RESUMEN
Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.
Asunto(s)
Linfoma de Burkitt , Vectores Genéticos , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Transposasas , Humanos , Transposasas/genética , Transposasas/metabolismo , Animales , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Inmunoterapia Adoptiva/métodos , Ratones , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Linfoma de Burkitt/terapia , Linfoma de Burkitt/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Línea Celular Tumoral , Elementos Transponibles de ADN , Linfocitos T/inmunología , Linfocitos T/metabolismo , TransgenesRESUMEN
Exposure of human corneal limbal epithelial (HCLE) cells to UVB triggers rapid loss of K(+) and apoptosis via activation of caspases -9, -8 and -3. It has been shown that preventing loss of intracellular K(+) can inhibit apoptosis. The goal of this study was to investigate the effect of K(+) on the UVB-induced caspase activity. HCLE cells were exposed to 150 mJ/cm(2) UVB, followed by measurement of caspase activity in cell lysates. Caspase activity was measured in the presence and absence of 100 mM K(+) in the reaction buffer. UVB-induced activity of caspases -9, -8 and -3 all decreased in the presence of 100 mM K(+). These results suggest that a role of high [K(+)] in the cell is to inhibit caspase activity. Therefore, when cells lose K(+) in response to UVB, caspases are activated and cells go into apoptosis. This supports our hypothesis that K(+) inhibits caspase activity.
