Biology subjects

Karni, R.

Publications and source records attributed to Karni, R..

10 recordsLinked to original sources

bioRxiv2026

Targeting the 3 splice site by a decoy oligonucleotide attenuates U2AF1 splicing activity and inhibits leukemia

Recurrent mutations in spliceosomal genes are a hallmark of myeloid malignancies, with SF3B1, SRSF2, U2AF1 and ZRSR2 among the most frequently affected. These alterations are typically heterozygous, mutually exclusive missense mutations targeting highly conserved residues, reflecting a selective pressure to maintain a dysregulated yet essential splicing machinery. This constraint suggests that leukemic cells remain dependent on residual splicing activity, exposing a potential therapeutic vulnerability that extends beyond genetically defined subsets. For example, a previously developed therapeutic, Pladienolide B, is a potent cancer cell growth inhibitor targeting the SF3B1 subunit of the spliceosome1,2. Here we present an RNA decoy-based strategy to disrupt 3' splice site recognition by competitively engaging components of the spliceosomal machinery. We engineered a chemically stabilized RNA decoy that mimics the 3 splice site (3'SS decoy), thereby sequestering proteins involved in 3 splice site recognition from endogenous pre-mRNA targets. Although the decoy is expected to engage multiple components of the 3' splice site recognition complex, U2AF1 was used as the primary molecular readout to assess target engagement and downstream effects. To enable intracellular delivery, decoys were encapsulated in lipid nanoparticles (LNPs), facilitating efficient uptake in leukemic systems. We show that LNP-encapsulated decoys are efficiently delivered into leukemic cells, including established cell lines and patient-derived blasts, and directly engage components of the splicing machinery. Decoy treatment induces widespread alterations in RNA splicing programs and impairs leukemic cell fitness in vitro. Importantly, systemic administration of the 3SS decoy significantly reduces leukemia burden in an in vivo xenograft model. Notably, these effects are observed independently of spliceosomal mutational status, supporting a broader dependency of leukemic cells on intact splicing factor function. Together, our findings establish decoy-mediated disruption of splicing factor activity as a mechanistically targeted therapeutic strategy and identify LNPs as an effective platform for the delivery of RNA-based modulators of essential RNA-protein interactions in myeloid malignancies.

molecular biology↗

bioRxiv2026

Allele-specific antisense oligonucleotide treatment rescues atad3-associated phenotype in zebrafish

Pathogenic variants in ATAD3A cause a spectrum of multisystem disorders, with a recurrent dominant-negative variant (c.1582C>T; p.Arg528Trp) associated with neurodevelopmental disease. Given the tolerance of ATAD3A to heterozygous loss of function variants, allele-specific transcript reduction represents a promising therapeutic strategy. We designed and optimized allele-specific antisense oligonucleotides (ASOs) targeting the c.1582C>T transcript and evaluated their efficacy and specificity in affected fibroblasts using allele-specific primers and amplicon-based next generation sequencing. Therapeutic potential was further assessed in vivo in zebrafish embryos expressing human wild-type or mutant ATAD3A transcripts. An optimized gapmer ASO selectively reduced mutant ATAD3A transcripts while relatively sparing the wild-type allele. In addition to RNase H-mediated degradation, the ASO induced exon skipping, leading to degradation of the aberrant transcript without production of a truncated protein. In zebrafish, expression of mutant human ATAD3A in embryos caused developmental abnormalities including reduced eye size, which were robustly rescued by co-injection of the optimized ASO. Our findings provide proof of concept for allele-targeted ASO therapy for dominant-negative ATAD3A variants. This work highlights the therapeutic potential of ASOs for rare dominant disorders involving genes tolerant to heterozygous loss-of-function, and establishes zebrafish as a versatile platform for in vivo ASO optimization.

genetics↗

bioRxiv2026

FTO regulates centrosome function and mitotic fidelity in cancers with chromosome instability (CIN)

Centrosomes are essential for spindle assembly and accurate chromosome segregation during mitosis. We found that the RNA demethylase FTO localizes to centrosomes in both human and mouse cells. Disruption of FTO function through a newly developed small-molecule inhibitor impaired spindle organization and centrosome function. This was evidenced by chromosome misalignment during mitosis, prometaphase arrest, followed by mitotic slippage or apoptosis. Mechanistically, we found that FTO is essential for intact localization of NuMA and KIFC1 proteins required for chromosome localization during mitosis. Importantly, cancers characterized by chromosomal instability (CIN) showed increased sensitivity to FTO inhibition compared to normal cells. These findings underscore FTO as a promising therapeutic target in CIN cancer subsets.

cancer biology↗

bioRxiv2026

Molecular characterization of the heterozygous loss of function mutations in the X-linked PCDH19 gene causing PCDH19-Cluster Epilepsy

PCDH19-Cluster Epilepsy (PCDH19-CE) is a rare neurological disorder caused by mutations in the PCDH19 (Protocadherin-19) gene and is characterized by early-onset seizures and cognitive impairment. In contrast to most X-linked disorders, PCDH19 mutations predominantly affect heterozygous females, while hemizygous males are largely spared. Although advances have been made to understand the pathological mechanism underlying PCDH19-CE, key downstream targets and compensatory pathways are yet to be elucidated. Using CRISPR/Cas9 technology, we generated both a mouse model of PCDH19-CE and a human embryonic stem cell (ESC) model. Transcriptomic analysis identified genes that were differentially expressed in the brains of heterozygous (Pcdh19WT/mut) female mice compared with wildtype (WT) and homozygous (Pcdh19mut/mut) female mice. Pathway analysis of these differentially expressed genes (DEGs) revealed enrichment in pathways involved in neuronal development, ion channel activity, synaptic development and neuronal signalling. Neurons differentiated from human ESCs carrying a PCDH19 mutation exhibited similar gene expression patterns, with heterozygous neurons displaying a distinct expression pattern compared to both WT and homozygous mutant neurons. In contrast to the molecular phenotype, neurons derived from homozygous mutant cells showed highly elongated neurites while neurons from heterozygous cells showed intermediate neurite elongation. This suggests that neurite morphology correlates directly with levels of WT PCDH19. Overall, our findings indicate that heterozygous PCDH19 mutations are associated with defects in the expression of genes involved in developmental, signalling, and neuronal pathways in both mouse and human disease models, while certain morphological phenotypes appear to depend on the levels of WT PCDH19.

genetics↗

bioRxiv2025

RNA splicing dynamics in CD8 T cells uncovers isoforms that impact T cell-mediated cancer immunotherapy

Immune checkpoint blockade has transformed cancer therapy, yet many patients fail to respond, and few new targets have emerged beyond PD-1 and CTLA-4. Alternative splicing dramatically diversifies the T cell proteome, but the functional roles of most isoforms remain unknown. Here we constructed the first single-cell splicing atlas of human CD8 T cells, capturing dynamic isoform programs across activation and subset differentiation. This revealed distinct splicing footprints that refine conventional transcriptomic states and highlight receptor families with isoform-level regulation. To functionally interrogate these candidates, we developed SpliceSeek, a CRISPR-based pooled screening platform that perturbs splice sites to redirect isoform usage. Using SpliceSeek, we uncovered isoform-specific immune checkpoints whose perturbation enhanced effector function and tumor control, including the LRRN3-203 isoform, which augmented cytokine secretion and antitumor immunity in mice models. Together, our results establish alternative splicing as a targetable layer of immune regulation and demonstrate the potential of isoform-focused screening to expand the landscape of cancer immunotherapy.

immunology↗

bioRxiv2025

Augmentation of self-motion perception with synthetic auditory cues

People who suffer from vestibular loss or damage have difficulty maintaining balance and perceiving their own motion in space (self-motion). Sensory augmentation of vestibular information, via other senses, could improve these functions. Here, we tested whether synthetic auditory signals carrying self-motion information can be integrated with vestibular cues. Twenty healthy participants experienced self-motion stimuli in a 3D motion simulator, comprising vestibular (inertial motion), and/or synthetic auditory "motion" cues. The auditory cues, presented via stereo headphones, comprised a series of beeps, with motion speed encoded by beep rate, and heading direction (in the horizontal plane) encoded by simulating the sound to emanate from that direction. In each trial, participants experienced a single-interval self-motion stimulus (vestibular, auditory or combined), and their task was to discriminate its heading direction (two-alternative forced-choice, right/left of straight ahead). A slight heading conflict ({Delta} = {+/-}6{degrees}) was introduced in the combined-cue condition to measure empirical cue weights. Combined auditory-vestibular thresholds were significantly lower (improved) compared to vestibular alone (p < 0.001). But integration was suboptimal - vestibular cues were overweighted, and combined-cue thresholds were larger than predicted by Bayesian-optimal weighting. Interestingly, combined-cue thresholds were better predicted by the empirically observed weights. Thus, humans can integrate synthetic auditory cues with natural vestibular cues to improve self-motion perception. However, synthetic cues are underweighted. This suggests that weighting is determined not only by cue reliability but also by perceived relevance, with (unnatural) synthetic cues potentially considered less relevant to the task. Training might be required to achieve reliability-based integration of synthetic and natural cues.

animal behavior and cognition↗

bioRxiv2024

The prevalent NR2E3 c.932G>A mutation induces aberrant splicing that can be rescued using splice-shifting antisense oligonucleotides

Mutations in NR2E3 have been implicated in several progressive retinal disease phenotypes such as enhanced S-cone syndrome, Goldmann-Favre syndrome and retinitis pigmentosa. One of the most frequent mutations in NR2E3 is c.932G>A (p.R311Q), where pathogenicity is thought to stem from the resulting amino acid substitution. However, multiple studies that evaluated the effect of this substitution on the protein, did not elucidate the molecular basis underlying the pathogenicity. Primed by bioinformatic analyses, we hypothesized and experimentally validated that the NR2E3 c.932G>A mutation leads to aberrant splicing which results in a short, non-functional protein isoform. Using cell models expressing WT and mutant constructs of the full NR2E3 sequence (including exonic and intronic regions), we observed that the mutated transcript exhibits a high level (75%) of aberrant splicing through gain of a novel splice acceptor site within exon 6. This mis-splicing results in the in-frame loss of 186 base pairs that code for a portion of the protein ligand binding domain. We further designed and evaluated splice-shifting antisense oligonucleotides (ASOs), that circumvented the aberrant splicing. The best performing ASO successfully restored 70% of the total NR2E3 full-length isoform levels and demonstrated rescue of nuclear localization and rhodopsin transcriptional activation. This study demonstrates the importance of understanding splicing consequences of pathogenic mutations, allowing the design and development of ASO-based therapies. Our findings set the stage for the potential treatment of NR2E3-related retinal degeneration caused by the c.932G>A mutation using splice-shifting ASOs.

genomics↗

bioRxiv2022

S6K1 phosphorylates Cdk1 and MSH6 to regulate DNA repair.

The mTORC1 substrate, S6 Kinase 1 (S6K1), is involved in the regulation of cell growth, ribosome biogenesis, glucose homeostasis and adipogenesis. Accumulating evidence has suggested a role for mTORC1 signaling in the DNA damage response. This is mostly based on the findings that mTORC1 inhibitors sensitized cells to DNA damage. However, a direct role of the mTORC1-S6K1 signaling pathway in DNA repair and the mechanism by which this signaling pathway regulates DNA repair is unknown. In this study, we discovered a novel role for S6K1 in regulating DNA repair through the coordinated regulation of the cell cycle, homologous recombination (HR) DNA repair (HRR) and mismatch DNA repair (MMR) mechanisms. Here we show that S6K1 orchestrates DNA repair by phosphorylation of Cdk1 at serine 39, causing G2/M cell cycle arrest enabling homologous recombination and by phosphorylation of MSH6 at serine 309, enhancing MMR. Moreover, breast cancer cells harboring RPS6KB1 gene amplification show increased resistance to several DNA damaging agents and S6K1 expression is associated with poor survival of breast cancer patients treated with chemotherapy. Our findings reveal an unexpected function of S6K1 in the DNA repair pathway, serving as a tumorigenic barrier by safeguarding genomic stability.

cancer biology↗

bioRxiv2020

Overconfidence in visual perception in Parkinson's disease

BackgroundIncreased dependence on visual cues in Parkinsons disease (PD) can unbalance the perception-action loop, impair multisensory integration, and affect everyday function of PD patients. It is currently unknown why PD patients seem to be more reliant on their visual cues. ObjectivesWe hypothesized that PD patients may be overconfident in the reliability (precision) of their visual cues. In this study we tested coherent visual motion perception in PD, and probed subjective (self-reported) confidence in their visual motion perception. Methods20 patients with idiopathic PD, 21 healthy aged-matched controls and 20 healthy young adult participants were presented with visual stimuli of moving dots (random dot kinematograms). They were asked to report: (1) whether the aggregate motion of dots was to the left or to the right, and (2) how confident they were that their perceptual discrimination was correct. ResultsVisual motion discrimination thresholds were similar (unimpaired) in PD compared to the other groups. By contrast, PD patients were significantly overconfident in their visual perceptual decisions (p=0.002 and p<0.001 vs. the age-matched and young adult groups, respectively). ConclusionsThese results suggest intact visual motion perception, but overestimation of visual cue reliability, in PD. Overconfidence in visual (vs. other, e.g., somatosensory) cues could underlie accounts of increased visual dependence and impaired multisensory integration in PD, and could contribute to gait and balance impairments. Future work should investigate PD confidence in somatosensory function. A better understanding of altered sensory reliance in PD might open up new avenues to treat debilitating symptoms.

neuroscience↗

bioRxiv2020

Alternative splicing of SLAMF6 in human T cells creates a co-stimulatory isoform that counteracts the inhibitory effect of the full-length receptor

SLAMF6 is a homotypic receptor of the Ig-superfamily associated with progenitor exhausted T cells. In humans, SLAMF6 has three splice isoforms involving its V-domain. While the canonical 8-exon receptor inhibits T cell activation through SAP recruitment, the short isoform SLAMF6{Delta}17-65 has a strong agonistic effect. The costimulatory action depends on protein phosphatase SHP-1 and leads to a cytotoxic molecular profile governed by transcription factors Tbet, Runx3, and Tcf7. In T cells from individual patients treated with immune checkpoint blockade, a shift was noted towards SLAMF6{Delta}17-65. Splice-switching antisense oligonucleotides designed to target the SLAMF6 splice junction, enhanced SLAMF6{Delta}17-65 in human tumor-infiltrating lymphocytes and improved their capacity to inhibit human melanoma in mice. The possible emergence of two opposing isoforms from the SLAMF6 gene may represent an immune-modulatory mechanism that can be exploited for cancer immunotherapy.

immunology↗