Cutaneous gene therapy has the potential to treat a wide range of skin disorders, but effective delivery remains limited by the barrier properties and immune surveillance of the skin. Here, we identify AAVrh32.33 as a potent vector for targeting dermal stromal compartments. Following systemic administration in mice, AAVrh32.33 mediated robust and durable transgene expression, with preferential targeting of dermal fibroblasts and hair follicle bulge cells. Expression peaked at 1 month and persisted for up to 2 years, highlighting its suitability for chronic conditions. To reduce immunogenicity, a dominant CD8+ T cell epitope was disrupted, generating the IDPΔ variant. This modification attenuated peptide-specific T cell responses while preserving stromal transduction. In human skin explants, IDPΔ achieved high levels of gene expression, primarily in dermal fibroblasts and precursors, confirming translational relevance. Finally, vectors encoding CCL17, CCL20, and CCL22 demonstrated localized targeted therapeutic gene delivery in both healthy and inflamed skin, underscoring the feasibility of using this platform to reshape local immune responses. Together, these findings establish AAVrh32.33 and IDPΔ as promising platforms for durable cutaneous gene therapy, with direct applications in diseases such as vitiligo, where long-term modulation of the dermal microenvironment is essential.

Traditional skin sampling methods include punch or shave biopsies to produce a solid tissue sample for analysis. These biopsy procedures are painful, require anesthesia, and leave permanent scars. This unit describes a suction blister skin biopsy method that can be used in place of traditional biopsy methodologies as a minimally invasive, non-scarring skin sampling technique. The induction of suction blisters uses an instrument with a chamber that applies negative pressure and gentle heat to the skin. Blister formation occurs within 1 hr, producing up to five blisters, each 10 mm in diameter per biopsy site. Blister fluid can be extracted and centrifuged to retrieve cells from the epidermis and upper dermis for flow cytometry, single-cell RNA sequencing, cell culture, and more without the need for digestion protocols. In addition, the blister fluid can be used to measure soluble proteins and metabolites. This unit describes the preparation of supplies and subjects, the suction blister biopsy procedure and blister formation, fluid extraction, and post-blistering care. (c) 2024 Wiley Periodicals LLC. Basic Protocol 1: Preparation of supplies and subject Basic Protocol 2: Suction blister biopsy procedure and formation Basic Protocol 3: Blister fluid extraction Basic Protocol 4: Post-blister care and clean up

Vitiligo is an autoimmune skin disease caused by melanocyte-targeting autoreactive CD8+ T cells. Regulatory T cells (Tregs) have been implicated in restraining vitiligo severity in both mouse models and human patients; however, whether they must be present in the skin for their suppressive function is still unclear. We observed uneven distribution of Tregs within different anatomical locations of mouse skin, which correlated with reduced depigmentation after vitiligo induction. We specifically depleted Tregs in our mouse model of vitiligo and observed increased disease. Next, we found that Tregs contact CD8+ T effector cells in vitiligo lesional skin and that Treg recruitment to the skin inversely correlated with disease severity, suggesting a critical role for Treg suppression within the skin. When we investigated the signals facilitating Treg migration to the skin, we found that although CXCR3 was dispensable for Treg migration and function in vitiligo, Tregs lacking CCR6 exhibited a reduced capacity to migrate to the skin and suppress depigmentation, despite normal systemic numbers in the skin-draining lymph nodes. Our observations highlight a key role for cutaneous Tregs in disease suppression during vitiligo and identify CCR6 as a chemokine receptor that contributes to Treg migration to the skin.

AIM Sessile serrated adenomas (SSAs) are pre-malignant lesions driven by the BRAFV600E mutation to give rise to colorectal cancers (CRCs). They are often missed during white light colonoscopy because of their subtle appearance. Previously, a fluorescently-labeled 7mer peptide KCCFPAQ was shown to detect SSAs in vivo. We aim to identify the target of this peptide. RESULTS Peroxiredoxin-1 (Prdx1) was identified as the binding partner of the peptide ligand. In vitro binding assays and immunofluorescence staining of human colon specimens ex vivo supported this result. Prdx1 was overexpressed on the membrane of cells with the BRAFV600E mutation, and this effect was dependent on oxidative stress. RKO cells harboring the BRAFV600E mutation and human SSA specimens showed higher oxidative stress as well as elevated levels of Prdx1 on the cell membrane. Innovation and Conclusions: These results suggest that Prdx1 is overexpressed on the cell surface in the presence of oxidative stress, and can serve as an imaging biomarker for in vivo detection of SSAs.

Vitiligo is a disease of the skin characterized by the appearance of white spots. Significant progress has been made in understanding vitiligo pathogenesis over the past 30 years, but only through perseverance, collaboration, and open-minded discussion. Early hypotheses considered roles for innervation, microvascular anomalies, oxidative stress, defects in melanocyte adhesion, autoimmunity, somatic mosaicism, and genetics. Because theories about pathogenesis drive experimental design, focus, and even therapeutic approach, it is important to consider their impact on our current understanding about vitiligo. Animal models allow researchers to perform mechanistic studies, and the development of improved patient sample collection methods provides a platform for translational studies in vitiligo that can also be applied to understand other autoimmune diseases that are more difficult to study in human samples. Here we discuss the history of vitiligo translational research, recent advances, and their implications for new treatment approaches.

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Abstract Epigenomic changes are commonly observed in cancer. We developed a next-generation sequencing (NGS) assay which can identify subtle changes in global methylation as well as copy number alterations (CNAs). The assay measures the methylation level of some repeat elements, covering over 25% of all CpG in the genome. Genomic DNA extracted from 18 pairs of matched colorectal tumor to normal tissue was tested (four adenocarcinomas, including one associated with inflammatory bowel disease, and fourteen adenomas, including one adenoma from a Lynch syndrome patient, one familial adenomatous polyposis (FAP) and two sessile serrated adenomas (SSA)). We also assayed organoids established from these tissues at early (2 month) and late (more than 6 months) timepoints in culture. Briefly, bisulfite conversion and enrichment of the repeats was performed. The Illumina compatible products were sequenced on a HiSEQ 2500. Analysis was performed using Bismark (Krueger F., Babraham institute) and Nexus copy number (BioDiscovery) to determine the global methylation and the CNAs, respectively. Finally, somatic and germline variants were determined by targeted sequencing of 71 genes using the QIAseq colorectal cancer panel (QIAGEN). Our data showed that all adenocarcinoma presented hypomethylation and CNAs as well as multiple somatic variants in APC, KRAS, TP53, SMAD4 and PIK3CA. Six out of the fourteen adenomas presented CNAs and nine showed hypomethylation. The most frequently observed copy number gain affected the chromosomes 8q and 13. Eight adenomas presenting somatic mutations in APC also exhibited distinct global hypomethylation. Previous publications have reported that mutations in APC precede global hypomethylation. Interestingly, one adenoma showed significant hypomethylation but no detectable CNAs or APC mutations. The two SSA samples showed the characteristic BRAF V600E mutations and the FAP sample presented germline mutation in APC. The FAP and the SSA samples did not show hypomethylation or CNAs. Most organoids presented CNAs and somatic mutations similar to their matched tissue even after 2 years in culture. However, few organoids developed new CNAs and somatic variants. Monitoring the changes in organoid may provide important information on tumor progression. This assay measured CNAs and methylation in colorectal cancer samples, findings which may assist in determining the status of the disease and provide guidance to the appropriate action. Our NGS assay interrogates a significant portion of the genome leading to a more accurate and sensitive evaluation of the state of these cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Julie C. Laliberte, Michael K. Dame, Durga Attili, Bodrul Islam, Kevin Kim, Jessica Zhang, Erica L. Katz, Gina M. Newsome, Priya H. Dedhia, Adele Kruger, Tobias Mann, Tom Goodman, Jeffrey Buis, Dean E. Brenner, James Varani, Jason R. Spence, Justin A. Colacino, Jay Stoerker. Simultaneous measurement of global methylation and copy number alterations in human colorectal cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-093. doi:10.1158/1538-7445.AM2017-LB-093