Biology subjects

Bhatia, V.

Publications and source records attributed to Bhatia, V..

15 recordsLinked to original sources

bioRxiv2026

LNP Delivery of Prime Editors Enables Durable Correction of PAH R408W in Phenylketonuria Models

Phenylketonuria (PKU) is a monogenic metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene, the most common of which is c.1222C>T (p.R408W). Current treatment options, including dietary restriction and enzyme substitution, can be effective but are often challenging for patients to maintain and do not address the underlying genetic defect. Gene editing represents a compelling one-time treatment approach for PKU. Herein, we demonstrate mRNA:LNP-mediated delivery of prime editors that enable therapeutically-relevant levels of correction of the R408W mutation in both in vitro and in vivo model systems. Further, we found that co-delivery of the DNA repair modulator RAD18 and engineering of the prime editor protein, enabled 2-fold and 3-fold increases in prime editing relative to PE3 respectively, resulting in ~50% correction efficiency in a humanized PKU mouse model. Collectively, this work identifies novel potential approaches to enhancing prime editing and establishes LNP-delivered prime editing as a mutation-specific, non-viral therapeutic strategy for PKU, as well as providing a framework for extending prime editing to other inherited liver diseases.

pharmacology and toxicology↗

bioRxiv2025

Armoring STEAP1 CAR T cells with IL-18 potentiates antitumor activity in Ewing sarcoma

BackgroundEwing sarcoma (EwS) is a highly aggressive cancer driven by the EWS::FLI1 fusion oncoprotein affecting children, adolescents, and young adults. Six transmembrane epithelial antigen 1 (STEAP1) is a cell surface antigen transcriptionally controlled by EWS::FLI1 that is broadly expressed in EwS, positioning it as a rational immunotherapy target. However, translating CAR T therapy to solid tumors requires overcoming barriers to potency while maintaining safety. MethodsAnalyses of transcriptome and proteome data were performed to evaluate the effects of EWS::FLI1 perturbation on STEAP1 expression at the transcript and protein levels in EwS models. STEAP1 expression was validated in EwS patient tissues by immunohistochemistry. Second-generation STEAP1-BB{zeta} CAR T cells were tested in orthotopic and disseminated EwS xenograft models. To enhance antitumor activity, an IL-18-armored STEAP1 CAR was engineered. Dose-dependent therapeutic efficacy and safety were evaluated through measurement of tumor burden, survival, and observation for gross toxicities. ResultsSTEAP1 was expressed in [~]97% of primary EwS tumors and directly associated with EWS::FLI1 fusion protein expression in EwS cell lines. In orthotopic EwS models, STEAP1 CAR T cells induced complete tumor regression at 5 x 106 cells. In disseminated disease models, responses were dose-dependent with no evidence of antigen loss. Notably, IL-18 armored STEAP1 CAR T cells achieved complete responses in [~]80% of mice at a reduced dose of 106 cells without overt toxicity. ConclusionsThese data establish STEAP1 as a clinically relevant and highly expressed target in EwS and demonstrate that IL-18 armoring significantly improves CAR T cell efficacy by enhancing potency evident through antitumor activity at reduced cell dose. STEAP1 CAR T cells are currently under evaluation in a first-in-human phase 1/2 dose-escalation clinical trial for metastatic castration-resistant prostate cancer (NCT06236139) and these studies support future clinical translation of STEAP1 CAR T cell therapy for relapsed/refractory EwS.

cancer biology↗

bioRxiv2025

Collagen-binding IL-12 expressing STEAP1 CAR-T cells reduce toxicity and eradicate mouse prostate cancer in combination with checkpoint inhibitors

Immunosuppressive microenvironments, the lack of immune infiltration, and antigen heterogeneity pose significant challenges for chimeric antigen receptor (CAR)-T cell therapies to tackle solid tumors. CAR-T cells were armed with immunostimulatory payloads, such as interleukin-12 (IL-12), to overcome this issue, but faced intolerable toxicity during clinical development. Here, we show that collagen-binding domain-fused IL-12 (CBD-IL-12) was retained within syngeneic murine prostate tumors, after secretion from CAR-T cells targeting human six transmembrane epithelial antigen of the prostate 1 (STEAP1). This led to equivalently high intratumoral interferon-{gamma} levels without hepatotoxicity and infiltration of T cells into non-target organs, compared with unmodified IL-12. Both innate and adaptive immune compartments were dramatically activated and recognized diverse tumor antigens after CBD-IL-12 CAR-T cell treatment. Combination immunotherapy of CBD-IL-12 CAR-T cells and immune checkpoint inhibitors eradicated large tumors in an established prostate cancer model, without pre-conditioning chemotherapy. The therapy generated anti-tumor immunological memory. CBD-fusion to potent yet toxic payloads of CAR-T therapy may remove obstacles to their clinical translation towards elimination of solid tumors.

bioengineering↗

bioRxiv2025

Defined cellular reprogramming of androgen receptor-active prostate cancer to neuroendocrine prostate cancer

Neuroendocrine prostate cancer (NEPC) arises primarily through neuroendocrine transdifferentiation (NEtD) as an adaptive mechanism of therapeutic resistance. Models to define the functional effects of putative drivers of this process on androgen receptor (AR) signaling and NE cancer lineage programs are lacking. We adapted a genetically defined strategy from the field of cellular reprogramming to directly convert AR-active prostate cancer (ARPC) to AR-independent NEPC using candidate factors. We delineated critical roles of the pioneer factors ASCL1 and NeuroD1 in NEtD and uncovered their abilities to silence AR expression and signaling by remodeling chromatin at the somatically acquired AR enhancer and global AR binding sites with enhancer activity. We also elucidated the dynamic temporal changes in the transcriptomic and epigenomic landscapes of cells undergoing acute lineage conversion from ARPC to NEPC which should inform future therapeutic development. Further, we distinguished the activities of ASCL1 and NeuroD1 from the inactivation of RE-1 silencing transcription factor (REST), a master suppressor of a major neuronal gene program, in establishing a NEPC lineage state and in modulating the expression of genes associated with major histocompatibility complex class I (MHC I) antigen processing and presentation. These findings provide important, clinically relevant insights into the biological processes driving NEtD of prostate cancer.

cancer biology↗

bioRxiv2024

Bitter taste genetics and oral health in Canadian Longitudinal Study on Aging

This study aimed to investigate the association of single nucleotide polymorphisms (SNPs) in 25 Bitter Taste Receptor genes (TAS2Rs) and 12 TAS2R pseudogenes with self-reported oral health outcomes in the Canadian Longitudinal Study on Aging (CLSA) cohort. Following quality control, 124 SNPs with a minor allele frequency > 0.01 and 21,991 individuals of European ancestry were included in the analysis. Fifteen SNPs in TAS2R8, 9, 13, 14, 20, and 50 were significantly associated with self-reported sore jaw muscles, a symptom commonly linked to temporomandibular disorders (TMDs). TAS2R20 exhibited the highest number of associated SNPs. Structure-function analysis suggests that variants in TAS2R20 may contribute to this symptom by altering ligand interactions. These findings highlight the potential for TAS2R genetic screening to identify individuals at elevated risk for TMD, supporting the development of personalized treatment strategies and advancing our understanding of TMD genetic risk factors.

genetics↗

bioRxiv2024

Does the PolyJet Printing Technology Affect Mechanical Properties of 3D Printed Synthetic Tissue?

Within the medical field, there are multiple opportunities in which 3D printed anatomical models could be utilized such as physician training, surgical planning, education, and R&D testing. Because of this, it is vital to choose materials that most closely mechanically mimic the tissues that would be seen within the simulated model. Stratasys currently has a line of Digital Anatomy (DA) printers that contain predetermined compositions and mixtures called anatomical presets to mimic soft tissue materials such as liver, myocardium, aortic, and epicardium tissue. Different compositions of these printed materials have been previously printed on the J750 Digital Anatomy printer and have undergone mechanical testing. Stratasys is releasing a new printer called J5 Digital Anatomy printer that will also have the capabilities of printing anatomical presets. To compare any differences between the two printer sample types, these mechanical tests were repeated on the J5 DA printer. Mechanical testing included stiffness testing of printed liver samples, compliance testing of printed myocardium samples, and lubricity testing of printed cardiac tissue samples. Stiffness of J5 DA liver and J750 DA liver samples were similar with slight variation seen in more rigid J5 DA samples; however, both printer samples still fell within the stiffness range observed in porcine liver tissue. Compliance testing showed comparable stiffness between J5 DA and J750 DA myocardium samples; however, J5 DA samples tended to be slightly less stiff making them more similar to porcine myocardium stiffness. Thinner J5 DA samples were more variable when compared to J750 DA samples; however, this was minimal variability when compared to that experienced in porcine tissue. Lubricity testing showed comparable coefficients of friction between J5 DA and J750 DA samples with the exception of Mineral oil and Dry lubricants. J5 DA cardiac samples saw smaller coefficients of friction; however, these still fell within the range of aortic and epicardium coefficients of frictions. Because of these results, it can be concluded that the J5 DA printer samples are comparable to J750 DA printer samples and are alternatives to animal tissue benchtop testing depending on the application.

bioengineering↗

bioRxiv2024

T2R14 mediated antimicrobial responses through interactions with CFTR

Bitter taste receptors (T2Rs), are a subset of G protein-coupled receptors (GPCRs) that play a key role in responding to microbial presence at epithelial surfaces. In epithelia, the activities of ion channels and transporters, and of T2Rs, mutually affect each other. The normal function of one such anion channel, cystic fibrosis transmembrane conductance regulator (CFTR), is essential for the maintenance of healthy epithelia, not just in the respiratory but in the digestive and reproductive system as well. Based on evidence that T2R14 activity is affected upon mutations in CFTR, we explored the possibility that T2R14 and CFTR directly interact in cell membranes. The biophysical interaction between these proteins was mapped to specific regions of the CFTR, and was dependent on agonist stimulation of T2R14. Further, T2R14 was found to couple to Gq, in addition to the canonical Gi, in response to bacterial and fungal quorum sensing molecules. Whether the interaction with CFTR affects T2R14 driven responses to microbial signals is under investigation.

biochemistry↗

bioRxiv2023

Development of a robotic cluster for automated and scalable cell therapy manufacturing

The production of commercial autologous cell therapies such as chimeric antigen receptor T cells requires complex manual manufacturing processes. Skilled labor costs and challenges in manufacturing scale-out have contributed to high prices for these products. Here, we present a robotic system that uses industry-standard cell therapy manufacturing equipment to automate the steps involved in cell therapy manufacturing. The robotic cluster consists of a robotic arm and customized modules, allowing the robot to manipulate a variety of standard cell therapy instruments and materials such as incubators, bioreactors, and reagent bags. This system enables existing manual manufacturing processes to be rapidly adapted to robotic manufacturing, without having to adopt a completely new technology platform. Proof-of-concept for the robotic clusters expansion module was demonstrated by activating and expanding human CD8+ T cells. The robotic cultures showed comparable cell yields, viability, and identity to those manually performed. Such modular robotic solutions may support scale-up and scale-out of cell therapies that are developed using classical manual methods in academic laboratories and biotechnology companies.

bioengineering↗

bioRxiv2023

Combinatorial genetic strategy accelerates the discovery of cancer genotype-phenotype associations

Available genetically-defined cancer models are limited in genotypic and phenotypic complexity and underrepresent the heterogeneity of human cancer. Herein, we describe a combinatorial genetic strategy applied to an organoid transformation assay to rapidly generate diverse, clinically relevant bladder and prostate cancer models. Importantly, the clonal architecture of the resultant tumors can be resolved using single-cell or spatially resolved next-generation sequencing to uncover polygenic drivers of cancer phenotypes.

cancer biology↗

bioRxiv2022

Targeting advanced prostate cancer with STEAP1 chimeric antigen receptor T cell therapy

Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a compelling tumor-associated cell surface antigen for therapeutic targeting in solid tumors. We identified broad expression of STEAP1 (87% positive) in lethal metastatic prostate cancer, even more so than prostate-specific membrane antigen (PSMA, 60% positive) which is a clinically established diagnostic and therapeutic target. Second-generation chimeric antigen receptor (CAR) T cells were engineered for reactivity against STEAP1 and demonstrated substantial antitumor activity in metastatic human prostate cancer models in immunodeficient mice. Adoptive transfer of STEAP1 CAR T cells was associated with prolonged peripheral persistence and either disease eradication or substantial tumor growth inhibition with progressive disease demonstrating antigen loss. As STEAP1 CAR T cells were also highly active in antigen density conditions as low as [~]1,500 molecules/cell, we generated a human STEAP1 (hSTEAP1) knock-in (KI) mouse to evaluate the potential for on-target off-tumor toxicities. hSTEAP1-KI mice demonstrated a pattern of systemic hSTEAP1 expression akin to that observed in humans with the greatest expression found in the prostate gland. Mouse-in-mouse studies of STEAP1 CAR T cell therapy in immunocompetent hSTEAP1-KI mice engrafted with disseminated mouse prostate cancer showed preliminary safety without evidence of gross toxicity, cytokine storm, or architectural disruption and increased T cell infiltration at sites of systemic hSTEAP1 expression. Tumor responses and extension of survival were appreciated but antigen loss was identified in recurrent and progressive disease. In summary, we report the extent of STEAP1 expression in treatment-refractory metastatic prostate cancer, the generation of a STEAP1 CAR T cell therapy with promising potency and safety in preclinical studies of advanced prostate cancer, and antigen escape as a mechanism of resistance to effective STEAP1 CAR T cell therapy.

cancer biology↗

bioRxiv2020

PolyJet 3D Printing of Tissue Mimicking Materials: An Investigation of Characteristic Properties of 3D Printed Synthetic Tissue

Current anatomical 3D printing has been primarily used for education, training, and surgical planning purposes. This is largely due to the models being printed in materials which excel at replicating macro-level organic geometries; however, these materials have the drawback of unrealistic mechanical behavior and system properties compared to biological tissue. The new Digital Anatomy (DA) family of materials from Stratasys utilizes composite printed materials to more closely mimic mechanical behavior of biological tissue, potentially allowing more realistic models for design evaluation. Various experimental DA Solid Organ (SO) configurations were quantitatively evaluated under axial loading for comparison with porcine liver in terms of stiffness. Additionally, Structural Heart - Myocardium (Myo) configurations were quantitatively evaluated under different lubricant conditions for comparison with porcine epicardium and aorta in terms of lubricity. Finally, experimental DA Subcutaneous Tissue configurations were qualitatively evaluated by experts with significant pre-clinical implant experience for cutting, tunneling, and puncture procedures. In general, the experimental SO configurations showed promising compliance results when compared to porcine liver. The stiffness of DA configurations was either within the same range or on the lower bound of porcine tissue stiffness values. The lubricity of DA configurations with surface treatments was comparable with porcine epicardium and aorta. In terms of qualitative cutting, DA did not perform well for any of the configurations; however, tunneling and puncture were rated favorably for some of the experimental configurations. Despite some limitations, DA feels closer to real tissue than other commercially available 3D printed materials. Furthermore, the lower sample-to-sample variability of DA allows for repeatability not provided by biological tissue. The promising results and repeatability indicate that DA materials can be used to configure structures with similar characteristic mechanical properties to porcine liver, epicardium, and subcutaneous tissue, adding new value as not only an educational, training, and surgical tool, but also as a research tool.

bioengineering↗

bioRxiv2020

Transcriptional network involving ERG and AR orchestrates Distal-Less Homeobox 1 mediated prostate cancer progression.

Nearly half of the prostate cancer (PCa) cases show elevated levels of ERG oncoprotein due to TMPRSS2-ERG gene fusion. Here, we demonstrate ERG mediated upregulation of Distal-less homeobox-1 (DLX1), an established PCa biomarker. Using series of functional assays, we show DLX1 elicits oncogenic properties in prostate epithelial cells, and abrogating its function leads to reduced tumor burden in mouse xenografts. Clinically, [~]60% of the PCa patients exhibit high DLX1 levels, while [~]50% of these cases also harbor elevated ERG associated with aggressive disease and poor survival probability. Mechanistically, we show that ERG gets recruited onto DLX1 promoter and interacts with its enhancer-bound androgen receptor (AR) and FOXA1 to regulate DLX1 expression in TMPRSS2-ERG positive cases. Alternatively, in ERG-negative cases, DLX1 is regulated by AR/AR-V7 and FOXA1. Importantly, BET bromodomain inhibitors disrupt the transcriptional regulators of DLX1 and its associated oncogenic properties, signifying their efficacy in treatment of DLX1-positive PCa patients.

cancer biology↗

bioRxiv2019

Polyjet 3D printing of tissue-mimicking materials: how well can 3D printed synthetic myocardium replicate mechanical properties of organic myocardium?

Anatomical 3-D printing has potential for many uses in education, research and development, implant training, and procedure planning. Conventionally, the material properties of 3D printed anatomical models have often been similar only in form and not in mechanical response compared to biological tissue. The new Digital Anatomy material from Stratasys utilizes composite printed materials to more closely mimic the mechanical properties of tissue. Work was done to evaluate Digital Anatomy myocardium under axial loading for comparison with porcine myocardium regarding puncture, compliance, suturing, and cutting performance.\n\nIn general, the Digital Anatomy myocardium showed promising comparisons to porcine myocardium. For compliance testing, the Digital Anatomy was either within the same range as the porcine myocardium or stiffer. Specifically, for use conditions involving higher stress concentrations or smaller displacements, Digital Anatomy was stiffer. Digital Anatomy did not perform as strongly as porcine myocardium when evaluating suture and cutting properties. The suture tore through the printed material more easily and had higher friction forces both during needle insertion and cutting. Despite these differences, the new Digital Anatomy myocardium material was much closer to the compliance of real tissue than other 3D printed materials. Furthermore, unlike biological tissue, Digital Anatomy provided repeatability of results. For tests such as cyclic compression, the material showed less than two percent variation in results between trials and between parts, resulting in lower variability than tissue. Despite some limitations, the myocardium Digital Anatomy material can be used to configure structures with similar mechanical properties to porcine myocardium in a repeatable manner, making this a valuable research tool.

bioengineering↗

bioRxiv2019

Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer

The Serine Peptidase Inhibitor, Kazal type 1 (SPINK1) overexpression represents ~10-25% of the prostate cancer (PCa) cases associated with shorter recurrence-free survival and poor prognosis. Nonetheless, androgen-deprivation therapy (ADT) remains the mainstay treatment for locally advanced and metastatic PCa patients. However, majority of these individuals eventually progress to castration-resistant stage, and a subset of these patients develop ADT-induced neuroendocrine PCa. Despite adverse effects of ADT, possible role of androgen signaling in SPINK1-mediated prostate oncogenesis remains unexplored. Here, we show that androgen receptor (AR) and its corepressor, the RE1-silencing transcription factor (REST), occupy SPINK1 promoter and functions as a direct transcriptional repressor of SPINK1, thus blocking AR signaling via ADT relieves its repression, leading to SPINK1 upregulation. In agreement, an inverse association between SPINK1 levels and AR expression was observed across multiple PCa cohorts, and in neuroendocrine differentiated cells. While, lineage reprogramming factor SOX2 in turn binds to SPINK1 promoter leading to its transactivation in androgen-deprived conditions with concomitant increase in neuroendocrine markers. Additionally, we also confirm the role of SPINK1 in epithelial-mesenchymal transition, drug resistance, stemness and cellular plasticity. Moreover, we show that Casein Kinase 1 inhibitor stabilizes the REST levels, which in cooperation with AR, conjures transcriptional repression of SPINK1 expression, and impedes SPINK1-mediated oncogenesis. Collectively, our findings provide a plausible explanation to the paradoxical clinical outcomes of ADT, possibly due to increased SPINK1 levels. This study highlights the need to take a well-informed decision prior to ADT and develop alternative therapeutic strategies for castrate-resistant PCa patients.

cancer biology↗

bioRxiv2018

Polycomb Group protein EZH2-mediated transcriptional repression of microRNA-338/-421 drives SPINK1-positive prostate cancer

PurposeSerine Peptidase Inhibitor, Kazal type-1 (SPINK1) overexpression defines the second most recurrent and aggressive prostate cancer (PCa) subtype. However, the underlying molecular mechanism and pathobiology of SPINK1 in PCa remains largely unknown.\n\nExperimental DesignMicroRNA-prediction tools were employed to examine the SPINK1-3UTR for miRNAs binding. Luciferase reporter assays were performed to confirm the SPINK1-3UTR binding of shortlisted miR-338-5p/miR-421. Further, miR-338-5p/-421 overexpressing cancer cells (SPINK1-positive) were evaluated for oncogenic properties using cell-based functional assays and mice xenograft model. Global gene expression profiling was performed to unravel the biological pathways altered by miR-338-5p/-421. Immunohistochemistry and RNA in-situ hybridization was carried-out on PCa patients tissue microarray for SPINK1 and EZH2 expression respectively. Chromatin immunoprecipitation assay was performed to examine EZH2 occupancy on the miR-338-5p/-421 regulatory regions. Bisulfite sequencing and methylated DNA-immunoprecipitation was performed on PCa cell lines and patients specimens.\n\nResultsWe established a critical role of miRNA-338-5p/-421 in post-transcriptional regulation of SPINK1. Ectopic expression of miRNA-338-5p/-421 in SPINK1-positive PCa cells abrogate oncogenic properties including cell-cycle progression, stemness and drug resistance, and show reduced tumor burden and distant metastases in mice model. Importantly, we show SPINK1-positive PCa patients exhibit increased EZH2 expression, suggesting its role in miRNA-338-5p/-421 epigenetic silencing. Furthermore, presence of CpG dinucleotide DNA methylation marks on the regulatory regions of miR-338-5p/-421 in SPINK1-positive PCa cells and patients specimens confirms epigenetic silencing.\n\nConclusionOur findings revealed that miRNA-338-5p/-421 are epigenetically silenced in SPINK1-positive PCa, while restoring the expression of these miRNAs using epigenetic drugs or synthetic mimics could abrogate SPINK1-mediated oncogenesis.\n\nTRANSLATIONAL IMPACTWe establish a regulatory model involving the functional interplay between SPINK1, miRNA-338-5p/miRNA-421 and EZH2, thereby, revealing hitherto unknown mechanism of SPINK1 up-regulation in SPINK1-positive subtype. Our findings provide a strong rationale for the development of potential therapeutic strategies for SPINK1-positive malignancies. We demonstrate that restoring miRNA-338-5p/miRNA-421 expression using epigenetic drugs including DNMTs inhibitors in combination with HDACs or HKMTs inhibitors or miRNA synthetic mimics in SPINK1-positive prostate cancer abrogate SPINK1-mediated oncogenicity. The major findings of this study will not only advance the prostate cancer field, but will also be valuable for treatment and disease management of other SPINK1-positive malignancies.

cancer biology↗