Browsing by Subject "epidermolysis bullosa"

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    Identification of novel genes and compounds for the development of precision therapeutics for dystrophic epidermolysis bullosa and associated cutaneous squamous cell carcinoma
    (2021-08) Pickett-Leonard, Michael
    Dystrophic epidermolysis bullosa (DEB) is a skin blistering disease caused by dominant (DDEB) or recessive (RDEB) mutations in the COL7A1 gene, with the latter being more severe. COL7A1 encodes type VII collagen (C7), which aggregates into structures called anchoring fibrils that maintain skin integrity by securing the epidermis to the dermis. DEB is characterized by shearing and blistering of the skin (and various mucosae) at the level of the superficial dermis. In severe cases of RDEB, this leads to fibrosis, scarring, and aggressive cutaneous squamous cell carcinoma (RDEBSCC). Using a combination of high-throughput sequencing and genome-wide screening tools we identified novel (1) genes and compounds that increase C7 production in wild-type and RDEB keratinocytes, (2) genes that become progressively dysregulated with RDEB disease progression, and (3) genes that drive or inhibit proliferation in wild-type, RDEB, and RDEBSCC keratinocytes. In Chapter 2, we start by describing the creation and validation of a keratinocyte C7 reporter cell line, in which C7 production is linked to tdTomato fluorescence. We used this reporter line to perform a genome wide CRISPR activation (CRISPRa) screen to identify genes that increase C7 production in keratinocytes. There were 1544 CRISPRa single guide RNAs (sgRNAs), targeting 1464 distinct genes, whose abundance was substantially increased in the top 10% of tdTomato-expressing cells relative to the plasmid DNA library. Validation of the top two candidates identified in this screen, DENND4B and TYROBP, showed that CRISPRa-mediated upregulation of these genes significantly increased tdTomato fluorescence and C7 protein production, but not COL7A1 mRNA. Pathway analysis of the 1464 gene targets identified significantly enriched upstream regulators, signaling pathways, and biological functions. We performed a targeted drug screen using compounds that act on some of these upstream regulators and signaling pathways and found that kaempferol, a plant flavonoid, was able to increase COL7A1 mRNA and C7 protein in both wild-type and RDEB keratinocytes. In Chapter 3, we investigate potential mechanisms of RDEBSCC development. We performed RNA-sequencing on nine sibling pairs of wild-type and RDEB keratinocytes (WTK, RDEBK) and six RDEBSCC cell lines and identified numerous genes whose expression progressively increased or decreased from WTKs to RDEBKs to RDEBSCCs, suggesting that these genes could be involved in disease progression. To identify inhibitors of proliferation in keratinocytes and RDEBSCCs, we performed CRISPRi proliferation screens in one WTK (NTERT) and three RDEBSCC (RDEBSCC2, 53, and 70) cell lines. There were 53 sgRNAs that were enriched in all four CRISPRi screens, including all three sgRNAs targeting the TAFA3 gene. To identify drivers of proliferation in keratinocytes, we performed CRISPRa proliferation screens in one RDEBK (RDEBK8) and two WTK (NTERT and WTK1) cell lines. No sgRNAs were enriched in all three screens, but there was some overlap across each of the three possible pairs. We performed competition assays to validate some of the top hits from the CRISPRa and CRISPRi screens and found that inhibition of PTK2B, QPRT, STAT2, or TAFA3 expression and upregulation of ADAM2, CDYL2, CSF3R, DENND4B, FSTL1, GCSAM, ITGB1, ITGB3, KLHDC8A, KRT33B, SEMA5A, or TYROBP expression significantly increased proliferation in NTERT keratinocytes. Using a combination of RNA-sequencing and genome wide CRISPRai screens, we identified (1) genes that promote C7 production, (2) a novel strategy for increasing C7 production in RDEB skin, (3) genes that were progressively dysregulated from WTK to RDEBK to RDEBSCC, (4) genes that drive proliferation in wild-type and/or RDEB keratinocytes, and (5) genes that inhibit proliferation in keratinocytes and RDEBSCC cells. We also created and validated a keratinocyte C7 reporter cell line that could be used in future DEB research. Overall, this research has led to the discovery of numerous previously unexplored avenues of investigation in dystrophic epidermolysis bullosa research and a multitude of new gene targets for the development of novel targeted therapeutics.
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    Steps towards understanding cellular therapies for Recessive Dystrophic Epidermolysis Bullosa
    (2022-05) Riedl, Julia
    Chronic or recurrent cutaneous wounding is the phenotypic hallmark of the heterogeneous inherited skin disease epidermolysis bullosa (EB). In a severe generalized form of these genodermatoses, recessive dystrophic epidermolysis bullosa (RDEB), biallelic mutations in the COL7A1 gene result in absent or dysfunctional type VII collagen. In healthy individuals, secreted type VII collagen homotrimerizes to form anchoring fibrils in the basement membrane zone (BMZ) connecting the epidermal and dermal layers of the skin. Without these protein anchors to hold the two layers together, the skin easily separates into either blisters or wounds with mild mechanical trauma. There is no cure for RDEB, but improvement in clinical phenotype has been achieved with bone marrow transplant (BMT) and subsequent epidermal allografting from the BMT donor. Previous research into BMT for RDEB has shown that the bone marrow mesenchymal stem cells (BM-MSCs) can home to wounds and secrete collagen VII. Unfortunately, BMT does not fully cure RDEB and chronic wounds can persist after therapy. Therefore, epidermal allografting from the BMT donors of RDEB patients has been employed on chronic wounds. This combination therapy has decreased wound surface area for up to three years post treatment. Characterization of RDEB full-thickness skin biopsies after BMT with and without epidermal allografting with single-cell RNA sequencing uncovered that keratinocytes co-expressing collagen VII and basal stem cell marker keratin 15 might be a source of keratinocytes allowing the epidermal allograft to persist. Additionally, pro-inflammatory immune and fibroblast phenotypes were found in RDEB skin after BMT and subsequent epidermal allografting, which is potentially driven by the local environment of RDEB skin. This is further highlighted by the presence of a myofibroblast population, which has not been described in healthy control human skin. Additionally, while human bone-marrow derived MSC (BM-MSC) trials in RDEB demonstrate improvement in clinical severity, the mechanisms of MSC migration to and persistence in injured skin and their contributions to wound healing are not completely understood. A unique subset of MSCs expressing ATP-binding cassette subfamily member 5 (ABCB5) resides in the reticular dermis and exhibits similar immunomodulatory characteristics to BM-MSCs. This work aimed to test the hypothesis that skin-derived ABCB5+ dermal MSCs (DSCs) possess superior skin homing ability compared to BM-MSCs in immunodeficient NOD-scid IL2rgammanull (NSG) mice. Compared to BM-MSCs, peripherally injected ABCB5+ DSCs demonstrated superior homing and engraftment of wounds. Further, ABCB5+ DSCs versus BM-MSCs co-cultured with macrophages induced less anti-inflammatory interleukin-1 receptor antagonist (IL-1RA) production. RNA sequencing of ABCB5+ DSCs compared to BM-MSCs showed unique expression of Homeobox (Hox) genes, specifically HOXA3. Critical to inducing migration of endothelial and epithelial cells for wound repair, increased expression of HOXA3 may explain superior skin homing properties of ABCB5+ DSCs. Further discernment of the immunomodulatory mechanisms amongst MSC populations could have broader regenerative medicine implications beyond RDEB treatment. In conclusion, this thesis provides insights into the mechanisms of existing cellular treatments for RDEB and targets for future studies and treatments.