Gamma delta T (γδT) lymphocytes are primed for rapid function, including cytotoxicity toward cancer cells, and are a component of the immediate stress response. Following activation, they can function as professional antigen-presenting cells. Chimeric antigen receptors (CARs) work by focusing T cell function on defined cell surface tumor antigens and provide essential costimulation for robust activation. Given the natural tropism of γδT cells for the tumor microenvironment, we hypothesized that their transduction with CARs might enhance cytotoxicity while retaining their ability to migrate to tumor and act as antigen-presenting cells to prolong the intratumoral immune response. Using a GD2-targeting CAR as a model system, we showed that γδT cells of both Vδ1 and Vδ2 subsets could be expanded and transduced to sufficient numbers for clinical studies. The CAR added to the cells' innate cytotoxicity by enhancing GD2-specific killing of GD2-expressing cancer cell lines. Migration toward tumor cells in vitro was not impaired by the presence of the CAR. Expanded CAR-transduced Vδ2 cells retained the ability to take up tumor antigens and cross presented the processed peptide to responder alpha beta T (αβT) lymphocytes. γδ CAR-T cell products show promise for evaluation in clinical studies of solid tumors.

We use lentiviral-delivered RNA interference (RNAi) to inhibit the growth of a model of primary effusion lymphoma (PEL) in vitro and in vivo. RNAi is a phenomenon allowing the sequencespecific targeting and silencing of exogenous and endogenous gene expression and is being applied to inhibit viral replication both in vitro and in vivo. We show that silencing of genes believed to be essential for the Kaposi sarcomaassociated herpesvirus (KSHV) latent life cycle (the oncogenic cluster) has a varied effect in PEL cell lines cultured in vitro, however, concomitant silencing of the viral cyclin (vcyclin) and viral FLICE (Fasassociating protein with death domainlike interleukin-1␤-converting enzyme) inhibitory protein (vFLIP) caused efficient apoptosis in all PEL lines tested. We demonstrate that in a murine model of PEL, lentiviral-mediated RNA interference both inhibits development of ascites and can act as a treatment for established ascites. We also show that the administered lentiviral vectors are essentially limited to the peritoneal cavity, which has advantages for safety and dosage in a therapeutic setting. This shows the use of lentiviral-mediated RNA interference in vivo as a potential therapeutic against a virally driven human cancer. IntroductionRNA interference (RNAi) is being exploited to treat or prevent infection and as a therapeutic against cancer. 1,2 Attractive targets include foreign (eg, viral) and mutated, fused, or overexpressed genes (ie, cancer). [3][4][5] In vitro, RNAi has been shown to block infection and replication of various pathogens including human immunodeficiency virus (HIV-1), 6,7 influenza, 8 and viruses implicated in oncogenesis such as Epstein Barr virus (EBV), 9,10 hepatitis B, 11,12 hepatitis C, 13 and human papilloma virus (HPV). 14,15 In experimental in vivo models, RNAi has been shown to prevent chemical-and viral-induced hepatitis. [16][17][18] The efficient delivery of therapies that knock-down specific RNA remains one obstacle to translate RNAi into a realistic treatment option for human disease. 19 The direct delivery of antisense RNA to treat or prevent CMV retinitis is one of the few successful clinical applications thus far of RNA-targeted treatment. 20,21 Although antisense provides significantly less robust inhibition of gene expression compared to RNAi, the major problem with both therapies remains effective delivery to the site of disease. One use of RNAi is based around injecting large quantities of synthetic double-stranded RNA (dsRNA) or DNA encoding short hairpin RNA (shRNA) intravenously or using hydrodynamic transfection. 16,18 This approach is not realistic for treating human disease, except for diseases involving sites where delivery of synthetic dsRNA is more straightforward. The development of shRNA to knock-down gene expression 22,23 and the incorporation of shRNA into lentiviral- 24,25 or adenoviral-based 26,27 vectors offers the opportunity to use these vectors to target RNA efficiently in vivo. Vesicular stomatitis virus-g envelope (VSV-g)-pseudoty...

Summary Background Wilms tumour is the most common childhood renal cancer and is genetically heterogeneous. While several Wilms tumour predisposition genes have been identified, there is strong evidence that further predisposition genes are likely to exist. Our study aim was to identify new predisposition genes for Wilms tumour. Methods In this exome sequencing study, we analysed lymphocyte DNA from 890 individuals with Wilms tumour, including 91 affected individuals from 49 familial Wilms tumour pedigrees. We used the protein-truncating variant prioritisation method to prioritise potential disease-associated genes for further assessment. We evaluated new predisposition genes in exome sequencing data that we generated in 334 individuals with 27 other childhood cancers and in exome data from The Cancer Genome Atlas obtained from 7632 individuals with 28 adult cancers. Findings We identified constitutional cancer-predisposing mutations in 33 individuals with childhood cancer. The three identified genes with the strongest signal in the protein-truncating variant prioritisation analyses were TRIM28, FBXW7 , and NYNRIN . 21 of 33 individuals had a mutation in TRIM28 ; there was a strong parent-of-origin effect, with all ten inherited mutations being maternally transmitted (p=0·00098). We also found a strong association with the rare epithelial subtype of Wilms tumour, with 14 of 16 tumours being epithelial or epithelial predominant. There were no TRIM28 mutations in individuals with other childhood or adult cancers. We identified truncating FBXW7 mutations in four individuals with Wilms tumour and a de-novo non-synonymous FBXW7 mutation in a child with a rhabdoid tumour. Biallelic truncating mutations in NYNRIN were identified in three individuals with Wilms tumour, which is highly unlikely to have occurred by chance (p<0·0001). Finally, we identified two de-novo KDM3B mutations, supporting the role of KDM3B as a childhood cancer predisposition gene. Interpretation The four new Wilms tumour predisposition genes identified— TRIM28, FBXW7, NYNRIN , and KDM3B —are involved in diverse biological processes and, together with the other 17 known Wilms tumour predisposition genes, account for about 10% of Wilms tumour cases. The overlap between these 21 constitutionally mutated predisposition genes and 20 genes somatically mutated in Wilms tumour is limited, consisting of only four genes. We recommend that all individuals with Wilms tumour should be offered genetic testing and particularly, those with epithelial Wilms tumour should be offered TRIM28 genetic testing. Only a third of the familial...

Neuroblastoma is a childhood cancer that resembles developmental stages of the neural crest. It is not established what developmental processes neuroblastoma cancer cells represent. Here, we sought to reveal the phenotype of neuroblastoma cancer cells by comparing cancer (n = 19,723) with normal fetal adrenal single-cell transcriptomes (n = 57,972). Our principal finding was that the neuroblastoma cancer cell resembled fetal sympathoblasts, but no other fetal adrenal cell type. The sympathoblastic state was a universal feature of neuroblastoma cells, transcending cell cluster diversity, individual patients, and clinical phenotypes. We substantiated our findings in 650 neuroblastoma bulk transcriptomes and by integrating canonical features of the neuroblastoma genome with transcriptional signals. Overall, our observations indicate that a pan-neuroblastoma cancer cell state exists, which may be attractive for novel immunotherapeutic and targeted avenues.