Rakesh Naduvile Veedu

Background: Hepatic fibrosis is a common response to hepatocyte damage presenting a major global health challenge. Despite its prevalence, there are currently no clinically approved therapeutics. In a previous biomarker discovery, we identified dysregulated expression of microRNAs, particularly miR-25-3p (miR-25), in patients with cystic fibrosis, with or without associated liver disease. Further investigation revealed miR-25 acting as a negative regulator of cross-talk between Notch-1 and TGFβR1 in hepatic stellate cells (HSCs). Targeting key activators of Notch 1 signalling, ADAM-17 and FKBP14, miR-25 suppresses fibrillar collagen expression. Building on these findings, we designed a novel, chemically modified miR-25 mimic (miR-25-C3) with significantly enhanced anti-fibrotic activity compared to commercially available mimics. In this study, we evaluate the therapeutic efficacy of this proprietary miR-25-C3.

Methods: To assess miR-25-C3 efficacy, the human HSC line LX-2 was transfected, confirming downregulation of Notch1 activators and fibrillar collagens. A vitamin A-coupled lipid nanoparticle (VA-lipo) system was then optimized for targeted in vivo delivery to hepatic stellate cells. Cirrhosis was induced in C57Bl6J mice via thioacetamide (TAA; 300 mg/L in drinking water) over 8 weeks. Mice received tail vein injections of VA-lipo–miR-25C3 (0.75 mg/kg) every 3 days while continuing TAA exposure. Two treatment cohorts were established: a 2-week group (5 injections) and a 4-week group (10 injections). Livers were harvested for histopathological evaluation, METAVIR scoring, and qPCR analysis of target genes.

Results: miR-25-C3 mimic showed superior downregulation of Notch 1 activator genes ADAM-17 and FKBP14, compared to a commercially available miR-25 mimic, leading to significant inhibition of TGFβRI and fibrillar collagen expression. Additionally, vitamin A-coupled liposomes effectively targeted miR-25-C3 to HSCs in vivo, ensuring enhanced delivery to the liver (vs off-target organs) and minimizing off-target effects. In the TAA-induced liver fibrosis model, both males and females treated with VA-lipo-miR-25-C3 showed significant downregulation of collagen I, II, III, TGFβRI, ADAM-17, and FKBP14 gene expression, as a direct benefit of miR-25-C3 anti-fibrotic therapy. Notably, only male mice showed regression of fibrillar collagens at the protein level, as evidenced by reduced Sirius Red histochemistry and a significant decrease in METAVIR fibrosis scores.

Conclusion: These findings highlight the potential of miR-25-C3 as a novel and promising anti-fibrotic therapeutic. The use of vitamin A-coupled liposomes ensures the effective targeting of miR-25-C3 to HSCs in the liver. Future studies will focus on the underlying mechanisms driving the sex differences and further investigate the translational potential of miR-25-C3 in preclinical for both males and females.

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.

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.

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.

There is a significant need for fast, cost-effective, and highly sensitive protein target detection, particularly in the fields of food, environmental monitoring, and healthcare. The integration of high-affinity aptamers with metal-based nanomaterials has played a crucial role in advancing the development of innovative aptasensors tailored for the precise detection of specific proteins. Aptamers offer several advantages over commonly used molecular recognition methods, such as antibodies. Recently, a variety of metal-based aptasensors have been established. These metallic nanomaterials encompass noble metal nanoparticles, metal oxides, metal-carbon nanotubes, carbon quantum dots, graphene-conjugated metallic nanostructures, as well as their nanocomposites, metal–organic frameworks (MOFs), and MXenes. In general, these materials provide enhanced sensitivity through signal amplification and transduction mechanisms. This review primarily focuses on the advancement of aptasensors based on metallic materials for the highly sensitive detection of protein targets, including enzymes and growth factors. Additionally, it sheds light on the challenges encountered in this field and outlines future prospects. We firmly believe that this review will offer a comprehensive overview and fresh insights into metallic nanomaterials-based aptasensors and their capabilities, paving the way for the development of innovative point-of-care (POC) diagnostic devices.

The last decade (2013-2023) has seen unprecedented successes in the clinical translation of therapeutic antisense oligonucleotides (ASOs). Eight such molecules have been granted marketing approval by the United States Food and Drug Administration (US FDA) during the decade, after the first ASO drug, fomivirsen, was approved much earlier, in 1998. Splice-modulating ASOs have also been developed for the therapy of inborn errors of metabolism (IEMs), due to their ability to redirect aberrant splicing caused by mutations, thus recovering the expression of normal transcripts, and correcting the deficiency of functional proteins. The feasibility of treating IEM patients with splice-switching ASOs has been supported by FDA permission (2018) of the first "N-of-1" study of milasen, an investigational ASO drug for Batten disease. Although for IEM, owing to the rarity of individual disease and/or pathogenic mutation, only a low number of patients may be treated by ASOs that specifically suppress the aberrant splicing pattern of mutant precursor mRNA (pre-mRNA), splice-switching ASOs represent superior individualized molecular therapeutics for IEM. In this work, we first summarize the ASO technology with respect to its mechanisms of action, chemical modifications of nucleotides, and rational design of modified oligonucleotides; following that, we precisely provide a review of the current understanding of developing splice-modulating ASO-based therapeutics for IEM. In the concluding section, we suggest potential ways to improve and/or optimize the development of ASOs targeting IEM.

Chemically modified antisense oligonucleotide (ASO) has been established as a successful therapeutic strategy for treating various human diseases. To date, ten ASO drugs, which are capable of either inducing mRNA degradation via RNase H recruitment (fomivirsen, mipomersen, inotersen, volanesorsen and tofersen) or splice modulation (eteplirsen, nusinersen, golodirsen, viltolarsen and casimersen), have been approved by the regulatory agencies for market entry. Nonetheless, none of these approved drugs are prescribed as cancer therapy. Towards this, we have developed steric-blocking ASOs targeting BIRC5 – a well-validated oncogene. Initial screening was performed by transfection of HepG2 cells with seven BIRC5 exon-2 targeting, uniformly 2′-OMe-PS modified ASOs at 400 nM respectively, leading to the identification of two best-performing candidates ASO-2 and ASO-7 in reducing the production of BIRC5 mRNA. Subsequent dose–response assay was conducted via transfection of HepG2 cells by different concentrations (400, 200, 100, 50, 25 nM) of ASO-2 and ASO-7 respectively, showing that both ASOs consistently and efficiently inhibited BIRC5 mRNA expression in a dose-dependent manner. Furthermore, western blot analysis confirmed that ASO-7 could significantly repress survivin production on protein level. Based on our preliminary results, we believe that ASO-7 could be a useful BIRC5 inhibitor for both research purpose and therapeutic development.

Synthetic antisense oligonucleotides (ASOs) are emerging as an attractive platform to treat various diseases. By specifically binding to a target mRNA transcript through Watson–Crick base pairing, ASOs can alter gene expression in a desirable fashion to either rescue loss of function or downregulate pathogenic protein expression. To be clinically relevant, ASOs are generally synthesized using modified analogs to enhance resistance to enzymatic degradation and pharmacokinetic and dynamic properties. Phosphorothioate (PS) belongs to the first generation of modified analogs and has played a vital role in the majority of approved ASO drugs, mainly based on the RNase H mechanism. In contrast to RNase H-dependent ASOs that bind and cleave target mature mRNA, splice-switching oligonucleotides (SSOs) mainly bind and alter precursor mRNA splicing in the cell nucleus. To date, only one approved SSO (Nusinersen) possesses a PS backbone. Typically, the synthesis of PS oligonucleotides generates two types of stereoisomers that could potentially impact the ASO’s pharmaco-properties. This can be limited by introducing the naturally occurring phosphodiester (PO) linkage to the ASO sequence. In this study, towards fine-tuning the current strategy in designing SSOs, we reported the design, synthesis, and evaluation of several stereo-random SSOs on a mixed PO–PS backbone for their binding affinity, biological potency, and nuclease stability. Based on the results, we propose that a combination of PO and PS linkages could represent a promising approach toward limiting undesirable stereoisomers while not largely compromising the efficacy of SSOs.