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Towards New Generation of Hemoglobin-Based Blood Substitutes

Chakane, Sandeep LU (2017)
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 the
availability of the blood. Hemoglobin (Hb), owing to its oxygen carrying capacity, has been studied as a starting
material for the development of artificial blood substitutes/Hb-based oxygen carrier (HBOC). Several kinds of
HBOC products have been developed and tested for their safety at different stages of clinical trials with minimal
success. The failure of such products is mainly associated with intrinsic toxicity of cell-free Hb which damages
lipids, proteins, DNA and surrounding tissues.... (More)
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 the
availability of the blood. Hemoglobin (Hb), owing to its oxygen carrying capacity, has been studied as a starting
material for the development of artificial blood substitutes/Hb-based oxygen carrier (HBOC). Several kinds of
HBOC products have been developed and tested for their safety at different stages of clinical trials with minimal
success. The failure of such products is mainly associated with intrinsic toxicity of cell-free Hb which damages
lipids, proteins, DNA and surrounding tissues. This thesis describes two approaches aiming to gain further
knowledge of potential side effects of Hb molecules on genetic material. Additionally, genetic engineering
approach was used as an alternative to chemical modification of Hb molecule, which is essential for the
performance of HBOC product in cell-free environment.
Using the comet assay, we have evaluated the genotoxic effect of the penultimate tyrosine residues of the alpha
and beta chains. Replacement of a tyrosine residue with phenylalanine, in the alpha chain (α-Y140F) has shown
40% higher DNA damage compared to wildtype HbA. However, a similar mutant on the beta chain had negligible
effect on the genotoxicity of Hb molecule.
In a plasmid DNA cleavage assay, we have demonstrated that Hb itself can interact with DNA molecules and
initiate their cleavage. Conversion of supercoiled plasmid DNA (sc pDNA) into open circular (ocDNA) or linear
DNA (LDNA) was used to determine the DNA cleavage activity of Hb. Our investigation revealed that fetal
hemoglobin (HbF) was three-fold less active than adult hemoglobin (HbA). Thus, we have proposed HbF as a
potential starting material for creation of safe HBOC product.
In a second approach, we have demonstrated beneficial effects of a polypeptide tag (dubbed XTEN) genetically
attached to fusion fetal hemoglobin (fHbF), forming XTEN-HbF. The main purpose of this XTEN polymer is to
avoid 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, the
functional properties of a fHbF remains largely unchanged, reflected by identical oxygen affinity and absorption
spectra. XTEN-HbF was produced as a homogenous mixture of product and increased the molecular size of
fHbF 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, as
reflected by spectral properties of fHbF and characteristic fluorescence of the GFP molecule. Furthermore, the
adsorption properties of the molecularly imprinted polymers (MIP) have been estimated using fHbF, with or
without GFP. These MIPs have a capacity to facilitate the separation and purification of Hb molecules. (Less)
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author
opponent
  • Doktor D'Agnillo, Felice, Food and Drug Administration (FDA), USA
organization
publishing date
type
Thesis
publication status
published
subject
keywords
hemoglobin, Fetal hemoglobin, Hemoglobin-based oxygen carrier, DNA cleavage, comet assay
pages
143 pages
publisher
Department of Chemistry, Lund University
defense location
lecture hall B, Kemicentrum, Naturvetarvägen 16, Lund University, Faculty of Engineering LTH, Lund
defense date
2017-06-07 09:30
ISBN
978-91-7422-520-4
978-91-7422-521-1
language
English
LU publication?
yes
id
96f74e7f-45b8-4292-b3ea-6b8b3607bc92
date added to LUP
2017-05-11 09:39:36
date last changed
2017-05-11 13:20:18
@phdthesis{96f74e7f-45b8-4292-b3ea-6b8b3607bc92,
  abstract     = {Blood transfusion is a clinically significant and crucial process, which saves millions of lives every year. However,<br/>shortage of donated blood and the risk of virus transmission through transfusible blood seriously affect the<br/>availability of the blood. Hemoglobin (Hb), owing to its oxygen carrying capacity, has been studied as a starting<br/>material for the development of artificial blood substitutes/Hb-based oxygen carrier (HBOC). Several kinds of<br/>HBOC products have been developed and tested for their safety at different stages of clinical trials with minimal<br/>success. The failure of such products is mainly associated with intrinsic toxicity of cell-free Hb which damages<br/>lipids, proteins, DNA and surrounding tissues. This thesis describes two approaches aiming to gain further<br/>knowledge of potential side effects of Hb molecules on genetic material. Additionally, genetic engineering<br/>approach was used as an alternative to chemical modification of Hb molecule, which is essential for the<br/>performance of HBOC product in cell-free environment.<br/>Using the comet assay, we have evaluated the genotoxic effect of the penultimate tyrosine residues of the alpha<br/>and beta chains. Replacement of a tyrosine residue with phenylalanine, in the alpha chain (α-Y140F) has shown<br/>40% higher DNA damage compared to wildtype HbA. However, a similar mutant on the beta chain had negligible<br/>effect on the genotoxicity of Hb molecule.<br/>In a plasmid DNA cleavage assay, we have demonstrated that Hb itself can interact with DNA molecules and<br/>initiate their cleavage. Conversion of supercoiled plasmid DNA (sc pDNA) into open circular (ocDNA) or linear<br/>DNA (LDNA) was used to determine the DNA cleavage activity of Hb. Our investigation revealed that fetal<br/>hemoglobin (HbF) was three-fold less active than adult hemoglobin (HbA). Thus, we have proposed HbF as a<br/>potential starting material for creation of safe HBOC product.<br/>In a second approach, we have demonstrated beneficial effects of a polypeptide tag (dubbed XTEN) genetically<br/>attached to fusion fetal hemoglobin (fHbF), forming XTEN-HbF. The main purpose of this XTEN polymer is to<br/>avoid the chemical processing such as PEGylation, which often increases the production cost. Additionally,<br/>PEGylation also impair the structural and functional properties of Hb molecule. Using XTEN polypeptide, the<br/>functional properties of a fHbF remains largely unchanged, reflected by identical oxygen affinity and absorption<br/>spectra. XTEN-HbF was produced as a homogenous mixture of product and increased the molecular size of<br/>fHbF by a factor of 2.2 folds.<br/>In addition, we have produced fluorescent Hb, referred as GFP-HbF. It is composed of green fluorescent protein<br/>(GFP) linked to fHbF at the DNA level. The primary results suggest that the purified protein is fully functional, as<br/>reflected by spectral properties of fHbF and characteristic fluorescence of the GFP molecule. Furthermore, the<br/>adsorption properties of the molecularly imprinted polymers (MIP) have been estimated using fHbF, with or<br/>without GFP. These MIPs have a capacity to facilitate the separation and purification of Hb molecules.},
  author       = {Chakane, Sandeep},
  isbn         = {978-91-7422-520-4},
  keyword      = {hemoglobin,Fetal hemoglobin,Hemoglobin-based oxygen carrier,DNA cleavage,comet assay},
  language     = {eng},
  pages        = {143},
  publisher    = {Department of Chemistry, Lund University},
  school       = {Lund University},
  title        = {Towards New Generation of Hemoglobin-Based Blood Substitutes},
  year         = {2017},
}