Biology subjects

Saha, K.

Publications and source records attributed to Saha, K..

32 records · Page 2Linked to original sources

bioRxiv2022

Comprehensive Analysis of CRISPR Base Editing Outcomes for Multimeric Protein

Point mutations in the KCNJ13 gene cause an autosomal recessive, childhood blindness, Leber congenital amaurosis (LCA16) due to a loss-of-function Kir7.1 channel. In the present study, we investigated the etiology of LCA16 caused by a KCNJ13 missense mutation (c.431T>C, p.Leu144Pro) and explored the activity of two cytosine base editors mRNAs (CBEs, BE4max-WTCas9, and evoCDA-SpCas9-NG) as a proof-of-concept therapeutic option. We observed the KCNJ13-related retinopathy phenotype in patients harboring L144P mutation. Our in-silico prediction and in vitro validation demonstrated that L144P mutation affects the channel function. We observed high on-target efficiency in the CBEs treated L144P mutant gene expressing HEK-293 cells. Strikingly, our evaluation of base editing efficacy using electrophysiology showed negligible channel function. We found that the editing bystander Cs in the protospacer region led to a missense change (L143F) in evoCDA edited cells and only silent changes in BE4max edited cells. Upon investigation of the effect of the synonymous codon, our extended analysis revealed distortion of mRNA structure, altered half-life, and/or low abundance of the cognate tRNA. We propose that KCNJ13-L144P mutation or other genes that share similar genetic complexity may be challenging to correct with the current generation of CRISPR base editors, and a combinational therapy using CRISPR base editors with a tighter editing window and requisite cognate-tRNA supplementation could be an alternative therapeutic approach to restore Kir7.1 channel function in LCA16 patients. Other options for hard-to-rescue alleles could employ homology-directed repair using CRISPR/Cas9 nucleases, Prime editing, and AAV-mediated gene augmentation.

genetics↗

bioRxiv2022

Autophagy Increases Occludin Levels to Enhance Intestinal Paracellular Tight Junction Barrier.

Background and AimFunctional loss of paracellular tight junction (TJ) barrier of the gut epithelium and mutations in autophagy genes are factors potentiating inflammatory bowel disease (IBD). Previously we showed the role of autophagy in enhancing the TJ barrier via claudin-2 degradation, however, its role in the regulation of the barrier-forming protein occludin remains unknown. Here, we investigate the role of autophagy in the regulation of occludin and its role in inflammation-mediated TJ barrier loss. MethodsPharmacological and genetic tools were used to study the effect of autophagy on occludin levels and localization, and the role of the MAPK pathway. ResultsAutophagy induction using pharmacological activators and nutrient starvation increased total occludin levels in different epithelial cells. Starvation enriched membrane occludin levels and reduced paracellular inulin flux in Caco-2 cells. Starvation-induced TJ barrier enhancement was contingent on the presence of occludin as OCLN-/- nullified its TJ barrier enhancing effect. Autophagy inhibited the constitutive degradation of occludin and protected against inflammation-induced TJ barrier loss. Starvation-induced TJ barrier enhancement was prevented by inhibition of autophagy. Autophagy enhanced the phosphorylation of ERK-1/2. Inhibition of these kinases in Caco-2 cells and human intestinal mucosa inhibited the protective effects of autophagy. In-vivo, autophagy induction by rapamycin increased occludin levels in mouse intestines and protected against LPS and TNF--induced TJ barrier loss. Additionally, acute Atg7 knockout in adult mice decreased intestinal occludin levels, increasing baseline colonic TJ-permeability and exacerbating the effect of DSS-induced colitis. ConclusionOur data suggest a novel role of autophagy in promoting the intestinal TJ barrier by increasing occludin levels in an ERK1/2 MAPK-dependent mechanism. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/487876v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1e08d0dorg.highwire.dtl.DTLVardef@14c65f5org.highwire.dtl.DTLVardef@b9e393org.highwire.dtl.DTLVardef@bcd65_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Label-free imaging to track reprogramming of human somatic cells

The process of reprogramming patient samples to human induced pluripotent stem cells (iPSCs) is stochastic, asynchronous, and inefficient leading to a heterogeneous population of cells. Here, we track the reprogramming status of single patient-derived cells during reprogramming with label-free live-cell imaging of cellular metabolism and nuclear morphometry to identify high-quality iPSCs. Erythroid progenitor cells (EPCs) isolated from human peripheral blood showed distinct patterns of autofluorescence lifetime for the reduced form of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) during reprogramming. Random forest models classified starting EPCs, partially-reprogrammed intermediate cells, and iPSCs with [~]95% accuracy. Reprogramming trajectories resolved at the single cell level indicated significant reprogramming heterogeneity along different branches of cell state. This combination of micropatterning, autofluorescence imaging, and machine learning provides a unique non-destructive method to assess the quality of iPSCs in real-time for various applications in regenerative medicine, cell therapy biomanufacturing, and disease modeling.

bioengineering↗

bioRxiv2021

Subfamily-specific differential contribution of individual monomers and the tether sequence to mouse L1 promoter activity

BackgroundThe internal promoter in L1 5UTR is critical for autonomous L1 transcription and initiating retrotransposition. Unlike the human genome, which features one contemporarily active subfamily, four subfamilies (A_I, Gf_I and Tf_I/II) have been amplifying in the mouse genome in the last one million years. Moreover, mouse L1 5UTRs are organized into tandem repeats called monomers, which are separated from ORF1 by a tether domain. In this study, we aim to compare promoter activities across young mouse L1 subfamilies and investigate the contribution of individual monomers and the tether sequence. ResultsWe observed an inverse relationship between subfamily age and the average number of monomers among evolutionarily young mouse L1 subfamilies. The youngest subgroup (A_I and Tf_I/II) on average carry 3-4 monomers in the 5UTR. Using a single-vector dual-luciferase reporter assay, we compared promoter activities across six L1 subfamilies (A_I/II, Gf_I and Tf_I/II/III) and established their antisense promoter activities in a mouse embryonic fibroblast cell line. Using consensus promoter sequences for three subfamilies (A_I, Gf_I and Tf_I), we dissected the differential roles of individual monomers and the tether domain in L1 promoter activity. We validated that, across multiple subfamilies, the second monomer consistently enhances the overall promoter activity. For individual promoter components, monomer 2 is consistently more active than the corresponding monomer 1 and/or the tether for each subfamily. Importantly, we revealed intricate interactions between monomer 2, monomer 1 and tether domains in a subfamily-specific manner. Furthermore, using three-monomer 5UTRs, we established a complex nonlinear relationship between the length of the outmost monomer and the overall promoter activity. ConclusionsThe laboratory mouse is an important mammalian model system for human diseases as well as L1 biology. Our study extends previous findings and represents an important step toward a better understanding of the molecular mechanism controlling mouse L1 transcription as well as L1s impact on development and disease.

genomics↗

bioRxiv2021

Mechanisms of modulation of pre-mRNA 3D structural scaffolds for splicing substrate definition

We recently reported that serine-arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65, and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.

biochemistry↗

bioRxiv2021

CRISPR-mediated insertion of a chimeric antigen receptor produces nonviral T cell products capable of inducing solid tumor regression

BackgroundChimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures. MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within nine days using recombinant Cas9 protein and nucleic acids, without any viral vectors or animal components. The CAR was specifically targeted to the T Cell Receptor Alpha Constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, scRNA-seq, spectral cytometry, and ELISA assays. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model. ResultsIn comparison to traditional retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory phenotype, and low tonic signaling prior to infusion arising in part from the the knockout of the TCR. Upon exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo, with robust homing, persistence, and low exhaustion against a human neuroblastoma xenograft model. ConclusionsThis proof-of-principle study leveraging virus-free genome editing technology could enable flexible manufacturing of clinically relevant, high-quality CAR T cells to treat cancers, including solid tumors.

bioengineering↗

bioRxiv2020

Nano-Emulsion Adjuvant Enhances Cross-presentation by Lipid Body Formation Independent of Glycolysis

Here, we report that a carbomer-based adjuvant, Adjuplex(R) (ADJ), stimulated robust CD8 T-cell responses to subunit antigens by modulating multiple steps in the cytosolic pathway of cross-presentation, and afforded effective immunity against virus and intracellular bacteria. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs and NLRP3-driven caspase 1 activity. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, induction of ROS and lipid bodies (LBs) and alterations in LB composition mediated by ADJ were critical for DC cross-presentation. These findings challenge the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have implications in the rational development of novel adjuvants.

immunology↗

bioRxiv2019

Microglial homeostasis requires balanced CSF-1/CSF-2 receptor signaling

CSF-1R haploinsufficiency causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). Previous studies in the Csf1r+/- mouse model of ALSP hypothesized a central role of elevated cerebral Csf2 expression. Here we show that monoallelic deletion of Csf2 rescues most behavioral deficits and histopathological changes in Csf1r+/- mice by preventing microgliosis and eliminating most microglial transcriptomic alterations, including those indicative of oxidative stress and demyelination. We also show elevation of Csf2 transcripts and of several CSF-2 downstream targets in the brains of ALSP patients, demonstrating that the mechanisms identified in the mouse model are functional in man. Our data provide new insights into the mechanisms underlying ALSP. Since both increased CSF2 levels and decreased microglial Csf1r expression have also been reported in Alzheimers disease and multiple sclerosis, we suggest that the unbalanced CSF-1R/CSF-2 signaling we describe in the present study may contribute to the pathogenesis of other neurodegenerative conditions. HighlightsO_LIALSP is a CSF1R-deficiency dementia associated with increased CSF2 expression C_LIO_LIIn Csf1r+/- ALSP mice CSF-2 promotes microgliosis by direct signaling in microglia C_LIO_LITargeting Csf2 improves cognition, myelination and normalizes microglial function C_LIO_LICSF-2 is a therapeutic target in ALSP C_LI

neuroscience↗

bioRxiv2019

Human iPSC modeling elucidates mutation-specific responses to gene therapy in a genotypically diverse dominant maculopathy

Dominantly inherited disorders are not typically considered therapeutic candidates for gene augmentation. Here, we utilized patient-specific induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in iPSC-RPE models of recessive bestrophinopathy and dominant Best disease caused by two different ion binding domain mutations. A dominant Best disease iPSC-RPE model that did not respond to gene augmentation showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then tested gene editing in all three dominant Best disease iPSC-RPE models, which produced premature stop codons exclusively within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of RPE transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some dominant Best disease patients and that non-responders are candidates for alternate approaches such as genome editing. However, testing genome editing strategies for on-target efficiency and off-target events using patient-matched iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected patients. SignificanceDominantly inherited disorders pose distinct challenges for gene therapies, particularly in the face of extreme mutational diversity. We tested whether a broad gene replacement strategy could reverse the cellular phenotype of Best disease, a dominant blinding condition that targets retinal pigment epithelium (RPE). Using RPE generated from patient-specific induced pluripotent stem cells (iPSCs), we show that gene replacement functionally overcomes some, but not all, of the tested mutations. In comparison, all dominant Best disease models tested were phenotypically corrected after mutation-specific genome editing, although one off-target genomic alteration was discovered. Our results support a two-tiered approach to gene therapy for Best disease, guided by safety and efficacy testing in iPSC-RPE models to maximize personal and public health value.

genetics↗

bioRxiv2019

Label-free Method for Classification of T cell Activation

T cells have a range of cytotoxic and immune-modulating functions, depending on activation state and subtype. However, current methods to assess T cell function use exogenous labels that often require cell permeabilization, which is limiting for time-course studies of T cell activation and non-destructive quality control of immunotherapies. Label-free optical imaging is an attractive solution. Here, we use autofluorescence imaging of NAD(P)H and FAD, co-enzymes of metabolism, to quantify optical imaging endpoints in quiescent and activated T cells. Machine learning classification models were developed for label-free, non-destructive determination of T cell activation state. T cells were isolated from the peripheral blood of human donors, and a subset were activated with a tetrameric antibody against CD2/CD3/CD28 surface ligands. NAD(P)H and FAD autofluorescence intensity and lifetime of the T cells were imaged using a multiphoton fluorescence lifetime microscope. Significant differences in autofluorescence imaging end-points were observed between quiescent and activated T cells. Feature selection methods revealed that the contribution of the short NAD(P)H lifetime (1) is the most important feature for classification of activation state, across multiple donors and T cell subsets. Logistic regression models achieved 97-99% accuracy for classification of T cell activation from the autofluorescence imaging endpoints. Additionally, autofluorescence imaging revealed NAD(P)H and FAD autofluorescence differences between CD3+CD8+ and CD3+CD4+ T cells, and random forest models of the autofluorescence imaging endpoints achieved 97+% accuracy for four-group classification of quiescent and activated CD3+CD8+ and CD3+CD4+ T cells. Altogether these results indicate that autofluorescence imaging of NAD(P)H and FAD is a powerful method for label-free, non-destructive determination of T cell activation and subtype, which could have important applications for the treatment of cancer, autoimmune, infectious, and other diseases.

bioengineering↗

bioRxiv2018

A CRISPR/Cas9 based strategy to manipulate the Alzheimer’s amyloid pathway

The gradual accumulation of amyloid-{beta} (A{beta}) is a neuropathologic hallmark of Alzheimers disease (AD); playing a key role in disease progression. A{beta} is generated by the sequential cleavage of amyloid precursor protein (APP) by {beta}- and {gamma}-secretases, with BACE-1 ({beta}-site APP cleaving enzyme-1) cleavage as the rate limiting step 1-3. CRISPR/Cas9 guided gene-editing is emerging as a promising tool to edit pathogenic mutations and hinder disease progression 4,5,6 However, few studies have applied this technology to neurologic diseases 7-9. Besides technical caveats such as low editing efficiency in brains and limited in vivo validation 7, the canonical approach of mutation-correction would only be applicable to the small fraction of neurodegenerative cases that are inherited (i.e. < 10% of AD, Parkinsons, ALS); with a new strategy needed for every gene. Moreover, feasibility of CRISPR/Cas9 as a therapeutic possibility in sporadic AD has not been explored. Here we introduce a strategy to edit endogenous APP at the extreme C-terminus and reciprocally manipulate the amyloid pathway - attenuating {beta}-cleavage and A{beta}, while up-regulating neuroprotective a-cleavage. APP N-terminus, as well as compensatory APP homologues remain intact, and key physiologic parameters remain unaffected. Robust APP-editing is seen in cell lines, cultured neurons, human embryonic stem cells/iPSC-neurons, and mouse brains. Our strategy works by limiting the physical association of APP and BACE-1, and we also delineate the mechanism that abrogates APP/BACE-1 interaction in this setting. Our work offers an innovative cut and silence gene-editing strategy that could be a new therapeutic paradigm for AD.

neuroscience↗

bioRxiv2018

Exonic unpaired elements modulate pre-mRNA structure for splice site recognition

Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short ([lsim]70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.

biochemistry↗

bioRxiv2018

In vitro assembly of an early spliceosome defining both splice sites

The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of three-dimensional structural scaffold of AdML - a model pre-mRNA substrate - guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5' and 3' splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a three-dimensional structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable three-dimensional structural scaffolds to the splicing code across the mammalian transcriptome.

biochemistry↗

bioRxiv2017

Micropatterned Substrates To Promote And Dissect Reprogramming Of Human Somatic Cells

Reprogramming of human somatic cells to induce pluripotent stem cells (iPSCs) generates valuable precursors for disease modeling and regenerative medicine. However, the reprogramming process can be inefficient and noisy, creating many partially reprogrammed cells in addition to fully reprogrammed iPSCs. To address these shortcomings, we developed a micropatterned substrate that allows for dynamic live-cell microscopy of thousands of cell subpopulations undergoing reprogramming. Micropatterning facilitated a change in shape, size and clustering of nuclei to promote somatic identity erasure. Increased proliferation, cell density and decreased intercellular YAP signaling accompanied these nuclear changes. A combination of eight nuclear characteristics could be used to track reprogramming progression and distinguish partially reprogrammed cells from those that were fully reprogrammed.\n\nMicropatterned substrates constitute a new tool for facile iPSC production and can be used in high-throughput to probe and understand the subcellular changes that accompany human cell fate transitions.

bioengineering↗