Development of Effective Non-Viral Transfection Techniques to Improve Biomanufacturing of Cellular Immunotherapies – PI: Jonathan Kopechek

The recent development of adoptive cell transfer immunotherapies, such as chimeric antigen receptor-modified T cells (CAR T), is revolutionizing treatment of cancer and other diseases. Recent clinical trials in blood cancers (i.e., lymphoma and leukemia) have achieved impressive remission rates, but there are still significant challenges that limit the safety and availability of CAR T therapies. Life-threatening side effects are common, including cytokine release syndrome (>70% of patients) and neurotoxicity (>50% of patients) with delirium, seizures, and coma occurring in some patients. In addition, T cell processing techniques are currently very complex, inefficient, costly, and time-consuming. There is a significant need for improved methods to rapidly and precisely transfect T cells for adoptive cell transfer treatments such as CAR T. Current techniques to engineer T cells generally involve retroviral or lentiviral vectors which raises safety concerns, such as sustained transgene expression, overactivation of T cells, risk of insertional mutagenesis, and risk of adverse residual impurities from host cells. In addition, retrovirus and lentivirus production is very expensive and time-consuming, which can limit availability of this treatment. Therefore, there is a significant unmet need for effective nonviral transfection techniques that can increase safety, throughput, and scalability of cell therapy manufacturing. To address this unmet need, we are developing a novel 3D-printed acoustofluidic system which combines ultrasound waves with a continuous flow chamber to rapidly load biomolecules into T cells via “sonoporation.” Similar to electroporation, sonoporation is a phenomenon in which microbubble cavitation is driven by ultrasound waves and induces transient pores in cell membranes, enabling rapid delivery of compounds, such as nucleic acids or proteins, directly into the cytoplasm within seconds. The acoustofluidic system enables sequential transfection of T cells with high precision for adoptive cell transfer applications such as CAR T. We hypothesize that the acoustofluidic system can enable sustained CAR expression (using CRISPR/cas9) or transient CAR expression (using mRNA) in human T cells, which could potentially lead to future point-of-care treatments. Therefore, the objective of this proposal is to assess acoustofluidic delivery of CRISPR/cas9 or mRNA to human T cells for sustained or transient CAR expression to increase the safety and availability of CAR T therapy.