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Content archived on 2024-04-15

CONSTRUCTION OF ENZYMES LOADED ERYTHROCYTES AS BIO REACTORS

Objective

ASSESSMENT OF THE POSSIBILITY TO USE HUMAN HEXOKINASE ENTRAPED IN RED BLOOD CELLS AS A VALUABLE BIO-REACTOR PERFORMING UNUSUAL METABOLIC FUNCTIONS "IN VIVO".

IN PARTICULAR, IT WILL BE ATTEMPTED TO ACHIEVE SOME CONTROL OF MODERATE HYPERGLYCEMIES, TO INVESTIGATE ERYTHROCYTE AGING AND TO CONSTRUCT NEW BLOOD PRODUCTS FOR TRANSFUSION TECHNOLOGY.
It has been demonstrated that several enzymes can function normally when they are internalysed into erythrocytes. Hence, modified red blood cells (RBC) are able to behave as therapeutic agents for the correction of some metabolic disorders. The main enzymes which have been studied are: hexokinase, asparaginase, acetaldehyde dehydrogenase and bilirubinoxidase. The methodology may also be extended to incorporate various other drugs.

Targeted manipulation of red blood cells (RBC) can provide unique opportunities for the treatment of many diseases in human and veterinary medicine. Procedures of hypotonic hemolysis isotonic resealing have been optimized. Several heterologous (including human hexokinase (HK)) enzyme proteins were encapsulated in human and mouse RBC. The new properties of engineered RBC were characterized. Cloning of human HK was undertaken with the goal of obtaining large scale expression of this protein. Prodrug loaded RBC were also constructed which behave as bioreactors for the time programmed and organ targeted release of active drugs useful in cancer and acquired immune deficiency syndrome (AIDS) therapy.

Human and mouse RBC were loaded with the following enzyme proteins: glucose oxidase ((GOD), producing glucose overconsumption and splenic targeting); DT-diaphorase (detoxification); L-asparaginase (therapy of acute lymphoblastic leukaemias); bilirubin oxidase (bilirubin consumption); acetaldehyde dehydrogenase (detoxification in alcoholism). Human HK, normally present in low amounts, was also encapsulated in human RBC, significantly improving their metabolic properties. A complementary deoxyribonucleic acid (cDNA) clone corresponding to the human HK gene was isolated and characterized, and expression systems for this and for other genes were developed. Selective targeting of carrier RBC to liver versus spleen was obtained and successfully tested for antitumour therapy in the mouse. Prodrug loaded RBC were obtained which perform as 'intelligent' bioreactors for the formation and delivery of active drugs.
INVESTIGATION OF THE USE OF RED BLOOD CELLS AS VEHICLES FOR ENTRAPED HUMAN HEXOKINASE TO BE DISSEMINATED VIA CIRCULATION.

THE FOLLOWING LINES WILL BE PURSUED IN COLLABORATION WITH THE FOUR LABORATORIES PARTICIPATING IN THIS RESEARCH:

1. PURIFICATION OF HUMAN HEXOKINASE
2. PRODUCTION OF SPECIFIC ANTIBODIES
3. PARTIAL AMINO ACID SEQUENCE
4. CONSTRUCTION OF CDNA LIBRARIES AND THEIR SCREENING USING ANTIBODIES OR OLIGODEXYNUCLIOTIDE PROBES.
5. CLONING OF THE HEXOKINASE GENE AND PRODUCTION OF HUMAN RDNA FOR HEXOKINASE.
6. ENTRAPMENT OF HEXOKINASE IN HUMAN RED BLOOD CELLS USING LARGE SCALE PROCEDURES.
7. ANALYSIS OF PROPERTIES OF HEXOKINASE - LOADED RED BLOOD CELLS.
8. TRANSFUSION OF HEXOKINASE-LOADED RED BLOOD CELLS.

Topic(s)

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Call for proposal

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Coordinator

Université de Tours (Université François Rabelais)
EU contribution
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Address
2 boulevard Tonnelle
37044 Tours
France

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Total cost
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Participants (3)