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As a visionary leader in the field of scientific R&D/CMC, with a specialization in iPSC…

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Publications

  • The inhibitor of histone acetyltransferases Nir regulates epidermis development

    Development

    Nir/ Noc2l is an inhibitor of histone acetyl-transferases (INHAT). In addition to its function on histone acetylation, Nir binds to p53 and TAp63 to regulate their activity. Here, we show that epidermis-specific ablation of Nir impairs epidermal stratification and barrier function, thereby causing perinatal lethality. Nir-deficient epidermis lacks appendages and remains single layered during embryogenesis. Cell proliferation is inhibited, whereas apoptosis and p53 acetylation is increased…

    Nir/ Noc2l is an inhibitor of histone acetyl-transferases (INHAT). In addition to its function on histone acetylation, Nir binds to p53 and TAp63 to regulate their activity. Here, we show that epidermis-specific ablation of Nir impairs epidermal stratification and barrier function, thereby causing perinatal lethality. Nir-deficient epidermis lacks appendages and remains single layered during embryogenesis. Cell proliferation is inhibited, whereas apoptosis and p53 acetylation is increased, indicating that Nir is controlling cell proliferation by limiting p53 acetylation. Transcriptome analysis revealed that Nir regulates expression of essential factors of epidermal development, such as keratins, integrins, and laminins. Furthermore, Nir binds to and controls expression of p63 and limits H3K18ac at the p63 promoter. Corroborating stratification defects, asymmetric cell divisions were virtually absent in Nir-deficient mice, suggesting that Nir is required for correct mitotic spindle orientation. In summary, our data define Nir as a key regulator of skin development.

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  • The histone demethylase LSD1 regulates inner ear progenitor differentiation through interactions with Pax2 and the NuRD repressor complex

    PLOS ONE

    The histone demethylase LSD1 plays a pivotal role in cellular differentiation, particularly in silencing lineage-specific genes. However, little is known about how LSD1 regulates neurosensory differentiation in the inner ear. Here we show that LSD1 interacts directly with the transcription factor Pax2 to form the NuRD co-repressor complex at the Pax2 target gene loci in a mouse otic neuronal progenitor cell line (VOT-N33). VOT-N33 cells expressing a Pax2-response element reporter were…

    The histone demethylase LSD1 plays a pivotal role in cellular differentiation, particularly in silencing lineage-specific genes. However, little is known about how LSD1 regulates neurosensory differentiation in the inner ear. Here we show that LSD1 interacts directly with the transcription factor Pax2 to form the NuRD co-repressor complex at the Pax2 target gene loci in a mouse otic neuronal progenitor cell line (VOT-N33). VOT-N33 cells expressing a Pax2-response element reporter were GFP-negative when untreated, but became GFP positive after forced differentiation or treatment with a potent LSD inhibitor. Pharmacological inhibition of LSD1 activity resulted in the enrichment of mono- and di-methylation of H3K4, upregulation of sensory neuronal genes and an increase in the number of sensory neurons in mouse inner ear organoids. Together, these results identify the LSD1/NuRD complex as a previously unrecognized modulator for Pax2-mediated neuronal differentiation in the inner ear.

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  • CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa

    npj Regenerative Medicine

    Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the COL7A1 gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for…

    Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the COL7A1 gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for multilineage engineering of therapeutic cell types. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system facilitated correction of an RDEB-causing COL7A1 mutation in primary fibroblasts that were then used to derive induced pluripotent stem cells (iPSCs). The resulting iPSCs were subsequently re-differentiated into keratinocytes, mesenchymal stem cells (MSCs) and haematopoietic progenitor cells using defined differentiation strategies. Gene-corrected keratinocytes exhibited characteristic epithelial morphology and expressed keratinocyte-specific genes and transcription factors. iPSC-derived MSCs exhibited a spindle morphology and expression of CD73, CD90 and CD105 with the ability to undergo adipogenic, chondrogenic and osteogenic differentiation in vitro in a manner indistinguishable from bone marrow-derived MSCs. Finally, we used a vascular induction strategy to generate potent definitive haematopoietic progenitors capable of multilineage differentiation in methylcellulose-based assays. In totality, we have shown that CRISPR/Cas9 is an adaptable gene-editing strategy that can be coupled with iPSC technology to produce multiple gene-corrected autologous cell types with therapeutic potential for RDEB.

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  • CRISPR/Cas9 Targeted Gene Editing and Cellular Engineering in Fanconi Anemia

    Stem Cells and Development

    The ability to rationally target disease-causing mutations has been made possible with programmable nucleases with the clustered, regularly interspaced short palindromic repeats/Cas9 system representing a facile platform for individualized gene-based medicine. In this study we employed footprint-free reprogramming of fibroblasts from a patient with mutations to the Fanconi anemia I (FANCI) gene to generate induced pluripotent stem cells (iPSCs). This process was accomplished without gene…

    The ability to rationally target disease-causing mutations has been made possible with programmable nucleases with the clustered, regularly interspaced short palindromic repeats/Cas9 system representing a facile platform for individualized gene-based medicine. In this study we employed footprint-free reprogramming of fibroblasts from a patient with mutations to the Fanconi anemia I (FANCI) gene to generate induced pluripotent stem cells (iPSCs). This process was accomplished without gene complementation and the resultant iPSCs were able to be gene corrected in a robust manner using the Cas9 nickase. The self-renewing iPSCs that were maintained under feeder-free conditions were differentiated into cells with characteristics of definitive hematopoiesis. This defined and highly efficient procedure employed small molecule modulation of the hematopoietic differentiation pathway and a vascular induction technique to generate hematopoietic progenitors. In sum, our results demonstrate the ability to induce patient-derived FA cells to pluripotency for patient-specific therapeutic cell derivation.

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  • Tlx3 promotes glutamatergic neuronal subtype specification through direct interactions with the chromatin modifier CBP

    PLOS ONE

    Nervous system development relies on the generation of precise numbers of excitatory and inhibitory neurons. The homeodomain transcription factor, T-cell leukemia 3 (Tlx3), functions as the master neuronal fate regulator by instructively promoting the specification of glutamatergic excitatory neurons and suppressing the specification of gamma-aminobutyric acid (GABAergic) neurons. However, how Tlx3 promotes glutamatergic neuronal subtype specification is poorly understood. In this study, we…

    Nervous system development relies on the generation of precise numbers of excitatory and inhibitory neurons. The homeodomain transcription factor, T-cell leukemia 3 (Tlx3), functions as the master neuronal fate regulator by instructively promoting the specification of glutamatergic excitatory neurons and suppressing the specification of gamma-aminobutyric acid (GABAergic) neurons. However, how Tlx3 promotes glutamatergic neuronal subtype specification is poorly understood. In this study, we found that Tlx3 directly interacts with the epigenetic co-activator cyclic adenosine monophosphate (cAMP)-response element-binding protein (CREB)-binding protein (CBP) and that the Tlx3 homeodomain is essential for this interaction. The interaction between Tlx3 and CBP was enhanced by the three amino acid loop extension (TALE)-class homeodomain transcription factor, pre-B-cell leukemia transcription factor 3 (Pbx3). Using mouse embryonic stem (ES) cells stably expressing Tlx3, we found that the interaction between Tlx3 and CBP became detectable only after these Tlx3-expressing ES cells were committed to a neural lineage, which coincided with increased Pbx3 expression during neural differentiation from ES cells. Forced expression of mutated Tlx3 lacking the homeodomain in ES cells undergoing neural differentiation resulted in significantly reduced expression of glutamatergic neuronal subtype markers, but had little effect on the expression on pan neural markers. Collectively, our results strongly suggest that functional interplay between Tlx3 and CBP plays a critical role in neuronal subtype specification, providing novel insights into the epigenetic regulatory mechanism that modulates the transcriptional efficacy of a selective set of neuronal subtype-specific genes during differentiation.

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  • Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture.

    Nature

    The inner ear contains sensory epithelia that detect head movements, gravity and sound. It is unclear how to develop these sensory epithelia from pluripotent stem cells, a process that will be critical for modelling inner ear disorders or developing cell-based therapies for profound hearing loss and balance disorders. So far, attempts to derive inner ear mechanosensitive hair cells and sensory neurons have resulted in inefficient or incomplete phenotypic conversion of stem cells into…

    The inner ear contains sensory epithelia that detect head movements, gravity and sound. It is unclear how to develop these sensory epithelia from pluripotent stem cells, a process that will be critical for modelling inner ear disorders or developing cell-based therapies for profound hearing loss and balance disorders. So far, attempts to derive inner ear mechanosensitive hair cells and sensory neurons have resulted in inefficient or incomplete phenotypic conversion of stem cells into inner-ear-like cells. A key insight lacking from these previous studies is the importance of the non-neural and preplacodal ectoderm, two critical precursors during inner ear development. Here we report the stepwise differentiation of inner ear sensory epithelia from mouse embryonic stem cells (ESCs) in three-dimensional culture. We show that by recapitulating in vivo development with precise temporal control of signalling pathways, ESC aggregates transform sequentially into non-neural, preplacodal and otic-placode-like epithelia. Notably, in a self-organized process that mimics normal development, vesicles containing prosensory cells emerge from the presumptive otic placodes and give rise to hair cells bearing stereocilia bundles and a kinocilium. Moreover, these stem-cell-derived hair cells exhibit functional properties of native mechanosensitive hair cells and form specialized synapses with sensory neurons that have also arisen from ESCs in the culture. Finally, we demonstrate how these vesicles are structurally and biochemically comparable to developing vestibular end organs. Our data thus establish a new in vitro model of inner ear differentiation that can be used to gain deeper insight into inner ear development and disorder.

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  • Phosphorylation of histone H3T6 by PKCbI controls demethylation at histone H3K4

    Nature

    Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation1, 2. As a component of co-repressor complexes, LSD1 contributes to target gene repression by removing mono- and dimethyl marks from lysine 4 of histone H3 (H3K4)1, 3. In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9)2. Yet, the mechanisms that control this dual…

    Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation1, 2. As a component of co-repressor complexes, LSD1 contributes to target gene repression by removing mono- and dimethyl marks from lysine 4 of histone H3 (H3K4)1, 3. In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9)2. Yet, the mechanisms that control this dual specificity of demethylation are unknown. Here we show that phosphorylation of histone H3 at threonine 6 (H3T6) by protein kinase C beta I (PKCβI, also known as PRKCβ) is the key event that prevents LSD1 from demethylating H3K4 during AR-dependent gene activation. In vitro, histone H3 peptides methylated at lysine 4 and phosphorylated at threonine 6 are no longer LSD1 substrates. In vivo, PKCβI co-localizes with AR and LSD1 on target gene promoters and phosphorylates H3T6 after androgen-induced gene expression. RNA interference (RNAi)-mediated knockdown of PKCβI abrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of PKCβI requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1)4. Notably, increased levels of PKCβI and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKCβI blocks AR-induced tumour cell proliferation in vitro and cancer progression of tumour xenografts in vivo. Together, our data establish that androgen-dependent kinase signalling leads to the writing of the new chromatin mark H3T6ph, which in consequence prevents removal of active methyl marks from H3K4 during AR-stimulated gene expression.

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  • Mutations associated with retinopathies alter mitogen-activated protein kinase-induced phosphorylation of neural retina leucine-zipper

    Molecular Vision

    Neural retina leucine-zipper (NRL), a member of the basic motif leucine zipper family of transcription factors, is preferentially expressed in rod photoreceptors of the mammalian retina. Mutations in NRL are associated with retinopathies; many of these are suggested to change phosphorylation status and alter NRL-mediated transactivation of rhodopsin promoter. The purpose of this study was to identify potential kinases responsible for the phosphorylation of NRL and determine if such…

    Neural retina leucine-zipper (NRL), a member of the basic motif leucine zipper family of transcription factors, is preferentially expressed in rod photoreceptors of the mammalian retina. Mutations in NRL are associated with retinopathies; many of these are suggested to change phosphorylation status and alter NRL-mediated transactivation of rhodopsin promoter. The purpose of this study was to identify potential kinases responsible for the phosphorylation of NRL and determine if such kinase-dependent phosphorylation is altered in disease-associated NRL mutations.

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  • Contextual reprogramming of CAR-T cells for treatment of HER2+ cancers

    Journal of J Transl Med . 2021 Nov 7;19(1):459. doi: 10.1186/s12967-021-03132-6

    Background

    Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities.

    Methods

    To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon…

    Background

    Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities.

    Methods

    To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS.

    Results

    Here, we show that RB-340-1 consistently demonstrated higher production of homeostatic cytokines, enhanced expansion of CAR-T cells in vitro, prolonged in vivo persistence and more efficient suppression of HER2+ FaDu oropharyngeal cancer growth compared to the respective conventional CAR-T cell product.

    Conclusions

    As the first application of CRISPRi toward a clinically relevant product, RB-340-1 with the conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.

    Supplementary Information

    The online version contains supplementary material available at 10.1186/s12967-021-03132-6.

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Courses

  • Neuroscience at National Brain Research Centre

    -

Honors & Awards

  • Associate Faculty member in Hematology Oncology at Faculty of 1000 (F1000)

    Faculty of 1000

    The F1000Prime Faculty comprises peer-nominated, internationally-renowned researchers across biology and medicine, who review and recommend the articles they consider of greatest interest and merit. I am passionate associate faculty member at F1000 and wrote recommendation of several research articles.Some of my recommendation includes work of others published in high impact factor journals
    such as Cell, Nature Biotechnology, Nature Cell Biology and Stem Cell Reports.

  • Travel Award by The European Molecular Biology Laboratory and Cold Spring Harbor Laboratories

    The European Molecular Biology Organization (EMBO)

    I have been awarded a travel award by EMBO for presenting my work on "CREB-1 heterodimerize with NRL and repress NRL mediated activation of rhodopsin gene. Poster presentation and abstract published in EMBO/CSHL jointly organized Conference on “Protein Synthesis and Translational Control” at EMBL, Heidelberg, Germany.

  • Junior and senior research fellowships from Department of Biotechnology

    Department of Biotechnology, India

    The central government Department of Biotechnologies provides very competitive up to five years fellowships to 100 biotechnology students to pursue doctoral research in universities/research institutions across the country. The students are selected (over millions appeared for test) through Biotechnology Eligibility Test (BET) now being co-ordinated by the National Centre for Cell Sciences.

  • Secured position in National Eligibility Test for Assistant Professor in all over India

    Council for Scientific and Industrial Research and University Grant Commission, Govt. of India

    Selected among top 5% candidates in All India CSIR-UGC (Council for Scientific and
    Industrial Research and University Grant Commission) combined NET-LS, 2003 (National
    Eligibility Test for Assistant Professor in all over India)

  • Associate Editor in the Nature journal Leukemia

    -

Languages

  • English

    Professional working proficiency

  • Gujarati

    Native or bilingual proficiency

  • Hindi

    Native or bilingual proficiency

Organizations

  • Society of Immunotherapy for Cancer

    member

    - Present

  • International Society for Stem Cell Research

    Associate Member

  • The American Association of Immunologists

    Member

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