Bal Hari Poudel

Messenger RNA (mRNA) therapeutics have significantly transformed contemporary medicine, particularly through their role as the active component in the SARS-CoV-2 vaccine. This remarkable achievement is the culmination of extensive research conducted over many years by scientists. The widespread administration of the COVID-19 vaccine has further accelerated research into the precise therapeutic potential of mRNA technologies. Since mRNA doesn’t integrate with the host genome, the safety and versatility of mRNA-based therapeutics make them an iconic candidate in targeted therapies. Due to a surge in innovation efforts, biomodification of the molecular signatures of mRNAs like the 5′cap, untranslated regions (UTRs), and the poly(A) tail are being developed to increase translation efficacy. Recent advancements in chemical modifications, codon optimization techniques, and targeted delivery methods have significantly enhanced the stability of synthetic mRNAs while concurrently reducing their immunogenicity. Various mRNA manufacturing and synthesizing methods are investigated in this review, focusing on their scalability and limitations. mRNA therapeutic strategies can be divided into protein replacement, immune modulation, and cellular modulation. This review explores mRNA’s molecular landscape and comprehensive utility, including applications in both clinical trials and commercial sectors.

Antisense oligonucleotides (ASOs) have emerged as powerful tools for gene modulation; however, their clinical application is often hindered by inefficient intracellular delivery. Recent advances suggest that conjugation to biomolecules such as aptamers, lipids, or vitamins may enhance uptake and efficacy. This study investigates the potential of aptamer, vitamin E, and cholesterol-conjugated ASOs to improve delivery and functional activity in cancer cell models. The ASO PNAT524 was conjugated to two DNA aptamers-AS1411 and S2.2-via thiol and triethylene glycol (TEG) linkers, respectively. In parallel, PNAT524 was modified with vitamin E and cholesterol moieties. The conjugates were evaluated for cellular uptake, exon-skipping activity, and cytotoxicity in cancer cell lines. Fluorescence microscopy was used to determine subcellular localization. Aptamer conjugation (AS1411, S2.2) did not significantly enhance exon-skipping efficiency compared to unconjugated PNAT524, consistent with previous findings. In contrast, vitamin E and cholesterol conjugates demonstrated potent, dose-dependent exon-skipping activity and cytotoxic effects. Among all formulations, the cholesterol-conjugated ASO (524-Chol) showed the highest efficacy, with superior splice-modulating and cytotoxic outcomes. Fluorescence microscopy confirmed nuclear and cytoplasmic localization of lipid-conjugated ASOs. These findings indicate that aptamer conjugation provides minimal benefit for ASO delivery, while cholesterol and vitamin E conjugation significantly enhance intracellular delivery and therapeutic activity. The 524-Chol conjugate holds strong potential for adaptation in ASOs targeting EGFR and other oncogenes, representing a promising avenue for ASO-based cancer therapeutics.

Herein, we report a systematic exploration of 2′-O-methyl (2′-OMe) antisense oligonucleotides (ASOs) with a stereo-random phosphodiester–phosphorothioate (PO–PS) backbone regarding their design, RNA binding affinity, nuclease resistance, and exon skipping efficacy. The results showed that ASOs having PO linkages arranged at the 3′-end exhibited higher exon skipping efficacy and slightly higher target binding affinity than their counterparts with PO linkages arranged at the 5′-end; and the residual length of ASOs can be still well protected from 3′-exonuclease after initial hydrolysis of as many as four successive PO linkages from the 3′-end. These findings provide insights and guidance for rational design of splice-switching ASOs with limited PS linkages.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder presenting progressive weakness of the bulbar and extremity muscles, leading to a wide-ranging clinical phenotype. More than 30 genes have been associated to genetically inherited ALS yet, approximately 85%–90% of ALS cases are sporadic. Short tandem repeats expansions, have recently been found in clinically diagnosed ALS patients and are currently investigated as potential genetic biomarkers. In this paper we compare the investigation of pathological tandem repeat expansions on a group of ALS patients by comparing the standard short-read sequencing (SRS) technique with a long-read-sequencing (LRS) method which has recently become more accessible. Blood samples from 47 sporadic ALS cases were subjected to SRS by Illumina Whole Genome Sequencing. The genome-wide tandem repeat expansions were genotyped using GangSTR, while wANNOVAR was used for variant annotation. Uncertain cases were further explored using LRS. SRS identified pathological expansions in HTT , ATXN2 , and CACNA1A genes in one patient, which were not confirmed with LRS. The latter identified large tandem repeat expansions in the C9orf72 gene of one patient that were missed by SRS. Our findings suggest that LRS should be preferred to SRS for accurate identification of pathological tandem repeat expansions.

Antisense oligonucleotides (ASOs) have been utilized for developing RNA-targeting agents that act as an inhibitor of microtubule-associated protein tau (MAPT) for the treatment of tauopathies. Although several anti-tau ASO candidates have been reported that could reduce MAPT expression either through RNase H-mediated mRNA degradation or splice switching, novel designs of chemically modified ASOs are still needed to improve their activity and safety profile. Moreover, the development of a stereodefined anti-tau ASO is highly desirable due to differences in efficacy and toxicity between diastereomers. Kunihiko Kanatsu et al.1 identified two best-performing fully stereocontrolled phosphorodiamidate morpholino oligomer (PMO) gapmers (ASO-486-R5-S and ASO-486-R5-R) targeting MAPT mRNA after performing a screening of optimal ASO sequence and subsequent screening of optimal phosphorous stereochemistry (Figure 1). Surprisingly, a dramatic difference in safety profiles between stereoisomers (ASO-409-R3-S versus ASO-409-R3-R, and ASO-409-SSR2-S versus ASO-409-SSR2-R), which only differ in one single phosphorous stereochemistry, was also observed (Figure 1). Fundamentally, this work not only revolutionizes ASO design by adopting PMO as the chemistry of wing regions in a gapmer but also highlights the importance of stereopattern screening in identifying ASO leads since as few as one phosphorous stereogenic center (generating two possible stereoisomers) matters in determining in vivo toxicity.

Osteosarcoma is a form of bone cancer that predominantly impacts osteoblasts, the cells responsible for creating fresh bone tissue. Typical indications include bone pain, inflammation, sensitivity, mobility constraints, and fractures. Utilising imaging techniques such as X-rays, MRI scans, and CT scans can provide insights into the size and location of the tumour. Additionally, a biopsy is employed to confirm the diagnosis. Analysing genes with distinct expression patterns unique to osteosarcoma can be valuable for early detection and the development of effective treatment approaches. In this research, we comprehensively examined the entire transcriptome and pinpointed genes with altered expression profiles specific to osteosarcoma. The study mainly aimed to identify the molecular fingerprint of osteosarcoma. In this study, we processed 90 FFPE samples from PathWest with an almost equal number of osteosarcoma and healthy tissues. RNA was extracted from Paraffin-embedded tissue; RNA was sequenced, the sequencing data was analysed, and gene expression was compared to the healthy samples of the same patients. Differentially expressed genes in osteosarcoma-derived samples were identified, and the functions of those genes were explored. This result was combined with our previous studies based on FFPE and fresh samples to perform a meta-analysis. We identified 1,500 identical differentially expressed genes in PathWest osteosarcoma samples compared to normal tissue samples of the same patients. Meta-analysis with combined fresh tissue samples identified 530 differentially expressed genes. IFITM5, MMP13, PANX3 , and MAGEA6 were some of the most overexpressed genes in osteosarcoma samples, while SLC4A1, HBA1, HBB, AQP7 genes were some of the top downregulated genes. Through the meta-analysis, 530 differentially expressed genes were identified to be identical among FFPE (105 FFPE samples) and 36 fresh bone samples. Deconvolution analysis with single-cell RNAseq data confirmed the presence of specific cell clusters in FFPE samples. We propose these 530 DEGs as a molecular fingerprint of osteosarcoma.

Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene (DYSF) can result in rare forms of muscular dystrophy; Miyoshi myopathy; limb girdle muscular dystrophy type 2B (LGMD2B); and distal myopathy. These conditions are collectively known as dysferlinopathies and are caused by more than 600 mutations that have been identified across the DYSF gene to date. In this review, we discuss the key molecular and clinical features of LGMD2B, the causative gene DYSF, and the associated dysferlin protein structure. We also provide an update on current approaches to LGMD2B diagnosis and advances in drug development, including splice switching antisense oligonucleotides. We give a brief update on clinical trials involving adeno-associated viral gene therapy and the current progress on CRISPR/Cas9 mediated therapy for LGMD2B, and then conclude by discussing the prospects of antisense oligomer-based intervention to treat selected mutations causing dysferlinopathies.

Background: Various primers have been used for detection of Mycobacterium tuberculosis complex (MTBC) DNA using Polymerase chain reaction (PCR). The MPB64 gene has been demonstrated to be a highly specific target for MTBC detection.

Aims & Objectives: To detect MTBC DNA using MPB64 primers in clinical specimens of Nepalese patients suspected to have tuberculosis (TB).

Materials and Methods: DNA was extracted from 44 clinical specimens of patients suspected of having TB and MPB64 targeted PCR was performed.

Results: Bands comprising 240 base pair (bp) of MPB64 sequence were present in 15 of the 44 clinical samples, of which 4 were pulmonary and the remainders were extra-pulmonary samples. The overall positivity of MPB64 targeted PCR was 34.1%.

Conclusion: PCR targeting the MPB64 fragment has a potential of detecting the MTBC DNA and has potentially valuable clinical applications in early detection of TB in Nepal.