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CONSTRUCTION OF ENZYME LOADED ERYTHROCYTES AS BIO REACTORS

Objective

ASSESSMENT OF THE POSSIBILITY TO USE HUMAN HEXOKINASE ENTRAPED IN A 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.
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.
PRODUCTION OF HUMAN HEXOKINASE BY GENETIC ENGINEERING FOR ENCAPSIDATION IN RED BLOOD CELLS.

1. THE GENE CODING FOR HUMAN HEXOKINASE WILL BE CLONED FROM A CDNA LIBRARY IN THE EXPRESSION VECTOR GT 11.
2. THE POSITIVE CLONES WILL BE CHARACTERIZED BY ANTIGENIC PROPERTIES AND SEQUENCING.
3. TRANSCRIPTION PRODUCTS AND POLYMORPHISM OF THE GENE WILL BE CHARACTERIZED.
4. THE GENOMIC HUMAN KEXOKINASES CLONES WILL BE ISOLATED.
5. THE EXPRESSION OF CLONED GENES IN E.COLI AND TRANSFECTED MAMMALIAN CELLS WILL BE OPTIMIZED.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Università degli Studi di Torino
Address
Via Santena 5Bis
10126 Torino
Italy

Participants (3)

Centre National de la Recherche Scientifique (CNRS)
France
Address

37200 Tours
UNIVERSITÀ DEGLI STUDI DI URBINO
Italy
Address
Via Saffi 2
61029 Urbino
Università degli Studi di Genova
Italy
Address
Viale Benedetto Xv 1-3
16126 Genova