Sherif G Ahmed1, Farnaz Hadaegh1, Gary J Brenner2. 1. Department of Anesthesiology, Critical Care, and Pain Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, United States. 2. Department of Anesthesiology, Critical Care, and Pain Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, United States. Electronic address: gjbrenner@mgh.harvard.edu.
Abstract
BACKGROUND: Schwannomas are peripheral nerve sheath tumors composed entirely of Schwann-lineage cells that cause pain and sensory-motor dysfunction through compression of peripheral nerves, the spinal cord, and/or the brain stem. Treatment of schwannoma is largely limited to resection which itself has limited value. The goal of this study is to establish a technique to identify the most efficient and tissue-specific promoter for use in a schwannoma gene therapy construct. NEW METHOD: This work involves transfection of schwannoma cells with adeno-associated viral vector plasmids expressing GFP under different myelin cell specific promoters. The transfected cells were evaluated for green fluorescence intensity in vitro, and in vivo after implantation into sciatic nerves of nude mice. RESULTS: Our data demonstrate that myelin protein zero (MPZ, P0) and peripheral myelin protein 22 (PMP22) promoters produce greater GFP expression in schwannoma cell lines than myelin basic protein (MBP) promoter. In vitro, P0 promoter activity in schwannoma cell lines was shown to be less active than the cytomegalovirus and chicken β-actin (CBA) promoter. However, we did not observe any significant difference between the activity of the CBA and P0 promoters in a xenograft schwannoma model. COMPARISON WITH EXISTING METHODS(S): We show here the influence of the peripheral nerve microenvironment on promoter efficacy in expressing transgenes using simple transfection by lipofection followed by prompt implantation of the transfected cells into the sciatic nerve of nude mice. CONCLUSIONS: We demonstrate that of the myelin specific promoters evaluated, P0 is optimal for driving expression of transgenes in schwannoma cells.
BACKGROUND:Schwannomas are peripheral nerve sheath tumors composed entirely of Schwann-lineage cells that cause pain and sensory-motor dysfunction through compression of peripheral nerves, the spinal cord, and/or the brain stem. Treatment of schwannoma is largely limited to resection which itself has limited value. The goal of this study is to establish a technique to identify the most efficient and tissue-specific promoter for use in a schwannoma gene therapy construct. NEW METHOD: This work involves transfection of schwannoma cells with adeno-associated viral vector plasmids expressing GFP under different myelin cell specific promoters. The transfected cells were evaluated for green fluorescence intensity in vitro, and in vivo after implantation into sciatic nerves of nude mice. RESULTS: Our data demonstrate that myelin protein zero (MPZ, P0) and peripheral myelin protein 22 (PMP22) promoters produce greater GFP expression in schwannoma cell lines than myelin basic protein (MBP) promoter. In vitro, P0 promoter activity in schwannoma cell lines was shown to be less active than the cytomegalovirus and chicken β-actin (CBA) promoter. However, we did not observe any significant difference between the activity of the CBA and P0 promoters in a xenograft schwannoma model. COMPARISON WITH EXISTING METHODS(S): We show here the influence of the peripheral nerve microenvironment on promoter efficacy in expressing transgenes using simple transfection by lipofection followed by prompt implantation of the transfected cells into the sciatic nerve of nude mice. CONCLUSIONS: We demonstrate that of the myelin specific promoters evaluated, P0 is optimal for driving expression of transgenes in schwannoma cells.
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