Fig. 4: Targeting strategies using an antisense oligonucleotide (ASO)-based approach.

A shows a schematic diagram of two strategies describing therapeutic approaches to modulate HMMR splicing in MM patients. Strategy 1: SNPs that induce aberrant HMMR splicing can be used as targets for splice-switching antisense oligonucleotides (SSOs) by degrading pre-mRNA transcripts without recruiting RNase H1. Strategy 2: use of RNase H1-active ASOs. DNA-like antisense oligonucleotides, which span exon junctions of mis-spliced mRNA, interact with their target mRNA in the cytoplasm or pre-mRNA in the nucleus of a cell, recruit RNase H1, and mediate the selective degradation of the target RNA molecule within the DNA:RNA duplex. B is a summary of the experiments that demonstrate HMMR transcript and protein inhibitions by ASOs. HMMR-Fl and HMMR-V3 knockdown efficiency was evaluated at protein and mRNA levels. After gymnosis, cell lysates were collected from each sample for western blotting analyses and total RNA was isolated for RT-PCR DNA fragment analyses and capillary electrophoresis. B The results are presented as RFU (relative fluorescent units) to GAPDH RFU. Protein lysates were subjected to immunoblotting using anti-HMMR antibodies; GAPDH served as a loading control. Bar graphs show a densitometric analysis of the HMMR-FL and V3 protein bands measured by ImageJ software. Fold expressions on the Y axis shows the protein expression level compared to loading controls. C is a summary of the genomic instability assay. After 48 h of gymnosis, cells were collected and viable cells were separated using Ficoll-Paque PLUS. Micronuclei were quantified by flow cytometry staining using In Vitro MicroFlow Kit (Litron Labs). Images of micronuclei (flow cytometry plots) and bar graphs showing the percentage of micronuclei are presented.