Liposomal CRISPR/Cas9-Mediated Local Genome Editing for Joint Disease in Mucopolysaccharidosis Type I

<b>Background/Objectives</b>: Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by α-L-iduronidase (IDUA) deficiency, leading to progressive glycosaminoglycan (GAG) accumulation and severe joint involvement. Gene editing represents a promising alternative to restore localized enzyme production. Therefore, this study aimed to evaluate the feasibility, efficacy, and safety of in situ genome editing through intra-articular administration of a nonviral CRISPR/Cas9 system to increase localized IDUA expression in an MPS I mouse model. <b>Methods</b>: Cationic liposomes were formulated to deliver plasmids encoding the CRISPR/Cas9 system targeted to the <i>ROSA26</i> locus along with an <i>IDUA</i> donor sequence. In vitro assays were performed in fibroblast-like synoviocytes (FLSs) isolated from MPS I mice to assess cytotoxicity, gene editing efficiency, and IDUA activity. In vivo, MPS I mice received intra-articular injections in the knee joints, either as a single dose (short-term study) or monthly for three months (long-term study). IDUA activity, GAG levels, and genome editing efficiency were evaluated in joint tissues, synovial fluid, serum, and major organs. <b>Results</b>: Gene-edited FLS showed sustained IDUA activity for up to 30 days with low cytotoxicity. In vivo, intra-articular administration resulted in a significant increase in IDUA activity in joint tissue and synovial fluid without detectable systemic IDUA. Long-term treatment led to persistent joint-localized IDUA activity, significant reductions (>50%) in GAG levels, and detectable genome editing in joint DNA. <b>Conclusions</b>: Intra-articular delivery of CRISPR/Cas9 via cationic liposomes enables safe and effective localized genome editing, representing a promising strategy for treating joint manifestations of MPS I.