CENTRAL-NERVOUS-SYSTEM DELIVERY OF RECOMBINANT CILIARY NEUROTROPHIC FACTOR BY POLYMER ENCAPSULATED DIFFERENTIATED C2C12 MYOBLASTS

Neurotrophic factors hold promise for the treatment of neurodegenerati ve diseases. Intrathecal transplantation of polymer encapsulated cell lines genetically engineered to release neurotrophic factors provides a means to deliver them continuously behind the blood-brain barrier. L ong-term delivery, however, may benefit from the use of conditionally mitotic cells to avoid the overgrowth observed with continuously divid ing cell lines. Myoblast lines have all the advantages of dividing cel l lines, i.e., unlimited availability, possibility for in vitro screen ing for the presence of pathogens, suitability for stable gene transfe r and clonal selection. Furthermore they can be differentiated into a nonmitotic stage upon exposure to low-serum-containing medium. Zn this study, mouse C2C12 myoblasts were transfected with a pNUT expression vector containing the human ciliary neurotrophic factor (CNTF) gene. h CNTF expression and bioactivity were demonstrated by Northern blot, EL ISA assay, and measurement of choline acetyltransferase (ChAT) activit y in embryonic spinal cord motor neuron cultures. One C2C12 clone was found to secrete 200 ng of CNTF/10(6) cells per day. The rate of secre tion of hCNTF was not altered upon differentiation of C2C12 myoblasts. A bromodeoxyuridine (BrdU) proliferation assay indicated that approxi mately 12% of the myoblasts continue to divide after 4 days in low-ser um-containing medium. The presence of the herpes simplex thymidine kin ase gene (HSV-tk) in the expression vector, however, provides a way to eliminate these dividing myoblasts upon exposure to ganciclovir, ther efore increasing the safety of the encapsulation technology using esta blished cell lines. Encapsulated hCNTF-C2C12 cells can partially rescu e motor neurons from axotomy-induced cell death. In adult rats, intrat hecal implantation of encapsulated hCNTF-C2C12 cells or control C2C12 confirmed the long-term survival of these cells and their potential us e as a source of neurotophic factors for the treatment of neurodegener ative diseases.