RBCs and sortase tagging, demonstrating proof of concept and paving the way for future therapeutic possibilities (Shi et al., 2014).
Sortase refers to a group of prokaryotic enzymes that are used to anchor secreted proteins on the surface of gram-positive bacteria. A secreted protein will reach the external membrane of the bacterium and will be momentarily retained, as the C-terminus is usually hydrophobic. Sortase is anchored to the membrane nearby and will cleave the protein at the LPXT | G motif found at the C-terminus of the secreted protein. This forms an intermediate thioester bond between sortase and the protein – shortly after, an N-terminal glycine of a peptidoglycan peptide attacks this bond, thereby releasing sortase and forming an amide bond with the threonine of the LPXTG motif. This mechanism has a wide variety of applications and lends to a number of exciting possibilities; sortase tagging can be used to attach peptides to cell surfaces as well as solid supports. Shi and colleagues have developed engineered human red blood cells that are able to carry a variety of payloads systemically using the sortase mechanism, with the potential for future therapeutic applications (Shi et al., 2014).
Engineered red blood cells confer several advantages for therapeutic delivery over other methods. For example, erythroid precursors are modified to express sortase-modifiable proteins that are retained on the plasma membrane of mature red blood cells. Since mature red blood cells lack a nucleus, there is no trace of the previous genetic alterations, eradicating the possibility of tumorigenicity which provides a substantial risk in stem cell therapies (Gruen & Grabel, 2006). In addition, red blood cells have a lifespan of around 120 days and populate both the macro- and microcirculation (Shi et al., 2014). This means that engineered red blood cells could allow for the persistence of potential therapies in the body as well as the coverage of a wide area. Payloads can also be delivered in a site-specific manner through the addition of a cell type-specific recognition module on the cell surface as well as the therapeutic payload (Shi et al., 2014). Critically, engineered RBCs can be labelled with a large variety of probes while cell survival in vivo remains unaffected; proving their potential as a mode of therapy.
Shi and colleagues engineered erythroid precursors to express proteins that can be modified by sortase and retained on the plasma membrane of mature RBCs (Shi et al., 2014). Sortase A from Staphylococcus aureus can be used to recognise an LPXTG motif at the C terminus of the substrate protein, cleaving between the T and G to form a covalent acyl-enzyme intermediate. Subsequent nucleophilic attack by the N-terminal glycine of a chosen probe allows for peptide bond formation between substrate and probe. Alternatively, the protein substrate can be labelled at its N-terminus by extension with glycine residues and reaction with a probe that contains the LPXTG motif. The two variations of sortase tagging allow for the use of RBC membrane proteins that either have an exposed C-terminus (in which case they can be modified to contain the LPXTG motif), or an exposed N-terminus (in this instance they may be extended with a series of glycine residues). With this in mind, the research group chose the proteins Kell and glycophorin A (GPA) for modification (Shi et al., 2014). Kell is a type II membrane protein that has an extracellularly exposed C terminus, thereby allowing for extension of the sortase-recognition motif LPETG. GPA is a type I membrane protein with an extracellular N-terminus, allowing for N-terminal modification by extension with three glycine residues. Reaction of the kell-modified RBC with a biotin containing glycine-based probe led to the successful conjugation of the probe onto the Kell C-terminus in the presence of sortase. Similarly, a biotin containing probe with a C-terminal sortase-recognition motif, LPETG, could be conjugated onto the GPA-modified RBC. Preincubation of the probe with sortase was required for formation of the acyl-enzyme intermediate between cysteine on sortase and threonine on the probe. The intermediate was resolved by nucleophilic attack of the N-terminal glycine on GPA, conjugating the probe and GPA.
To test the potential application of engineered RBCs in the targeted delivery to specific tissue types, Shi and colleagues conjugated an antibody to modified GPA on the surface of RBCs (Shi et al., 2014). Specifically, they used VHH7-LPETG, a modified single-domain antibody specific for murine class II MHC molecules as the probe. Using sortase, the research group covalently attached this to RBCs with glycine-extended GPA on their surface. Demonstrating the efficacy of site-specific delivery, they found that incubation of the modified RBCs with class II MHC-positive B resulted in binding of the RBCs to B cells. With the concept successfully demonstrated, the possibilities of future applications of engineered RBCs are exciting and could pave the way for targeted, systemic therapies that address the tumorigenicity concerns associated with other modes of therapy.
Gruen, L. & Grabel, L. (2006) Concise review: scientific and ethical roadblocks to human embryonic stem cell therapy. Stem Cells. 24 (10), 2162-2169.
Shi, J., Kundrat, L., Pishesha, N., Bilate, A., Theile, C., Maruyama, T., Dougan, S. K., Ploegh, H. L. & Lodish, H. F. (2014) Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes. Proceedings of the National Academy of Sciences. 111 (28), 10131-10136.