Engineered red blood cells as therapeutic agents (2017)

Human red blood cells (RBC) are naturally endowed of unique properties and, once properly engineered, may serve as therapeutic agents or carriers of active payloads with many advantages over conventional treatments. A long survival in circulation (up to 120 days) associated with a removal based on physiological senescence (random destruction doesn't exceed 0.06–0.4% of total cells), the absence of cellular compartments, and the availability of methods allowing the encapsulation of selected agents within the mature erythrocytes or their coupling on the RBC membrane, have prompted a number of researchers to develop original and innovative cell-based therapeutic agents. More recently, the availability of procedures that permit the manipulation of the erythroid precursors by way of viral vectors or targeted genome editing, and the identification of factors necessary for the ex vivo expansion of erythroid stem cell precursors, have add an additional impulse to the design of more complex, red blood cell-based, therapeutics. Pioneering studies by Garry Ihler in 19731 prompted the idea of RBC as carriers for enzymes to be used in enzyme replacement therapies. Although this approachwas not translated into clinical applications, it was inspiring for many academic groups. The concept has subsequently been extended to a number of other enzymes for the potential treatment of diverse conditions, and finally an application has now advanced to the clinic.2 More recently, RBC have been also developed as carriers for drugs to be released in circulation.3 The most advanced application concerns the release of the glucocorticoid analogue dexamethasone that, after many investigators' driven and company sponsored trial, is now in Phase III for the treatment of Ataxia Telangiectasia (Clinical Trials.gov Id.: NCT02770807) and has received the designation of orphan drug both by the FDA and EMA. These and similar applications are possible thanks to the peculiar osmotic properties of the erythrocytes. When placed into a hypotonic solution RBC swell increasing their volume and pores, transiently, open on the erythrocyte membranes allowing the exchange of macromolecules. As originally investigated by Joe Hoffman4 the pores close with an active process as the saline solution is brought to physiological values and the entrapped agents remain confined within the erythrocytes. This procedure can now be performed by dedicated medical devices that allow to process small volumes of autologous blood (50 mL) at the bedsise or in large volumes of allogenic donations (400 mL) in a centralized facility. In these applications the active agent is the drug released or the entrapped enzyme while the erythrocyte is the carrier. When the active agent, instead, is coupled to the RBC surface, different chemistries have been exploited with alternate success. Early methods of surface loading compromised the biocompatibility of the constructs causing activation of complement or other pathways. Most of these problems have now been solved. Surface loading could be especially useful for the delivery of agents that modulate hemostasis prolonging circulation and specially restricting diffusion into the cerebral nervous system.5 Interestingly, it was shown that antigens bound to the erythrocyte membrane by way of a peptide selectively recognizing glycophorin were very effective in inducing immunotollerance.6 This approach can be likely applied to a number of antigenic peptides developing a protection against autoimmune disease. A significant step forward was realized by Harvey Lodish et al.7 by the introduction of genes encoding surface proteins that can be covalently and specifically modified to became useful sites for the generation of substrates recognized by a traspeptidase able to attach peptides and proteins to RBC. Because mature RBC does not possess nucleus and other transcription and translation pathways, the modification must be introduced at the stage of erythroid precursors which are then differentiated in vitro at least up to enucleated reticulocytes. These erythrocytes were also shown to be able to induce protection in animal models of autoimmune diseases.8 The article by Sun et al.9 that appears in this issue of The American Journal of Hematology combines the biological peculiarities of sickle erythrocytes with a genetic method consisting in the transduction of sickle hematopoietic stem cells with a lentiviral vector with optimized Locus Control Region/promoters and enhancers of β-globin and armed with autologous cytotoxics (perforin and granzyme) or with immune modulating superantigens. In fact, sickle erythrocytes under hypoxic conditions are able to home into tumor niches, to shut down blood flow and induce tumoricidal response. The novelty of the approach is, in my opinion, to be identified in the synergistic combination of years of knowledge of the well-known biological property of sickle erythrocytes (sickling of HbS in the hypoxic nice) with the knowledge gained in years of research on the regulatory sequences linked with the beta globin gene. This way of reasoning should likely be extended in the future to other conditions including the knowledge gained by years of investigations on a wide spectrum of erythrocyte abnormalities arising by selected gene defects, the mechanisms of RBC physiological senescence and the recognition by professional phagocytic cells and the mechanisms that modulate RBC homing and bio distribution of normal or pathogenic cells. Integration of this knowledge will likely open the door to additional new developments to the use of engineered erythrocytes for new therapeutic applications. A number of studies and clinical investigations have demonstrated that the transient opening of pores on the RBC membrane followed by a controlled resealing, does not modify the rheological, immunological and biochemical properties of the erythrocytes and that, once these manipulated cells are administered back to the original donor, they show a near-normal survival in circulation. More attention, instead, should be devoted to surface loaded cargoes or surface perturbations induced by transmembrane proteins in engineered erythrocytes. Red blood cells engineered by modification of the external membrane surface may induce tolerance but cases of induced polyclonal immunization by transfusion of RBC differing only in one amino acid on a single membrane protein, are also known. Thus, engineering of the RBC membrane for drug delivery should be further investigated in a case-by-case modification, taking into account also the condition of the transfusion recipient, and upon repeated infusions. In conclusion, after many years of research on the physiology and pathology of RBC, and many attempts in extending the use of erythrocytes over the conventional transfusion practice, we are finally at the clinical staged with some mature applications and a large platform that need to be fully exploited.10 During these years we have learned that RBC are natural carriers, abundantly available, amenable to a wide range of reproducible modifications, and with an immense potential for serving as therapeutic agents in a number of conditions with high medical needs. It is hoped that by this way hematology, as a science, could contribute to the broadening of red cell based therapies for the benefit of many other diseases. MM hold shares in, and is Chief Scientific Officer and board member of EryDel SpA.