(1060-A) High aspect ratio nanostraws for high throughput primary immune cell transfection

Abstract: Chimeric Antigen Receptor-T (CAR-T) cell immunotherapy relies on genetically modifying CD3+ T lymphocytes to express a tumor targeting receptor for effective tumor clearance in patients. Autologous CAR-T cells manufactured using patient-derived T cells are prone to production failures and poor persistence arising from patients’ prior therapies and old age. Allogeneic, off-the-shelf CAR-T cells manufactured from healthy donors have a higher product quality and is readily available to patients, without the constraints of patient health status. Other immune effector cells like Gamma-Delta (γδ) T cells, Dendritic Cells (DCs), macrophages and Natural Killer (NK) cells are also considered for allogeneic CAR immunotherapies to reduce risk of immune rejection. However, transfection of CAR transgene into immune cells and ex vivo expansion of CAR-immune cells are critical bottlenecks in cell manufacturing. The gold standard transfection methods, such as viral transduction and bulk electroporation suffer tremendously from cell toxicity, low efficiency, and cell perturbations leading to altered immune cell function in primary human T lymphocytes.
Using advanced nanofabrication method, we developed hollow, high aspect-ratio nanostraws to precisely inject biomolecule cargo into cells by applying localized nano-electric fields. We named this technique nano-electro-injection (NEI). Here, we demonstrate the ability of NEI to deliver proteins (Bovine Serum Albumin, Cas9-GFP), large polysaccharides (Dextran), and eGFP mRNA with more than 80% efficiency into primary human CD8+ T cells using low voltage ( < 40V) electric pulses. NEI platform also achieved CRISPR/Cas9 RNPs mediated gene editing of CXCR4 gene in primary human CD3+ T cells. CXCR4 knockout in CAR-T cells can help overcome CXCL12 mediated immunosuppressive tumor microenvironment and improve the treatment efficacy. NEI is also a versatile or universal method to transfect a plethora of other primary immune cell types such as CD4+ T cells, macrophages, DCs, and γδ-T cells, without affecting proliferative capacity and cell viability after transfection. Importantly, NEI did not adversely perturb critical biological attributes including cell migration, activation and differentiation into T-cell subsets, cytotoxic granules secretion and cytotoxic function compared to non-transfected samples.
To overcome the limitation of low transfection throughput and to enhance automation, we further engineered a NEI-based high-throughput multiwell transfection system capable of delivering various cargo species into both adherent and suspension cell types including CD4+ T cells, CD8+ T cells, DC cells, γδ-T cells, Jurkat cells, and HEK293T cells at one go.
As it is recommended that each CAR-T dose requires 10^6 cells/kg of patient, our high throughput transfection system dramatically increases the yield of transfected cells, reducing cell expansion time and overall manufacturing costs to improve patient access to this groundbreaking cell therapy. In conclusion, our work on multiwell NEI shows promise to generate significant impact on CAR-immune cell manufacturing and therapy, particularly to bring allogeneic cell therapy closer to the clinics.