Immediate transfection of patient-derived leukemia: a novel source for generating cell-based vaccines

Jill A Gershan,James Weber,Karen Burns,Kelly W Maloney,Dennis W Schauer,Bryon D Johnson,Anne B Warwick,Stephanie Behnke,Rimas J Orentas,Natalia Natalia

doi:10.1186/1479-0556-3-4

Jill A Gershan, James Weber + Show 8 more

Open Access

https://doi.org/10.1186/1479-0556-3-4

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Abstract

BackgroundThe production of cell-based cancer vaccines by gene vectors encoding proteins that stimulate the immune system has advanced rapidly in model systems. We sought to develop non-viral transfection methods that could transform patient tumor cells into cancer vaccines, paving the way for rapid production of autologous cell-based vaccines.MethodsAs the extended culture and expansion of most patient tumor cells is not possible, we sought to first evaluate a new technology that combines electroporation and chemical transfection in order to determine if plasmid-based gene vectors could be instantaneously delivered to the nucleus, and to determine if gene expression was possible in a cell-cycle independent manner. We tested cultured cell lines, a primary murine tumor, and primary human leukemia cells from diagnostic work-up for transgene expression, using both RFP and CD137L expression vectors.ResultsCombined electroporation-transfection directly delivered plasmid DNA to the nucleus of transfected cells, as demonstrated by confocal microscopy and real-time PCR analysis of isolated nuclei. Expression of protein from plasmid vectors could be detected as early as two hours post transfection. However, the kinetics of gene expression from plasmid-based vectors in tumor cell lines indicated that optimal gene expression was still dependent on cell division. We then tested to see if pediatric acute lymphocytic leukemia (ALL) would also display the rapid gene expression kinetics of tumor cells lines, determining gene expression 24 hours after transfection. Six of 12 specimens showed greater than 17% transgene expression, and all samples showed at least some transgene expression.ConclusionGiven that transgene expression could be detected in a majority of primary tumor samples analyzed within hours, direct electroporation-based transfection of primary leukemia holds the potential to generate patient-specific cancer vaccines. Plasmid-based gene therapy represents a simple means to generate cell-based cancer vaccines and does not require the extensive infrastructure of a virus-based vector system.

Highlights

The production of cell-based cancer vaccines by gene vectors encoding proteins that stimulate the immune system has advanced rapidly in model systems
Optimization of Transfection by Electroporation To determine differences in the kinetics and strength of expression of a transfected reporter gene using either an electroporation method or a cationic liposome-based methodology, U2OS human osteosarcoma cells were transfected with 0.5 μg pDSRed2C-1 (RFP) plasmid per 106 cells, and red fluorescent protein (RFP) protein expression measured over time by flow cytometry
By 12 hours, 60% of the nucleofected cells expressed RFP. When this rate was adjusted for the cell death associated with nucleofection, the transfection rate dropped to 18% of the total cells initially transfected expressing RFP at 12 hours

Summary

Introduction

The production of cell-based cancer vaccines by gene vectors encoding proteins that stimulate the immune system has advanced rapidly in model systems. The efficacy of cell-based tumor vaccines in murine models of malignancy is well established. The advantage of working in model systems is that unlimited amounts of tumor are available to produce cell-based vaccines. Cell-based vaccines from tumor-derived material have been prepared and administered in either an allogeneic or autologous fashion, recently reviewed by Mocellin, et al [1]. An allogeneic vaccine usually features the expansion of a single tumor cell line that can grow well in culture, genetic transduction by the desired vector, and preparation of large vaccine stocks that can be qualified for clinical use. The disadvantage of a single cell line approach is that each malignancy is to some degree unique, and perhaps the most immunogenic antigens, or the most relevant ones for a given patient, will fail to be expressed by the allogeneic vaccine

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