Achieving Therapeutic Efficacy with a High Performance, Electroporation-based Cellular Engineering Technology
Peter Gee, Joseph Abad, Rama Shivakumar, and James Brady. MaxCyte, Gaithersburg, MD, USA
Advances in cell-based therapies for treating cancer and other diseases have created a demand for rapid and flexible manufacturing processes. While virus-based delivery methods have provided the foundation for the cell therapy revolution, the costs of manufacturing viral vectors and related safety issues have created an urgent need for non-viral approaches to cellular engineering that can be implemented at scale in a GMP environment. In this poster, we present a GMP-compliant, scalable electroporation-based technology for engineering primary human cells, stem cells and cell lines with high efficiency while maintaining high cell viability. Examples include introducing precise genomic modifications into primary T cells and hematopoietic stem cells via transfection of mRNA or RNPs encoding zinc finger nucleases or CRISPR-Cas9 components. We also include data on redirecting the specificity of T cells and NK cells by expressing recombinant receptors. Finally, we provide examples of cellular engineering with induced pluripotent stem cells (iPSCs) and other types of progenitor cells.
Universal, High Performance Primary & Stem Cell Engineering
Figure 1: High GFP Expression Post Electroporation in Primary and Stem Cells.
Human primary or stem cells were electroporated with GFP plasmid or mRNA using the MaxCyte STx®. Transfection efficiency and viability were assessed at 24-48 hours post electroporation.
Advancing CAR Therapy using NK Cell Engineering
High Performance, Clinical-Scale Anti-CD19 CAR Expression
Figure 2: Development of a Non-Viral, Clinically Adaptable Method to Enhance NK Cell Cytotoxicity Against B Cell Malignancies via CAR Expression.
NK cells from healthy human donors were expanded in vitro and electroporated with anti-CD19-BB-ζ mRNA using the MaxCyte GTx®. Transfection efficiency and consistency were significantly higher than those following retroviral transduction (87% ± 6 vs. 60% ± 20). Anti-CD19 CAR expression correlated with increased cytokine production and killing of CD19+ tumor cell lines. In vivo anti-tumor activity was demonstrated using a mouse model of acute lymphoblastic leukemia (ALL). These studies opened the path to an ongoing clinical trial NCT01914479 using large-scale MaxCyte GTx engineered NK cells. Cytotherapy, 14(7), 830-840, 2012.
Clinically Relevant Levels of Ex Vivo Gene Correction in X-linked Chronic Granulomatous Disease (X-CGD) Patient Stem Cells
High Performance, Clinical-scale Anti-CD19 CAR Expression
Figure 3: Therapeutic CYBB Gene Correction.
X-CGD is caused by a mutation in the CYBB gene which encodes a critical component (gp91-phox) of NADPH oxidase, a key enzyme for the anti-microbial activity of phagocytes. Mutation correction offers a new curative treatment for X-CGD patients. The patient’s own cells are harvested, the mutated gene corrected using CRISPR-mediated gene editing, and the cells returned to the patient. The engrafted cells multiply to create a new population of cells displaying ‘normal’ function and eliminating disease. In these studies, CD34+ hematopoietic stem cells (HSPCs) were isolated from X-CGD patients and electroporated with CRISPR-Cas9, guide RNA and the gene-correcting, oligo template using the MaxCyte GTx. A portion of the cells were differentiated in vitro into myeloid cells and gene correction rates determined to be 31%. The other portion of corrected HSPCs were introduced into immunodeficient mice. After 20 weeks the engrafted human cells expressed the corrected gp91 gene at 14% in the mouse peripheral blood and 10% – 20% in the bone marrow. These correction rates are within clinically beneficial potency thresholds. Sci. Transl. Med., 9(372), Jan 2017.
Advancement of an HIV Clinical Program for CCR5 Gene Disruption
Rapid Development & IND-Enabling Pre-Clinical Studies Support Progression of Clinical Trial
Figure 4: Therapeutic CCR5 Gene Disruption.
Researchers at Sangamo developed a CCR5-targeted zinc finger nuclease that was active in a variety of CD4 T cells and HSPCs and conferred resistance to HIV infection. This therapy advanced to the clinic using adenovirus delivery of ZFN constructs. The phase 1/2 trials showed that CD4 cells with a disrupted CCR5 gene could be engrafted, were safe and persisted, but toxicity related to the adenoviral vector precluded the trials from progressing. To rescue the therapy, the company turned to mRNA delivery of the CCR5-specific ZFN using the MaxCyte GTx. This demonstrates the rapid progression from process development of ZFN delivery, through manufacturing qualification runs, pre-clinical toxicity studies and initiation of clinical trial NCT02500849 using the MaxCyte GTx. Mol Ther. Methods Clin. Dev., 3, 2016.
High Efficiency, High Viability iPSC Transfection
CRISPR-Mediated Gene Deletion Recreates Cardiac Arrhythmia
Figure 5: High GFP Expression in iPSCs.
Human iPSCs (iXCells Biotechnologies) were treated with Accutase® and dissociated into single cells before electroporation. Cells were electroporated with pGFP using the MaxCyte STx and re-plated on Matrigel®-coated plates. Images were taken 24, 48 or 120 hours post electroporation. The transfection efficiency and viability were determined using a NovoCyte® flow cytometer. These data were compared to historic results using iXCells’ previously optimized lipid-based transfection method.
Cardiac “Disease-in-a-Dish” Using iPSC-Derived Cardiomyocytes
CRISPR Gene Deletion Recreates Cardiac Arrhythmia
Figure 6: Deletion of a ~200kb Fragment Involved in Cardiac Arrhythmia.
Two CRISPR constructs targeting opposite ends of the sequence for deletion were electroporated into hiPSCs using the MaxCyte STx. Electroporated cells were cultured on Matrigel-coated plates and single cell colonies screened by genomic PCR (Primer F+R). 30% of the isolated clones had the desired deletion. Mutant and wild-type iPSCs were subsequently differentiated into cardiomyocytes using iXCells proprietary methods and beating monitored. Cardiomyocytes from wild-type iPSCs exhibited regular beating while those derived from mutant colonies beat irregularly mimicking cardiac arrhythmia.
• MaxCyte’s ExPERT® platform provides for engineering of primary and stem cells with high efficiency and cell viability enabling:
– improved, more powerful disease modeling
– high efficiency gene editing (correction, deletion or insertion)
– development of high potency, highly efficacious human therapeutics
• Flow Electroporation® technology meets the stringent demands of cell & gene therapy:
– highly efficient and reproducible transfection
– payload flexibility
– clinical-scale manufacturing
• Platform scalability and established regulatory-compliance supports rapid and seamless advancements from concept through clinic
• Supported by publications, clinical trials and 100+ partnered clinical development programs