CLONING AND EXPRESSION OF HUMAN INTERLEUKIN-3

THE GOAL OF THIS PROJECT IS TO DEVELOP A COMMERCIALLY FEASIBLE SYSTEM FOR THE PRODUCTION OF THE HUMAN EQUIVALENT OF INTERLEUKIN-3 (IL-3) FOR USE AS A THERAPEUTIC AGENT IN THE TREATMENT OF PATIENTS WHOSE HEMATOPOIESIS HAS BEEN SUPPRESSED AS A RESULT OF CHEMOTHERAPY OR RADIATION THERAPY,OR WHO ARE RECOVERING FROM BONE MARROW TRANSPLANTATION. IN PHASE I, CDNA WILL BE CLONED INTO A GEM-1 IN VITRO TRANSCRIPTION VECTOR. A PLASMID LIBRARY WILL BE ESTABLISHEDIN E. COLI. AFTER REPLICA PLATING, PLASMID DNA WILL BE PREPARED FROM SUBSETS OF THE LIBRARY AND LINEARIZED WITH RESTRICTION ENDONUCLEASES. RNA WILL BE TRANSCRIBED FROM THECDNA IN EACH POOL USING SP6 OR T7 RNA POLYMERASE. THE RNA POOLS WILL BE TRANSLATED IN FROG OOCYTES. EXPRESSION OF IL-3 WILL BE ASSESSED BY TISSUE CULTURE BIOLOGICAL ASSAYS. THOSE LIBRARY SUBSETS WHICH PRODUCE IL-3 ACTIVITY WILL BE FURTHER SUBDIVIDED UNTIL PURE CLONES OF IL-3 ARE ISOLATED. EXPRESSION OF BIOLOGICALLY ACTIVE IL-3 IN PHASE IWILL PROVIDE INFORMATION FOR DETERMINING THE FEASIBILITY OF OBTAINING PRODUCTION LEVEL EXPRESSION IN PHASE II. FUTURE RESEARCH WILL INVOLVE SCALE-UP OF PRODUCTION AND PURIFICATION OF IL-3 TO COMMERCIAL LEVELS AND PERFORMANCE OF TRIALS TO DETERMINE SAFETY, DOSAGE, AND USEFULNESS OF THEDRUG.