Biology subjects

Kandasamy, K.

Publications and source records attributed to Kandasamy, K..

6 recordsLinked to original sources

bioRxiv2025

Modeling Adeno-Associated Viral Vector 6-mediated In Vivo Gene Delivery to Expanded Non-Mobilized Haemopoietic Stem Cells from Transfusion dependent Thalassemia Patients in a Humanized Mouse

Hematopoietic stem cells (HSC) are important targets for gene modification therapies (GMT) as they originate several serious genetic conditions including the {beta}-haemoglobinopathies. Potentially curative ex vivo GMT pose the barriers of accessibility, myeloablation-associated morbidity and prohibitive cost. In vivo GMT using non-integrating single-strand adeno-associated viral vectors (ssAAV) are a promising alternative that address these challenges directly, although the small ssAAV payload limits the capacity to package much larger gene or base editors. We investigated the feasibility of targeting human HSC in vivo with a dual-ssAAV6 strategy, which in future may be useful to deliver split-intein editing tools to overcome this limitation. We engrafted NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ humice with human hCD45+CD34+ HSC from transfusion-dependent {beta}-thalassemic patients to test in vivo targeting of hCD45 cells with ssAAV6, then administered 5E+12 genomes/kg of ssAAV6-GFP/ssAAV6-mCherry. Humice showed peak single-transgene expression (GFP+ or mCh+) of 1.96-10.17%, and dual-transgene expression (GFP+mCh+) of 31.77% in circulating hCD45+ cells. Nested hCD45+ from liver, spleen and bone marrow showed single-and dual-transgene expression of 36.13-68.14% and 21.91-59.44% respectively. Secondary transplantation experiments demonstrated long-term persistence of AAV6-transduced hCD45 cells showing single-and dual-transgene expression of 9.19-60.72% and 7.15-9.19% respectively, with significant increase in expression from circulating cells. Minimal pro-inflammatory cytokine expression was observed following ssAAV6 administration in thalassemia humice compared with humice carrying non-thalassemia HSC. Our model demonstrates the efficiency of in vivo ssAAV6-mediated targeting of thalassaemia HSC, potential long-term survivability of transduced cells, and feasibility of a dual-AAV strategy for gene editing, which offers a promising alternative to ex vivo GMT for {beta}-haemoglobinopathies.

molecular biology↗

bioRxiv2023

Small RNA Sequencing Reveals a Distinct MicroRNA Signature between Glucocorticoid Responder and Glucocorticoid Non-responder Primary Human Trabecular Meshwork Cells after Dexamethasone Treatment

The present study aimed to understand the role of miRNAs in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using small RNA sequencing. For this, total RNA was extracted from cultured HTM cells with known GC responsiveness using Human organ-cultured anterior segment (HOCAS) (GC-responder GC-R; n=4) and GC-non-responder (GC-NR; n=4) after treatment with either 100nM dexamethasone (DEX) or ethanol (ETH) for 7 days. Differentially expressed miRNAs (DEMIRs) were compared among 5 groups and validated by RT-PCR. There were 13 and 21 DEMIRs identified in Group #1 (ETH vs DEX-treated GC-R) and Group #2 (ETH vs DEX-treated GC-NR) respectively. Seven miRNAs were found as common miRNAs dysregulated in both GC-R and GC-NR (Group #3). There were 6 and 14 unique DEMIRs were identified in GC-R (Gropu#4) and GC-NR (Group#5) HTM cells respectively. Ingenuity Pathway Analysis identified enriched pathways and biological processes associated with differential GC responsiveness in HTM cells. Integrative analysis of miRNA-mRNA of the same set of HTM cells revealed several molecular regulators for GC non-responsiveness. This is the first study revealed a unique miRNA signature between GC-R and GC-NR HTM cells which raises the possibility of developing new molecular targets for the management of steroid-OHT/glaucoma.

cell biology↗

bioRxiv2022

Targeting caveolae to pump bispecific antibody to TGF-β into diseased lungs enables ultra-low dose therapeutic efficacy

The long-sought-after "magic bullet" in systemic therapy remains unrealized for disease targets existing inside most tissues, theoretically because vascular endothelium impedes passive tissue entry and full target engagement. We engineered the first "dual precision" bispecific antibody with one arm pair to precisely bind to lung endothelium and drive active delivery and the other to precisely block TGF-{beta} effector function inside lung tissue. Targeting caveolae for transendothelial pumping proved essential for delivering most of the injected intravenous dose precisely into lungs within one hour and for enhancing therapeutic potency by >1000-fold in a rat pneumonitis model. Ultra-low doses (g/kg) inhibited inflammatory cell infiltration, edema, lung tissue damage, disease biomarker expression and TGF-{beta} signaling. The prodigious benefit of active vs passive transvascular delivery of a precision therapeutic unveils a new promising drug design, delivery and therapy paradigm ripe for expansion and clinical testing.

bioengineering↗

bioRxiv2021

A Comparative Genome-wide Transcriptome Analysis of Glucocorticoid Responder and Non-Responder Primary Human Trabecular Meshwork Cells

The genome-wide gene expression analysis of primary human trabecular meshwork (HTM) cells with known glucocorticoid (GC) responsiveness was not reported earlier. Therefore, the purpose of this study was to investigate genes and pathways involved in the GC responsiveness in human trabecular meshwork (HTM) cells using RNA sequencing. A perfusion cultured human anterior segment ex vivo model was utilized to identify the induction of GC-induced ocular hypertension in one eye of a paired eyes after dexamethasone treatment based on the maximum intraocular pressure response and in the contralateral eye, HTM cells were isolated to classify GC-responder and non-responder cells. Some previously reported and unique genes and their associated pathways were identified in HTM cells in response to dexamethasone treatment versus vehicle control and more significantly in GC-responder and non-responder cells. This study will open up the possibility of identifying suitable molecular targets which have the potential to treat GC-induced ocular hypertension/glaucoma.

genomics↗

bioRxiv2021

Comparative Analysis of non-coding small-RNAs in P. aeruginosa Keratitis Strains with Different Antibiotic Susceptibility

Pseudomonas aeruginosa, is a gram-negative bacterium causes opportunistic or nosocomial infections in immunocompromised individuals. In recent years, a steady increase in human corneal infections of P. aeruginosa has been reported with increased multi-drug resistance (MDR) or extensively drug resistance (XDR). Several non-coding sRNAs, has been identified to regulate various physiological processes in P. aeruginosa, including biofilm formation, quorum sensing. However, the regulatory mechanism of sRNAs in MDR/XDR pathways of P. aeruginosa keratitis strains is not yet studied. In this study, we identified bacterial sRNAs in publicly available P. aeruginosa keratitis genomes and investigated their regulatory role in MDR/XDR pathways using bioinformatic analysis. Totally, 46 P. aeruginosa keratitis strains from different geographical regions were included. Of 46, Eight (30%) out of Twenty-seven and Nine (52) out of Nineteen P. aeruginosa strains from India and Australia were identified as not-MDR. Whereas, 10 (38%) Indian and 9 (47%) Australian strains were identified as MDR. Eight Indian strains were identified as XDR. Out of 46 strains, 23 (50%) carried ExoU, 21(45%) carried ExoS and two (5%) strains carried both ExoU and ExoS, exotoxins for their virulence. The sRNA, SPA0021 was identified in 18 MDR/XDR and 6 not-MDR strains along with UCBPP-PA14. Interestingly, majority of the imipenem resistant P. aeruginosa keratitis strains from the present study was found to be carried SPA0023 sRNA (18 out of 30 strains). The outer membrane porin protein OprD, identified as binding target of SPA0023. Negative regulation or inactivation of OprD, reported in increased imipenem resistance in P. aeruginosa. Mutation analysis revealed that SPA0023 carrying P. aeruginosa keratitis strains contains a lesser number of amino acid changes in OprD protein than other strains. These findings indicate, imipenem resistance in SPA0023 carried strains might arose from the negative regulation or inhibition of OprD by SPA0023. However, functional studies are warranted with large number of P. aeruginosa keratitis strains to confirm the negative regulation of OprD by SPA0023 and imipenem resistance.

genomics↗

bioRxiv2020

Comparative genomics of ocular Pseudomonas aeruginosa strains from keratitis patients with different clinical outcomes

Bacterial keratitis caused by Pseudomonas aeruginosa is a destructive disease of cornea. Pseudomonas keratitis progresses rapidly and leads to vision loss if untreated. Even with adequate treatment, many patients show poor visual outcomes. The virulence factors or multiple drug-resistant (MDR) mechanisms of the ocular strains responsible for poor clinical outcomes remain largely unknown. Here, we performed whole-genome sequencing of five P. aeruginosa strains cultured from corneal scrapings of the healed and corneal buttons of the poor outcome keratitis patients. We investigated the distribution of virulence factors, resistance genes and resistance-associated mutations, the efflux-pump system in all five genomes, and as groups between poor and good clinical outcome as well as MDR vs. non-MDR. We detected several resistance genes and mutations associated with drug resistance in MDR groups; however, a large number of virulence genes were detected in all our genomes. Among the virulence genes, exoU and exoS exotoxin of the Type III secretion system detected in MDR and non-MDR strains, respectively, considered as main virulence contributors of keratitis pathogenesis. Despite this fact, this study did not show an association between MDR with exoU and poor clinical outcomes. However, strain-specific resistance and virulence genes were observed in this study, suggesting their role in the clinical outcome. Mainly, the flagellar genes fliC and fliD, reported to altering the host immune response, might impact the clinical outcome. This comparative study may provide new insights into the genome of ocular strains and requires further functional studies.

microbiology↗