Optimized two-step electroporation process to achieve efficient nonviral-mediated gene insertion into primary T cells.

The development of gene editing technologies over the past years has allowed the precise and efficient insertion of transgenes into the genome of various cell types. Knock-in approaches using homology-directed repair and designer nucleases often rely on viral vectors, which can considerably impact the manufacturing cost and timeline of gene-edited therapeutic products. An attractive alternative would be to use naked DNA as a repair template. However, such a strategy faces challenges such as cytotoxicity from double-stranded DNA (dsDNA) to primary cells. Here, we sought to study the kinetics of transcription activator-like effector nuclease (TALEN)-mediated gene editing in primary T cells to improve nonviral gene knock-in. Harnessing this knowledge, we developed a rapid and efficient gene insertion strategy based on either short single-stranded oligonucleotides or large (2 Kb) linear naked dsDNA sequences. We demonstrated that a time-controlled two-step transfection protocol can substantially improve the efficiency of nonviral transgene integration in primary T cells. Using this approach, we achieved modification of up to ˜ 30% of T cells when inserting a chimeric antigen receptor (CAR) at the T-cell receptor alpha constant region (TRAC) locus to generate 'off-the shelf' CAR-T cells.

Genome-wide cell-free DNA mutational integration enables ultra-sensitive cancer monitoring.

In many areas of oncology, we lack sensitive tools to track low-burden disease. Although cell-free DNA (cfDNA) shows promise in detecting cancer mutations, we found that the combination of low tumor fraction (TF) and limited number of DNA fragments restricts low-disease-burden monitoring through the prevailing deep targeted sequencing paradigm. We reasoned that breadth may supplant depth of sequencing to overcome the barrier of cfDNA abundance. Whole-genome sequencing (WGS) of cfDNA allowed ultra-sensitive detection, capitalizing on the cumulative signal of thousands of somatic mutations observed in solid malignancies, with TF detection sensitivity as low as 10-5. The WGS approach enabled dynamic tumor burden tracking and postoperative residual disease detection, associated with adverse outcome. Thus, we present an orthogonal framework for cfDNA cancer monitoring via genome-wide mutational integration, enabling ultra-sensitive detection, overcoming the limitation of cfDNA abundance and empowering treatment optimization in low-disease-burden oncology care.

KDM2B, an H3K36-specific demethylase, regulates apoptotic response of GBM cells to TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells. TRAIL resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at the epigenetic level is not understood. In this study, we investigated novel epigenetic mechanisms regulating TRAIL response in glioblastoma multiforme (GBM) cells by a short-hairpin RNA loss-of-function screen. We interrogated 48 genes in DNA and histone modification pathways and identified KDM2B, an H3K36-specific demethylase, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, the activation of caspase-8, -3 and -7 and PARP cleavage. KDM2B knockdown also accelerated the apoptosis, as revealed by live-cell imaging experiments. To decipher the downstream molecular pathways regulated by KDM2B, levels of apoptosis-related genes were examined by RNA-sequencing upon KDM2B loss, which revealed derepression of proapoptotic genes Harakiri (HRK), caspase-7 and death receptor 4 (DR4) and repression of antiapoptotic genes. The apoptosis phenotype was partly dependent on HRK upregulation, as HRK knockdown significantly abrogated the sensitization. KDM2B-silenced tumors exhibited slower growth in vivo. Taken together, our findings suggest a novel mechanism, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells.

Identification of Mitoxantrone as a TRAIL-sensitizing agent for Glioblastoma Multiforme.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) has tremendous promise in treating various forms of cancers. However, many cancer cells exhibit or develop resistance to TRAIL. Interestingly, many studies have identified several secondary agents that can overcome TRAIL resistance. To expand on these studies, we conducted an extensive drug-re-profiling screen to identify FDA-approved compounds that can be used clinically as TRAIL-sensitizing agents in a very malignant type of brain cancer, Glioblastoma Multiforme (GBM). Using selected isogenic GBM cell pairs with differential levels of TRAIL sensitivity, we revealed 26 TRAIL-sensitizing compounds, 13 of which were effective as single agents. Cardiac glycosides constituted a large group of TRAIL-sensitizing compounds, and they were also effective on GBM cells as single agents. We then explored a second class of TRAIL-sensitizing drugs, which were enhancers of TRAIL response without any effect on their own. One such drug, Mitoxantrone, a DNA-damaging agent, did not cause toxicity to non-malignant cells at the doses that synergized with TRAIL on tumor cells. We investigated the downstream changes in apoptosis pathway components upon Mitoxantrone treatment, and observed that Death Receptors (DR4 and DR5) expression was upregulated, and pro-apoptotic and anti-apoptotic gene expression patterns were altered in favor of apoptosis. Together, our results suggest that combination of Mitoxantrone and TRAIL can be a promising therapeutic approach for GBM patients.

Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase.

The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, > 30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number.