Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression.

BACKGROUND: Chimeric 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. METHODS: Anti-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. RESULTS: In 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. CONCLUSIONS: This 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.

Comparison of seven methods for DNA extraction from prosomata of the acorn barnacle, Amphibalanus amphitrite.

Next generation sequencing (NGS) technologies can provide an understanding of the molecular processes involved in marine fouling by Amphibalanus spp. barnacles. Here, seven methods for extracting DNA from A. amphitrite prosomata were assessed with respect to recovery, purity and size distribution. Methods incorporating organic extractions generally resulted in low recovery of fragmented DNA. The most promising method was the commercial E.Z.N.A. Blood DNA Mini kit, which provided tens of micrograms of DNA of sufficient molecular weight for use in long-read NGS library preparation. Other kits resulted in DNA preps suitable for short read length NGS platforms.

Correction: Effect of hydrogel material composition on hBMSC differentiation into zone-specific neo-cartilage: engineering human articular cartilage-like tissue with spatially varying properties.

Correction for 'Effect of hydrogel material composition on hBMSC differentiation into zone-specific neo-cartilage: engineering human articular cartilage-like tissue with spatially varying properties' by Kirsten Parratt et al., J. Mater. Chem. B, 2017, 5, 6237-6248.

Effect of hydrogel material composition on hBMSC differentiation into zone-specific neo-cartilage: engineering human articular cartilage-like tissue with spatially varying properties.

Biological tissues are complex structures with spatially distinct cellular compositions, architecture, and biochemical and mechanical properties. Therefore, it is imperative that biomaterial scaffolds which serve as frameworks for engineering tissue structures contain spatially-varying cues to differentiate encapsulated progenitor cells into distinct, spatially-organized phenotypes. Human articular cartilage consists of three spatially distinct zones: superficial, transitional, and middle, which have unique extracellular matrix (ECM) compositions, chondrocyte phenotypes, and mechanical properties. To identify material compositions that can differentiate human bone marrow stromal cells (hBMSCs) into these zone-specific cells, we studied nine different composite hydrogel materials under normoxic and hypoxic conditions, and determined their collagen composition, sulfated glycosoaminoglycan (sGAG) levels, and mechanical properties. A combined collagen-sGAG index was used to identify three material compositions that yielded superficial, transitional, and middle zone-like cells. These materials were then used to generate a composite tri-layer scaffold and hBMSC differentiation into spatially-varying cartilage-like tissue was evaluated. Our results show that material composition alone can be used to direct hBMSCs into distinct, zone-specific cell phenotypes and that spatially-varying, multi-layered material scaffolds can generate complex, patterned tissue structures.