Biology subjects

Vaidyanathan, S.

Publications and source records attributed to Vaidyanathan, S..

11 recordsLinked to original sources

bioRxiv2026

Cholesterol remodels the endoplasmic reticulum to control myofibroblastic CAF function

Chronic exposure of local fibroblasts to the reactive tumour niche results in the emergence of cancer-associated fibroblasts (CAFs), which can be classed into multiple subtypes with distinct functional and molecular characteristics. Of these subtypes, myofibroblastic CAFs (myCAFs), contribute to tumour progression primarily by depositing and remodelling extracellular matrix (ECM) within the tumour microenvironment (TME). ECM production is known to be a metabolically demanding process, but specific metabolic pathways that are reprogrammed during myCAF activation are poorly understood. Here, we show that cholesterol biosynthesis is elevated in myCAFs relative to normal fibroblasts. We find that cholesterol is specifically enriched in the myCAF endoplasmic reticulum (ER) membrane and is associated with augmented ER mass and enhanced ER secretory function. Importantly, we demonstrate that pharmacological or genetic inhibition of cholesterol biosynthesis plays a critical role in regulating the ECM-secreting and ECM remodelling function of myCAFs. Moreover, we show that inhibition of cholesterol biosynthesis in myCAFs markedly constrains mammary tumour metastasis in vivo. These studies suggest that cholesterol biosynthesis represents a metabolic vulnerability that can be exploited to normalise myCAF-mediated ECM dysfunction in the TME.

cell biology↗

bioRxiv2025

Robust Integration of Sparse Single-Cell Alternative Splicing and Gene Expression Data with SpliceVI

Alternative splicing (AS) and gene expression (GE) are tightly related regulatory processes, critical for defining cell types and states, yet are rarely modeled together in single-cell analyses. This hinders a comprehensive understanding of cellular identity. We address this by introducing SpliceVI, adapted from MultiVI (Multi-modal Variational Inference) to specifically handle AS. Applied to a large multisample mouse Smart-seq2 dataset (n = 142, 315 cells/nuclei), SpliceVI jointly learns from both AS and GE using a partial variational autoencoder that effectively handles the sparsity and missingness of splicing data. We show that SpliceVIs joint embeddings are more expressive and informative of biological correlates like age than a GE-only approach (scVI). SpliceVI also uncovers splicingbased differences between neuronal subclusters. This approach reveals the distinct yet synergistic relationship between AS and GE in shaping cellular diversity in mouse.

systems biology↗

bioRxiv2025

REMEDY: REpair of heterozygous Mutations independent of Exogenous Donor template with high efficiency using allele specific CRISPR targeting and HDR enhancers

We report the development of REMEDY (REpair of heterozygous Mutations independent of Exogenous Donor template with high efficiencY), a genome editing strategy that allows efficient repair of heterozygous mutations in human and mouse cells without necessitating an exogenous donor DNA template. Here, we used in-PAM or near-PAM CRISPR strategies to induce a double-strand break (DSB) in mutant alleles. Following the DSB, the wild-type homologous chromosome itself serves as an endogenous DNA donor template and initiates the correction of the mutant allele. Concurrently treating the cells with HDR enhancers, such as AZD7648, further improved the efficiency of the correction. We demonstrated the utility of REMEDY in the context of six different diseases with heterozygous mutations such as IBMPFD, cystic fibrosis, progeria, ITPR3-associated combined immunodeficiency, ACTA1, and TBCD in human patient derived primary cells and complementary mouse model cell lines. O_FIG O_LINKSMALLFIG WIDTH=91 HEIGHT=200 SRC="FIGDIR/small/641201v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@5f6c41org.highwire.dtl.DTLVardef@bc50d5org.highwire.dtl.DTLVardef@15edca2org.highwire.dtl.DTLVardef@e74ada_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

genetics↗

bioRxiv2025

Durable reconstitution of sinonasal epithelium by transplant of CFTR gene corrected airway stem cells

Modulator agents that restore cystic fibrosis transmembrane conductance regulator (CFTR) function have revolutionized outcomes in cystic fibrosis, an incurable multisystem disease. Barriers exist to modulator use, making local CFTR gene and cell therapies attractive, especially in the respiratory tract. We used CRISPR to gene-correct CFTR in upper airway basal stem cells (UABCs) and show durable local engraftment into recipient murine respiratory epithelium. Interestingly, the human cells recapitulate the in vivo organization and differentiation of human sinus epithelium, with little expansion or contraction of the engrafted population over time, while retaining expression of the CFTR transgene. Our results indicate that human airway stem cell transplantation with locoregional restoration of CFTR function is a feasible approach for treating CF and potentially other diseases of the respiratory tract.

bioengineering↗

bioRxiv2024

DNA-PKcs Inhibition Improves Sequential Gene Insertion of the Full-Length CFTR cDNA in Airway Stem Cells

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which the CFTR cDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two halves of the CFTR cDNA and an enrichment cassette into the CFTR locus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improve CFTR cDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558 which inhibit non-homologous end joining (NHEJ) and micro-homology mediated end joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2-3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations.

genetics↗

bioRxiv2024

Single-Stranded DNA with Internal Base Modifications Mediates Highly Efficient Gene Insertion in Primary Cells

Single-stranded DNA (ssDNA) templates along with Cas9 have been used for gene insertion but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 10-17% of internal bases (eDNA) is compatible with the homologous recombination machinery. Moreover, eDNA templates improve gene insertion by 2-3 fold compared to unmodified and end-modified ssDNA in airway basal stem cells (ABCs), hematopoietic stem and progenitor cells (HSPCs), T-cells and endothelial cells. Over 50% of alleles showed gene insertion in three clinically relevant loci (CFTR, HBB, and CCR5) in ABCs using eDNA and up to 70% of alleles showed gene insertion in the HBB locus in HSPCs. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. Knocking out TREX1 nuclease improved gene insertion using unmodified ssDNA but not eDNA suggesting that chemical modifications inhibit TREX1. This approach can be used for therapeutic applications and biological modeling.

molecular biology↗

bioRxiv2024

Dual DNA/RNA-binding factor regulates dynamics of hnRNP splicing condensates

How RNA splicing events are targeted to the correct genomic locations in specific cellular contexts to generate context-specific transcript diversity and prevent deleterious cryptic splicing remains very poorly understood. We show that a functionally conserved GA-rich DNA-binding transcription factor (TF), CLAMP, targets distinct RNA molecules in male and female cells to precisely regulate sex-specific splicing events through physical and functional interactions with RNA and RNA-binding proteins (RBPs). The prion-like domain of CLAMP (PrLD) and a stem-loop region in the target RNA are important for CLAMP-RNA interaction. Moreover, the CLAMP PrLD domain regulates sex-specific splicing by modulating the dynamics of an hnRNPA2/B1 family protein that regulates alternative splicing. Thus, we demonstrate that a TF targets co-transcriptional splicing to the correct genomic locations by directly linking DNA binding sites to RNA targets and modulating the dynamics of RBP partners that drive alternative splicing.

genomics↗

bioRxiv2022

Prioritizing landscapes to reconcile biodiversity conservation, ecosystem services, and human well-being in India

Biodiversity conservation and human well-being are tightly interlinked; yet mismatches in the scale at which both priorities are planned and implemented have exacerbated biodiversity loss, erosion of ecosystem services, and declining human quality of life. India houses the second largest human population on the planet, while <5% of the countrys land area is effectively protected for conservation. This warrants landscape-level conservation planning through a judicious mix of land-sharing and land-sparing approaches, and co-production of ecosystem services. Through a multi-faceted assessment, we prioritize spatial extents of land parcels that, in the face of anthropogenic threats, can safeguard conservation landscapes across Indias biogeographic zones. We find that only a fraction (~15%) of such priority areas identified here are encompassed under Indias extant PA network, and several landscapes of high importance were omitted in all previous global-scale assessments. We then examined the spatial congruence of priority areas with administrative units earmarked for economic development by the Indian government, and propose management-zoning through state-driven and participatory approaches. Our spatially explicit insights can help meet the twin goals of biodiversity conservation and sustainable development in India and other countries across the Global South.

ecology↗

bioRxiv2021

Targeted replacement of full-length CFTR in human airway stem cells by CRISPR/Cas9 for pan-mutation correction in the endogenous locus

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR/Cas9 and two adeno-associated viruses (AAV) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial basal stem cells (HBECs). The modified cells were enriched to obtain 60-80% tCD19+ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

bioengineering↗

bioRxiv2019

Highly Efficient Repair of the ΔF508 Mutation in Airway Stem Cells of Cystic Fibrosis Patients with Functional Rescue of the Differentiated Epithelia

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel. CF results in multiorgan dysfunction and ultimately mortality from respiratory sequelae. Although pharmacologic approaches have demonstrated efficacy in reducing symptoms and respiratory decline, a curative treatment modality remains elusive. Gene therapy, a promising curative strategy, has been limited due to poor correction efficiencies both in vitro and in vivo. Here, we use Cas9 and adeno-associated virus 6 (AAV6) to correct the {Delta}F508 mutation (found in [~]70% of CF alleles and [~]90% of CF patients in North America) in upper airway basal stem cells (UABCs) obtained from CF and non-CF patients undergoing functional endoscopic sinus surgery (FESS). In UABCs from homozygous ({Delta}F508/{Delta}F508) and compound heterozygous ({Delta}F508/Other) CF patients, we achieved 28 {+/-} 5 % and 42 {+/-} 15% correction, respectively. In homozygous human bronchial epithelial cells (HBECs), we achieved 41{+/-} 4 % correction. Upon differentiation in air-liquid interface (ALI), cultures of corrected CF cells displayed partial restoration of CFTRinh-172 sensitive Cl- currents relative to non-CF controls: 31{+/-} 5 % in UABCs and 51 {+/-} 3 % in HBECs (both from subjects homozygous for {Delta}F508 CFTR). Finally, gene edited cells embedded successfully and retained expression of cytokeratin 5 (KRT5), a basal cell marker, on a FDA-approved porcine small intestinal submucosal (pSIS) membrane previously shown to improve re-mucosalization after FESS. In summary, we present an efficient, feeder-free, selection-free and clinically compatible approach to generate cell-based therapies for CF from autologous airway stem cells. This approach represents a first step towards developing patient-specific autologous airway stem cell transplant as a curative treatment for CF.

bioengineering↗

bioRxiv2016

Protected corridors preserve tiger genetic diversity and minimize extinction into the next century

Habitat loss is the greatest threat to large carnivores around the world. Maintenance of functional connectivity in fragmented landscapes will be important for long-term species persistence. Here, we merge landscape genetics analyses and spatially-explicit simulations to understand future persistence and extinction of tigers (Panthera tigris) in Central India. Tigers in this landscape are restricted to Protected Areas (PAs) and forest fragments embedded within a mosaic of agricultural fields and human settlements. We examined current population connectivity of tigers across nine reserves (using 116 non-invasively sampled individuals and 12 microsatellites). Genetic data was used to infer resistance-to-movement. Our results suggest that dense human settlements and roads with high traffic are detrimental to tiger movement. We used landscape genetic simulations to model 86 different scenarios that incorporated impacts of future land-use change on inferred population connectivity and extinction. Our results confirm that genetic variability (heterozygosity) will decrease in the future and small and/or isolated PAs will have a high risk of local extinction. The average extinction risk of small PAs reduced by 23-70% on adding a 5 km buffer around exiting boundaries. Unplanned development results in 35% lower heterozygosity and 56% higher average extinction probability for tigers even within protected areas. Increasing tiger numbers in such a scenario decreases extinction probability just by 12 % (from 56% to 44%). Scenarios where habitat connectivity was enhanced and maintained, stepping-stone populations were introduced/maintained, and tiger numbers were increased, led to low overall extinction probability (between 3-21%). Our simulations provide a means to quantitatively evaluate the effects of different land-use change scenarios on connectivity and extinction, linking basic science to land-use change policy and planned infrastructure development.

genetics↗