Nucleic acid therapeutics最新文献

Oligonucleotides have emerged as valuable new therapeutics. Presently, oligonucleotide manufacturing consists in a series of stepwise additions until the full-length product is obtained. Deprotection of the phosphorus backbone before cleavage and deprotection (C&D) by ammonolysis is necessary to control the 3-(2-cyanoethyl) thymidine (CNET) impurity. In this study, we demonstrate that the use of piperazine as a scavenger of acrylonitrile allows phosphorus deprotection and C&D to be combined in a single step. This reduces solvent consumption, processing time, and CNET levels. Additionally, we showed that substitution of piperazine for triethylamine in the phosphorus deprotection step of supported-synthesis leads to reduced reaction times and lower levels of CNET impurities.

RNA interference (RNAi)-based therapeutics hold the potential for dominant genetic disorders, enabling sequence-specific inhibition of pathogenic gene products. We aimed to direct RNAi for the selective suppression of the heterozygous GNAO1 c.607 G > A variant causing GNAO1 encephalopathy. By screening short interfering RNA (siRNA), we showed that GNAO1 c.607G>A is a druggable target for RNAi. The si1488 candidate achieved at least twofold allelic discrimination and downregulated mutant protein to 35%. We created vectorized RNAi by incorporating the si1488 sequence into the short hairpin RNA (shRNA) in the adeno-associated virus (AAV) vector. The shRNA stem and loop were modified to improve the transcription, processing, and guide strand selection. All tested shRNA constructs demonstrated selectivity toward mutant GNAO1, while tweaking hairpin structure only marginally affected the silencing efficiency. The selectivity of shRNA-mediated silencing was confirmed in the context of AAV vector transduction. To conclude, RNAi effectors ranging from siRNA to AAV-RNAi achieve suppression of the pathogenic GNAO1 c.607G>A and discriminate alleles by the single-nucleotide substitution. For gene therapy development, it is crucial to demonstrate the benefit of these RNAi effectors in patient-specific neurons and animal models of the GNAO1 encephalopathy.

RNA-based medicines have potential to treat a large variety of diseases, and research in the field is very dynamic. Proactively, The European Medicines Agency (EMA) organized a virtual conference on February 2, 2023 to promote the development of RNA-based medicines. The initiative addresses the goal of the EMA Regulatory Science Strategy to 2025 to "catalyse the integration of science and technology in medicines development." The conference focused on RNA technologies (excluding RNA vaccines) and involved different stakeholders, including representatives from academia, industry, regulatory authorities, and patient organizations. The conference comprised presentations and discussion sessions conducted by panels of subject matter experts. In this meeting report, we summarize the presentations and recap the main themes of the panel discussions.

The triantennary N-acetylgalactosamine (GalNAc₃) cluster has demonstrated the utility of receptor-mediated uptake of ligand-conjugated antisense drugs targeting RNA expressed by hepatocytes. GalNAc₃-conjugated 2'-O-methoxyethyl (2'MOE) modified antisense oligonucleotides (ASOs) have demonstrated a higher potency than the unconjugated form to support lower doses for an equivalent pharmacological effect. We utilized the Ionis integrated safety database to compare four GalNAc₃-conjugated and four same-sequence unconjugated 2'MOE ASOs. This assessment evaluated data from eight randomized placebo-controlled dose-ranging phase 1 studies involving 195 healthy volunteers (79 GalNAc₃ ASO, 24 placebo; 71 ASO, 21 placebo). No safety signals were identified by the incidence of abnormal threshold values in clinical laboratory tests for either ASO group. However, there was a significant increase in mean alanine transaminase levels compared with placebo in the upper dose range of the unconjugated 2'MOE ASO group. The mean percentage of subcutaneous injections leading to local cutaneous reaction was 30-fold lower in the GalNAc₃-conjugated ASO group compared with the unconjugated ASO group (0.9% vs. 28.6%), with no incidence of flu-like reactions (0.0% vs. 0.7%). Three subjects (4.2%) in the unconjugated ASO group discontinued dosing. An improvement in the overall safety and tolerability profile of GalNAc₃-conjugated 2'MOE ASOs is evident in this comparison of short-term clinical data in healthy volunteers.

The ability to reverse the binding of aptamers to their target proteins has received considerable attention for developing controllable therapeutic agents. Recently, use of aptamers as reversible cell-sorting ligands has also sparked interest. Antibodies are currently utilized for isolating cells expressing a particular cell surface receptor. The inability to remove antibodies from isolated cells following sorting greatly limits their utility for many applications. Previously, we described how a particular aptamer-antidote oligonucleotide pair can isolate cells and clean them. Here, we demonstrate that this approach is generalizable; aptamers can simultaneously recognize more than one cell type during fluorescent activated cell sorting (FACS). Moreover, we describe a novel approach to reverse aptamer binding following cell sorting using a nuclease. This alternative strategy represents a cleaning approach that does not require the generation of antidote oligonucleotides for each aptamer and will greatly reduce the cost and expand the utility of Clean FACS.

Antisense oligonucleotides (AONs) are promising therapeutic candidates, especially for neurological diseases. Intracerebroventricular (ICV) injection is the predominant route of administration in mouse studies, while in clinical trials, intrathecal (IT) administration is mostly used. There is little knowledge on the differences in distribution of these injection methods within the same species over time. In this study, we compared the distribution of splice-switching AONs targeting exon 15 of amyloid precursor protein pre-mRNA injected via the ICV and IT route in mice. The AON was labeled with radioactive indium-111 and mice were imaged using single-photon emission computed tomography (SPECT) 0, 4, 24, 48, 72, and 96 h after injection. In vivo SPECT imaging showed ¹¹¹In-AON activity diffused throughout the central nervous system (CNS) in the first hours after injection. The ¹¹¹In-AON activity in the CNS persisted over the course of 4 days, while signal in the kidneys rapidly decreased. Postmortem counting in different organs and tissues showed very similar distribution of ¹¹¹In-AON activity throughout the body, while the signal in the different brain regions was higher with ICV injection. Overall, IT and ICV injection have very similar distribution patterns in the mouse, but ICV injection is much more effective in reaching the brain.

Recent FDA approvals of mRNA vaccines, short-interfering RNAs, and antisense oligonucleotides highlight the success of oligonucleotides as therapeutics. Aptamers are excellent affinity reagents that can selectively label protein biomarkers, but their clinical application has lagged. When formulating a given aptamer for in vivo use, molecular design details can determine biostability and biodistribution; therefore, extensive postselection manipulation is often required for each new design to identify clinically useful reagents harboring improved pharmacokinetic properties. Few methods are available to comprehensively screen such aptamers, especially in vivo, constituting a significant bottleneck in the field. In this study, we introduce barcoded aptamer technology (BApT) for multiplexed screening of predefined aptamer formulations in vitro and in vivo. We demonstrate this technology by simultaneously investigating 20 aptamer formulations, each harboring different molecular designs, for targeting Non-Small Cell Lung Cancer cells and tumors. Screening in vitro identified a 45 kDa bispecific formulation as the best cancer cell targeting reagent, whereas screening in vivo identified a 30 kDa monomeric formulation as the best tumor-specific targeting reagent. The multiplexed analysis pipeline also identified biodistribution phenotypes shared among formulations with similar molecular architectures. The BApT approach we describe here has the potential for broad application to fields where oligonucleotide-based targeting reagents are desired.

The ABCA4 gene, involved in Stargardt disease, has a high percentage of splice-altering pathogenic variants, some of which cause complex RNA defects. Although antisense oligonucleotides (AONs) have shown promising results in splicing modulation, they have not yet been used to target complex splicing defects. Here, we performed AON-based rescue studies on ABCA4 complex splicing defects. Intron 13 variants c.1938-724A>G, c.1938-621G>A, c.1938-619A>G, and c.1938-514A>G all lead to the inclusion of different pseudo-exons (PEs) with and without an upstream PE (PE1). Intron 44 variant c.6148-84A>T results in multiple PE inclusions and/or exon skipping events. Five novel AONs were designed to target these defects. AON efficacy was assessed by in vitro splice assays using midigenes containing the variants of interest. All screened complex splicing defects were effectively rescued by the AONs. Although varying levels of efficacy were observed between AONs targeting the same PEs, for all variants at least one AON restored splicing to levels comparable or better than wildtype. In conclusion, AONs are a promising approach to target complex splicing defects in ABCA4.