Target-specific genome editing using engineered nucleases has become widespread in various fields. Long gene knock-in and single-base substitutions can be performed by homologous recombination (HR), but the efficiency is usually very low. To improve the efficiency of knock-in with single-stranded oligo DNA nucleotides (ssODNs), we have investigated optimal design of ssODNs in terms of the blocking mutation, orientation, size, and length of homology arms to explore the optimal parameters of ssODN design using reporter systems for the detection of single-base substitutions. We have also investigated the difference in knock-in efficiency among the delivery forms and methods of Cas9 and sgRNA. The knock-in efficiencies for optimized ssODNs were much higher than those for ssODNs with no blocking mutation. We have also demonstrated that Cas9 protein/sgRNA ribonucleoprotein complexes (Cas9-RNPs) can dramatically reduce the re-cutting of the edited sites.
The recent success of chimeric antigen receptor (CAR)-T cell therapy for treatment of hematologic malignancies supports further development of treatments for both liquid and solid tumors. However, expansion of CAR-T cell therapy is limited by the availability of surface antigens specific for the tumor while sparing normal cells. There is a rich diversity of tumor antigens from intracellularly expressed proteins that current and conventional CAR-T cells are unable to target. Furthermore, adoptively transferred T cells often suffer from exhaustion and insufficient expansion, in part, because of the immunosuppressive mechanisms operating in tumor-bearing hosts. Therefore, it is necessary to develop means to further activate and expand those CAR-T cells in vivo. The Wilms tumor 1 (WT1) is an intracellular oncogenic transcription factor that is an attractive target for cancer immunotherapy because of its overexpression in a wide range of leukemias and solid tumors, and a low level of expression in normal adult tissues. In the present study, we developed CAR-T cells consisting of a single chain variable fragment (scFv) specific to the WT1/HLA-A*2402 complex. The therapeutic efficacy of our CAR-T cells was demonstrated in a xenograft model, which was further enhanced by vaccination with dendritic cells (DCs) loaded with the corresponding antigen. This enhanced efficacy was mediated, at least partly, by the expansion and activation of CAR-T cells. CAR-T cells shown in the present study not only demonstrate the potential to expand the range of targets available to CAR-T cells, but also provide a proof of concept that efficacy of CAR-T cells targeting peptide/major histocompatibility complex can be boosted by vaccination.
Adoptive immunotherapy using TCR gene-modified T-lymphocytes is an attractive strategy for targeting malignancies. However, TCR mispairings between endogenous and introduced TCR chains are a major concern, as they may induce mixed TCRs with unknown specificities and may reduce the expression of therapeutic TCRs. To overcome these problems, we have recently established a novel retroviral siTCR vector encoding small-interfering RNAs (siRNAs) to knockdown endogenous TCR genes for the efficient expression of therapeutic TCRs. In this study, to improve the efficacy of siTCR vectors, we developed 2A peptide-based siTCR vectors that could increase the expression levels of transduced TCRs compared with internal promoter-based siTCR vectors. We also evaluated the efficacy of an siTCR strategy and the addition of a new interchain disulfide bond created by cysteine modification. We found that the effect of the cysteine modification depended on TCR variations, while the siTCR strategy improved the expression of all TCRs tested. Furthermore, the combined effect of the siTCR and cysteine modification strategies was highly significant for certain TCRs. Therefore, our novel siTCR technology, in isolation or in combination with another strategy, may open the door to effective immunotherapy for cancer patients.
Carcinoembryonic antigen (CEA) is a cell surface antigen highly expressed in various cancer cell types and in healthy tissues. It has the potential to be a target for chimeric antigen receptor (CAR)-modified T-cell therapy; however, the safety of this approach in terms of on-target/off-tumor effects needs to be determined. To address this issue in a clinically relevant model, we used a mouse model in which the T cells expressing CEA-specific CAR were transferred into tumor-bearing CEA-transgenic (Tg) mice that physiologically expressed CEA as a self-antigen. The adoptive transfer in conjunction with lymphodepleting and myeloablative preconditioning mediated significant tumor regression but caused weight loss in CEA-Tg, but not in wild-type mice. The weight loss was not associated with overt inflammation in the CEA-expressing gastrointestinal tract but was associated with malnutrition, reflected in elevated systemic levels of cytokines linked to anorexia, which could be controlled by the administration of an anti-IL-6 receptor monoclonal antibody without compromising efficacy. The apparent relationship between lymphodepleting and myeloablative preconditioning, efficacy, and off-tumor toxicity of CAR-T cells would necessitate the development of CEA-specific CAR-T cells with improved signaling domains that require less stringent preconditioning for their efficacy. Taken together, these results suggest that CEA-specific CAR-based adoptive T-cell therapy may be effective for patients with CEA+ solid tumors. Distinguishing the fine line between therapeutic efficacy and off-tumor toxicity would involve further modifications of CAR-T cells and preconditioning regimens.
Glucosylceramide and galactosylceramide were detected in three Aspergillus species: Aspergillus oryzae, Aspergillus sojae and Aspergillus. awamori, using borate-coated TLC. The cerebrosides from A. oryzae were further purified by ion exchange and iatrobeads column chromatographies with or without borate, and determined the composition of sugar, fatty acid and sphingoid base by GC/MS, MALDI-TOF/MS and (1)H-NMR. We identified them as β-glucosylceramide and β-galactosylceramide. The ceramide moiety of both cerebrosides consisted mainly of 2-hydroxystearic acid and either 9-methyl-octadeca-4, 8-sphingadienine or octadeca-4, 8-sphingadienine. To our knowledge, this is the first study to provide evidence for the presence of β-galactosylceramide in A. oryzae.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.