Red blood cells as vehicles for the introduction of novel therapeutics, immunomodulatory agents, and diagnostic imaging probes into the human body.
Red blood cells possess many unique characteristics that make them attractive candidates for in vivo delivery of natural and synthetic payloads. They have a long circulatory half-life (~120 days in humans and ~50 days in mice), and old or damaged RBCs are removed and degraded by cells of the reticuloendothelial system. They are biocompatible and have a large surface area of ~ 140 µm2 with a favorable surface to volume ratio. Importantly, they contain no DNA; any genes introduced into red cell precursors will no longer be present in the enucleated red cells introduced into a recipient.
A large DARPA- supported project, in collaboration with Prof. Hidde Ploegh, involves the generation in culture of both murine and human red blood cells that have on their surface monoclonal antibodies that inactivate a variety of toxic substances, or receptors that can bind and remove unwanted materials from the blood. Gene- modified red cells can be targeted to specific sites in the vasculature where they can deliver drugs or reagents or serve as imaging modalities.
Engineered red blood cells that bind and neutralize toxic proteins
Single domain antibodies (VHH) are the antigen- binding domains of the unique functional heavy- chain- only camelid antibodies. These VHHs have equivalent binding activities to their cognate antigens compared to conventional IgGs and these VHHs are more stable and easier to make. VHHs that can target botulinum neurotoxin (BoNT) have been generated and studied intensively by our collaborator Dr. Charles B. Shoemaker of Tufts University Veterinary School; these can neutralize botulinum neurotoxins in cell culture and in vivo. Nai-Jia Huang and Nova Pishesha generated genes encoding four chimeric proteins, fusing the cDNAs encoding a chimeric VHH for BoNT/A or the VHH for BoNT/B, with either the N- terminus of glycophorin A or the C- terminus of Kell. Both types of red cells expressing the VHH BoNT/A chimeras effectively neutralized BoNT/A in an in vitro nerve- protection assay; in this assay primary neuronal cells are incubated with the toxin and the cleavage of the SNAP-25 protein by the toxin is quantified. They calculated that it should be possible to protect mice from a 1 LD100 toxin dose by administering as few as 2000 engineered red cell – VNA conjugates.
More recently they generated and tested the in vitro neutralization ability of VHH BoNT/A -engineered human red blood cells.. As few as 5 x106 human red cells expressing GPA-VNA/A and 106 cells expressing Kell-VNA/A effectively neutralized the botulinum toxin.
In collaboration with the Shoemaker laboratory Nai-Jia and Nova transplanted murine hematopoietic stem/progenitor cells expressing the VHH BoNT/A glycophorin and the Kell - VHH BoNT/A chimeric proteins into irradiated CD1 mice to produce normal red blood cells bearing these chimeras. These mice, together with control transplanted mice, were challenged with BoNT/A. All control mice died after challenge with 10 LD50 of BoNT/A. In contrast, all mice bearing red cells expressing either GPA-VNA/A or Kell-VNA/A survived successive challenges, at one - week intervals, of up to 1,000 LD50 BoNT/A treatment.
They are currently testing the ability of their engineered human red blood cells to neutralize botulinum toxin A in immune-compromised mouse strains, and also quantifying the in vivo efficacy, potency, and persistence of both mouse and human red cells, generated in culture and expressing the VHH BoNT/A – glycophorin chimeric protein. In collaboration with the Shoemaker lab they are testing whether similarly generated red cells can neutralize other bacterial toxins and also several pathogenic viruses.
Engineered red blood cells that induce immune tolerance
Nova Pishesha, in a collaborative project with Hidde Ploegh’s laboratory, is currently exploring the possibility of applying the aforementioned methods to generate red cells that have on their surface any of several covalently linked foreign or proteins. She aims to use these red cells to induce immune tolerance rather than an immune reaction, offering promise for novel treatments of several autoimmune disorders. Her current work has shown that administration of red blood cells that carry a relevant peptide epitope indeed leads to the drastic reduction in the number of transferred T cells that can specifically recognize this epitope. Building on the encouraging results of this T cell adoptive transfer model, she is adapting the system to several animal models of autoimmune diseases.