Biology subjects

Wang, J. L.

Publications and source records attributed to Wang, J. L..

3 recordsLinked to original sources

bioRxiv2024

Oncolytic vaccinia virus as a precision cancer vaccine platform

Oncolytic viruses are immunotherapeutic agents that selectively replicate in and kill tumour cells with the goal of promoting anti-cancer immune responses. Vaccinia virus (VACV) is a strong oncolytic virus candidate as it infects a wide range of cancer cells, is amenable to genetic tailoring and induces potent, long-lasting immunity. Here, we examine the use of genetically engineered oncolytic VACVs (oVACV) to express a variety of MHC-I complexes and the co-stimulatory ligand CD80 to stimulate anti-tumour CD8+ T cell responses in two syngeneic cancer models. Tumour antigen specific CD8+ T cells were detected in both the tumour and spleen following oVACV treatment, demonstrating the ability to induce tumour targeted T cell responses with viral therapy. oVACV expression of peptide-MHC-I complexes enhanced the efficacy of primary tumour clearance with tumour specific antigen targeting showing the greatest efficacy. Initial tumour clearance following treatment with oVACVs led to variable anti-tumour immune memory depending on the tumour model. Depletion of CD8+ T cells prevented therapeutic efficacy of oVACV while combination with PD-L1 immune checkpoint blockade enhanced tumour clearance. Overall, we demonstrate the ability to generate an oncolytic virus capable of inducing recognition and elimination of tumour cells through VACV-mediated expression of defined peptide-MHC-I complexes.

immunology↗

bioRxiv2022

Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation

Streptococcus pyogenes CRISPR-Cas9 (SpCas9) stabilizes each strand of a DNA bubble via distinct interactions, forming a stable ~20 bp R-loop on the complementary strand but relying on narrower protein-DNA interactions to bind the non-complementary strand with 5 NGG protospacer adjacent motif (PAM). The enzymes HNH endonuclease domain cleaves DNA within the R-loop, and its RuvC endonuclease cleaves 5 to the NGG and opposite the HNH cleavage site to generate blunt DNA ends. We show that this nucleoprotein complex can be mechanically strained by shifting the RNA:DNA hybrid and that the HNH cleavage site tracks this shift but the RuvC cleavage site does not, resulting in overhanging DNA ends. This is observed using cleavage in living cells and sequencing assays to characterize overhangs generated by stressed complexes, as well as single-molecule cleavage assays to characterize differential cleavage kinetics of HNH and RuvC endonucleases in stressed complexes as well as complex disassembly. Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the SpCas9 complex responds to internal mechanical strain by robustly generating overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by increasing or decreasing the spacing between the RNA:DNA hybrid and the downstream canonical PAM. Up to two-base 3 overhangs can be robustly generated via a two-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR-SpCas9 reaction in real-time, monitoring and quantifying the R-loop formation, the first and second DNA incision events, and the post-catalytic complex dissociation. Complex dissociation and release of broken DNA ends appears to be a rate-limiting step of the reaction, and strained SpCas9 are sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends.

genomics↗

bioRxiv2020

Single cell profiling reveals novel tumor and myeloid subpopulations in small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively), which are associated with distinct therapeutic vulnerabilities. To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 54,523 cellular transcriptomes from 21 human biospecimens. Our single-cell SCLC atlas reveals tumor diversity exceeding lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discovered a PLCG2-high tumor cell population with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival, and manipulation of PLCG2 expression in cells confirms correlation with key metastatic markers. Treatment and subtype are associated with substantial phenotypic changes in the SCLC immune microenvironment, with greater T-cell dysfunction in SCLC-N than SCLC-A. Moreover, the recurrent, PLCG2-high subclone is associated with exhausted CD8+ T-cells and a pro-fibrotic, immunosuppressive monocyte/macrophage population, suggesting possible tumor-immune coordination to promote metastasis.

cancer biology↗