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

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 KRASG12C-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 KD 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.

CRISPR-Cas deployment in non-small cell lung cancer for target screening, validations, and discoveries.

Continued advancements in CRISPR-Cas systems have accelerated genome research. Use of CRISPR-Cas in cancer research has been of great interest that is resulting in development of orthogonal methods for drug target validations and discovery of new therapeutic targets through genome-wide screens of cancer cells. CRISPR-based screens have also revealed several new cancer drivers through alterations in tumor suppressor genes (TSGs) and oncogenes inducing resistance to targeted therapies via activation of alternate signaling pathways. Given such dynamic status of cancer, we review the application of CRISPR-Cas in non-small cell lung cancer (NSCLC) for development of mutant models, drug screening, target validation, novel target discoveries, and other emerging potential applications. In addition, CRISPR-based approach for development of novel anticancer combination therapies is also discussed in this review.

Aggregation of Respiratory Complex Subunits Marks the Onset of Proteotoxicity in Proteasome Inhibited Cells.

Proteostasis is maintained by optimal expression, folding, transport, and clearance of proteins. Deregulation of any of these processes triggers protein aggregation and is implicated in many age-related pathologies. In this study, using quantitative proteomics and microscopy, we show that aggregation of many nuclear-encoded mitochondrial proteins is an early protein destabilization event during short-term proteasome inhibition. Among these, respiratory chain complex (RCC) subunits represent a group of functionally related proteins consistently forming aggregates under multiple proteostasis stresses with varying aggregation propensities. Sequence analysis reveals that several RCC subunits, irrespective of the cleavable mitochondrial targeting sequence, contain low-complexity regions at the N-terminus. Using different chimeric and mutant constructs, we show that these low-complexity regions partially contribute to the intrinsic instability of multiple RCC subunits. Taken together, we propose that physicochemically driven aggregation of unassembled RCC subunits destabilizes their functional assembly inside mitochondria. This eventually deregulates the biogenesis of respiratory complexes and marks the onset of mitochondrial dysfunction.