Biology subjects

Kilic, O.

Publications and source records attributed to Kilic, O..

5 recordsLinked to original sources

bioRxiv2026

A Spatio-Temporal Analysis Framework for Characterizing Radiation-Induced Genomic Instability

Chronic low-dose ionizing radiation induces complex genomic instability encompassing both structural variants and point mutations, yet these alterations are typically analyzed as independent events, limiting detection of mechanistic coupling between rearrangement formation and localized mutagenesis at breakpoint junctions. This gap is particularly consequential given the widespread occupational and environmental exposure contexts such as nuclear energy, medical imaging, and environmental contamination, where coupled genomic alterations may contribute to cancer risk through mechanisms invisible to type-agnostic analyses. We developed an integrated analytical framework combining temporal pattern tracking, breakpoint-proximal mutation enrichment analysis, and systematic testing across all structural variant types to resolve these coupled dynamics across dose and time. Applying this framework to whole-genome sequencing data from primary human endothelial cells (HUVEC) exposed to chronic low-dose gamma radiation (0.20-2.62 mGy/hr) over three weeks, we identified inversion-specific mutagenic coupling; doublet base substitutions (DBS) were 7.13-fold enriched within 10bp of inversion breakpoints, a signal absent from other structural variant types, with sharp distance-dependent decay indicating localized mutagenesis at these junctions. Temporal analysis further revealed divergent fates of co-occurring alterations: inversions appeared transiently while DBS mutations showed greater persistence. These results illustrate how systematic integration of dose, time, and variant-type dimensions can uncover coupled mutagenic mechanisms that remain invisible in static or type-agnostic analyses. The framework is broadly applicable to longitudinal sequencing studies of genotoxic exposures, with applications to cancer genomics, radiation risk assessment, and mechanistic studies of DNA repair fidelity.

bioinformatics↗

bioRxiv2025

A differentiated and durable allogeneic strategy applicable to cell therapies

Autologous T cell therapies have shown profound clinical responses; however, their widespread use has been limited primarily due to their individualized manufacturing requirements. To develop a persistent "off-the-shelf" allogeneic (Allo) approach, a multiplex Nme2Cas9-based cytosine base editor was deployed to knockout select HLA Class I and II alleles (HLA-A, HLA-B, and the class II transactivator (CIITA)), while retaining HLA-C to protect from NK cell rejection. Matching the residual HLA-C allele from homozygous donors to the host prevented rejection of the donor T cells by allogeneic host T and NK cells. Site-specific integration of a tumor-specific CAR or TCR into the TRAC locus using SpyCas9 nuclease and an adeno-associated virus (AAV) template allowed for high localized insertion rate while simultaneously removing the endogenous TCR and preventing GvHD. Using a lipid nanoparticle (LNP)-based delivery system of the editing components enabled a robust cell engineering process, achieving high editing rates and cell expansion. These allogeneic T cells demonstrated comparable functional activity to their autologous counterparts in preclinical assays. Moreover, this gene editing approach generated cells with minimal chromosomal aberrations. The Allo strategy has also been applied to induced pluripotent stem cells (iPSCs), suggesting potential applications in regenerative medicine applications.

bioengineering↗

bioRxiv2023

Enhancing In Vitro SMN Protein Expression and Cell Viability through Xeno-Nucleic Acid-Based ASOs in Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA) stands as a devastating ailment arising from the dearth of functional SMN (Survival Motor Neuron) protein due to genetic anomalies within the SMN1 gene. This condition is marked by the consequential attrition of motor neurons, precipitating a progressive decline in muscular strength and culminating in the disruption of neuromuscular junctions. Existing therapeutic approaches encompassing Zolgensma, Nursinersen, and Evrysdi employ innovative genetic therapeutic strategies involving transgene delivery, Antisense Oligonucleotide (ASO) technology, and modulation of pre-mRNA processing to enhance functional SMN protein expression. However, the ASO therapeutics remain suboptimal in establishing a sustained panacea for SMA, as they inadequately maintain consistent levels of functional SMN protein expression. In this study, we present a discerning inquiry into focusing on XNA-DNA-ASO products that exhibit enhanced safety and stability compared to conventional DNA/RNA-ASO sequences. Through precise targeting of the ISSN-1 region within SMN2 genes intron 7, our approach seeks to amplify SMN protein expression. Employing Xeno Nucleic Acid (XNA) bases, known for their augmented hydrophobicity and stability, our strategy surmounts previous limitations associated with chemical modifications, showcasing heightened endonuclease resistance. Comparative analyses with conventional DNA/RNA-ASO products substantiate the superiority of XNA-DNA-ASO sequences, underscoring elevated SMN protein expression and reduced toxicity. In a comprehensive evaluation, our gene therapy paradigm is scrutinized within a type 1 SMA fibroblast cell line. Utilizing diverse analytical methodologies, encompassing Annexin V-PI analysis for cytotoxicity, MTT assays for mitochondrial activity, and flow cytometry for SMN protein expression profile, we gauge therapeutic impact and potential toxicity. In conclusion, our investigation not only spotlights the promise of XNA-DNA-ASO sequences but also holds implications for refining SMA treatment strategies, converging on minimized dosages, lowered toxicity, and heightened therapeutic efficacy, thus shaping the landscape of gene therapy for SMA.

genetics↗

bioRxiv2022

Macro-Chemical Biology: Engineering Biomimetic Trogocytosis with Farnesylated Chemically Self-Assembled Nanorings

With the recent success of cell-based therapies, there has been a rapidly emerging interest in the engineering of cell-cell interactions and communications. Inspired by the natural intercellular material transfer process of trans-endocytosis or trogocytosis, we proposed that targeted farnesylated chemically self-assembled nanorings (farnesyl-CSANs) could serve as a biomimetic trogocytosis vehicle for engineering directional cargo transfer between cells; thus, allowing cell-cell interactions to be monitored, as well as facilitating communication between the cells by delivery of bioactive species. The membranes of sender cells were stably modified by hydrophobic insertion with the targeted farnesyl-CSANs and to be efficiently transferred to receiver cells expressing the appropriate receptor by endocytosis. CSAN-assisted cell-cell cargo transfer (C4T) was demonstrated to be receptor-specific and dependent on direct cell-cell interactions, the rate of receptor internalization and the amount of receptor expression. In addition, C4T was shown to facilitate cell-to-cell delivery of an apoptosis inducing drug, as wells as antisense oligonucleotides (ASO). Taken together, the C4T approach is a potentially versatile biomimetic trogocytosis platform that can be used to monitor cell-cell interactions, as well as the engineering of cell-cell communications, such as cell-based drug delivery.

bioengineering↗

bioRxiv2020

Anti-EGFR Fibronectin Bispecific Chemically Self-Assembling Nanorings (CSANs) Induce Potent T cell Mediated Anti-Tumor Response and Downregulation of EGFR Signaling and PD-1/PD-L1 Expression

Numerous approaches have targeted the Epidermal Growth Factor Receptor (EGFR) for the development of anti-cancer therapeutics, since it is over-expressed on a variety of cancers. Recently, EGFR chimeric antigen receptor (CAR)-T cells have shown potential promise for the immunological control of tumors. Our laboratory has recently demonstrated that bispecific chemically self-assembled nanorings (CSANs) can modify T cell surfaces and function as prosthetic antigen receptors (PARs). This technology allows selective targeting of tumor antigens due to high avidity of the multimeric rings, while incorporating a mechanism to dissociate the rings to prevent further T cell stimulation. Previously, PARs with single-chain variable fragments (scFvs) have been successful in vitro and in vivo, activating T cells selectively at the tumor site. Alternatively, here we report fibronectin (FN3)-based PARs with improved properties such as increased protein yield, rapid protein production, increased protein stability and predicted low immunogenicity due to the human origin of fibronectins. We examined the cytotoxicity of EGFR-targeting PARs in vitro in which the affinities of the EGFR fibronectins, the EGFR/ CD3 valency of the CSANs and the antigen expression levels were varied. Based on these selective in vitro cytotoxicity results, we conducted an in vivo study of FN3-PARs using an orthotopic breast cancer model. The FN3-PARs demonstrated potent tumor growth suppression with no adverse effects. Furthermore, these results demonstrated that FN3-PARs modulated the tumor microenvironment by downregulating EGFR signaling resulting in decreased PD-L1 expression. In addition, the expression of PD-1 was also found to be reduced. Collectively, these results demonstrate that FN3-PARs have the potential to direct selective T cell targeted tumor killing and that EGFR FN3-PARs may enhance anti-tumor T cell efficacy by modulating the tumor microenvironment.

bioengineering↗