Biology subjects

Kannan, S.

Publications and source records attributed to Kannan, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Disrupting SORL1-retromer endosomal recycling in mice drives the age-dependent neurodegenerative signature of Alzheimer's disease

MRI studies have mapped the atrophic signature that distinguishes Alzheimer's disease (AD), isolating regions differentially vulnerable to its age-dependent neurodegenerative process. An endosomal recycling pathway regulated by the interaction of SORL1 with retromer is pathogenically disrupted in AD. Here we test the hypothesis, that the pathway might drive the neurodegenerative process, via multiple yearlong studies in over 140 wildtype and SORL1 deficient mice, modeling SORL1's causative mutations. MRI studies revealed that SORL1 deficiency induced an age-dependent AD-pattern of atrophy, centered on the trans-entorhinal cortex, and that atrophy was prevented by SORL1 restoration, secondary to overexpressing the retromer protein VPS26b. Postmortem studies validated that the atrophy reflects neuronal loss. Proteomic profiling revealed that the atrophy is associated with reductions in cell death suppressors, one of which was then found deficient in the trans-entorhinal cortex of AD patients. Besides supporting the hypothesis, when taken together with other findings the results suggest that SORL1-retromer disruptions represent a therapeutically-amenable common 'neurodegeneration pathway' in AD.

neuroscience↗

bioRxiv2026

Convergent IGHV3-53/3-66 antibodies elicited by Omicron BA.1 infection broadly neutralize emerging SARS-CoV-2 variants

Natural SARS-CoV-2 infections or vaccinations induce neutralizing antibodies (nAbs) offer protection from severe disease. The shared use of IGHV3-53/3-66 genes makes this class of monoclonal antibodies (mAbs) a public clonotype and is well established, but the evolution and structural basis of how these public antibodies maintain broad binding and acquire potent neutralizing activity is not completely understood. To understand how these features are facilitated by the IGHV3-53/3-66 germline segments and enhanced by somatic mutations, we investigated the biology of a panel of 242 human mAbs isolated from an individual infected with SARS-CoV-2 BA.1 strain and recovered. Interestingly, a mAb designated COV2-3731 encoded by IGHV3-53/IGKV1-33 retained potent neutralizing activity against SARS-CoV-2 variants BA.2.86, JN.1, KP.2, BA.3.2, and, to some extent, KP.3. Studies using deep mutational scanning with a BA.2 lentiviral library and determination of the structural complex of the BA.2 S protein and COV2-3731 Fab fragments using cryo-EM revealed key contact residues. Further, germline revertant analysis of the COV2-3731 mAb provided additional insights into how this COV2-3731 and other IGHV3-53/3-66-encoded public antibodies evolve to gain breadth against antigenically distinct SARS-CoV-2 variants such as BA.2.86, JN.1, KP.2, and BA.3.2.

immunology↗

bioRxiv2026

Genomic repeats for single-cell molecular recording

Genomic recording enables transient biological signals to be indelibly captured through DNA alterations, creating a permanent record of cellular history retrievable by sequencing. However, current methods are limited by scarce writing space, typically targeting only one or a few amenable genomic sites and requiring large cell populations for signal reconstruction. Here, we establish Repeats for Genomic Recording (RGRs): sequences with up to 400 copies targetable by a single CRISPR guide RNA, readable with a common primer pair, and predicted to have minimal functional impact. We demonstrate that RGRs enable both signal deconvolution in single cells and high-resolution recording in cell populations. Individual RGR sites exhibit distinct response kinetics; thus, combining them improves recording resolution beyond what redundancy alone provides, analogous to diversity reception in wireless communication. We develop a computational pipeline for systematic RGR identification, revealing 15,000 to 25,000 candidates per species across human, mouse, and zebrafish, thereby markedly expanding recording capacity and enabling cell-type-specific applications. Finally, we validate RGRs in live mice by recording long-term immediate early gene activity across the brain following epilepsy induction. This work establishes genomic repeats as a high-capacity platform for single-cell molecular recording in vivo.

synthetic biology↗

bioRxiv2026

Mucosal-associated invariant T cells support IL-15-dependent Treg response in skin injury and promote resolution of skin inflammation

Mucosal-associated invariant T (MAIT) cells are enriched at barrier sites, but their role in autoimmune skin inflammation remains unknown. Using cutaneous lupus as a model, we identify MAIT cells as protective regulators of skin inflammation and as critical upstream modulators of regulatory T cells (Treg). Topical MAIT cell activation with 5-OP-RU induced durable resolution of spontaneous skin lesions in MRL/lpr mice and suppressed TLR7-driven skin inflammation. MAIT cell activation selectively expanded and activated Treg populations in both healthy and lupus-like skin, while suppressing effector T cell cytokine production and cytotoxic programs. This MAIT-Treg axis was also activated in UV light-driven barrier injury in healthy murine and human skin, where MAIT cells were required for UV-elicited Treg expansion and function. In lupus-like skin, local MAIT cell activation restored the defective UVB-induced Treg response and limited CD8+ T cell expansion. Mechanistically, CCR2+ monocyte-derived antigen-presenting cells and IL-15 signaling were required for MAIT cell-driven Treg accumulation and therapeutic benefits of MAIT cells in inflamed skin. These studies identify a MAIT-IL-15-Treg axis that links barrier injury sensing to immune regulation, which is disrupted in cutaneous lupus, and nominate therapeutic MAIT cell activation as an unappreciated strategy for restoring immune homeostasis in inflamed skin.

immunology↗

bioRxiv2026

Comparison of Automated White Matter Lesion Segmentation Approaches for Use in Large, Multi-Site Data Analyses in Parkinson's Disease

BackgroundParkinsons disease (PD) is the second most common neurodegenerative disorder. PD currently lacks effective disease-modifying treatments, likely due to its diverse clinical features and underlying neuropathology. The vascular role in PD is emerging, with vascular mechanisms increasingly implicated, yet the literature remains conflicted, motivating large-data analyses with greater statistical power. White matter lesions (WML) are an accepted imaging marker of small vessel disease. Accurate automated WML segmentation techniques are crucial for large-scale studies in PD due to the impracticality of manual segmentation for extensive datasets and to ensure consistency. Evaluation of the optimum approach in PD for large-scale analysis is lacking. This study aimed to evaluate various automated WML segmentation algorithms to determine the most accurate and reliable method, among those selected, for assessing WML for multi-site large data analysis in PD. MethodsWe assessed whole-brain volumetric T1-weighted and FLAIR images from 201 PD patients (mean age, 66.6 {+/-} 7.86 years) and 64 healthy controls (HC; mean age, 66.3 {+/-} 8.67) across three datasets: the Parkinsons Progression Markers Initiative (PPMI), the University of Pennsylvania (UPenn) and the Montreal Neurological Institute Biobank: Clinical Biological Imaging and Genetic Repository (C-BIG). The sample included different scanners, imaging parameters and lesion loads, as would be expected for multi-site data. WML were manually segmented to provide the gold standard, and four freely available automated algorithms were evaluated: FSLs BIANCA, FreeSurfer, SPMs LST-LPA and U-Net-pgs using the performance metrics: Dice score, Hausdorff distance, recall, precision, F1 score, log absolute volume difference (LOGAVD) and intraclass correlation coefficient (ICC). Subgroup analyses were performed based on lesion load and lobar regions. The associations of data from these automated approaches with age, and with Fazekas and Wahlund visual rating scales, were assessed through partial correlation analysis. ResultsU-Net-pgs performed best overall, with the highest Dice score (PD: 0.46 {+/-} 0.21; HC: 0.39 {+/-} 0.21), recall (PD: 0.76 {+/-} 0.25; HC: 0.62 {+/-} 0.31), precision (PD: 0.49 {+/-} 0.25; HC: 0.63 {+/-} 0.27), F1 score (PD: 0.54 {+/-} 0.22; HC: 0.56 {+/-} 0.22) and ICC (PD: 0.965; HC: 0.967) and lowest Hausdorff distance (PD: 8.89 {+/-} 3.96; HC: 6.33 {+/-} 2.91). U-Net-pgs achieved the lowest LOGAVD in the PD group (0.31 {+/-} 0.31) whereas BIANCA-LOO with a threshold of 0.9 was lowest in HC (0.27 {+/-} 0.30). U-Net also showed superior performances in all lesion loads for PD and overall across various brain regions in both PD and HC. ConclusionOverall, U-Net-pgs emerged as the best performing automated method, of those we evaluated, for WML segmentation in PD and HC within a dataset collected with various scanner and image acquisition parameters. U-Net-pgs consistently outperformed other automated approaches across lesion loads and brain regions, for most metrics. The accuracy and reliability of U-Net-pgs make it a promising tool for large-scale analyses, facilitating future research investigating WML in PD.

neuroscience↗

bioRxiv2026

RhoGEF12 regulates endosomal SORL1-retromer and its inhibition is therapeutic in human neuronal models of Alzheimer's disease

The interaction of the endosomal sorting protein SORL1 with the retromer complex at endosomal membranes controls a recycling pathway whose dysfunction is pathogenic in Alzheimers disease (AD) and is linked to other neurodegenerative disorders. To search for novel therapeutic targets, we hypothesize that endosomal SORL1-retromer might be regulated by SORL1s cytoplasmic tail. We begin by completing an in vitro analysis of the tail and show that its phosphorylation by ROCK2 (Rho-associated kinase 2) reduces SORL1s affinity to retromer. Since RhoGEF12 (Rho guanine nucleotide exchange factor 12) is an upstream activator of ROCK2 that is upregulated in AD, we used a RhoGEF12 pharmacological inhibitor to mechanistically and therapeutically validate the findings in neuronal cultures. First, in mouse neurons we confirm that the inhibitor increases endosomal SORL1-retromer. Next, we turned to human iPSC-derived neurons to show that the inhibitor reduces A{beta}40 and A{beta}42, an indicator of pathway upregulation, in a SORL1-dependent manner. Finally, we validate its therapeutic potential by applying the RhoGEF12 inhibitor to human iPSC-derived neurons expressing AD-associated mutations in either APP or SORL1. Collectively, our results identify a novel and therapeutically amenable mechanism that regulates endosomal SORL1-retromer and preclinically validate RhoGEF12 as a therapeutic target for AD and potentially other neurodegenerative disorders. One sentence summaryPharmacological inhibition of RhoGEF12 increases endosomal SORL1-retromer recycling and reduces pathogenic amyloid secretion in human neuronal models, identifying a novel, targetable pathway for treating Alzheimers disease.

neuroscience↗

bioRxiv2026

An ER retention motif controls the heteromeric stoichiometry of hERG1a/1b channels

The human ether-a-go-go related gene (hERG) encodes a potassium channel essential for cardiac repolarization and neuronal excitability. In the heart, heteromeric assemblies of hERG1a and hERG1b subunits produce cardiac IKr, and mutations in either subunit are associated with long QT syndrome. Although hERG1a and 1b contain identical transmembrane and C-terminal cytosolic domains, they differ in N-terminal cytosolic domains, with hERG1b harboring an arginine-based endoplasmic reticulum (ER) retention/retrieval motif that limits its surface expression in the absence of hERG1a. While the association of hERG1a and 1b subunits is known to influence channel function, the stoichiometry of heteromeric hERG channels and mechanisms that regulate it have remained unresolved. Here, using single molecule photobleaching step analysis in HeLa cells, we show that heteromeric hERG1a/1b channels assemble predominantly with a fixed 2:2 stoichiometry. Mutation of the hERG1b ER retention motif disrupts this bias, resulting in a broader, near-random distribution of subunit compositions. Independent functional assays using dominant-negative pore mutants in Xenopus oocytes yielded quantitative current suppression consistent with a 2:2 assembly and similarly revealed loss of stoichiometric bias upon RXR mutation. Together, these results establish the oligomeric composition of hERG1a/1b channels and identify ER retention as a previously unrecognized determinant of heteromeric stoichiometry. Statement of SignificanceSubunit stoichiometry is a key determinant of ion channel function, yet how defined stoichiometries are established during biogenesis remains poorly understood. Here we demonstrate that heteromeric hERG1a/1b channels assemble with a fixed 2:2 stoichiometry and that an arginine-based ER retention motif in hERG1b is required to maintain this assembly bias. Using single-molecule photobleaching and independent functional assays, we show that disrupting this motif leads to a broadened, near-random distribution of tetrameric subunit compositions. These findings reveal that ER retention contributes not only to quality control and trafficking but also specifies subunit stoichiometry during ion channel assembly.

biophysics↗

bioRxiv2025

In silico discovery of pathogenic PD-L1 nsSNVs with altered glycosylation and immunotherapy binding

Immune checkpoint inhibitors (ICIs), particularly anti-PD-L1 monoclonal antibodies, block extracellular interactions between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) to enhance antitumour immunity. Here, we present a streamlined workflow integrating public databases, bioinformatics tools, and in silico molecular dynamics (MD) simulations and in vitro experimental assays to identify consequential pathogenic nonsynonymous PD-L1 variants (PD-L1SNVs). Four variants predicted as pathogenic across six bioinformatic tools (SIFT, PolyPhen-2, PANTHER, SNP-PhD, SNP&GO, and Pmut) destabilized the anti-PD-L1 ICI atezolizumab-drug binding epitope in MD studies, correlating with reduced drug affinity. Live-cell assays directly linked PD-L1SNVs to intracellular trafficking defects and aberrant glycosylation, even distal from N-glycosylation sites. Allostery modeling (AlloSigMA) linked these disruptions to long-range structural perturbations. Our work establishes bioinformatics-driven predictions of functionally impactful pathogenic PD-L1SNVs and highlight how glycosylation/trafficking defects may serve as key mechanisms compromising therapeutic efficacy. These insights in predicting and validating pathogenic PD-L1SNVs has significant implications for guiding the personalized selection of effective PD-L1-targeted therapies in clinical settings. One Sentence Summary: Uncovering the implication of non-synonymous single nucleotide variants (nsSNVs) on the structure and function of Programmed Death Ligand-1.

bioinformatics↗

bioRxiv2025

Random survival forests identify myocardial gene signatures associated with survival in heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) continues to be poorly understood at the molecular level. While previous studies have identified gene expression signatures unique to HFpEF, genes associated with clinical decompensation have not been determined. Here, we performed exploratory analysis of myocardial RNA-seq data to identify genes associated with event-free survival in HFpEF. We analyzed previously published RNA sequencing data of right ventricular septal endomyocardial biopsies from HFpEF patients (n=41) with paired clinical, echocardiographic, and outcome data (including mortality and heart failure hospitalizations). We constructed random survival forests with forward stepwise regularization (the "variable hunting" method) to determine genes associated with time to first event using a combined end-point of heart failure hospitalization or all-cause death. Selection of candidate forest variables was tested with both random and weighted sampling methods. We identified 33 genes that are predictive of survival in HFpEF. This set includes genes previously implicated in heart failure, including ADAMTSL2, ADRB1, BMP6, and METRNL. Survival forests constructed using these genes outperform those constructed from clinical and hemodynamic parameters alone (out-of-bag C-index 0.894 vs 0.545). Moreover, in survival forests constructed from both gene data and hemodynamic measurements, individual survival genes consistently showed higher variable importance than clinical/hemodynamic parameters. Our study shows that random survival forests identify myocardial gene signatures that may better model HFpEF prognosis than clinical measurements. Further studies are warranted to validate findings in independent cohorts.

genomics↗

bioRxiv2025

A privileged ER compartment for post-translational heteromeric assembly of an ion channel

Mechanisms underlying heterotypic subunit assembly of ion channels and other oligomeric complexes are poorly understood. In the human heart, heteromeric assembly of two isoforms encoded by the human ether-a-go-go related gene (hERG) is essential for the normal function of cardiac IKr in ventricular repolarization, with loss of hERG1b contributing to arrhythmias associated with long QT-syndrome (LQTS). While hERG1a homomers traffic efficiently to the plasma membrane, hERG1b homomers are retained in the endoplasmic reticulum (ER). When expressed together, the two subunits avidly associate during biogenesis. Seeking rules specifying heteromeric association, we characterized the fate of hERG1b proteins using confocal and superresolution imaging in fixed and live HeLa cells. We found hERG1b sequestered in punctate intracellular structures when expressed alone in HeLa cells. These puncta, which depend on the presence of an N-terminal "RXR" ER retention signal, represent a privileged ER sub-compartment distinct from that containing ER-retained, type 2 (hERG-based) LQTS mutant proteins, which were rapidly degraded by the proteasome. Introducing hERG1a to cells with preformed hERG1b puncta dissolved these puncta by rescuing extant hERG1b. Rescue occurred by association of fully translated hERG1b with 1a, a surprising finding given previous studies demonstrating cotranslational heteromeric association. We propose that sequestration limits potentially deleterious surface expression of hERG1b homomeric channels while preserving hERG1b for an alternative mode of heteromeric hERG1a/1b channel assembly post-translationally. These findings reveal a surprising versatility of biosynthetic pathways promoting heteromeric assembly. Significance StatementhERG potassium channels are essential for repolarizing the ventricular action potential. Heteromeric hERG channels, which conduct cardiac IKr, are formed by association of hERG1a and 1b subunits. However, mechanisms governing the assembly of hERG and other heteromeric protein complexes are poorly understood. We identify a noncanonical ER compartment that sequesters hERG1b, distinct from previously described ER quality control pathways that degrade misfolded proteins, including LQTS-associated hERG variants. Instead, this compartment preserves hERG1b and facilitates its post-translational assembly with hERG1a, challenging the current view of simultaneous, cotranslational heteromeric assembly. Our findings redefine ER retention as an active regulatory step in protein complex formation that may broadly govern hetero-oligomeric assembly in many systems and facilitate therapeutic approaches to arrhythmia and other diseases.

cell biology↗

bioRxiv2024

Recontextualizing Eukaryogenesis via Computational Analysis of RNA Processing in 16,449 Archaeal Genomes

The analysis of proteins related to RNA processing reveals intriguing aspects of the evolutionary transition from archaea to eukaryotes. Eukaryagenesis is the process in which the first eukaryote came into existence, with multiple hypotheses on the order of events. These hypothesis often focus on the mitochondria and cell skeletal structure, without much discussion on eukaryotic signature proteins(ESP). ESP is integral in increasing the longevity of RNA and for the increase in the variety of proteins able to be produced which ultimately increases fitness of eukaryotes. 16,449 genomes and 10 proteins were acquired and BLAST was run for each superclass for each protein. BLAST scores were compared between superclasses and analyzed. Results for proteins such as Prp9, Rex3, Histone H2A, H2B, and Histone 3 indicate that there is no substantial difference between BLAST results implying a transitional state consistent with E3 model. The results for Smd3 and Ceg1 highlight that a group of Asgardarchaeota and Diaforarchaearchaea were different to other types of archaea. These groups likely underwent similar environmental pressures giving organisms with these genes higher fitness. These early genes evolved into their eukaryotic versions, while other genes like Histone 4, Abd1, and Lsm2 may have had ancestral prototypes present across archaea. Gene prototypes likely served different purposes, but the presence of such prototypes imply that evolution of the nucleus was likely independent from the presence of the mitochondria.

evolutionary biology↗

bioRxiv2024

Modulation of Ideonella sakaiensis PETase active site flexibility and activity on morphologically distinct substrates by surface charge engineering

Enzymatic hydrolysis of polyethylene terephthalate (PET) waste is a compelling strategy for environmentally friendly recycling of a major pollutant. Here, we investigate the effects of surface charge point mutations both proximal and distal to the active site of the mesophilic PET-degrading enzyme from Ideonella sakaienses (IsPETase) and an engineered thermostable variant with superior activity, STAR PETase. The vicinal K95A mutation significantly inhibited IsPETase activity on mechanically processed PET powder. Conversely, this mutation significantly increased hydrolysis of PET powder in the STAR PETase background. Activity of both enzymes on PET film was inhibited by the K95A mutation, highlighting complex interplay between mutation context and substrate morphology. Further installing the distal R132N and R280A surface charge mutations potentiated activity of STAR on all substrates tested. This variant afforded 100% degradation of bottle-grade PET powder in 3 days at 40{degrees}C reaction temperature, a 3-fold improvement over IsPETase. Molecular dynamics simulations reveal modulation of active site flexibility in mutants, which differentially impacts both hydrolysis of morphologically distinct PET substrates and the concentration-dependent inhibition phenomenon observed for PETase.

bioengineering↗

bioRxiv2024

Rarγ-Foxa1 signaling promotes luminal identity in prostate progenitors and is disrupted in prostate cancer

Retinoic acid (RA) signaling is a master regulator of vertebrate development with crucial roles in directing body axis orientation and tissue differentiation, including in the reproductive system. However, a mechanistic understanding of how RA signaling promotes cell lineage identity in different tissues is often missing. Here, leveraging prostate organoid technology, we demonstrated that RA signaling orchestrates the commitment of adult mouse prostate progenitors to glandular identity, epithelial barrier integrity, and ultimately, proper specification of the prostatic lumen. Mechanistically, RA-dependent RAR{gamma} activation promotes the expression of the pioneer factor Foxa1, which synergizes with the androgen pathway for proper luminal expansion, cytoarchitecture and function. FOXA1 nucleotide variants are common in human prostate and breast cancers and considered driver mutations, though their pathogenic mechanism is incompletely understood. Combining functional genetics experiments with structural modeling of FOXA1 folding and chromatin binding analyses, we discovered that FOXA1F254E255 is a loss-of-function mutation leading to compromised transcriptional function and lack of luminal fate commitment of prostate progenitors. Overall, we define RA as a crucial instructive signal for glandular identity in adult prostate progenitors. We propose deregulation of vitamin A metabolism as a risk factor for benign and malignant prostate disease, and identified cancer associated FOXA1 indels affecting residue F254 as loss-of-function mutations promoting dedifferentiation of adult prostate progenitors. Summary: Retinoic acid signaling orchestrates luminal differentiation of adult prostate progenitors

cancer biology↗

bioRxiv2023

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97

The t(X,17) chromosomal translocation, generating the ASPSCR1-TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCC), frustrating efforts to identify therapeutic targets for these rare cancers. Proteomic analysis showed that VCP/p97, an AAA+ ATPase with known segregase function, was strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1-TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1-TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributed with ASPSCR1-TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrated the oncogenic transcriptional signature of ASPSCR1-TFE3, by facilitating assembly of higher-order chromatin conformation structures as demonstrated by HiChIP. Finally, ASPSCR1-TFE3 and VCP demonstrated co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCPs potential as a novel therapeutic target.

cancer biology↗

bioRxiv2023

Expanding the DNA editing toolbox: novel lambda integrase variants targeting microalgal and human genome sequences.

Recombinase enzymes are extremely efficient at integrating very large DNA fragments into target genomes. However, intrinsic sequence specificities curtail their use to DNA sequences with sufficient homology to endogenous target motifs. Extensive engineering is therefore required to broaden applicability and robustness. Here, we describe the directed evolution of novel lambda integrase variants capable of editing exogenous target sequences identified in the diatom Phaeodactylum tricornutum and the algae Nannochloropsis oceanica. These microorganisms hold great promise as conduits for green biomanufacturing and carbon sequestration. The evolved enzyme variants show >1000-fold switch in specificity towards the non-natural target sites when assayed in vitro. A single-copy target motif in the human genome with homology to the Nannochloropsis oceanica site can also be efficiently targeted using an engineered integrase, both in vitro and in human cells. The developed integrase variants represent useful additions to the DNA editing toolbox, with particular application for targeted genomic insertion of large DNA cargos.

bioengineering↗

bioRxiv2023

A Gene Therapy for Hereditary Nonpolyposis Colorectal Cancer using CRISPR-Cas9 Nickase

Hereditary non-polyposis colorectal cancer (HNPCC) is an inherited disorder characterized by an increased risk of developing colorectal cancer before age 50. HNPCC is predominantly caused by genetic mutations in MLH1 and MSH2, which are involved in DNA mismatch repair. Current standard practice is to perform prophylactic colectomy, resulting in debilitating aftereffects for life. Though the genetic cause of HNPCC is well-known, there are currently no available treatments that target these mutations. Herein we describe a novel treatment protocol using a CRISPR-Cas9n-based genetic therapy to restore DNA mismatch repair. First, gRNA and template DNA targeting the most prevalent mutation clusters in MLH1 and MSH2 as well as CRISPR-Cas9n elements will be packaged into an integrase-deficient lentiviral vector. Then, the viral vector will be used to transduce human colonic tumor-derived organoids as well as administered systemically in mouse models of HNPCC. Mice will be monitored clinically and for signs of disease progression. At termination, colonic tissue will be harvested and analyzed for restoration of the wild-type MLH1 and MSH2 sequence and biochemical markers of HNPCC. This protocol offers an alternative strategy using CRISPR-Cas9n-based gene therapy to prevent tumor formation in patients, avoid morbid surgery, and significantly improve quality of life.

cancer biology↗

bioRxiv2023

Single-cell RNA sequencing identifies response of renal lymphatic endothelial cells to acute kidney injury

The inflammatory response to acute kidney injury (AKI) likely dictates future renal health. Lymphatic vessels are responsible for maintaining tissue homeostasis through transport and immunomodulatory roles. Due to the relative sparsity of lymphatic endothelial cells (LECs) in the kidney, past sequencing efforts have not characterized these cells and their response to AKI. Here we characterized murine renal LEC subpopulations by single-cell RNA sequencing and investigated their changes in cisplatin AKI. We validated our findings by qPCR in LECs isolated from both cisplatin-injured and ischemia reperfusion injury, by immunofluorescence, and confirmation in in vitro human LECs. We have identified renal LECs and their lymphatic vascular roles that have yet to be characterized in previous studies. We report unique gene changes mapped across control and cisplatin injured conditions. Following AKI, renal LECs alter genes involved endothelial cell apoptosis and vasculogenic processes as well as immunoregulatory signaling and metabolism. Differences between injury models are also identified with renal LECs further demonstrating changed gene expression between cisplatin and ischemia reperfusion injury models, indicating the renal LEC response is both specific to where they lie in the lymphatic vasculature and the renal injury type. How LECs respond to AKI may therefore be key in regulating future kidney disease progression.

pathology↗

bioRxiv2023

Single B cell transcriptomics identifies multiple isotypes of broadly neutralizing antibodies against flaviviruses

Sequential dengue virus (DENV) infections often generate neutralizing antibodies against all four DENV serotypes and sometimes, Zika virus. Characterizing cross-flavivirus broadly neutralizing antibody (bnAb) responses can inform countermeasure strategies that avoid infection enhancement associated with non-neutralizing antibodies. Here, we used single cell transcriptomics to mine the bnAb repertoire following secondary DENV infection. We identified several new bnAbs with comparable or superior breadth and potency to known bnAbs, and with distinct recognition determinants. Unlike all known flavivirus bnAbs, which are IgG1, one newly identified cross-flavivirus bnAb (F25.S02) was derived from IgA1. Both IgG1 and IgA1 versions of F25.S02 and known bnAbs displayed neutralizing activity, but only IgG1 enhanced infection in monocytes expressing IgG and IgA Fc receptors. Moreover, IgG-mediated enhancement of infection was inhibited by IgA1 versions of bnAbs. We demonstrate a role for IgA in flavivirus infection and immunity with implications for vaccine and therapeutic strategies.

microbiology↗