Biology subjects

Thangavel, S.

Publications and source records attributed to Thangavel, S..

7 recordsLinked to original sources

bioRxiv2026

Guide RNA truncation as a tunable approach for modulating base editing outcomes

Base editing enables programmable nucleotide conversion but can be limited by bystander editing and unintended genomic alterations. Here, we investigated guide RNA (gRNA) truncation as a strategy to modulate base editing outcomes. On editing across multiple genomic loci using ABE8e in HUDEP-2 cells, we found that truncated gRNAs retained measurable editing activity at lengths as short as 15 nucleotides, although activity depended strongly on target sequence, gRNA length, and delivery method. Importantly, truncation altered editing efficiencies at individual nucleotides within the editing window, enabling selective reduction of bystander editing while preserving on-target conversion at several loci, including a therapeutically relevant HbE target. Truncated gRNAs also eliminated detectable large deletions at homologous globin loci, consistent with loss of Cas9 nickase activity at short gRNAs lengths. Similar length dependent effects were observed with cytosine base editing, PAM relaxed ABE8e, and in primary hematopoietic stem and progenitor cells (HSPCs), although the optimal gRNA length varied between contexts. Together, these findings establish gRNA length as an additional parameter for tuning nucleotide level base editing outcomes and reducing bystander editing.

bioengineering↗

bioRxiv2025

STAT1-Mediated Regulation of IL-17A/CEBPB/NF-κB Axisin HIV-1 Infected Human Cerebral Organoids RevealsTherapeutic Targets for Neuroprotection

HIV-associated neurological complications remain a major concern in people living with HIV (PLWH), even under effective viral suppression with combination antiretroviral therapy (cART), underscoring unresolved mechanisms driving HIV-related neurodegeneration. To address this gap, we used HIV-1-infected human cerebral organoids (hCOs) containing microglia as a physiologically relevant three-dimensional model of the central nervous system (CNS) and validated key findings in simian immunodeficiency virus (SIV)-infected, cART-treated rhesus macaque model. Integrating single-nucleus RNA sequencing with ATAC sequencing, we identified cell-type-specific alterations in dual innate immune-driven inflammatory signaling pathways across neural and glial populations. Microglia were preferentially infected and activated, initiating IL-17A-mediated cascades involving IFN-{gamma}/STAT1, CEBPB, and NF-{kappa}B pathways, which converged with cGAS-STING and Notch-NEURL1 signaling, both associated with neuroinflammation and synaptic dysfunction. cART suppressed viral replication, reduced IL-17A-driven neuroinflammation, improved mitochondrial function, and restored synaptic gene expression. Collectively, these findings identify IL-17A, STAT1, CEBPB, and cGAS-STING as key molecular drivers of HIV-associated neuroinflammation and establish a novel mechanistic link between IL-17A signaling and microglial remodeling, providing a translational framework for the development of CNS-targeted therapies in PLWH.

neuroscience↗

bioRxiv2023

Editing of highly homologous gamma-globin genes by nickase deficient Base Editor mitigates large intergenic deletions

Base editing in gamma-globin promoter is a promising approach for reactivation of fetal-hemoglobin. Recent studies have shown that base editing could result in genotoxic events at the gamma globin locus including 4.9 kb large deletion of intervening region due to nicking in the paralogous HBG1 and HBG2 genes. Although the deletion frequency is less than what is observed with Cas9, it could diminish the therapeutic potential. We sought to evaluate if large deletion could be overcome while maintaining the editing efficiency by replacing the nCas9 of ABE8e with a catalytically inactive deadCas9 (dCas9). Using 3 therapeutically relevant gRNAs targeting the gamma globin promoter, we performed a comprehensive evaluation of the editing outcome and frequency of large deletion using dCas9, nCas9, dCas9-ABE8e and nCas9-ABE8e. Our findings indicate that while nicking in itself induced large deletions, the frequency reduced upon efficient base editing. Notably, there was no appreciable deletion with the use of dCas9-ABE8e making it a safer approach, in terms of genome integrity, for therapeutic genome editing in the gamma-globin locus. Further, we also demonstrate that dCas9 ABE8e can edit efficiently in primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) to achieve therapeutic benefits.

molecular biology↗

bioRxiv2022

Correlating the differences in the receptor binding domain of SARS-CoV-2 spike variants on their interactions with human ACE2 receptor

Spike protein of SARS-CoV-2 variants play critical role in the infection and transmission through its interaction with hACE2 receptor. Prior findings using molecular docking and biomolecular studies reported varied findings on the difference in the interactions among the spike variants with hACE2 receptor. Hence, it is a prerequisite to understand these interactions in a more precise manner. To this end, firstly, we performed ELISA with trimeric spike proteins of Wild (Wuhan Hu-1), Delta, C.1.2 and Omicron variants. Further, to study the interactions in a more specific manner by mimicking the natural infection, we developed hACE2 receptor expressing HEK-293T cell line and evaluated binding efficiencies of the variants and competitive binding of spike variants with D614G spike pseudotyped virus. In lines with the existing findings, we observed that Omicron had higher binding efficiency compared to Delta in both ELISA and Cellular models. Intriguingly, we found that cellular models could differentiate the subtle differences between the closely related C.1.2 and Delta in their binding to hACE2. From the analysis in receptor binding domain (RBD) revealed that a single common modification, N501Y, present in both Omicron and C.1.2 is driving the enhanced spike binding to the receptor and showed two-fold superior competitive binding than Delta. Our study using cellular model provides a precise method to evaluate the binding interactions between spike sub-lineages to hACE2 receptors and signifies the role of single common modification N501Y in RBD towards imparting superior binding efficiencies. Our approach would be instrumental in understanding the disease progression and developing therapeutics. Author SummarySpike proteins of evolving SARS-CoV2 variants demonstrated their signature binding to hACE2 receptor, in turn contributed to driving the infection and transmission. Prior studies to scale the binding efficiencies between the spike variant and the receptor had consensus in distinct variants, but discrepancies in the closely related ones. To this end, we compared spike variants-receptor interactions with ELISA, from cells expressing hACE2 receptor. Intriguingly, we found that cellular models could differentiate the subtle differences between the closely related C.1.2 and Delta in their binding to hACE2. More importantly, competitive binding studies in presence of pseudovirus, demonstrated that a single common modification, N501Y, present in both Omicron and C.1.2 showed two fold superior competitive binding than Delta. Collectively, our study suggests a precise approach to evaluate the binding interactions between spike sub-lineages to hACE2 receptor. This would be instrumental in understanding the disease progression and developing therapeutics.

pharmacology and toxicology↗

bioRxiv2022

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is the commonly adapted strategy to ameliorate {beta}-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits the HbF tetramer production affecting the therapeutic benefits. Here, we tested various deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified a 11 kb sequence, encompassing Putative Repressor Region (PRR) to {beta}-globin Exon-1 ({beta}E1), as the core deletion that ablates HbA and exhibit superior production of HbF compared to HPFH or other well-established targets. The PRR-{beta}E1 edited hematopoietic stem and progenitor cells (HSPCs) retained engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice generating HbF+ cells in vivo. Importantly, the editing induces therapeutically relevant levels of HbF to reverse the phenotypes of both sickle cell disease and {beta}-thalassemia major. These results indicate that the PRR-{beta}E1 gene editing in patient HSPCs can potentially lead to superior therapeutic outcomes for {beta}-hemoglobinopathies gene therapy.

biochemistry↗

bioRxiv2022

Precise modelling and correction of a spectrum of β-thalassemic mutations in human erythroid cells by base editors

{beta}-thalassemia and HbE result from mutations in the {beta}-globin locus that impedes the production of functional {beta}-hemoglobin and represents one of the most common genetic disorders worldwide. Recent advances in genome engineering have opened up new therapeutic opportunities to directly correct these pathogenic mutations using base editors that install transition mutations (A>G and C>T) in the target region with minimal generation of indels. Herein, for the first time, we demonstrate the usage of base editor in the correction of point mutations spanning multiple regions of the HBB gene, including promoter, intron and exon. To this end, we have engineered human erythroid cells harbouring the diverse HBB mutations, thus eliminating the requirement of patient CD34+ HSPCs with desired mutations for the primary screening by base editors. We further performed precise creation and correction of individual HBB point mutations in human erythroid cells using base editors, which were effectively corrected in the HBB-engineered erythroid model. Intriguingly, most bystander effects produced by the base editor at the target site were reported to exhibit normal hemoglobin variants. Overall, our study provides the proof-of-concept for the precise, efficient and scarless creation and correction of various pathogenic mutations at the coding and non-coding regions of HBB gene in human erythroid cells using base editors and establishes a novel therapeutic platform for the treatment of {beta}-thalassemia/HbE patients. This study can be further explored in correcting the other monogenic disorders caused due to single base substitutions.

genomics↗

bioRxiv2020

Identification of novel HPFH-like mutations by CRISPR base editing that elevates the expression of fetal hemoglobin

Switching hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin is an effective strategy for the treatment of beta-hemoglobinopathies. Fetal hemoglobin expression is down-regulated in the postnatal period due to the interplay of transcription regulators with the HBG promoters. However, in the hereditary persistence of fetal hemoglobin (HPFH) condition, naturally occurring point mutations in the HBG promoter causes continued expression of fetal globin even during adulthood. Inspired by this natural phenomenon, we screened the proximal promoter of human HBG genes using adenine and cytosine base editors to identify other nucleotide substitutions that could potentially lead to elevated levels of fetal globin. Both the base editors efficiently and precisely edited at the target sites with a minimal generation of indels and no deletion of one of the duplicated HBG genes. Through systematic tiling across the HBG proximal promoter, we identified multiple novel target sites that resulted in a significant increase in fetal globin levels. Further, we individually validated the top eight potential target sites from both the base editors and observed robust elevation in the fetal globin levels up to 47 %, without any detrimental effects on erythroid differentiation. Our screening strategy resulted in the identification of multiple novel point mutations and also validated the known non-deletional HPFH mutations that could elevate the fetal globin expression at therapeutically relevant levels. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter that normally mediates fetal globin silencing and identify additional targets for therapeutic upregulation of fetal hemoglobin.

genomics↗