Objectif
ASSESSMENT OF THE POSSIBILITY TO USE RED BLOOD CELLS WITH ENTRAPPED ENZYMES (I.E. HUMAN HEXOKINASE) 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.
We have synthesised 2',3'-dideoxycytidine-5'-phosphate (ddCMP) as a prodrug, encapsulated it in human erythrocytes and found that it is dephosphorylated by endogenous pyrimidine nucleotidases and subsequently released by the cells as 2',3'-dideoxycytidine (ddCyd). Encapsulated ddCMP does not affect erythrocyte metabolism and was not deaminated by cytidine deaminase. Thus, ddCMP loaded erythrocytes might be used as endogenous bioreactors for ddCyd delivery in the treatment of human immunodeficiency virus (HIV) infection. The process we describe for the delivery of ddCyd permits, by a single administration, the maintenance of a virustatic ddCyd concentration for 24 hours while its toxicity is strongly reduced.
Red blood cells have been engineered by entrapment of enzyme proteins which degrade membrane diffusible blood metabolites or improve their metabolic and/or storage properties. Red blood cells as circulating bioreactors have the main advantages of biocompatibility and a long lasting effect and can provide a good alternative to many pharmacological treatments.
Human and mouse red blood cells (RBC) have been loaded with:
acetaldehyde dehydrogenase for the removal of blood acetaldehyde;
hexokinase to improve the stability of 2,3-BPG during blood storage;
hexokinase and glucose oxidase for blood glucose overconsumption.
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 BIOREACTORS WITH ENTRAPED ENZYMES OR DRUGS 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 OLIGODEOXYNUCLIOTIDE PROBES.
5. CLONING OF 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.
Champ scientifique
- engineering and technologyenvironmental biotechnologybioremediationbioreactors
- agricultural sciencesveterinary sciences
- medical and health scienceshealth sciencesinfectious diseasesRNA virusesHIV
- medical and health sciencesclinical medicineoncologyleukemia
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes
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Thème(s)
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CSC - Cost-sharing contractsCoordinateur
61029 Urbino
Italie