Human Fetal Astrocytes as an Ex Vivo Gene Therapy Vehicle for Delivering Biologically Active Nerve Growth Factor

Abstract

The therapeutic use of neurotrophic factors for neurodegenerative diseases is promising, however, optimal methods for continuous delivery of these substances to the human central nervous system (CNS) remains problematic. One approach would be to graft genetically engineered human cells that continuously secrete high levels of a biologically produced and processed neurotrophic factor. This ex vivo gene therapy approach has worked well in animal models of neurodegenerative diseases using a variety of nonneuronal cell types to deliver the transgene. In our studies, we have been investigating the potential of astrocytes, a cell type normally present in the CNS, as a vehicle for ex vivo gene therapy. Here, we demonstrate that astrocytes in the human fetal cortex can be isolated and efficiently infected with an amphotropic retrovirus harboring mouse β-nerve growth factor (NGF). These transduced astrocytes express high levels of NGF mRNA and secrete bioactive NGF protein as demonstrated by stimulation of neurite outgrowth from adrenal chromaffin cells. NGF ELISA showed that these astrocytes secrete NGF protein at a rate of 41 ng/day per 10⁵ cells after 2 weeks in vitro, whereas NGF is undetectable in medium conditioned by normal astrocytes. These data suggest that human fetal astrocytes can be used for delivering biologically produced neurotrophic factors to the human CNS.

Overview summary

Astrocytes, a central nervous system (CNS) cell type, can be isolated from human fetal cortex and efficiently transduced with an amphotropic retrovirus harboring mouse β-nerve growth factor (NGF). These astrocytes express high levels of NGF mRNA as determined by ribonuclease protection assay, whereas NGF mRNA is not detected in normal human astrocytes. After 2 weeks in vitro, the transduced astrocytes secrete NGF at a rate of 41 ng/day per 10⁵ cells which is bioactive in stimulating neurite outgrowth from chromaffin cells. These results suggest genetically modified human fetal astrocytes as a potential cellular vehicle for delivering NGF and other trophic factors to the human CNS.