Developing myelin specific promoters for schwannoma gene therapy

Abstract

Background: Schwannomas are peripheral nerve sheath tumors composed entirely of Schwannlineage 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 Method(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.

Schwannoma Gene Therapy via Adeno-Associated Viral Vector Delivery of Apoptosis-Associated Speck-like Protein Containing CARD (ASC): Preclinical Efficacy and Safety

“…injection of an adeno-associated virus serotype 1 vector (AAV1) encoding proapoptotic transgenes including as caspase-1 ( ICE ) [ 12 ], N terminal fragment of gasdermin-d [ 13 ] or apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain), known as ASC or PYCARD [ 14 ] in each case under control of rP0 (r = rat) cells resulted in significant reduction in tumor growth in human-derived schwannoma xenograft mouse models [ 12 ]. In these and other studies, we demonstrated that rP0 is optimal for both tissue specificity, and critically, protecting neuronal tissue from potential off-target toxicity [ 12 , 14 , 15 , 16 ].…”

Gene replacement therapy in a schwannoma mouse model of neurofibromatosis type 2

“… 30 , 31 Recent studies also support regression of schwannomas using proteins that induce cell death under a Schwann cell-specific promoter. 32 , 33 , 34 The current study demonstrates that AAV-based delivery of functional merlin produces inhibition of mTORC1 activation in human NF2 -null ACs and SCs in culture. Further, we describe a new sciatic nerve xenograft model in nude mice for schwannomas using human NF2 -null immortalized SCs for which a single intratumoral injection of the AAV-merlin vector leads to regression of tumors.…”

“…Still, more detailed biodistribution and toxicity studies would be required in follow-up preclinical toxicology studies before consideration of clinical trials. If toxicity to other cell types is found, merlin could be expressed under a SC-specific promoter, such as for P0 30 , 34 or periostin. 39 …”