Twin prime editing mediated exon skipping/reinsertion for restored collagen VII expression in recessive dystrophic epidermolysis bullosa.

Gene editing nucleases, base editors, and prime editors are potential locus specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa (RDEB); however, many RDEB COL7A1 mutations are unique, making the development of personalized editing reagents challenging. 270 of the ∼320 COL7A1 EB mutations reside in exons that can be skipped, and antisense oligonucleotides (ASO) and gene editing nucleases have been used to create in-frame deletions. ASOs are transient and nucleases generate deleterious double stranded DNA breaks (DSB) and uncontrolled mixtures of allele products. We developed a twin prime editing (twinPE) strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon five. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in RDEB fibroblasts, keratinocytes, and iPSC with minimal DSBs, and collagen type VII (C7) protein was restored. TwinPE can replace the target exon with recombinase attachment sequences, and we exploited this to re-insert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twinPE can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few DSBs as a strategy that may enable a single therapeutic agent to treat multiple RDEB patient cohorts.

Phosphoproteomic Analysis and Organotypic Cultures for the Study of Signaling Pathways.

Signaling pathways are involved in key cellular functions from embryonic development to pathological conditions, with a pivotal role in tissue homeostasis and transformation. Although most signaling pathways have been intensively examined, most studies have been carried out in murine models or simple cell culture. We describe the dissection of the TGF-ß signaling pathway in human tissue using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) in a 3D organotypic skin model combined with quantitative proteomics and phosphoproteomics mass spectrometry. The use of human 3D organotypic cultures and genetic engineering combined with quantitative proteomics and phosphoproteomics is a powerful tool providing insight into signaling pathways in a human setting. The methods are applicable to other gene targets and 3D cell and tissue models. Key features • 3D organotypic models with genetically engineered human cells. • In-depth quantitative proteomics and phosphoproteomics in 2D cell culture. • Careful handling of cell cultures is critical for the successful formation of the organotypic cultures. • For complete details on the use of this protocol, please refer to Ye et al. 2022.

Glycoengineered keratinocyte library reveals essential functions of specific glycans for all stages of HSV-1 infection.

Viral and host glycans represent an understudied aspect of host-pathogen interactions, despite potential implications for treatment of viral infections. This is due to lack of easily accessible tools for analyzing glycan function in a meaningful context. Here we generate a glycoengineered keratinocyte library delineating human glycosylation pathways to uncover roles of specific glycans at different stages of herpes simplex virus type 1 (HSV-1) infectious cycle. We show the importance of cellular glycosaminoglycans and glycosphingolipids for HSV-1 attachment, N-glycans for entry and spread, and O-glycans for propagation. While altered virion surface structures have minimal effects on the early interactions with wild type cells, mutation of specific O-glycosylation sites affects glycoprotein surface expression and function. In conclusion, the data demonstrates the importance of specific glycans in a clinically relevant human model of HSV-1 infection and highlights the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures.

Galectin-1 induces a tumor-associated macrophage phenotype and upregulates indoleamine 2,3-dioxygenase-1.

Galectins are a group of carbohydrate-binding proteins with a presumed immunomodulatory role and an elusive function on antigen-presenting cells. Here we analyzed the expression of galectin-1 and found upregulation of galectin-1 in the extracellular matrix across multiple tumors. Performing an in-depth and dynamic proteomic and phosphoproteomic analysis of human macrophages stimulated with galectin-1, we show that galectin-1 induces a tumor-associated macrophage phenotype with increased expression of key immune checkpoint protein programmed cell death 1 ligand 1 (PD-L1/CD274) and immunomodulator indoleamine 2,3-dioxygenase-1 (IDO1). Galectin-1 induced IDO1 and its active metabolite kynurenine in a dose-dependent manner through JAK/STAT signaling. In a 3D organotypic tissue model system equipped with genetically engineered tumorigenic epithelial cells, we analyzed the cellular source of galectin-1 in the extracellular matrix and found that galectin-1 is derived from epithelial and stromal cells. Our results highlight the potential of targeting galectin-1 in immunotherapeutic treatment of human cancers.

Targeting Solid Cancers with a Cancer-Specific Monoclonal Antibody to Surface Expressed Aberrantly O-glycosylated Proteins.

The lack of antibodies with sufficient cancer selectivity is currently limiting the treatment of solid tumors by immunotherapies. Most current immunotherapeutic targets are tumor-associated antigens that are also found in healthy tissues and often do not display sufficient cancer selectivity to be used as targets for potent antibody-based immunotherapeutic treatments, such as chimeric antigen receptor (CAR) T cells. Many solid tumors, however, display aberrant glycosylation that results in expression of tumor-associated carbohydrate antigens that are distinct from healthy tissues. Targeting aberrantly glycosylated glycopeptide epitopes within existing or novel glycoprotein targets may provide the cancer selectivity needed for immunotherapy of solid tumors. However, to date only a few such glycopeptide epitopes have been targeted. Here, we used O-glycoproteomics data from multiple cell lines to identify a glycopeptide epitope in CD44v6, a cancer-associated CD44 isoform, and developed a cancer-specific mAb, 4C8, through a glycopeptide immunization strategy. 4C8 selectively binds to Tn-glycosylated CD44v6 in a site-specific manner with low nanomolar affinity. 4C8 was shown to be highly cancer specific by IHC of sections from multiple healthy and cancerous tissues. 4C8 CAR T cells demonstrated target-specific cytotoxicity in vitro and significant tumor regression and increased survival in vivo. Importantly, 4C8 CAR T cells were able to selectively kill target cells in a mixed organotypic skin cancer model having abundant CD44v6 expression without affecting healthy keratinocytes, indicating tolerability and safety.

CRISPR-screen identifies ZIP9 and dysregulated Zn2+ homeostasis as a cause of cancer-associated changes in glycosylation.

INTRODUCTION: In epithelial cancers, truncated O-glycans, such as the Thomson-nouveau antigen (Tn) and its sialylated form (STn), are upregulated on the cell surface and associated with poor prognosis and immunological escape. Recent studies have shown that these carbohydrate epitopes facilitate cancer development and can be targeted therapeutically; however, the mechanism underpinning their expression remains unclear. METHODS: To identify genes directly influencing the expression of cancer-associated O-glycans, we conducted an unbiased, positive-selection, whole-genome CRISPR knockout-screen using monoclonal antibodies against Tn and STn. RESULTS AND CONCLUSIONS: We show that knockout of the Zn2+-transporter SLC39A9 (ZIP9), alongside the well-described targets C1GALT1 (C1GalT1) and its molecular chaperone, C1GALT1C1 (COSMC), results in surface-expression of cancer-associated O-glycans. No other gene perturbations were found to reliably induce O-glycan truncation. We furthermore show that ZIP9 knockout affects N-linked glycosylation, resulting in upregulation of oligo-mannose, hybrid-type, and α2,6-sialylated structures as well as downregulation of tri- and tetra-antennary structures. Finally, we demonstrate that accumulation of Zn2+ in the secretory pathway coincides with cell-surface presentation of truncated O-glycans in cancer tissue, and that over-expression of COSMC mitigates such changes. Collectively, the findings show that dysregulation of ZIP9 and Zn2+ induces cancer-like glycosylation on the cell surface by affecting the glycosylation machinery.

Characterization of TGF-ß signaling in a human organotypic skin model reveals that loss of TGF-ßRII induces invasive tissue growth.

Transforming growth factor-ß (TGF-ß) signaling regulates various aspects of cell growth and differentiation and is often dysregulated in human cancers. We combined genetic engineering of a human organotypic three-dimensional (3D) skin model with global quantitative proteomics and phosphoproteomics to dissect the importance of essential components of the TGF-ß signaling pathway, including the ligands TGF-ß1, TGF-ß2, and TGF-ß3, the receptor TGF-ßRII, and the intracellular effector SMAD4. Consistent with the antiproliferative effects of TGF-ß signaling, the loss of TGF-ß1 or SMAD4 promoted cell cycling and delayed epidermal differentiation. The loss of TGF-ßRII, which abrogates both SMAD4-dependent and SMAD4-independent downstream signaling, more strongly affected cell proliferation and differentiation than did loss of SMAD4, and it induced invasive growth. TGF-ßRII knockout reduced cell-matrix interactions, and the production of matrix proteins increased the production of cancer-associated cell-cell adhesion proteins and proinflammatory mediators and increased mitogen-activated protein kinase (MAPK) signaling. Inhibiting the activation of the ERK and p38 MAPK pathways blocked the development of the invasive phenotype upon the loss of TGF-ßRII. This study provides a framework for exploring TGF-ß signaling pathways in human epithelial tissue homeostasis and transformation using genetic engineering, 3D tissue models, and high-throughput quantitative proteomics and phosphoproteomics.

Global mapping of GalNAc-T isoform-specificities and O-glycosylation site-occupancy in a tissue-forming human cell line.

Mucin-type-O-glycosylation on proteins is integrally involved in human health and disease and is coordinated by an enzyme family of 20 N-acetylgalactosaminyltransferases (GalNAc-Ts). Detailed knowledge on the biological effects of site-specific O-glycosylation is limited due to lack of information on specific glycosylation enzyme activities and O-glycosylation site-occupancies. Here we present a systematic analysis of the isoform-specific targets of all GalNAc-Ts expressed within a tissue-forming human skin cell line, and demonstrate biologically significant effects of O-glycan initiation on epithelial formation. We find over 300 unique glycosylation sites across a diverse set of proteins specifically regulated by one of the GalNAc-T isoforms, consistent with their impact on the tissue phenotypes. Notably, we discover a high variability in the O-glycosylation site-occupancy of 70 glycosylated regions of secreted proteins. These findings revisit the relevance of individual O-glycosylation sites in the proteome, and provide an approach to establish which sites drive biological functions.

Intra-pulpal connective tissue formation and the advanced carious lesion: Is chondrogenesis and heterotopic ossification a response to pulpal inflammation?

AIMS: (a) The aim of this study was to investigate both the formation of dense connective tissue within the dental pulp, and its association with pulpal inflammation in teeth with advanced carious lesions; and (b) to investigate in vitro whether inflammation affects the expression of markers related to chondrogenesis/osteogenesis in pulp cells. MATERIALS AND METHODS: Radiology and Histology: Forty-six teeth with advanced carious lesions were radiographically investigated for intra-pulpal radiodense structures. Specimens were processed for histology and stained with haematoxylin/eosin and proteoglycan-specific stains. The intra-pulpal connective tissue was scored as pulp stones or ectopic connective tissue. Cell culture: pulpal cells from human third molars (n = 5) were cultured in chondrogenic medium +/- TLR2/4 agonists. Expression of the genes IL6, TLR2/4, SOX9, COL1A1, COL2A1, TGFB1, RUNX2 and ALPL was assessed by qPCR. Proteoglycan content within cultures was assessed spectrophotometrically. RESULTS: Radiodense structures were discovered in about half of all pulps. They were associated with ectopic connective tissue (χ2  = 8.932, p = .004, OR = 6.80, 95% CI: [1.84, 25.19]) and with pulp stones (χ2  = 12.274, df = 1, p < .001, OR = 22.167, 95% CI: [2.57, 200.00]). The morphology of the ectopic tissue resembled cartilage and was associated with inflammatory infiltration of the pulp (χ2  = 10.148, p = .002, OR = 17.77, 95% CI: [2.05, 154.21]). After continuous stimulation of cultured cells with TLR2/4 agonists, the expression of two inflammatory markers increased: IL6 at Days 7 (p = .020) and 14 (p = .008); TLR2 at Days 7 (p = .023) and 14 (p = .009). Similarly, expression of chondrogenic markers decreased: SOX9 at Day 14 (p = .035) and TGFB1 at Day 7 (p = .004), and the osteogenic marker COL1A1 at Day 7 (p = .007). Proteoglycan content did not differ between unstimulated and stimulated cells. CONCLUSIONS: Ectopic connective tissue resembling cartilage can form in teeth affected by advanced carious lesions. This tissue type is radiographically visible and is associated with inflammatory infiltration of the pulp. Although TLR2/4 agonists led to an inflammatory response in cell culture of pulp cells, the effect on the expression of osteogenic/chondrogenic markers was limited, suggesting that immune cells are needed for connective tissue formation in vivo.

In-Depth Profiling of O-Glycan Isomers in Human Cells Using C18 Nanoliquid Chromatography-Mass Spectrometry and Glycogenomics.

O-Glycosylation is an omnipresent modification of the human proteome affecting many cellular functions, including protein cleavage, protein folding, and cellular signaling, interactions, and trafficking. The functions are governed by differentially regulated O-glycan types and terminal structures. It is therefore essential to develop analytical methods that facilitate the annotation of O-glycans in biological material. While various successful strategies for the in-depth profiling of released O-glycans have been reported, these methods are often limitedly accessible to the nonspecialist or challenged by the high abundance of O-glycan structural isomers. Here, we developed a high-throughput sample preparation approach for the nonreductive release and characterization of O-glycans from human cell material. Reducing-end labeling allowed efficient isomer separation and detection using C18 nanoliquid chromatography coupled to Orbitrap mass spectrometry. Using the method in combination with a library of genetically glycoengineered cells displaying defined O-glycan types and structures, we were able to annotate individual O-glycan structural isomers from a complex mixture. Applying the method in a model system of human keratinocytes, we found a wide variety of O-glycan structures, including O-fucose, O-glucose, O-GlcNAc, and O-GalNAc glycosylation, with the latter carrying both elongated core1 and core2 structures and varying numbers of fucoses and sialic acids. The method, including the now well-characterized standards, provides the opportunity to study glycomic changes in human tissue and disease models using rather mainstream analytical equipment.

Protocol for CRISPR-Cas9 modification of glycosylation in 3D organotypic skin models.

Glycosylation is one of the most common protein modifications in living organisms and has important regulatory roles in animal tissue development and homeostasis. Here, we present a protocol for generation of 3D organotypic skin models using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) to study the role of glycans in epithelial tissue formation. This strategy is also applicable to other gene targets and organotypic tissue models. Careful handling of the cell cultures is critical for the successful formation of the organoids. For complete details on the use and execution of this protocol, please refer to Dabelsteen et al. (2020).

Mapping of truncated O-glycans in cancers of epithelial and non-epithelial origin.

BACKGROUND: Novel immunotherapies targeting cancer-associated truncated O-glycans Tn (GalNAcα-Ser/Thr) and STn (Neu5Acα2-6GalNacα-Ser/Thr) are promising strategies for cancer treatment. However, no comprehensive, antibody-based mapping of truncated O-glycans in tumours exist to guide drug development. METHODS: We used monoclonal antibodies to map the expression of truncated O-glycans in >700 tissue cores representing healthy and tumour tissues originating from breast, colon, lung, pancreas, skin, CNS and mesenchymal tissue. Patient-derived xenografts were used to evaluate Tn expression upon tumour engraftment. RESULTS: The Tn-antigen was highly expressed in breast (57%, n = 64), colorectal (51%, n = 140) and pancreatic (53%, n = 108) tumours, while STn was mainly observed in colorectal (80%, n = 140) and pancreatic (56%, n = 108) tumours. We observed no truncated O-glycans in mesenchymal tumours (n = 32) and low expression of Tn (5%, n = 87) and STn (1%, n = 75) in CNS tumours. No Tn-antigen was found in normal tissue (n = 124) while STn was occasionally observed in healthy gastrointestinal tissue. Surface expression of Tn-antigen was identified across several cancers. Tn and STn expression decreased with tumour grade, but not with cancer stage. Numerous xenografts maintained Tn expression. CONCLUSIONS: Surface expression of truncated O-glycans is limited to cancers of epithelial origin, making Tn and STn attractive immunological targets in the treatment of human carcinomas.

Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model.

The glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.

O-glycan initiation directs distinct biological pathways and controls epithelial differentiation.

Post-translational modifications (PTMs) greatly expand the function and potential for regulation of protein activity, and O-glycosylation is among the most abundant and diverse PTMs. Initiation of O-GalNAc glycosylation is regulated by 20 distinct GalNAc-transferases (GalNAc-Ts), and deficiencies in individual GalNAc-Ts are associated with human disease, causing subtle but distinct phenotypes in model organisms. Here, we generate a set of isogenic keratinocyte cell lines lacking either of the three dominant and differentially expressed GalNAc-Ts. Through the ability of keratinocytes to form epithelia, we investigate the phenotypic consequences of the loss of individual GalNAc-Ts. Moreover, we probe the cellular responses through global transcriptomic, differential glycoproteomic, and differential phosphoproteomic analyses. We demonstrate that loss of individual GalNAc-T isoforms causes distinct epithelial phenotypes through their effect on specific biological pathways; GalNAc-T1 targets are associated with components of the endomembrane system, GalNAc-T2 targets with cell-ECM adhesion, and GalNAc-T3 targets with epithelial differentiation. Thus, GalNAc-T isoforms serve specific roles during human epithelial tissue formation.

Base Editor Correction of COL7A1 in Recessive Dystrophic Epidermolysis Bullosa Patient-Derived Fibroblasts and iPSCs.

Genome editing represents a promising strategy for the therapeutic correction of COL7A1 mutations that cause recessive dystrophic epidermolysis bullosa (RDEB). DNA cleavage followed by homology-directed repair (HDR) using an exogenous template has previously been used to correct COL7A1 mutations. HDR rates can be modest, and the double-strand DNA breaks that initiate HDR commonly result in accompanying undesired insertions and deletions (indels). To overcome these limitations, we applied an A•T→G•C adenine base editor (ABE) to correct two different COL7A1 mutations in primary fibroblasts derived from RDEB patients. ABE enabled higher COL7A1 correction efficiencies than previously reported HDR efforts. Moreover, ABE obviated the need for a repair template, and minimal indels or editing at off-target sites was detected. Base editing restored the endogenous type VII collagen expression and function in vitro. We also treated induced pluripotent stem cells (iPSCs) derived from RDEB fibroblasts with ABE. The edited iPSCs were differentiated into mesenchymal stromal cells, a cell population with therapeutic potential for RDEB. In a mouse teratoma model, the skin derived from ABE-treated iPSCs showed the proper deposition of C7 at the dermal-epidermal junction in vivo. These demonstrate that base editing provides an efficient and precise genome editing method for autologous cell engineering for RDEB.

The Skin Reservoir Model: A Tool for Evaluating Microdialysis Sampling of Large Biomarkers from Human Skin.

Microdialysis is a well-established technique for sampling of small molecules from the human skin, but larger molecules are more difficult to recover. Consequently, sampling feasibility must be evaluated before microdialysis is used in vivo. This report presents a tool for estimating the recovery of large biomarkers from human skin by microdialysis, using previously frozen human skin specimens as reservoirs for biomarker reference solutions. Recovery of the following 17 biomarkers was assessed: CCL27/CTACK, CXCL1/GROα, CXCL7/NAP-2, CXCL10/IP-10, EGF, GM-CSF, IFN-γ, IL-1α, IL-6, IL-8, IL-17, IL-22, IL-23, MIF, TNF-α, TSLP and VEGF. The relative skin recoveries of 13/17 biomarkers were successfully determined in the range 4.0-18.4%. Sampling in the skin reservoir model was not associated with probe leakage, as fluid recovery was stable, at between 80% and 110%. Furthermore, the skin reservoir model enabled studies and optimization of different parameters known to affect biomarker recovery, including flow rate and perfusate composition.

Interleukin-26 (IL-26) is a novel anti-microbial peptide produced by T cells in response to staphylococcal enterotoxin.

Anti-microbial peptides are produced at outer and inner surfaces by epithelia and innate immune cells in response to bacterial infection. Staphylococcus aureus is an enterotoxin producing, Gram-positive pathogen, which is a major cause of soft tissue infections and life-threatening bacteremia and sepsis. Here we show that (i) skin T cells in chronic wounds infected with S. aureus express interleukin-26 (IL-26) in situ, (ii) staphylococcal enterotoxins (SE) trigger IL-26 expression in T cell lines and primary skin T cells, and (iii) IL-26 triggers death and inhibits biofilm formation and growth of S. aureus. Thus, we provide novel evidence that IL-26 is an anti-microbial peptide produced by T cells in response to SE. Accordingly, we propose that IL-26 producing T cells take part in the innate immune response to SE producing S. aureus and thus play a novel role in the primary innate immune defense in addition to their classical role in adaptive immunity.

De novo expression of human polypeptide N-acetylgalactosaminyltransferase 6 (GalNAc-T6) in colon adenocarcinoma inhibits the differentiation of colonic epithelium.

Aberrant expression of O-glycans is a hallmark of epithelial cancers. Mucin-type O-glycosylation is initiated by a large family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) that target different proteins and are differentially expressed in cells and organs. Here, we investigated the expression patterns of all of the GalNAc-Ts in colon cancer by analyzing transcriptomic data. We found that GalNAc-T6 was highly up-regulated in colon adenocarcinomas but absent in normal-appearing adjacent colon tissue. These results were verified by immunohistochemistry, suggesting that GalNAc-T6 plays a role in colon carcinogenesis. To investigate the function of GalNAc-T6 in colon cancer, we used precise gene targeting to produce isogenic colon cancer cell lines with a knockout/rescue system for GALNT6 GalNAc-T6 expression was associated with a cancer-like, dysplastic growth pattern, whereas GALNT6 knockout cells showed a more normal differentiation pattern, reduced proliferation, normalized cell-cell adhesion, and formation of crypts in tissue cultures. O-Glycoproteomic analysis of the engineered cell lines identified a small set of GalNAc-T6-specific targets, suggesting that this isoform has unique cellular functions. In support of this notion, the genetically and functionally closely related GalNAc-T3 homolog did not show compensatory functionality for effects observed for GalNAc-T6. Taken together, these data strongly suggest that aberrant GalNAc-T6 expression and site-specific glycosylation is involved in oncogenic transformation.

A novel BLK-induced tumor model.

B-lymphoid tyrosine kinase (BLK) is a non-receptor tyrosine kinase belonging to the SRC family kinases. BLK is known to be functionally involved in B-cell receptor signaling and B-cell development. New evidence suggests that B-lymphoid tyrosine kinase is ectopically expressed and is a putative oncogene in cutaneous T-cell lymphoma and other T-cell malignancies. However, little is known about the role of BLK in lymphomagenesis, and the oncogenic function seems to depend on the cellular context. Importantly, BLK is also ectopically expressed in other hematological and multiple non-hematological malignancies including breast, kidney, and lung cancers, suggesting that BLK could be a new potential target for therapy. Here, we studied the oncogenic potential of human BLK. We found that engrafted Ba/F3 cells stably expressing constitutive active human BLK formed tumors in mice, whereas neither Ba/F3 cells expressing wild type BLK nor non-transfected Ba/F3 cells did. Inhibition of BLK with the clinical grade and broadly reacting SRC family kinase inhibitor dasatinib inhibited growth of BLK-induced tumors. In conclusion, our study provides evidence that human BLK is a true proto-oncogene capable of inducing tumors, and we demonstrate a novel BLK activity-dependent tumor model suitable for studies of BLK-driven lymphomagenesis and screening of novel BLK inhibitors in vivo.