ASTROCYTES INFECTED WITH REPLICATION-DEFECTIVE ADENOVIRUS CONTAINING A SECRETED FORM OF CNTF OR NT3 SHOW ENHANCED SUPPORT OF NEURONAL POPULATIONS IN-VITRO

Neurotrophic factors have been shown to ameliorate neuronal death in s everal in vitro and in vive models of neurodegenerative disease. Howev er, delivery of polypeptide growth factors to compromised neurons in t he CNS is problematic as the blood-brain barrier prevents systemic del ivery, and chronic in-dwelling cannulae are required for intraparenchy mal delivery. To circumvent these problems and specifically target neu rotrophic factors to the environment surrounding degenerating neurons in the CNS, we have generated replication-defective adenovirus (Ad) ve ctors that contain a secretable form of ciliary neurotrophic factor (s CNTF) or neurotrophin-3 (NT-3). In this study, we demonstrate that sCN TF/Ad and NT-3/Ad can efficiently infect primary astrocytes, resulting in gene transcription and the production of functional protein. Using Northern blot analysis, dose dependent expression of sCNTF or NT-3 mR NA was detected 7 days after infection. The levels of mRNA expressed i n transgenic astrocytes was dependent on virus titer and increased wit h increasing virus concentration, sCNTF or NT-3 protein was also detec ted in astrocyte supernatants by immunoblot analysis and 2-site ELISA. ELISA indicated that astrocytes infected with sCNTF/Ad or NT-3/Ad sec reted neurotrophic factors at a rate of approximately 120 pg/10(6) cel ls/h and 350 pg/10(6) cells/h, respectively. To test for secretion of bioactive sCNTF or NT-3 protein, E8 chick ciliary ganglion or nodose g anglion neurons were grown in medium conditioned by control astrocytes or astrocytes treated with sCNTF/Ad or NT-3/Ad, showing a robust and dose-dependent increase in neuronal survival when compared to control supernatant. In addition, motor neurons plated onto astrocyte monolaye rs pretreated with sCNTF/Ad showed a two- to fourfold increase in ChAT activity when compared to those grown on astrocytes pretreated with L ac-Z/Ad. This study demonstrates that, using replication-defective ade novirus, primary astrocytes can be efficiently engineered to secrete b ioactive sCNTF or NT-3, resulting in enhanced survival of responsive p eripheral and central neuronal populations. (C) 1996 Academic Press, I nc.