Regional Finalist, SARC 2025
By Micaela Andia, Peru
Abstract:
Atopic Dermatitis is a chronic, relapsing skin condition affecting up to 30% of children worldwide, characterized by intense itching and inflamed, eczematous lesions (NIH, 2023). Approximately 40% of cases are associated with loss-of-function mutations in the filaggrin gene, a key regulator of skin barrier integrity. Current treatments, such as topical corticosteroids and monoclonal antibodies like dupilumab, only alleviate symptoms transiently and fail to address the genetic defect. We propose a new strategy that utilizes a CRISPR-engineered strain of Staphylococcus epidermidis to deliver modified FLG-mRNA via lipid nanoparticles. Unlike viral vectors or transient topical creams, this probiotic-based system offers a non-viral, sustained delivery system, aiming to restore FLG expression and improve skin barrier function. This approach could provide a long-term cure for AD patients with FLG mutations, offering efficacy and improved accessibility over biologics.
Introduction:
Atopic dermatitis is a chronic skin disorder marked by itching, dryness, and eczematous lesions, compromising the skin's barrier and making patients vulnerable to allergens, irritants, and infections. Symptoms often include intense itching, sleep disturbances, and an increased risk of secondary infections, significantly impacting quality of life. AD is linked to mutations in the filaggrin (FLG) gene, which plays a vital role in skin barrier integrity, and about 40% of AD patients carry these mutations. Current treatments like steroids and immunosuppressants only provide temporary symptom relief without addressing the underlying genetic cause. Modified messenger RNA (modRNA) therapies, encapsulated in lipid nanoparticles (LNPs), offer a promising approach by improving stability and targeted delivery to skin cells. Additionally, CRISPR-Cas9 technology, adapted for use with commensal bacteria like Staphylococcus epidermidis, enables continuous, localized delivery of FLG-modRNA, providing a novel solution for AD treatment and potential gene therapy for other genetic skin disorders.
Literature Review:
Atopic Dermatitis is a chronic inflammatory skin disorder with no cure, and current treatments only provide temporary relief (Wollenberg et al., 2018). Standard therapies like corticosteroids and biologics target inflammation but don’t address the underlying genetic or barrier defects (Eichenfield et al., 2014). While murine models have contributed to research, they lack full relevance to human AD (Ewald et al., 2017). Advances using iPSC-derived keratinocytes from AD patients have allowed modeling of key disease features like FLG deficiency and immune dysregulation (Guo et al., 2025). CRISPR-Cas9 shows potential in correcting FLG mutations, with some success in restoring barrier function (Samek et al., 2021). However, challenges such as off-target effects and immune responses remain (Chehelgerdi et al., 2024). ModRNA offers a safer alternative, enhancing transfection efficiency and reducing immune activation (Sahin et al., 2014; Hou et al., 2021). Studies in other skin disorders have shown its potential to restore critical proteins (Subramaniam et al., 2022). Despite these advances, current treatments lack sustained FLG correction, and viral vectors carry mutagenesis risks (Keeler et al., 2017). Our proposed solution—CRISPR-engineered S. epidermidis delivering LNP-modRNA—offers a novel approach for continuous FLG expression, providing a long-term solution for AD and other genetic skin disorders.
Methodology:​
Establishing FLG-Deficient Skin Equivalents
Primary keratinocytes from eczema patients with FLG mutations (e.g., R501X or 2282del4) will be obtained via skin biopsies or biobank samples and cultured with EGF and bovine pituitary extract (Palomo et al., 2018). FLG mutations will be introduced into healthy keratinocytes using CRISPR-Cas9 for isogenic controls (Zhang et al., 2014). These cells will be co-cultured with human fibroblasts in a 3D air-liquid interface (ALI) system to form stratified epidermis, promoting differentiation with calcium and retinoic acid (Jang et al., 2023). Model validation will include immunohistochemistry for FLG, loricrin, and involucrin, along with TEWL assays to assess barrier dysfunction. Three conditions will be tested: S. epidermidis delivering FLG-CRISPR/LNPs, AAV-FLG therapy, and untreated controls, each with patient-derived and CRISPR-edited FLG-KO keratinocytes.
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CRISPR Delivery via Engineered S. epidermidis
Guide RNAs (gRNAs) targeting exon 3 of the FLG gene will be designed using Benchling’s algorithm to minimize off-target effects (Zhang et al., 2014). The CRISPR-Cas9 system, optimized for S. epidermidis with codon-adapted Cas9 and an active sarA promoter, will be cloned into a shuttle plasmid with the FLG-specific gRNA. The engineered bacteria will be cultured in chloramphenicol-supplemented broth, and CRISPR components will be encapsulated in lipid nanoparticles (LNPs) for enhanced epidermal delivery (Mout et al., 2018). Upon application to FLG-deficient skin equivalents, the probiotic will release LNPs to deliver Cas9-gRNA complexes to keratinocytes, editing the FLG locus via nonhomologous end joining. Editing success will be confirmed by Sanger sequencing and filaggrin staining.
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Analysis of FLG Restoration and Barrier Function
To confirm FLG gene editing, genomic DNA from treated keratinocytes will be analyzed via PCR and Sanger sequencing to detect frameshift mutation repair. FLG mRNA and protein expression will be measured by quantitative PCR and Western blotting. Skin barrier function will be assessed through transepidermal water loss (TEWL), with successful therapy indicated by reduced water loss. Histological analysis of H&E-stained sections will evaluate epidermal stratification, and immunofluorescence will visualize cornified envelope reassembly. Cytokine profiling (IL-4, IL-13, TSLP) will assess Th2 inflammation reduction. These assays will determine if probiotic-delivered CRISPR therapy corrects FLG mutations and addresses eczema’s molecular and physiological hallmarks.
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Statistical Analysis of Therapeutic Efficacy
Gene-editing efficiency will be assessed by comparing the percentage of FLG alleles corrected between the S. epidermidis CRISPR and AAV-FLG groups using two-sample t-tests (p < 0.05). Transepidermal water loss (TEWL) values, recorded daily for three weeks, will be analyzed with repeated-measures ANOVA, followed by Tukey post-hoc tests for group comparisons. Cytokine profiles (IL-4, IL-13, TSLP) will be analyzed with one-way ANOVA on log-transformed ELISA data to assess normalization of Th2 inflammation. Epidermal thickness and stratum corneum integrity, assessed by H&E staining, will be analyzed using Kruskal-Wallis tests. Multiple comparisons will be adjusted using the Benjamini-Hochberg method. If efficacy is demonstrated, the framework will be expanded to target other eczema-related genes (e.g., SPINK5, KLK7) for personalized therapies.
Conclusion:
Ultimately, CRISPR-engineered S. epidermidis delivers FLG therapy more safely and effectively than current treatments by targeting eczema’s genetic cause. This probiotic-based approach represents a critical step toward curing—not just managing—the disease.
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