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Multiplexed epigenetic memory editing using CRISPRoff sensitizes glioblastoma to chemotherapy
Katie Lin1,2, Christopher Zou1,2, Akane Hubbard1,2
1Department of Neurological Surgery, University of California San Francisco, San Francisco, California, USA.
Background:
Glioblastoma (GBM) carries a poor prognosis, and new therapeutic strategies are necessary to improve outcomes for patients with this disease. Alkylating chemotherapies including temozolomide (TMZ) and lomustine (CCNU) are critical for treating GBM, but resistance mechanisms, including hypomethylation of O6-methylguanine-DNA methyltransferase (MGMT) promoter, undermine treatment. CRISPRoff is a programmable epigenetic memory editor that can induce stable and heritable gene silencing after transient delivery, and we hypothesize that CRISPRoff could potentiate the activity of TMZ and CCNU through long-term suppression of target genes.
Methods:
We transiently delivered CRISPRoff mRNA along with sgRNAs against target genes using both electroporation and lipid nanoparticles (LNPs) into established GBM cell lines, patient-derived primary GBM cultures, and orthotopic GBM xenografts. Gene repression, specificity, and stability were measured by RT-qPCR, Western blot, bisulfite sequencing, and RNA sequencing. Sensitivity to chemotherapies was measured by cell viability dose-response, microscopy, and bioluminescence imaging. Genome-wide mapping of CCNU sensitizers was performed using CRISPRi screens.
Results:
CRISPRoff induced complete suppression of MGMT and sensitization to TMZ that was stable for over 8 months of continuous cell propagation. GBM orthotopic tumors treated with CRISPRoff against MGMT demonstrated sensitivity to TMZ in vivo, and CRISPRoff delivery resulted in chemosensitivity in patient-derived primary GBM. Genome-wide CRISPRi screens identified combinatorial genetic vulnerabilities (BRIP1, FANCE) that were targetable by multiplexed CRISPRoff to achieve sensitization to CCNU.
Conclusion:
Transient delivery of a site-specific epigenetic memory can induce stable, complete, and multiplexed suppression of target genes for therapeutic application in GBM.
Insights
CRISPRoff epigenetically silences MGMT, enhancing chemotherapy for glioblastoma. This programmable gene silencing offers a stable, long-term strategy to improve treatment outcomes for this aggressive brain cancer.
Area of Science:
- Epigenetics
- Gene editing
- Oncology
Background:
- Glioblastoma (GBM) has a poor prognosis, necessitating novel therapeutic strategies.
- Resistance to alkylating chemotherapies like temozolomide (TMZ) and lomustine (CCNU) is a major challenge in GBM treatment.
- O6-methylguanine-DNA methyltransferase (MGMT) promoter hypomethylation is a key resistance mechanism.
Purpose of the Study:
- To investigate the potential of CRISPRoff, a programmable epigenetic editor, to enhance the efficacy of TMZ and CCNU in GBM.
- To assess the stability and heritability of gene silencing induced by CRISPRoff.
- To explore CRISPRoff's ability to overcome chemotherapy resistance in GBM.
Main Methods:
- Transient delivery of CRISPRoff mRNA and sgRNAs via electroporation and lipid nanoparticles (LNPs) into GBM cell lines, primary cultures, and xenografts.
- Assessment of gene repression, specificity, and stability using RT-qPCR, Western blot, bisulfite sequencing, and RNA-sequencing.
- Evaluation of chemotherapy sensitivity through cell viability assays, microscopy, and bioluminescence imaging; genome-wide CRISPRi screens identified CCNU sensitizers.
Main Results:
- CRISPRoff achieved stable, complete suppression of MGMT for over 8 months, leading to TMZ sensitization in vitro.
- In vivo studies showed CRISPRoff-mediated MGMT suppression sensitized orthotopic GBM xenografts to TMZ.
- CRISPRoff delivery enhanced chemosensitivity in patient-derived GBM, and multiplexed CRISPRoff targeted identified vulnerabilities for CCNU sensitization.
Conclusions:
- Transient delivery of CRISPRoff establishes stable, site-specific epigenetic memory for therapeutic gene silencing in GBM.
- This approach offers a promising strategy for potentiating chemotherapy and overcoming resistance in glioblastoma.
- CRISPRoff enables multiplexed gene suppression for combinatorial therapeutic applications.
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