Central Nervous System Delivery of Recombinant Ciliary Neurotrophic Factor by Polymer Encapsulated Differentiated C 2 C 12 Myoblasts

Abstract

Neurotrophic factors hold promise for the treatment of neurodegenerative 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. Long-term delivery, however, may benefit from the use of conditionally mitotic cells to avoid the overgrowth observed with continuously dividing cell lines. Myoblast lines have all the advantages of dividing cell lines, i.e., unlimited availability, possibility for in vitro screening for the presence of pathogens, suitability for stable gene transfer and clonal selection. Furthermore they can be differentiated into a nonmitotic stage upon exposure to low-serum-containing medium. In this study, mouse C₂C₁₂ myoblasts were transfected with a pNUT expression vector containing the human ciliary neurotrophic factor (CNTF) gene. hCNTF expression and bioactivity were demonstrated by Northern blot, ELISA assay, and measurement of choline acetyltransferase (ChAT) activity in embryonic spinal cord motor neuron cultures. One C₂C₁₂ clone was found to secrete 200 ng of CNTF/10⁶ cells per day. The rate of secretion of hCNTF was not altered upon differentiation of C₂C₁₂ myoblasts. A bromodeoxyuridine (BrdU) proliferation assay indicated that approximately 12% of the myoblasts continue to divide after 4 days in low-serum-containing medium. The presence of the herpes simplex thymidine kinase gene (HSV-tk) in the expression vector, however, provides a way to eliminate these dividing myoblasts upon exposure to ganciclovir, therefore increasing the safety of the encapsulation technology using established cell lines. Encapsulated hCNTF-C₂C₁₂ cells can partially rescue motor neurons from axotomy-induced cell death. In adult rats, intrathecal implantation of encapsulated hCNTF-C₂C₁₂ cells or control C₂C₁₂ confirmed the long-term survival of these cells and their potential use as a source of neurotophic factors for the treatment of neurodegenerative diseases.

Overview summary

Previous studies have established the suitability and safety of transplanting encapsulated genetically engineered cell lines for the delivery of neurotrophic factors to the central nervous system. The use of a cell line that can be conditionally differentiated to a nonmitotic state upon simple manipulation avoids the potential long-term cellular overgrowth observed with a continuously dividing cell line. In the present study, the use of encapsulated differentiated myoblasts as a delivery system for neurotrophic factors was investigated. The ability of differentiated C₂C₁₂ cells, a mouse myoblast line, to produce bioactive human ciliary neurotrophic factor (hCNTF) was established both in vitro and in vivo. The analysis of encapsulated cells implanted intrathecally into rats for 3 months confirmed the long-term survival and transgene expression of differentiated C₂C₁₂ cells.