Towards New Generation of Hemoglobin-Based Blood Substitutes

Abstract: Blood transfusion is a clinically significant and crucial process, which saves millions of lives every year. However,shortage of donated blood and the risk of virus transmission through transfusible blood seriously affect theavailability of the blood. Hemoglobin (Hb), owing to its oxygen carrying capacity, has been studied as a startingmaterial for the development of artificial blood substitutes/Hb-based oxygen carrier (HBOC). Several kinds ofHBOC products have been developed and tested for their safety at different stages of clinical trials with minimalsuccess. The failure of such products is mainly associated with intrinsic toxicity of cell-free Hb which damageslipids, proteins, DNA and surrounding tissues. This thesis describes two approaches aiming to gain furtherknowledge of potential side effects of Hb molecules on genetic material. Additionally, genetic engineeringapproach was used as an alternative to chemical modification of Hb molecule, which is essential for theperformance of HBOC product in cell-free environment.Using the comet assay, we have evaluated the genotoxic effect of the penultimate tyrosine residues of the alphaand beta chains. Replacement of a tyrosine residue with phenylalanine, in the alpha chain (α-Y140F) has shown40% higher DNA damage compared to wildtype HbA. However, a similar mutant on the beta chain had negligibleeffect on the genotoxicity of Hb molecule.In a plasmid DNA cleavage assay, we have demonstrated that Hb itself can interact with DNA molecules andinitiate their cleavage. Conversion of supercoiled plasmid DNA (sc pDNA) into open circular (ocDNA) or linearDNA (LDNA) was used to determine the DNA cleavage activity of Hb. Our investigation revealed that fetalhemoglobin (HbF) was three-fold less active than adult hemoglobin (HbA). Thus, we have proposed HbF as apotential starting material for creation of safe HBOC product.In a second approach, we have demonstrated beneficial effects of a polypeptide tag (dubbed XTEN) geneticallyattached to fusion fetal hemoglobin (fHbF), forming XTEN-HbF. The main purpose of this XTEN polymer is toavoid the chemical processing such as PEGylation, which often increases the production cost. Additionally,PEGylation also impair the structural and functional properties of Hb molecule. Using XTEN polypeptide, thefunctional properties of a fHbF remains largely unchanged, reflected by identical oxygen affinity and absorptionspectra. XTEN-HbF was produced as a homogenous mixture of product and increased the molecular size offHbF by a factor of 2.2 folds.In addition, we have produced fluorescent Hb, referred as GFP-HbF. It is composed of green fluorescent protein(GFP) linked to fHbF at the DNA level. The primary results suggest that the purified protein is fully functional, asreflected by spectral properties of fHbF and characteristic fluorescence of the GFP molecule. Furthermore, theadsorption properties of the molecularly imprinted polymers (MIP) have been estimated using fHbF, with orwithout GFP. These MIPs have a capacity to facilitate the separation and purification of Hb molecules.