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Cetuximab-siRNA Conjugate Linked Through Cationized Gelatin Knocks Down KRAS G12C Mutation in NSCLC Sensitizing the Cells Toward Gefitinib.

ABSTRACT: Delivery of small-interfering RNA (siRNA) has been of great interest in the past decade for effective gene silencing. To overcome synthetic and regulatory challenges posed by nanoparticle-mediated siRNA delivery, antibody-siRNA conjugate (ARC) platform is emerging as a potential siRNA delivery system suitable for clinical translation. Herein, we have developed a delivery technology based on the ARC platform for stable delivery of siRNA called as Gelatin-Antibody Delivery System (GADS). In GADS, positively charged gelatin acts as a linker between antibody-siRNA and enables the endosomal escape of siRNA for gene silencing postcellular internalization. For proof of concept, we synthesized a scalable GADS conjugate comprising of Cetuximab (CTB), cationized gelatin (cGel) and NSCLC KRAS_G12C-specific siRNA. CTB was chemically conjugated to cGel through an amide link to form the CTB-cGel complex. Thereafter, siRNA was chemically conjugated to the cGel moiety of the complex through the thioether link to form CTB-cGel-siRNA conjugate. RP-HPLC analysis was used to monitor the reaction while gel retardation assay was used to determine siRNA loading capacity. SPR analysis showed the preservation of ligand binding affinity of antibody conjugates with K_D of ∼0.3 nM. Furthermore, cellular internalization study using florescent microscopy revealed receptor-mediated endocytosis. The conjugate targeted EGFR receptor of KRAS mutant NSCLC to specifically knockdown G12C mutation. The oncogene knockdown sensitized the cells toward small molecule inhibitor-Gefitinib causing ∼70% loss in cell viability. Western blot analysis revealed significant downregulation for various RAS downstream proteins postoncogene knockdown. Comparison of the efficiency of GADS vis-à-vis positive siRNA control and CRISPR-Cas9-based knockout of KRAS Exon 2 in the NCI-H23 NSCLC cell line suggests GADS as a potential technology for clinical translation of gene therapy.

SUBMITTER: Sreedurgalakshmi K

PROVIDER: S-EPMC8461128 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundBoron neutron capture therapy (BNCT) is a cell-selective radiation therapy that uses the alpha particles and lithium nuclei produced by the boron neutron capture reaction. BNCT is a relatively safe tool for treating multiple or diffuse malignant tumors with little injury to normal tissue. The success or failure of BNCT depends upon the 10B compound accumulation within tumor cells and the proximity of the tumor cells to the body surface. To extend the therapeutic use of BNCT from surface tumors to visceral tumors will require 10B compounds that accumulate strongly in tumor cells without significant accumulation in normal cells, and an appropriate delivery method for deeper tissues.Hemagglutinating Virus of Japan Envelope (HVJ-E) is used as a vehicle for gene delivery because of its high ability to fuse with cells. However, its strong hemagglutination activity makes HVJ-E unsuitable for systemic administration.In this study, we developed a novel vector for 10B (sodium borocaptate: BSH) delivery using HVJ-E and cationized gelatin for treating multiple liver tumors with BNCT without severe adverse events.MethodsWe developed cationized gelatin conjugate HVJ-E combined with BSH (CG-HVJ-E-BSH), and evaluated its characteristics (toxicity, affinity for tumor cells, accumulation and retention in tumor cells, boron-carrying capacity to multiple liver tumors in vivo, and bio-distribution) and effectiveness in BNCT therapy in a murine model of multiple liver tumors.ResultsCG-HVJ-E reduced hemagglutination activity by half and was significantly less toxic in mice than HVJ-E. Higher 10B concentrations in murine osteosarcoma cells (LM8G5) were achieved with CG-HVJ-E-BSH than with BSH. When administered into mice bearing multiple LM8G5 liver tumors, the tumor/normal liver ratios of CG-HVJ-E-BSH were significantly higher than those of BSH for the first 48 hours (p < 0.05). In suppressing the spread of tumor cells in mice, BNCT treatment was as effective with CG-HVJ-E-BSH as with BSH containing a 35-fold higher 10B dose. Furthermore, CG-HVJ-E-BSH significantly increased the survival time of tumor-bearing mice compared to BSH at a comparable dosage of 10B.ConclusionCG-HVJ-E-BSH is a promising strategy for the BNCT treatment of visceral tumors without severe adverse events to surrounding normal tissues.

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Cetuximab-siRNA Conjugate Linked Through Cationized Gelatin Knocks Down KRAS G12C Mutation in NSCLC Sensitizing the Cells Toward Gefitinib.

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