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CD19-targeted chimeric antigen receptor (CAR) engineered T/natural killer (NK)-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been much less successful in solid cancers, in part due to “on-target off-tumor” toxicity related to expression of target tumor antigens on normal tissue. Based on preliminary observations of safety and clinical activity in proof-of-concept clinical trials, tumor antigen-specific messenger RNA (mRNA) CAR transfection into selected, activated, and expanded T/NK cells may permit prospective control of “on-target off-tumor” toxicity. To develop a commercial product for solid tumors, mesothelin was selected as an antigen target based on its association with poor prognosis and overexpression in multiple solid cancers. It was hypothesized that selecting, activating, and expanding cells ex vivo prior to mRNA CAR transfection would not be necessary, thus simplifying the complexity and cost of manufacturing. Now, the development of anti-human mesothelin mRNA CAR transfected peripheral blood lymphocytes (CARMA-hMeso) is reported, demonstrating the manufacture and cryopreservation of multiple cell aliquots for repeat administrations from a single human leukapheresis. A rapid, automated, closed system for cGMP-compliant transfection of mRNA CAR in up to 20 × 10(9) peripheral blood lymphocytes was developed. Here we show that CARMA-hMeso cells recognize and lyse tumor cells in a mesothelin-specific manner. Expression of CAR was detectable over approximately 7 days in vitro, with a progressive decline of CAR expression that appears to correlate with in vitro cell expansion. In a murine ovarian cancer model, a single intraperitoneal injection of CARMA-hMeso resulted in the dose-dependent inhibition of tumor growth and improved survival of mice. Furthermore, repeat weekly intraperitoneal administrations of the optimal CARMA-hMeso dose further prolonged disease control and survival. No significant off-target toxicities were observed. These data support further investigation of CARMA-hMeso as a potential treatment for ovarian cancer and other mesothelin-expressing cancers.

Natural killer (NK) cells hold promise for cancer therapy. NK cytotoxicity can be enhanced by expression of chimeric antigen receptors that re-direct specificity toward target cells by engaging cell surface molecules expressed on target cells. We developed a regulatory-compliant, scalable non-viral approach to engineer NK cells to be target-specific based on transfection of mRNA encoding chimeric receptors. Transfection of eGFP mRNA into ex vivo expanded NK cells (N=5) or purified unstimulated NK cells from peripheral blood (N=4) resulted in good cell viability with eGFP expression in 85+/-6% and 86+/-4%, 24 h after transfection, respectively. An mRNA encoding a receptor directed against CD19 (anti-CD19-BB-z) was also transfected into NK cells efficiently. Ex vivo expanded and purified unstimulated NK cells expressing anti-CD19-BB-z exhibited enhanced cytotoxicity against CD19(+) target cells resulting in > or =80% lysis of acute lymphoblastic leukemia and B-lineage chronic lymphocytic leukemia cells at effector target ratios lower than 10:1. The target-specific cytotoxicity for anti-CD19-BB-z mRNA-transfected NK cells was observed as early as 3 h after transfection and persisted for up to 3 days. The method described here should facilitate the clinical development of NK-based antigen-targeted immunotherapy for cancer.

Using a nonviral, electroporation-based gene transfection approach, we demonstrate the efficient and consistent transfection of two poorly immunogenic tumor cell lines: B16F10 melanoma and renal carcinoma (RENCA). Three genes, IL-12, angiostatin (AS), and an endostatin:angiostatin fusion protein (ES:AS) were subcloned into a DNA plasmid containing EBNA1-OriP, which was then transfected into B16F10 and RENCA cells. Significant levels of protein were secreted into the culture supernatants of transfected cells in vitro. Transfected tumor cells were injected subcutaneously into mice. All the three transgenes were capable of significantly delaying and reducing the formation of primary B16F10 and RENCA tumors, as well as B16F10 lung metastases. By day 11 post-injection, all control mice that received either mock-transfected or empty vector DNA-transfected B16F10 tumor cells had developed large primary tumors. In contrast, mice that received IL-12-transfected B16F10 cells did not develop appreciable tumors until day 17, and these were significantly smaller than controls. Similar results were observed for the RENCA model, in which only one of the IL-12 mice had developed tumors out to day 31. Expression of AS or ES:AS also significantly delayed and reduced primary tumors. Overall, ES:AS was more effective than AS alone. Furthermore, 25% of the AS mice and 33% of the ES:AS mice remained tumor-free at day 17, by which point all control mice had significant tumors. Mouse survival rates also correlated with the extent of tumor burden. Importantly, no lung metastases were detected in the lungs of mice that had received either AS or ES:AS-transfected B16F10 tumor cells and significantly fewer metastases were found in the IL-12 group. The consistency of our transfection results highlight the feasibility of directly electroporating tumor cells as a means to screen, identify, and validate in vivo potentially novel antiangiogenic and/or antineoplastic genes.

Lentiviral vectors are beginning to emerge as a viable choice for human gene therapy. Here, we describe a method that combines the convenience of a suspension cell line with a scalable, nonchemically based, and GMP-compliant transfection technique known as flow electroporation (EP). Flow EP parameters for serum-free adapted HEK293FT cells were optimized to limit toxicity and maximize titers. Using a third generation, HIV-based, lentiviral vector system pseudotyped with the vesicular stomatitis glycoprotein envelope, both small- and large-volume transfections produced titers over 1×108 infectious units/mL. Therefore, an excellent option for implementing large-scale, clinical lentiviral productions is flow EP of suspension cell lines.

Viral vectors show promise as gene therapy treatments for a number of diseases. However, it has been difficult to develop a robust system for generating large volumes of high-titer viral vectors based on lentivirus, alphavirus and adeno-associated virus (AAV) that are needed for clinical applications. Problems stem from the lack of stable producer cell lines and inherent inefficiencies associated with introducing multiple plasmids into adherent monolayer cells by conventional transient transfection techniques (i.e., calcium phosphate precipitation or cationic lipids). Other researchers have demonstrated that suspension cells of lymphoid origin, which can be grown at much higher density than adherent cells, can be converted into efficient retroviral vector producer cell lines (Chan et al., 2001. Gene Ther. 8: 697). However, commercially available transient transfection systems are not optimal for large volume co-transfection of suspension cells, especially hematopoietic cells. MaxCyte's proprietary high-throughput electroporation technology provides a closed, robust, scalable system for high efficiency transfection of both adherent and suspension cells. The MaxCyte system previously was used to produce high titer alphaviral vectors from >10 x109 Vero cells and lentiviral vectors from >1 x109 adherent HEK 293T cells. To improve the efficiency of lentiviral vector production at large scale and to demonstrate the feasibility of lentivector production in suspension cells, we used the MaxCyte system to transfect large volumes (38 x109 cells in 380 mL) of K562 human hematopoietic cells rapidly (within 20 minutes) with a plasmid encoding the green fluorescent protein (GFP) marker gene. The transfected cells were collected in fractions of 35-40 mL. Cells were cultured from each fraction or pooled samples to monitor consistency of the high- throughput electroporation. Greater than 90% of the cells expressed GFP when analyzed by flow cytometry at 48 hrs post-electroporation for all fractionated and pooled cell samples. Propidium iodide (PI) exclusion revealed the cell viability of all the fractionated and pooled cell samples to be greater than 90%. In addition, we were able to produce lentivirus in K562 cells by small- scale electroporation of 10 x106 cells with four plasmids encoding components of a lentiviral vector derived from HIV carrying the eGFP marker gene. In conclusion, these studies will demonstrate that use of suspension cells processed by MaxCyte's high- throughput electroporation-based transfection system is a feasible approach for generating large volumes of high-titer viral vectors suitable for clinical / commercial applications in human gene therapy.

Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporation-based gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64+/-15%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in long-term cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system.

Primary pulmonary hypertension (PPH) is a progressive disease of unknown pathogenesis with a bleak prognosis for affected patients. The available treatment options utilize vasorelaxants such as continuously infused prostaglandin analogs or inhaled nitric oxide (NO). We are developing a cell-based gene therapy approach that administers NO in situ and utilizes a non-viral gene delivery system. We present here preclinical in vivo data on a model system that utilizes PH induced by a chemical agent, monocrotaline (MCT), in the rat and intravenous (IV) administration of fibroblasts transfected with endothelial nitric oxide synthase (eNOS).

The identification of the chemokine receptor CCR5 as the major co-receptor required for HIV entry into CD4 T-cells has made the targeted disruption of CCR5 an attractive potential therapeutic approach for treating HIV-infected patients. Yet, no current method permits the efficient therapeutic disruption of a chosen gene in the human genome. We have developed designed zinc-finger protein nucleases (ZFNs) that specifically target the coding sequence of the CCR5 gene. Using standard delivery methods, we have shown these ZFNs can efficiently stimulate the formation of a double strand break (DSB) in the CCR5 locus to allow the natural DNA repair pathways, including Non-Homologous End Joining (NHEJ), to subsequently repair the DSB. Importantly, however, NHEJ is error prone and thus can result in frameshift mutations, leading to permanent disruption in >10% of K562 cells.

We are developing the Sleeping Beauty (SB) transposon system as a non-viral gene therapy for treatment of blood related disorders. The goal of this study is to characterize the efficacy of SB transposon vectors to elicit long-term gene expression after introduction into hematopoietic cells by electroporation. Ex vivo, non-viral gene therapy strategies have the potential to be safer than viral strategies because of (1) reduced potential to generate an immune response, (2) the ability to more stringently control the manufacturing process, and (3) the essentially random insertion into the genome. The SB transposase mediates integration of the associated transposon into chromosomes via a precise cut and paste mechanism. To evaluate the potential of SB transposon plasmids to mediate long-term expression in hematopoietic cell populations, we constructed a transposon containing a PGK promoter to regulate transcription of a bicistronic mRNA encoding eGFP and neomycin phosphotransferase. SB transposase was provided by a separate plasmid carrying the SB gene regulated by an ubiquitin promoter. These two plasmids were transfected into K562 - undifferentiated myeloid cells at 1:1 ratio via a clinically approved electroporation-based cell loading system (MaxCyte GT). Greater than 90% of the cells expressed GFP when analyzed by flow cytometry at 24 hrs post-electroporation. Propidium iodide (PI) exclusion revealed the transfected cell viability to be greater than 95%. To study subpopulations of K562 cells that exhibit long-term expression of eGFP, the transfected cells were plated in methylcellulose in the presence or absence of G418. The results showed that cotransfection of the transposon and SB gene gave rise to 40% 9.5% G418 resistant colony formation, while G418 resistant colonies were not observed after cotransfection of the transposon with an SB deleted plasmid. Several G418 resistant colonies were isolated and are currently being characterized molecularly for transposition vs. random recombination.

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.