Efficient and precise genome editing is essential for clinical applications and generating animal models, which requires engineered nucleases with high editing ability while low off-target activity. Here we present a high-throughput sequencing method, primer-extension-mediated sequencing (PEM-seq), to comprehensively assess both editing ability and specificity of engineered nucleases. We showed CRISPR/Cas9-generated breaks could lead to chromosomal translocations and large deletions by PEM-seq. We also found that Cas9 nickase possessed lower off-target activity while with some loss of target cleavage ability. However, high-fidelity Cas9 variants, including both eCas9 and the new FeCas9, could significantly reduce the Cas9 off-target activity with no obvious editing retardation. Moreover, we found AcrIIA4 inhibitor could greatly reduce the activities of Cas9, but off-target loci were not so effectively suppressed as the on-target sites. Therefore, PEM-seq fully evaluating engineered nucleases could help choose better genome editing strategy at given loci than other methods detecting only off-target activity.
BackgroundThe clinical benefit of immune checkpoint blockade (ICB) therapy is often limited by the lack of pre-existing CD8+ T cells infiltrating the tumor. In principle, CD8+ T-cell infiltration could be promoted by therapeutic vaccination. However, this remains challenging given the paucity of vaccine platforms able to induce the strong cytotoxic CD8+ T-cell response required to reject tumors. A therapeutic cancer vaccine that induces a robust cytotoxic CD8+ T-cell response against shared tumor antigens and can be combined with ICB could improve the outcome of cancer immunotherapy.MethodsHere, we developed a heterologous prime-boost vaccine based on a chimpanzee adenovirus (ChAdOx1) and a modified vaccinia Ankara (MVA) encoding MAGE-type antigens, which are tumor-specific shared antigens expressed in different tumor types. The mouse MAGE-type antigen P1A was used as a surrogate to study the efficacy of the vaccine in combination with ICB in murine tumor models expressing the P1A antigen. To characterize the vaccine-induced immune response, we performed flow cytometry and transcriptomic analyses.ResultsThe ChAdOx1/MVA vaccine displayed strong immunogenicity with potent induction of CD8+ T cells. When combined with anti-Programmed Cell Death Protein 1 (PD-1), the vaccine induced superior tumor clearance and survival in murine tumor models expressing P1A compared with anti-PD-1 alone. Remarkably, ChAdOx1/MVA P1A vaccination promoted CD8+ T-cell infiltration in the tumors, and drove inflammation in the tumor microenvironment, turning ‘cold’ tumors into ‘hot’ tumors. Single-cell transcriptomic analysis of the P1A-specific CD8+ T cells revealed an expanded population of stem-like T cells in the spleen after the combination treatment as compared with vaccine alone, and a reduced PD-1 expression in the tumor CD8+ T cells.ConclusionsThese findings highlight the synergistic potency of ChAdOx1/MVA MAGE vaccines combined with anti-PD-1 for cancer therapy, and establish the foundation for clinical translation of this approach. A clinical trial of ChadOx1/MVA MAGE-A3/NY-ESO-1 combined with anti-PD-1 will commence shortly.
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