Construction of recombinant human nerve growth factor beta adenovirus and evaluation of its function An in vitro and in vivo study

Abstract

Exogenous delivery of nerve growth factor (NGF) promotes neural regeneration. However, the short half-life limits delivery efficacy. Therefore, a long-term, efficient, local delivery tool or scheme is needed. The purpose of this study was to construct a functioning, recombinant, adenoviral vector carrying human NGF-beta (hNGF-beta) DNA, and to measure expression of the constructed vector in vitro and in vivo. rhNGF-beta adenoviral vector containing full-length hNGF-beta cDNA was generated by homologous recombination in Escherichia Coli. The rhNGF-beta adenovirus was packaged and amplified in human embryonic kidney HEK293 cells. Transformation efficiency, expression and function of rhNGF-beta adenovirus for primary Schwann cells, Schwann cell lines, human embryonic kidney HEK 293 cells, CRH myoblasts, and NIH3T3 fibroblasts were evaluated. Subsequently, expression of rhNGF-beta adenovirus at the peripheral nerve of rat was also assessed. Recombinant adenoviral vector carrying hNGF-beta was successfully constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. Green fluorescent protein expression was observed in 90% of rhNGF-beta adenovirus-infected cells (primary Schwann cells, Schwann cell line, human embryonic kidney HEK 293 cells, CRH myoblasts, and NIH3T3 fibroblasts) compared with non-infected cells. Total mRNA isolated from rhNGF-beta adenovirus-infected cells exhibited strong expression. Maximum NGF release was induced by primary cultured Schwann cells at 4 days after infection, which steadily continued for 14 days. PC-12 cells exposed to media conditioned with rhNGF-beta adenovirus-infected Schwann cells exhibited increased neurite extension. In vivo experiment revealed that the injected rhNGF-beta adenovirus was transfected into the cells at the injected site and promoted expression of NGF, p75NTR and brain derived neurotrophic factor at the sciatic nerve and dorsal root ganglia.