CRISPR/Cas-mediated activation of genes associated with inherited retinal dystrophies in human cells for diagnostic purposes

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Abstract

Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease-related gene transcripts from patient cells would improve detection of mutations that have been missed or misinterpreted in terms of pathogenicity during routine genome sequencing. However, the analysis of transcripts is complicated by the fact that a biopsy of the affected tissue is often not appropriate, and many disease-associated genes are not expressed in tissues or cells that can be easily obtained from patients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRD). We were able to efficiently activate several IRD-linked genes and detect the corresponding transcripts using different diagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read RNA sequencing. The detection and analysis of known and unknown mRNA isoforms demonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. These results will contribute to ceasing the critical gap in the genetic diagnosis of IRD patients and other inherited diseases.

Authors

Valentin J. Weber, Alice Reschigna, Maximilian J. Gerhardt, Thomas Heigl, Klara S. Hinrichsmeyer, Sander van den Engel, Dina Y. Otify, Zoran Gavrilov, Frank Blaser, Isabelle Meneau, Christian Betz, Hanno J. Bolz, Martin Biel, Stylianos Michalakis, Elvir Becirovic

Extracellular matrix alterations in chronic ischemic cardiomyopathy revealed by quantitative proteomics

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Abstract

Ischemic cardiomyopathy (ICM) is a leading cause of heart failure characterized by extensive remodeling of the cardiac extracellular matrix (ECM). While initially adaptive, ECM deposition following ischemic injury eventually turns maladaptive, promoting adverse cardiac remodeling. The strong link between the extent of fibrosis and adverse clinical outcomes has led to growing interest in ECM targeted therapies to prevent or reverse maladaptive cardiac remodeling in ICM; yet, the precise composition of the ECM in ICM remains poorly defined. In this study, we employed a sequential protein extraction enabled by the photocleavable surfactant Azo to enrich ECM proteins from left ventricular tissues of patients with end-stage ICM (n=16) and nonfailing donor hearts (n=16). High-resolution mass spectrometry-based quantitative proteomics identified and quantified over 6,000 unique protein groups, including 315 ECM proteins. We discovered significant upregulation of key ECM components, particularly glycoproteins, proteoglycans, collagens, and ECM regulators. Notably, LOXL1, FBLN1, and VCAN were among the most differentially expressed. Functional enrichment analyses revealed enhanced TGFβ signaling, integrin-mediated adhesion, and complement activation in ICM tissues, suggesting a feedback loop driving continued ECM deposition in the end-stage failing heart. Together, our findings provide a comprehensive proteomic landscape of ECM alterations in the end-stage ICM myocardium and identify promising molecular targets for therapeutic intervention.

Authors

Kevin M. Buck, Holden T. Rogers, Zachery R. Gregorich, Morgan W. Mann, Timothy J. Aballo, Zhan Gao, Emily A. Chapman, Andrew J. Perciaccante, Scott J. Price, Ienglam Lei, Paul C. Tang, Ying Ge

Urine Proteomic Signatures of Kidney Function Decline after Hospitalization

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Abstract

BACKGROUND. Urine proteomics may provide mechanistic insights on why patients experience a higher risk of kidney function decline after hospitalization. METHDOS. In 174 patients with and without acute kidney injury (AKI) from the Assessment, Serial Evaluation, and Subsequent Sequelae in AKI (ASSESS-AKI) cohort, we used Olink to profile 2783 urine proteins collected at 3 months post-hospitalization and determined their association with estimated glomerular filtration rate (eGFR) decline during median [IQR] of 5.1[4.0-6.0] years follow-up. In four independent cohorts including the Kidney Precision Medicine Project (KPMP), we determined if proteins were differentially expressed with AKI. We used weighted correlation network analysis to determine proteins’ cellular enrichment in the kidney transcriptome (single-cell and spatial transcriptomics) in patients with AKI receiving research kidney biopsy. RESULTS. We identified 387 and 10 proteins associated with faster and slower eGFR decline, respectively, most of which were differentially expressed in patients at the time of AKI. Among these proteins, 283 (71%) were expressed by kidney cells in participants with AKI from KPMP. The expression formed 3 clusters enriched in the proximal tubule, degenerative tubule and myeloid cells, and stromal cells, and correlated with histopathological features of AKI, such as tubular injury, interstitial inflammation, and fibrosis, respectively. CONCLUSION. Urinary proteins reflecting degenerative tubular injury, inflammation, and fibrosis are associated with eGFR decline in recently hospitalized patients. FUNDING. The Kidney Precision Medicine Project (KPMP) is supported by the National Institute of Diabetes and Digestive Kidney Diseases (NIDDK) through the following grantsU01DK133081, U01DK133091, U01DK133092, U01DK133093, U01DK133095, U01DK133097, U01DK114866, U01DK114908, U01DK133090, U01DK133113, U01DK133766, U01DK133768, U01DK114907, U01DK114920, U01DK114923, U01DK114933, U24DK114886, UH3DK114926, UH3DK114861, UH3DK114915, and UH3DK114937 We gratefully acknowledge the essential contributions of our patient participants and support of the American public though their tax dollars. SM is supported by NIDDK Grant K23DK128358.

Authors

Yumeng Wen, Steven Menez, Heather Thiessen Philbrook, Dennis Moledina, Steven G. Coca, Jiashu Xue, James Kaufman, Vernon Chinchillil, Paul L. Kimmel, T. Alp Ikizler, Chi-yuan Hsu, Tanika Kelly, Ana Ricardo, Jonathan Himmelfarb, Chirag R. Parikh

Living human lung slices for ex vivo modelling of lung cancer

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Abstract

The tumor microenvironment (TME) significantly impacts cancer progression, yet traditional animal models do not fully recapitulate the situation in humans. To address this, we developed tumor-derived precision lung slices (TD-PCLS), an ex vivo platform for studying the lung TME and evaluating therapies. TD-PCLS, viable for 8 to 10 days, preserve the heterogeneity and metabolic activity of primary tumors, as confirmed by seahorse analysis. Using multispectral FACS and phenocycler multiplex imaging, we spatially profiled TME components and cancer cell functionality. Additionally, TD-PCLS revealed patient-specific responses to chemo- and immunotherapies. To complement TD-PCLS, we established tumor-cell-seeded PCLS (TCS-PCLS) by introducing tumor and immune cells into healthy lung slices. This model highlighted macrophage-tumor interactions as critical for tumor cell proliferation, migration, and immune modulation. Together, these platforms provide a robust tool for lung cancer research, enabling precision medicine and advancing therapeutic discovery.

Authors

Siavash Mansouri, Annika Karger, Clemens Ruppert, Marc A. Schneider, Andreas Weigert, Rajender Nandigama, Blerina Aliraj, Lisa Strotmann, Anoop V. Cherian, Diethard Pruefer, Peter Dorfmuller, Ludger Fink, Ibrahim Alkoudmani, Stefan Gattenlöhner, Bastian Eul, Andre Althoff, Peter Kleine, Hauke Winter, Andreas Guenther, Ardeschir Ghofrani, Soni S. Pullamsetti, Friedrich Grimminger, Werner Seeger, Rajkumar Savai

Device-free isolation of photoreceptor cells from patient iPSC-derived retinal organoids

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Abstract

Autologous photoreceptor cell replacement is one of the most promising strategies currently being developed for the treatment of patients with inherited retinal degenerative blindness. Induced pluripotent stem cell (iPSC) derived retinal organoids, which faithfully recapitulate the structure of the neural retina, are an ideal source of transplantable photoreceptors required for these therapies. However, retinal organoids contain other retinal cell types, including bipolar, horizontal and amacrine cells, which are unneeded and may reduce the potency of the final therapeutic product. Therefore, approaches for isolating fate committed photoreceptor cells from dissociated retinal organoids are desirable. In this work, we present partial dissociation, a technique which leverages the high level of organization found in retinal organoids to enable selective enrichment of photoreceptor cells without the use of specialized equipment or reagents such as antibody labels. We demonstrate up to 90% photoreceptor cell purity by simply selecting cell fractions liberated from retinal organoids during enzymatic digestion in the absence of mechanical dissociation. As the presented approach relies on the use of standard plasticware and commercially available cGMP compliant reagents, we believe that it is ideal for use in the preparation of clinical photoreceptor cell replacement therapies.

Authors

Nicholas E. Stone, Laura R. Bohrer, Nathaniel K. Mullin, Alexander Berthold, Allison T. Wright, Ian C. Han, Edwin M. Stone, Robert F. Mullins, Budd A. Tucker

Label-free single cell phenotyping to determine tumor cell heterogeneity in pancreatic cancer in real-time

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Abstract

Resistance to chemotherapy of pancreatic ductal adenocarcinoma (PDAC) is largely driven by intratumoral heterogeneity (ITH) due to tumor cell plasticity and clonal diversity. In order to develop novel strategies to overcome this defined mechanism of resistance, tools to monitor and quantify ITH in a rapid and scalable fashion are needed urgently. Here, we employed label-free digital holographic microscopy (DHM) to characterize ITH in PDAC. We established a robust experimental and machine learning analysis pipeline to perform single cell phenotyping based on DHM-derived phase images of PDAC cells in suspension. Importantly, we are able to detect dynamic changes in tumor cell differentiation and heterogeneity of distinct PDAC subtypes upon induction of epithelial-to-mesenchymal transition and under treatment-imposed pressure in murine and patient-derived model systems. This platform allows us to assess phenotypic ITH in PDAC on a single cell level in real-time. Implementing this technology into the clinical workflow has the potential to fundamentally increase our understanding of tumor heterogeneity during evolution and treatment response.

Authors

Katja Wittenzellner, Manuel Lengl, Stefan Röhrl, Carlo Maurer, Christian Klenk, Aristeidis Papargyriou, Laura Schmidleitner, Nicole Kabella, Akul Shastri, David E. Fresacher, Farid Harb, Nawal Hafez, Stefanie Bärthel, Daniele Lucarelli, Carmen Escorial-Iriarte, Felix Orben, Rupert Öllinger, Ellen Emken, Lisa Fricke, Joanna Madej, Patrick Wustrow, I. Ekin Demir, Helmut Friess, Tobias Lahmer, Roland M. Schmid, Roland Rad, Günter Schneider, Bernhard Kuster, Dieter Saur, Oliver Hayden, Klaus Diepold, Maximilian Reichert

Cellular immunophenotyping in human and primate tissues during healthy conditions and Ebola and Nipah infections

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Abstract

We developed a 29-color spectral cytometry panel to enhance nonhuman primate (NHP) models for cross-reactive immunophenotyping. This panel is suitable for biosafety level 4 (BSL-4) viruses and can be used with both human and NHP samples in BSL-2 research settings. Tissues from humans, rhesus monkeys (RhMs), crab-eating macaques (CEMs), and green monkeys (GMs) were stained with a 29-color immunophenotyping panel requiring only two clone substitutions. Comparable staining was observed for all samples. Unbiased analysis showed acceptable overlap in T-cell phenotypes across samples, with differences in human and NHP B cells and granulocytes. In CEMs, most circulating CD8+ T cells were from effector memory cells, with significantly higher levels than in humans (p<0.0001), RhMs (p<0.05), and GMs (p<0.01). Analysis of samples from various anatomical sites revealed distinct location-specific phenotypes. In Nipah-virus-exposed animals, splenocytes showed a substantial increase in IgM+ B cells (p<0.0001) and a reduction in effector memory CD8+ T cells (p<0.0001) compared to unexposed controls. Lymph nodes from Ebola-virus-exposed animals showed a loss of CXCR3+CD8+ T cells vs unexposed controls. This panel may guide the development of additional multi-color panels in preclinical and clinical settings and potentially increase understanding of the pathogenesis of diseases caused by emerging and re-emerging viruses.

Authors

Andrew P. Platt, Bobbi Barr, Anthony Marketon, Rebecca Bernbaum, Deja F.P. Rivera, Vincent J. Munster, Daniel S. Chertow, Michael R. Holbrook, Scott M. Anthony, Bapi Pahar

Intravital imaging of peritubular microcirculation impairment in cisplatin-induced acute kidney injury

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Abstract

Despite the accumulation of cisplatin in proximal tubules, direct visualization of the surrounding peritubular microcirculation, including its alteration in cisplatin-induced acute kidney injury (AKI), is lacking. Here, using fluorescence and cellular angiography through video-rate high-resolution intravital microscopy, progressive disturbance of peritubular microcirculation in cisplatin-induced AKI in mice was demonstrated. Fluorescence angiography revealed increasing perfusion defects, with a stepwise rise in time to peak (TTP), originating from capillaries surrounding S1 segments. Cellular angiography demonstrated a progressive decrease in the velocity and track length of individual erythrocytes during AKI progression, accompanied by a sequential decrease in the functional capillary ratio (FCR). Alterations in the perfusion area, TTP, and FCR preceded significant changes in blood urea nitrogen and cystatin C, suggesting the potential for early diagnosis. Although neutrophil infiltration near proximal tubules increased throughout the progression, it did not cause obstruction of the peritubular microcirculation. Depletion of neutrophils increased mortality due to systemic side effects, whereas functional inactivation of neutrophils using an anti-CD11b antibody improved peritubular microcirculation in cisplatin-induced AKI. This approach enables direct visualization and quantification of peritubular microcirculation and immune cell dynamics, providing insights into renal pathophysiology and potential therapeutic strategies.

Authors

Inwon Park, Seonghye Kim, Young Woo Um, Hee Eun Kim, Jae Hyuk Lee, Sejoong Kim, PILHAN KIM, You Hwan Jo

Vitamin A-Retinoic Acid Contributes to Muscle Stem Cell and Mitochondrial Function Loss in Old Age

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Abstract

Adult stem cells decline in number and function in old age and identifying factors that can delay or revert age-associated adult stem cell dysfunction are vital for maintaining healthy lifespan. Here we show that Vitamin A, a micronutrient that is derived from diet and metabolized into retinoic acid, acts as an antioxidant and transcriptional regulator in muscle stem cells. We first show that obstruction of dietary Vitamin A in young animals drives mitochondrial and cell cycle dysfunction in muscle stem cells that mimics old age. Next, we pharmacologically targeted retinoic acid signaling in myoblasts and aged muscle stem cells ex vivo and in vivo and observed reductions in oxidative damage, enhanced mitochondrial function, and improved maintenance of quiescence through fatty acid oxidation. We next detected the receptor for vitamin A derived retinol, stimulated by retinoic acid 6 or Stra6, was diminished with muscle stem cell activation and in old age. To understand the relevance of Stra6 loss, we knocked down Stra6 and observed an accumulation of mitochondrial reactive oxygen species, as well as changes in mitochondrial morphology and respiration. These results demonstrate that Vitamin A regulates mitochondria and metabolism in muscle stem cells and highlight a unique mechanism connecting stem cell function with vitamin intake.

Authors

Paula M. Fraczek, Pamela Duran, Benjamin A. Yang, Valeria Ferre, Leanne Alawieh, Jesus A. Castor-Macias, Vivian T. Wong, Steve D. Guzman, Celeste Piotto, Klimentini Itsani, Jacqueline A Larouche, Carlos A. Aguilar

Tape strip expression profiling of juvenile dermatomyositis skin reveals mitochondrial dysfunction contributing to disease endotype

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Abstract

Skin inflammation in juvenile dermatomyositis (JDM) can signal disease onset or flare, and the persistence of cutaneous disease can prevent complete disease remission. The non-invasive study of cutaneous expression signatures through tape stripping (TS) holds the potential to reveal mechanisms underlying disease heterogeneity and organ-specific inflammation. The objectives of this study were to 1) define TS expression signatures in lesional and non-lesional JDM skin, 2) analyze TS signatures to identify JDM disease endotypes and 3) compare TS and blood signatures. While JDM lesional skin demonstrated interferon signaling as the top upregulated pathway, non-lesional skin uniquely highlighted pathways involved in metabolism, angiogenesis and calcium signaling. Both lesional and non-lesional skin shared inflammasome pathway dysregulation. Using unsupervised clustering of skin expression data, we identified a treatment-refractory JDM subgroup distinguished by upregulation of genes associated with mitochondrial dysfunction. The treatment-refractory JDM subgroup also demonstrated higher interferon, angiogenesis and innate immune expression scores in skin and blood, although scores were more pronounced in skin as compared to blood. Tape-stripping expression signatures in JDM provided insight into disease mechanisms and molecular subgroups. Skin, as compared to blood, transcriptional profiles served as more sensitive markers to classify disease subgroups and identify candidate treatment targets.

Authors

Jessica L. Turnier, Sarah M.H. Vandenbergen, Madison E. McClune, Christine Goudsmit, Sophia Matossian, Meredith Riebschleger, Nadine Saad, Jacqueline A. Madison, Smriti Mohan, Johann E. Gudjonsson, Lam C. Tsoi, Celine C. Berthier, J. Michelle Kahlenberg