583. Efficient Non-Viral Gene Delivery to Primary, Leukemia B Cells by mRNA Transfection

Abstract

Top of pageAbstract There is considerable interest in the use of genetically modified activated or malignant B cells as antigen presenting cells for immunotherapy applications. However, these cells are difficult targets for gene transfer by both viral and non-viral methodologies. Under optimal conditions, we previously demonstrated approximately 50% transfection efficiency by electroporating chronic lymphocytic leukemia (CLL) patients' malignant B cells with DNA plasmid encoding the eGFP marker gene. We next investigated whether transgene expression could be further increased by transfecting mRNA reasoning that in vitro transcribed RNA can be translated simultaneously when it is delivered to the cytosol. Here, we report that transfecting CLL-B cells with mRNA resulted in superior transgene expression. We first electroporated CLL -B cells with 5′-end capped mRNA encoding for the marker gene, eGFP, which was obtained by in vitro transcription of the full length cDNA (on pCI backbone) with a commercially available T7 polymerase kit (Ambion). The transfected cells were analyzed by FACS for transfection efficiency measuring eGFP expression and for cell viability by PI exclusion. Data showed that both cell viability and transfection efficiency were 90% at 3 hr post transfection. CLL patients' malignant B are ineffective antigen-presenting cells, due to the decreased cell surface expression of the co-stimulatory molecules needed to activate T cells after the engagement of T-cell receptor. Forced expression of hCD40L has been shown to up-regulate the expression of co-stimulatory and adhesion molecules and render them to be more efficient antigen-presenting cells, however, its expression has been difficult to achieve in human CLL-B cells. Full length hCD40L mRNA was obtained by the same procedure as described for eGFP and electroporated into CLL-B cells. The transfected cells were immunostained using a FITC-conjugated monoclonal antibody to hCD40L (BD Pharmingen) and analyzed by FACS. The cell surface expression of hCD40L was achieved in greater than 50% of the CLL-B cells as early as 2 hrs post transfection and persisted for at least 48 hrs. Cell viability, when normalized against the control cells was 90%. FACS analysis of the co-stimulatory molecules revealed that hCD40L expression correlated with an up-regulation of both CD80/CD86. Significant up-regulation of CD86 was detected as early as 2 – 4 hr post transfection. The mean fluorescence intensity of CD86 expression was increased approximately 10 fold vs. control cells. mRNA transfection eliminates the concern of genomic modification, gives rise to superior gene expression, safety and cell viability, and offers significant advantages over other gene transfer methodology. Transient gene expression by mRNA transfection should be sufficient and possibly preferable for both in vivo and ex vivo (CTL generation, adoptive) immunotherapy. A scalable, electroporation based system has been developed which will be a useful tool for clinical applications that require large volume mRNA transfection.