Complete restoration of p53 in p53 mutant cancers through co-delivery of p53 siRNA and mRNA using a targeted LNP platform

p53, the "Guardian of the Genome," maintains cellular integrity by inducing cell cycle arrest or apoptosis. While p53 mRNA delivery is an emerging therapeutic approach, its efficacy is often limited to p53-null backgrounds. In p53-mutant cancers, the effectiveness of wild-type mRNA alone is severely restricted by the dominant-negative and gain-of-function (GOF) effects of endogenous mutants. Consequently, active elimination of diverse mutants is essential for successful restoration. Although small-molecule refolding agents exist, they target only specific hotspots and fail to address the vast diversity of clinical p53 mutations. To overcome these challenges, we developed RePair53(Restoration of p53 via Paired interference [siRNA] and replacement [mRNA]), a "Delete-and-Replace" platform designed for broad-spectrum p53 restoration through the synergistic co-delivery of a pan-p53 siRNA and a codon-optimized p53 mRNA. Furthermore, leveraging our previous research, the lipid nanoparticle (LNP) surface was functionalized with tumor-associated antigen (TAA)-targeted ApoA1 fusion antibodies to achieve precise and selective delivery to cancer cells. We designed a pan-p53 siRNA capable of eliminating multiple hotspot mutations and a codon-optimized p53 mRNA that restores functional p53 while evading siRNA-mediated degradation. The RNAs were co-encapsulated into lipid nanoparticles (LNPs) and functionalized with Panitumumab-ApoA1 for precise EGFR targeting. Independent functionality was validated via qPCR and Western blot. Antitumor efficacy and targeting efficiency were evaluated in a CAL-27 xenograft mouse model. qPCR analysis confirmed that the pan-p53 siRNA achieved over 80% knockdown of endogenous mutant p53, an efficiency that remained consistent even during mRNA co-delivery. Western blot verified that the codon-optimized mRNA successfully restored p53 protein expression without interference. In vitro assays showed a 60% increase in antitumor efficacy compared to mRNA treatment alone. In the CAL-27 xenograft model, RePair53 demonstrated significantly enhanced tumor suppression. The RePair53 platform establishes a new paradigm in p53-targeted therapy by overcoming the critical limitations of conventional approaches restricted to p53-null backgrounds or specific mutation types. By integrating pan-mutant silencing with robust functional restoration, this "Delete-and-Replace" strategy effectively addresses the vast diversity and heterogeneity of p53 mutations encountered in clinical settings. Consequently, RePair53 provides a universal and versatile foundation for targeted gene therapy, offering a transformative solution for a wide spectrum of malignancies driven by diverse p53 mutations.