隱性失養型表皮溶解水皰症是一種由 COL7A1 基因突變引起的嚴重疾病，導致 VII 型膠原蛋白的缺失或功能障礙，而VII 型膠原蛋白是真皮－表皮連接處錨定纖維的關鍵成分。目前的治療策略是利用局部給藥的方式將病毒載體將全長 COL7A1 cDNA 直接遞送至皮膚傷口，以恢復局部蛋白質表現。雖然此類方法已在臨床上取得成功，但仍受載體相關免疫原性、組織穿透性不足以及基因組整合導致插入突變風險的限制。為了克服這些障礙，我們研究了一種基於非病毒 mRNA 的治療方法。我們將經過 N1-甲基-假尿苷和 AG Clean Cap 修飾的 COL7A1 mRNA，透過電穿孔與脂質奈米顆粒傳送至 COL7A1 基因敲除的 HaCaT 角質形成細胞中。經由免疫螢光與西方墨點法分析證實VII 型膠原蛋白蛋白的成功表現，顯示其在蛋白質層面具有功能恢復能力。接著為了評估其體內遞送潛力，我們將包裹螢光素酶 mRNA 的脂質奈米顆粒注射至隱性失養型表皮溶解水皰症的小鼠，觀察到明顯的冷光表現，顯示該平台具備轉譯應用潛力。進一步地，我們將包裹 COL7A1 mRNA 的脂質奈米顆粒經由皮下注射施打至隱性失養型表皮溶解水皰症的小鼠傷口附近，免疫組織化學結果顯示VII 型膠原蛋白在局部表現有部分恢復。我們的研究結果凸顯了脂質奈米顆粒介導的 mRNA 療法作為隱性失養型表皮溶解水皰症下一代基因替代策略的潛力。與基於病毒載體的系統相比，該平台能夠實現瞬時、無整合的表達，降低免疫原性，並支持重複或全身給藥，從而為隱性失養型表皮溶解水皰症和其他遺傳性皮膚病提供了一種有前景且靈活的治療方式。

Recessive dystrophic epidermolysis bullosa (RDEB), also known as “butterfly disease,” is a severe genetic skin disorder caused by mutations in the COL7A1 gene, resulting in the absence or dysfunction of type VII collagen (COL7), a critical component of anchoring fibrils at the dermal–epidermal junction. Current therapies rely on viral vectors to deliver full-length COL7A1 cDNA directly to skin wounds, aiming to restore local protein expression. While clinical successes have been reported, these approaches are limited by vector-associated immunogenicity, inadequate tissue penetration, and the risk of insertional mutagenesis due to genomic integration. To address these limitations, we developed a non-viral mRNA-based therapeutic strategy. Chemically modified COL7A1 mRNA, incorporating N1-methylpseudouridine and an AG Clean Cap, was delivered into COL7A1-knockout HaCaT keratinocytes via electroporation and lipid nanoparticles (LNPs). Expression of functional COL7 protein was confirmed by immunofluorescence and Western blot, indicating protein restoration. To assess in vivo potential, luciferase mRNA-loaded LNPs were injected into the model RDEB mice, resulting in strong bioluminescence signals, confirming effective mRNA expression. Furthermore, LNPs encapsulating COL7A1 mRNA were subcutaneously administered near the wound sites of RDEB mice, and immunohistochemistry revealed partial restoration of COL7 expression in treated skin. Our findings demonstrate the potential of LNP-mediated mRNA therapy as a next-generation gene replacement strategy for RDEB. Compared with viral systems, this platform offers transient, integration-free expression with lower immunogenicity and supports repeated or systemic delivery, providing a safer and more versatile therapeutic approach for RDEB and other inherited skin disorders.

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