Genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin

Abstract

A short half-life in the circulation limits the application of therapeutics such as single-domain antibodies (VHHs). We utilize red blood cells to prolong the circulatory half-life of VHHs. Here we present VHHs against botulinum neurotoxin A (BoNT/A) on the surface of red blood cells by expressing chimeric proteins of VHHs with Glycophorin A or Kell. Mice whose red blood cells carry the chimeric proteins exhibit resistance to 10,000 times the lethal dose (LD50) of BoNT/A, and transfusion of these red blood cells into naive mice affords protection for up to 28 days. We further utilize an improved CD34+ culture system to engineer human red blood cells that express these chimeric proteins. Mice transfused with these red blood cells are resistant to highly lethal doses of BoNT/A. We demonstrate that engineered red blood cells expressing VHHs can provide prolonged prophylactic protection against bacterial toxins without inducing inhibitory immune responses and illustrates the potentially broad translatability of our strategy for therapeutic applications.

Enhanced phosphocholine metabolism is essential for terminal erythropoiesis

“…Enriched erythroid progenitors were purified from embryonic day 14.5 (E14.5) C57BL/6J mouse embryos, and cultured in vitro for erythroid differentiation following a protocol described in detail previously. 6 Briefly, pregnant C57BL/6J mice at E14.5 were sacrificed by CO 2 asphyxiation and their embryos were collected. The fetal livers were isolated and suspended in PBS with 2% FBS and 100 mM EDTA.…”

“…To strengthen our findings, we studied aspects of metabolism during the 23-day period of differentiation in vitro of human mobilized bone marrow CD34 1 stem/progenitor cells into enucleated erythrocytes. 6 The culture system comprises 5 stages and differentiation is highly synchronized. We analyzed metabolites from cells at the end of each of the differentiation stages (Diff1-Diff5).…”

Microfluidic label-free bioprocessing of human reticulocytes from erythroid culture

“…38,39 Large-scale synchronized axenic culture system based on peripheral CD34 + HSPCs has the highest yield and enucleation rate reported so far. 13 The entire erythroid culture consists of an expansion of HSPCs, followed by 5 differentiation phases with varying cell density, cytokines and growth factors resulting in a 14 700-fold expansion over 23 days ( Fig. S1, ESI †).…”

“…In vitro erythroid cultures of reticulocytes have progressed significantly achieving more than 60% purity with >300 000 fold expansion. 13 However, this process requires erythroblasts, to eject their nuclei resulting in a viscous culture medium due to the accumulation of the expelled nuclei contaminants. 6 Downstream applications such as cellfree protein translation, RBCs production and malaria invasion of reticulocytes require purified reticulocytes free from extra-cellular DNA.…”

Camelid VHH Antibodies that Neutralize Botulinum Neurotoxin Serotype E Intoxication or Protease Function

“…Bakherad et al [ 26 ] reported a VHH that neutralized BoNT/E and partially protected against intoxication in mice. VHH-based neutralizing agents (VNAs) consisting of two linked neutralizing VHH components have proven highly effective in protecting mice from BoNT/A and BoNT/B intoxication when administered as proteins [ 21 , 25 ], on red blood cells [ 27 ], or by gene therapy [ 12 , 20 ], offering novel therapeutic options for botulism.…”