BackgroundChimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work showed that precise insertion of a CAR at a defined locus improves treatment outcomes in the context of a CD19 CAR; however, it is unclear if such a strategy could also affect outcomes in solid tumors. Furthermore, CAR manufacturing generally relies on viral vectors for gene delivery, which comprise a complex and resource-intensive part of the manufacturing supply chain.MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within 9 days using recombinant Cas9 protein and nucleic acids, without any viral vectors. The CAR was specifically targeted to the T cell receptor alpha constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, targeted next-generation sequencing, scRNA-seq, spectral cytometry, and ELISA assays, respectively. Functionality was evaluated in vivo in an NSG™ xenograft neuroblastoma model.ResultsIn comparison to retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory-like phenotype, and low tonic signaling prior to infusion arising in part from the knockout of the T cell receptor. On exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibit specific cytotoxicity against GD2+ cells in vitro and induce solid tumor regression in vivo. VFC-CAR T cells demonstrate robust homing and persistence and decreased exhaustion relative to retroviral CAR T cells against a human neuroblastoma xenograft model.ConclusionsThis study leverages virus-free genome editing technology to generate CAR T cells featuring a TRAC-targeted CAR, which could inform manufacturing of CAR T cells to treat cancers, including solid tumors.
The next generation of therapeutic products to be approved for the clinic is anticipated to be cell therapies, termed “living drugs” for their capacity to dynamically and temporally respond to changes during their production ex vivo and after their administration in vivo. Genetically engineered chimeric antigen receptor (CAR) T cells have rapidly developed into powerful tools to harness the power of immune system manipulation against cancer. Regulatory agencies are beginning to approve CAR T cell therapies due to their striking efficacy in treating some hematological malignancies. However, the engineering and manufacturing of such cells remains a challenge for widespread adoption of this technology. Bioengineering approaches including biomaterials, synthetic biology, metabolic engineering, process control and automation, and in vitro disease modeling could offer promising methods to overcome some of these challenges. Here, we describe the manufacturing process of CAR T cells, highlighting potential roles for bioengineers to partner with biologists and clinicians to advance the manufacture of these complex cellular products under rigorous regulatory and quality control.
A significant clinical need exists to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, enabling tissue modeling for in vitro discovery of new drugs or cell-based therapies for heart repair in vivo. Chemical and mechanical microenvironmental factors are known to impact efficiency of stem cell differentiation, but cardiac differentiation protocols in hPSCs are typically performed on rigid tissue culture polystyrene (TCPS) surfaces which do not present a physiological mechanical setting. To investigate the temporal effects of mechanics on cardiac differentiation, we cultured human embryonic stem cells (hESCs) and their derivatives on polyacrylamide hydrogel substrates with a physiologically relevant range of stiffnesses. In directed differentiation and embryoid body culture systems, differentiation of hESCs to cardiac Troponin T-expressing (cTnT+) cardiomyocytes peaked on hydrogels of intermediate stiffness. Brachyury expression also peaked on intermediate stiffness hydrogels at day 1 of directed differentiation, suggesting that stiffness impacted the initial differentiation trajectory of hESCs to mesendoderm. To investigate the impact of substrate mechanics during cardiac specification of mesodermal progenitors, we initiated directed cardiomyocyte differentiation on TCPS and transferred cells to hydrogels at the Nkx2.5/Isl1+ cardiac progenitor cell stage. No differences in cardiomyocyte purity with stiffness were observed on day 15. These experiments indicate that differentiation of hESCs is sensitive to substrate mechanics at early stages of mesodermal induction, and proper application of substrate mechanics can increase the propensity of hESCs to differentiate to cardiomyocytes.
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