Tailored Yeast-derived Polyplexes for Oral CRISPR Cas9/dCas9 Delivery

Introduction: Effective delivery mechanisms are pivotal in unlocking the therapeutic potential of CRISPR-Cas9/dCas9 technologies. Despite global efforts to refine gene editing delivery, oral administration has remained a formidable challenge. In this study, we introduce a groundbreaking orally-administered, non-viral gene editing platform that promises to revolutionize both localized and systemic gene editing. Our innovative solution employs a novel oral nanoparticle platform, YF-CPNP, which incorporates yeast fragments into polyplexes enabling efficient navigation across the densely structured mucosal epithelium via intestinal microfold cells.

Upon administration, YF-CPNP is endocytosed by macrophages in local sites and Peyer’s patches, subsequently accumulating in mesenteric lymph nodes (MLN). Remarkably, YF-CPNP facilitates sustained gene editing across macrophages, dendritic cells, T cells, and B cells within lymph nodes. This mechanism not only maintains gene editing activities within the gastrointestinal tract and its associated lymphoid tissues but also extends nanoparticle transport across the lymphoid pathway into systemic circulation. This process enables the modulation of genomic expression in remote organs such as the liver. Significantly, we demonstrate the activation of the titin (TTN) gene in both the small intestine and liver, achieved through oral administration using YF-CPNP loaded with a dCas9 VPR plasmid. This novel oral delivery platform not only simplifies the process of gene editing but also opens new pathways for non-invasive, repeatable treatments with broad-ranging therapeutic implications.

Materials and

Methods: We manufactured the YF-CPNP nanoparticles using the flash nanocomplexation (FNC) technique. By adjusting the succinylation degree of chitosan, the polyethyleneimine grafting ratio, and N/P ratio, as well as the ratio of yeast fragments to plasmid DNA, we generated a diverse library of NP formulations. For in vitro validation, we employed the HEK-Ai9 reporter cell line to assess gene transfection and editing efficiencies across different NPs. Further in vivo validation was conducted using two reporter mouse models: Ai14 mice and IDEAS mice, which express Tdtomato and GFP respectively, upon excision of stop codons. The expression of these fluorescent proteins serves as a marker for successful gene editing by YF-CPNP carrying either Cre or Cas9 plasmids. To further elucidate the cellular uptake and response to YF-CPNP in MLN, single-cell RNA sequencing was performed, confirming efficient endocytosis and positive cellular responses, thus demonstrating the delivery system's effectiveness. Finally, we targeted the TTN gene for in vivo CRISPR-mediated transcriptional activation using our oral gene delivery system YF-CPNP. The successful activation of the TTN gene in the gastrointestinal tract and liver showcases the potential of YF-CPNP to enhance gene modulation efficiency, a significant achievement given the challenges associated with delivering large plasmids such as the dCas9/VPR/EGFP/gRNA-TTN pDNA, which exceeds 12 kb.

Results, Conclusions, and Discussions: Our findings underscore the enhanced efficacy of YF-CPNP, which incorporates yeast fragments into polyplexes, in improving the delivery of gene editing systems when administered orally. YF-CPNP outperforms non-coated CPNP, particularly in targeting M cells and macrophages within the GI tract and associated lymphoid tissues. A key feature of YF-CPNP is its pronounced affinity for intestinal M cells, which significantly boosts transepithelial absorption and subsequent localization in gut-associated lymphoid tissues. The superior performance of YF-CPNP is evident in its facilitation of robust gene editing across various immune cells within Peyer’s patches and mesenteric lymph nodes (MLN). This enhanced localization also suggests potential for systemic distribution, reaching distant organs such as the liver. Moreover, we achieved successful oral gene activation of the TTN gene using the YF-CPNP loaded with the dCas9 VPR plasmid, demonstrating effective gene modulation both in the GI tract and the liver. Collectively, our results reveal the potential of YF-CPNP as an efficient, orally-administered, nonviral platform for targeted gene editing within the GI tract and gut-associated lymphoid tissues, and for systemic gene activation. This approach offers a promising, patient-friendly, and non-invasive alternative for gene therapy applications.

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