Engineering a new generation of blood substitutes

Patients with blood loss suffer acute hypotension and a drop in oxygen content; oxygen delivery to tissue then falls with a catastrophic clinical outcome. Blood transfusions are therefore a major element of treating patients suffering blood loss through trauma or surgery. Globally around 85 million units of red blood cells are transfused annually in an industry worth ~$16Bn. However, there are inherent problems with transfusions of packed red blood cells. The need for blood group typing and the short shelf life limits the ability to stockpile the supplies necessary for major disasters. It also precludes routine transfusion support in ambulances and in population centres distant from major hospitals (including use by the military). Homologous blood transfusions are not always benign: they can result in acute transfusion reactions, either by mistyping accidents or in immune compromised individuals. Hence immune compromised individuals, as well as certain religious groups, are unable to benefit from blood transfusions.

Blood is a biological agent; it cannot be sterilised and therefore always has the potential for contamination with bacteria and blood-borne infectious agents such as HIV, hepatitis, vCJD and West Nile virus. Not all viruses are screened for in all countries, and in some cases (vCJD) no validated tests are currently available. New blood borne viruses are discovered on a regular basis; for those that are infectious and induce pathology, there will always be a pathological window prior to the introduction of a new screening test. Increased screening raises the cost of blood products as well as decreasing supply; the increasing age of populations adds to this problem by increasing the number of people requiring transfusions, whilst decreasing the fraction of the population able to donate blood. The combination of these factors limits the use of "safe" blood transfusions to those countries with a funded and developed medical system able both to manage a blood transfusion service and having a population that is relatively free of infectious blood borne viruses. In many counties, notably in Africa, these factors preclude the use of blood transfusions in trauma.

There is a clear global medical need for a long life, pathogen free "blood substitute" able to augment the use of red blood cells. A great deal of effort has been expended into producing products based on chemical or genetic modification to haemoglobin, the red cell oxygen carrying protein. Although several haemoglobin-based oxygen carriers (HBOC) have been shown to deliver oxygen they have failed to get approval for use in the major healthcare markets due to safety concerns. Three major mechanisms have been proposed for HBOC toxicity. One is a lack of product stability. The other two relate to molecular mechanisms: (i) outside the protective environment of the red cell HBOC can scavenge nitric oxide (NO) that controls blood pressure and blood flow (ii) HBOC can create toxic free radicals. All these problems can be addressed by genetic engineering techniques. At Essex we have been funded by BBSRC and the EU to investigate the basic science of toxic free radical production by haemoglobin. We have been able to develop novel modified haemoglobin molecules that are more readily detoxified by the body's antioxidants, and so induce less free radical damage.

We now wish to apply this knowledge to directly address clinical issues. Previous researchers have developed haemoglobin mutants that do not scavenge nitric oxide. We intend to combine these mutations with our own optimised mutations that decrease free radical production. Our base protein will be human foetal haemoglobin, which is a far more stable protein than the adult form. Solving these three issues (product stability, NO scavenging and free radical production) will form a basis for the next generation of blood substitute products, accessible to all countries and useable in all environments.

There is a clinical need, and a financial market, for a long-lasting sterile red blood cell substitute to be used in environments or patient groups where homologous blood is not available or appropriate. Synthetic hemoglobin (Hb) is the best candidate for the oxygen-carrying component of a blood replacement. However, cell-free Hb displays an inherent toxicity relating to its capacity to induce oxidative reactions, causing damage to DNA and cell membranes.

We have shown that tyrosine residues can enhance the safe removal of these oxidative intermediates through the formation of safe electron transfer pathways from the external environment of the protein to the heme iron. We have patents pending on novel bioengineered Hb mutants with additional electron pathways resulting in rapid and safe removal of toxic intermediates. Furthermore, we recently discovered an intrinsically less reactive form of Hb that is a better starting molecule to engineer a safe product.

The first aim is to generate new tyrosine mutants of this novel Hb species, optimising the ability of plasma reductants such as vitamin C to decrease oxidative toxicity as measured by cell free liposome studies and damage to primary aortic endothelial cells. (Milestone 1). Additional mutations, known to decrease NO scavenging at the heme oxygen binding site, will then be added. A lead candidate will emerge from this process and will then be converted into a candidate blood substitute via globin PEGylation to increase circulation time.

We will upgrade protein production (Milestone 2) to a scale for testing in models of trauma and transfusion. Real time monitoring and ex vivo biochemistry will be undertaken to obtain the maximal amount of information from each study.

Succesful tests in the model systems (Milestone 3) will greatly enhance our goal of securing further funding to further optimise our product for clinical trials.

Commercial Benefit.
The increasing probability of surgical intervention in older people and general longevity is driving growth in demand for blood. Blood substitutes are initially expected to complement donated blood and then progressively replace at least a proportion of the transfusion market. The US, Europe and Japan reflect 60% of current demand for transfused blood. Emerging economies reflect 40% of demand but are expected to follow a similar demographic trajectory. The market comprises two key segments, acute blood loss (60%) and chronic (surgery related, 40%). HBOCs are expected to make a significant impact on the acute market due to their ability to provide immediate treatment without the need to carry different blood types or undertake blood typing before administration. Initial work on market segmentation pointed towards a three prong strategy of approach with the new product: patients who have had a previous transfusion related incident; patients who are undergoing a clinical treatment where the immune system is compromised or there is an inherent infection; situations where red cells are required to be stored for lengthy period or in unrefrigerated settings before use. These 3 market segments account for $3Bn revenues.

Clinical Impact
As well as being of commercial benefit, the success of this project would impact on a range of groups who currently are unable to receive blood. These include those injured too distant from a blood bank (including the military), those who are immune compromised and those in developing countries where there is no blood bank service. Being sterile, it would also have great benefit for those countries without the resources to test blood for common blood borne viral pathogens.

Research and Professional Skills
The project would benefit the professional career development of the post-doctoral research assistants. The research assistants will be trained in a wide range of research skills, including gaining scientific practical skills in research and biotechnology, communication skills through the dissemination of the research results at meetings and in writing articles describing the project outcomes for scientific journals. Due to the nature of this project the research assistants will also be encouraged to develop their entrepreneurial skills. As well as courses organised by the University's research and Enterprise Office, one-to-one advice will be available from the industrial and commercial advisers to the HaemO2 project.

Many of the skills, such as data handling and analysis, independent planning and communication of results are transferable to other employment sectors. The post-doctoral research assistant will be made aware of the RCUK Concordat to support the career development of researchers and will also be encouraged to take advantage of the processional development opportunities by the Department and the University's Learning and Teaching Unit.