Biology subjects

Dogan, M.

Publications and source records attributed to Dogan, M..

8 recordsLinked to original sources

bioRxiv2025

Comparative Landscape of Small RNAs in Tissue and Liquid Biopsies for Liver Transplant Outcomes

BackgroundIschemia-reperfusion injury (IRI) is an inevitable consequence of liver transplantation, arising during donor organ procurement and reoxygenation. Severe IRI is a leading contributor to early allograft dysfunction (EAD), a post-transplant complication associated with reduced graft survival. Current postoperative biomarkers provide limited time for intervention, highlighting a need to identify preoperative biomarkers of IRI. Meanwhile, tRNA fragments (tRFs) have emerged as novel biomarkers in various diseases but remain unexplored in the context of liver transplant. ResultsWe performed small RNA sequencing on 96 paired donor liver biopsies from 48 patients to investigate IRI-associated transcript changes. In parallel, 161 donor liver perfusates were analyzed as a non-invasive surrogate for tissue. Across samples, microRNAs (miRNAs) and tRFs were the most abundant. Perfusate expression strongly correlated with biopsies, supporting their value as a non-invasive source of small RNAs. Comparison between post-reperfusion and pre-implantation biopsies revealed that IRI reprogrammed tRF expression. Stratification by clinical outcome showed that patients who developed EAD exhibited specific small RNA signatures in both biopsy and perfusate. Receiver operating characteristic (ROC) analysis revealed a miRNA-based model that achieved an AUC of 0.772, outperforming donor risk index alone (AUC = 0.665), representing a 10.7% increase in discriminative capacity. ConclusionsThese results are the first to establish tRFs as IRI-responsive biomolecules abundant in both donor liver tissue and non-invasive perfusate. In particular, various small RNAs emerged as promising candidate biomarkers for early detection of EAD. These results lay the foundation to further investigate the prognostic utility of tRFs/miRNAs in liver transplantation.

genomics↗

bioRxiv2024

Removal of the catalytic subunit of DNA-protein kinase in the proximal tubules promotes DNA and tubular damage during kidney injury

Tubular epithelial cell damage can be repaired through a series of complex signaling pathways. An early event in many forms of tubular damage is the observation of DNA damage, which can be repaired by specific pathways depending upon the type of genomic alteration.. In this study, we report that the catalytic subunit of DNA protein kinase (DNA-PKcs), a central DNA repair enzyme involved in sensing DNA damage and performing double stranded DNA break repair, plays an important role in the extent of tubular epithelial cell damage following exposure to injurious acute and chronic stimuli. Selective loss of DNA-PKcs in the proximal tubules led to increased markers of kidney dysfunction, DNA damage, and tubular epithelial cell injury in multiple models of acute kidney injury, specifically bilateral renal ischemia-reperfusion injury and single dose of cisplatin (15 mg/kg IP). In contrast, in a mouse model of kidney fibrosis and chronic kidney disease (UUO),the protective effects of DNA-PKcs was not as obvious histologically from the tissue sections. In the absence of proximal tubular DNA-PKcs, there was reduced levels of fibrotic markers, -SMA and fibronectin, which suggests that there may be a biphasic role of DNA-PKcs depending upon the conditions exerted upon the kidney. In conclusion, this study demonstrates that the catalytic subunit of DNA-PKcs plays a context-dependent role in the kidney to reduce DNA damage during exposure to various types of acute, but not chronic forms of injurious stimuli.

physiology↗

bioRxiv2024

Engineering programmable CAR-T cells with tunable controls as fail-safe mechanisms during cancer immunotherapy

Chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable efficacy in treating various cancers, but significant risks of severe adverse effects remain. In this study, we present a synthetic biology toolbox for engineering CAR-T cells with tunable and controllable features to mitigate these risks. Using this toolbox, we programmed cytotoxic CAR-T cells to be turned on and off using synthetic genes with tetracycline response elements, referred to as iOnCAR and iOffCAR. We achieved temporal regulation of CAR expression using the synthetic Notch receptor system and "AND-gate" programs, which allow for additional control using doxycycline. We also engineered cooperation among T cells through CD4 help to CD8 T cells in targeting cancer cells via synthetic Notch receptors controlled by doxycycline treatment. Additionally, we developed a T cell fratricide system by triggering a genetic "Battle Royale" mechanism that enables CAR-T cells to eliminate each other. In this system, surviving T cells become activated, proliferate, and display high cytotoxicity to target cells. In conclusion, our synthetic biology toolbox provides targeted fail-safe solutions for improving the safety and potency of CAR-T cell therapies by integrating different genetic circuits with a commonly used antibiotic.

immunology↗

bioRxiv2023

Unveiling APOL1 Haplotypes: A Novel Classification Through Probe-Independent Quantitative Real-Time PCR

IntroductionApolipoprotein-L1 (APOL1) is a primate-specific protein component of high- density lipoprotein (HDL). Two variants of APOL1 (G1 and G2), provide resistance to parasitic infections in African Americans but are also implicated in kidney-related diseases and transplant outcomes in recipients. This study aims to identify these risk variants using a novel probe- independent quantitative real-time PCR method in a high African American recipient cohort. Additionally, it aims to develop a new stratification approach based on haplotype-centric model. MethodsGenomic DNA was extracted from recipient PBMCs using SDS lysis buffer and proteinase K. Quantitative PCR assay with modified forward primers and a common reverse primer enabled us to identify single nucleotide polymorphisms (SNPs) and the 6-bp deletion quantitatively. Additionally, we used sanger sequencing to verify our QPCR findings. ResultsOur novel probe-independent qPCR effectively distinguished homozygous wild-type, heterozygous SNPs/deletion, and homozygous SNPs/deletion, with at least 4-fold differences. High prevalence of APOL1 variants was observed (18% two-risk alleles, 34% one-risk allele) in our recipient cohort. Intriguingly, up to 12-month follow-up revealed no significant impact of recipient APOL1 variants on transplant outcomes. Ongoing research will encompass more time points and a larger patient cohort, allowing a comprehensive evaluation of G1/G2 variant subgroups categorized by new haplotype scores, enriching our understanding. ConclusionsOur cost-effective and rapid qPCR technique facilitates APOL1 genotyping within hours. Prospective and retrospective studies will enable comparisons with long-term allograft rejection, potentially predicting early/late-stage transplant outcomes based on haplotype evaluation in this diverse group of kidney transplant recipients.

genetics↗

bioRxiv2022

Engineering human Mucosal Associated Invariant T (MAIT) cells with chimeric antigen receptors for cancer immunotherapy

Engineering immune cells with chimeric antigen receptors (CAR) is a promising technology in cancer immunotherapy. Besides classical cytotoxic CD8+ T cells, innate cell types such as NK cells have also been used to generate CAR-T or CAR-NK cells. Here we devised an approach to program a non-classical cytotoxic T cell subset called Mucosal Associated Invariant T (MAIT) cells into effective CAR-T cells against B cell lymphoma and breast cancer cells. Accordingly, we expressed anti-CD19 and anti-Her2 CARs in activated primary human MAIT cells and CD8+ T cells, expanded them in vitro and compared their cytotoxicity against tumor cell targets. We show upon activation through CARs, CAR-MAIT cells exhibit high levels of cytotoxicity towards target cells, comparable to CD8+ CAR-T cells, but interestingly expressed lower levels of IFN-{gamma} than conventional CAR CD8+ T cells. Additionally, in the presence of vitamin B2 metabolite 5-ARU, which is a conserved compound that activates MAIT cells through MHC-I related (MR1) protein, MAIT cells killed MR1-expressing target breast cancer and B cell lymphoma cell lines in a dose dependent manner. Thus, MAIT cells can be genetically edited as CAR-T cells or mobilized and expanded by MR1 ligands as an off-the-shelf novel approach to cell-based cancer immunotherapy strategies while being comparable to conventional methods in effectivity. Key PointsO_LIMAIT cells expressing CARs effectively kill CD19 and Her2 expressing targets C_LIO_LICAR-MAITs display strong cytotoxicity with lower TNF-{gamma} compared to CD8+ CAR-T cells C_LI

immunology↗

bioRxiv2022

3D Bioprinted perfusable and vascularized breast tumor model for dynamic screening of chemotherapeutics and CAR-T cells

Despite substantial advancements in development of cancer treatments, lack of standardized and physiologically-relevant in vitro testing platforms limit the rapid and early screening of anti-cancer agents. A major barrier in this endeavor, is the complex interplay between the tumor microenvironment and host immune response and lack of predictive biomarkers for clinical benefit. To tackle this challenge, we have developed a dynamic-flow based three-dimensionally (3D) bioprinted vascularized breast tumor model, responding to chemo and immunotherapeutic treatments. Heterotypic tumor spheroids, comprising metastatic breast cancer cells (MDA-MB-231), human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs), precisely bioprinted at pre-defined distances from a perfused vasculature, exhibited tumor angiogenesis and cancer invasion. Proximally bioprinted tumors ([~]100 m) exhibited enhanced capillary sprouting, anastomosis to perfused vasculature and increased cancer cell migration as compared to distally bioprinted spheroids ([~]500 m). Proximally bioprinted tumors treated with varying dosages of doxorubicin for 72 h enabled functional analysis of drug response, wherein, tumors portrayed a dose-dependent drug response behavior with [~]70% decrease in tumor volume for 1 M dose. Additionally, a cell based immune therapy approach was explored by perfusing HER2-targeting chimeric antigen receptor (CAR) modified CD8+ T cells for 24 or 72 h through the central vasculature. Extensive CAR-T cell recruitment to the endothelium and substantial T cell activation and infiltration in the tumor site, resulted in [~]70% reduction in tumor growth for high CAR treatment densities, after 72 h of treatment. The presented 3D model paves the way for a robust, precisely fabricated and physiologically-relevant 3D tumor microenvironment platform for future translation of anti-cancer therapies to personalized medicine for cancer patients. One Sentence SummaryA physiologically-relevant 3D bioprinted perfusable vascularized tumor model capable of dynamic screening of chemo and immunotherapeutics.

bioengineering↗

bioRxiv2022

Development of 3D breast cancer models with human T cells expressing engineered MAIT cell receptors

Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune-cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we developed proof-of-concept 3D tumor models with increasing complexity to study the cytotoxic responses of CD8+ T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast (HDF) tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3-day culture period, demonstrating that the engineered TCR recognized MR1 expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as IFN{gamma}, IL-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune-cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune-cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies.

immunology↗

bioRxiv2022

Targeting SARS-CoV-2 infection through CAR T cells and bispecific T cell engagers

Despite advances in antibody treatments and vaccines, COVID-19 caused by SARS-CoV-2 infection remains a major health problem resulting in excessive morbidity and mortality and the emergence of new variants has reduced the effectiveness of current vaccines. Here, as a proof-of-concept we engineered primary CD8 T cells to express SARS-CoV-2 Spike protein-specific CARs, using extracellular region of ACE2, and demonstrated their highly specific and potent cytotoxicity towards Spike-expressing target cells. To improve on this concept as a potential therapeutic, we developed a bispecific T cell engager combining ACE2 with an anti-CD3 scFv (ACE2-Bite) to target infected cells and the virus. Similar to CAR-T cell approach, ACE2-Bite endowed cytotoxic cells to selectively kill Spike-expressing targets. Furthermore, ACE2-Bite neutralized the pseudoviruses of SARS-CoV, SARS-CoV-2 wild-type and variants including Delta and Omicron, as a decoy protein. Remarkably, ACE2-Bite molecule showed a higher binding and neutralization affinity to Delta and Omicron variants compared to SARS-CoV-2 wild-type Spike proteins, suggesting the potential of this approach as a variant-proof, therapeutic strategy for future SARS-CoV-2 variants, employing both humoral and cellular arms of the adaptive immune response.

bioengineering↗