👤 Vignesh N Hariharan

Chemically modified small interfering RNAs (siRNAs) are a promising drug class that silences disease-causing genes via mRNA degradation. Both siRNA-specific features (e.g. sequence, modification pattern, and structure) and target mRNA-specific factors contribute to observed efficacy. Systematically defining the relative contributions of siRNA sequence, structure, and modification pattern versus the native context of the target mRNA is necessary to inform design considerations and facilitate the widespread application of this therapeutic platform. To address this, we synthesized a panel of ∼1260 differentially modified siRNAs and evaluated their silencing efficiency against therapeutically relevant mRNAs (APP, BACE1, MAPT, and SNCA) using both reporter-based and native expression assays. Our results demonstrate that the siRNA modification pattern (e.g. level of 2'-O-methyl content) significantly impacts efficacy, while structural features (e.g. symmetric versus asymmetric configurations) do not. Furthermore, we observed substantial differences in the number of effective siRNAs identified per target. These target-specific differences in hit rates are largely mitigated when efficacy is tested in the context of a reporter assay, confirming that native mRNA-specific features influence siRNA performance. Key target-specific factors, including exon usage, polyadenylation site selection, and ribosomal occupancy, partially explained efficacy variability. These insights led to a proposed framework of parameters for optimizing therapeutic siRNA design. Show less

A small, albeit significant, number of head and neck squamous cell carcinoma (HNSCC) patients has no history of tobacco and alcohol use. Such non-habits associated HNSCCs may represent a distinct clinical entity and exhibit increased aggressiveness. The objective of the study was to understand differences in molecular etiology of habits, and non-habits associated tongue carcinomas. High-throughput gene expression profiling of 22 tumor samples was carried out. This was followed by quantitative real-time PCR validation of four of the identified differentially expressed genes. Eighteen genes were identified that correlate strongly with the habits- and non-habits distinction. Among the genes significantly overexpressed in the non-habits group are CCND1, a key cell-cycle regulator, DACT3, a modulator of the Wnt/beta-catenin pathway, and three genes associated with the Notch signaling pathway. CCND1 and DACT3 overexpression in non-habits associated tongue carcinomas were subsequently validated by quantitative real-time PCR in an independent cohort (n = 18) of patient samples. Gene expression data were integrated with publicly available protein interaction data to build a small protein interaction network containing five of 18 differentially expressed genes. This suggested that a functional 'network module' can be implicated in the subgroup distinction. All the tumors analyzed here were human papillomavirus (HPV) negative samples. An association between CCND1 overexpression in oral tumors and poor prognosis has previously been reported. Thus, CCND1 overexpression in non-habits associated anterior tongue carcinomas may contribute to their increased clinical aggressiveness. Show less