Preparation of brain-derived neurotrophic factor-and neurotrophin-3-secreting schwann cells by infection with a retroviral vector

Abstract

One reason that the central nervous system of adult mammals does not regenerate after injury is that neurotrophic factors are present only in low concentrations in these tissues. Recent studies have shown that the application of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) acts to encourage the regrowth of motor and sensory fibers after spinal cord injury. Other studies have reported that the regrowth of axons after injury was enhanced by the implantation of Schwann cells, which normally secrete BDNF and NT-3. The purpose of the present study was to genetically modify Schwann cells to secrete increased amounts of BDNF or NT-3 by infection with a retroviral vector. Retroviral vectors were constructed by the ligation of BDNF or NT-3 cDNA to the LXSN vector. Viruses were generated from the plasmid forms of the vectors by transient transfection of PA317 amphotrophic retroviral packaging cells. Viruses were harvested and used to infect the human Schwann cell line designated NF-1T. Northern blot analysis of poly (A+) RNA prepared from Schwann cells that were infected with BDNF- or NT-3-containing virus showed the presence of BDNF or NT-3 mRNA. An enzyme-linked immunosorbent assay (ELISA) for BDNF and NT-3 was performed on media the cells were grown in, and on cellular extracts prepared from the BDNF- and NT-3-infected Schwann cells. The ELISA results demonstrated that the Schwann cells were secreting increased levels of immunologically active BDNF or NT-3. Immunocytochemical staining of these cells revealed the presence of these two neurotrophic factors located in perinuclear granules. These neurotrophic factor-secreting Schwann cells are currently being evaluated for their efficacy in the treatment of spinal cord injury.

Strategies to Create a Regenerating Environment for the Injured Spinal Cord

“…Adenoviral vector-mediated delivery of BDNF in combination with OEC implants increases axonal regrowth, reduces lesion size and rubrospinal tract degeneration, and improves locomotor function 4 months after SCI [107]. Retroviral vector mediated overexpression of BDNF and NT3 in Schwann cells can promote sensory fiber growth and remyelination [111]. Retrovirus transfected NGF-secreting fibroblasts also can induce robust axonal sprouting in the hemisected spinal cord of the rat [112,113].…”

From Molecular to Nanotechnology Strategies for Delivery of Neurotrophins: Emphasis on Brain-Derived Neurotrophic Factor (BDNF)

“…Local delivery of neurotrophins to neurons has been performed by gene therapy [ 12 , 32 , 41 , 118 , 140 , 161 , 184 , 185 , 186 , 187 , 188 , 189 , 190 , 191 , 192 , 193 , 194 , 195 , 196 , 197 , 198 , 199 , 200 , 201 , 202 , 203 , 204 , 205 , 206 , 217 ]. This method avoids the protein instability in circulation and may yield a lasting expression of BDNF [ 2 ].…”

Neural Transplantation in Spinal Cord Injury

“…86 To enhance the intrinsic ability of Schwann cells to secrete these neurotrophs, genetically modified Schwann cells have been used in grafting experiments. [87][88][89][90] Considerably more brain stem and hypothalamic labelling was observed in the modified Schwann cell grafts compared to the nonmodified Schwann cell grafts. 87 Furthermore, modified Schwann cell grafts were spontaneously arranged into regular arrays within the cord and showed evidence of enhanced axonal growth and remyelination when compared to untreated grafts.…”