Nucleic acid therapeutics最新文献

Antisense oligonucleotide technology is one of the most successful gene therapy (GT) approaches. However, low selectivity of antisense agents limits their application as anticancer drugs. To achieve activation of antisense agent selectively in cancer cells, herein, we propose the concept of binary antisense oligonucleotide (biASO) agent. biASO recognizes an RNA sequence of a gene associated with cancer development (marker) and then activates RNase H-dependent cleavage of a targeted messenger RNA. biASO was optimized to produce only the background cleavage of the targeted RNA in the absence of the activator. The approach lays the foundation for the development of highly selective and efficient GT agents.

We report two new 6-pyruvoyl-tetrahydropterin synthase splicing variants identified through genomic sequencing and transcript analysis in a patient with tetrahydrobiopterin deficiency, presenting with hyperphenylalaninemia and monoamine neurotransmitter deficiency. Variant c.243 + 3A>G causes exon 4 skipping. The deep-intronic c.164-672C>T variant creates a potential 5' splice site that leads to the inclusion of four overlapping pseudoexons, corresponding to exonizations of an antisense short interspersed nuclear element AluSq repeat sequence. Two of the identified pseudoexons have been reported previously, activated by different deep-intronic variants, and were also detected at residual levels in control cells. Interestingly, the predominant pseudoexon is nearly identical to a disease causing activated pseudoexon in the F8 gene, with the same 3' and 5' splice sites. Splice switching antisense oligonucleotides (SSOs) were designed to hybridize with splice sites and/or predicted binding sites for regulatory splice factors. Different SSOs corrected the aberrant pseudoexon inclusion, both in minigenes and in fibroblasts from patients carrying the new variant c.164-672C>T or the previously described c.164-716A>T. With SSO treatment PTPS protein was recovered, illustrating the therapeutic potential of the approach, for patients with different pseudoexon activating variants in the region. In addition, the natural presence of pseudoexons in the wild type context suggests the possibility of applying the antisense strategy in patients with hypomorphic PTS variants with the purpose of upregulating their expression to increase overall protein and activity.

Nucleic acids are an increasingly popular platform for the development of biotherapeutics to treat a wide variety of illnesses, including diseases where traditional drug development efforts have failed. To date, there are 14 short oligonucleotide therapeutics and 2 messenger RNA (mRNA) vaccines approved by the U.S. Food and Drug Administration (FDA), which demonstrates the potential of nucleic acids as a platform for the development of safe and effective medicines and vaccines. Despite the increasing popularity of nucleic acid-based drugs, there has been a paucity of high-resolution structural techniques applied to rigorously characterize these molecules during drug development. Here, we present application of nuclear magnetic resonance (NMR) methods to structurally "fingerprint" short oligonucleotide therapeutics at natural isotope abundance under full formulation conditions. The NMR methods described herein leverage signals arising from the native structural features of nucleic acids, including imino, aromatic, and ribose resonances, in addition to non-native chemistries, such as 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), and phosphorothioate (PS) modifications, introduced during drug development. We demonstrate the utility of the NMR methods to structurally "fingerprint" a model short interfering RNA (siRNA) and a sample that simulated the drug product Givosiran. We anticipate broad applicability of the NMR methods to other nucleic acid-based therapeutics due to the generalized nature of the approach and ability to monitor many quality attributes simultaneously.

Inherited retinal dystrophies are caused by mutations in more than 250 genes, each of them carrying several types of mutations that can lead to different clinical phenotypes. Mutations in Retinitis Pigmentosa GTPase-Regulator (RPGR) cause X-linked Retinitis pigmentosa (RP). A nucleotide substitution in intron 9 of RPGR causes the increase of an alternatively spliced isoform of the mature mRNA, bearing exon 9a (E9a). This introduces a stop codon, leading to truncation of the protein. Aiming at restoring impaired gene expression, we developed an antisense RNA-based therapeutic approach for the skipping of RPGR E9a. We designed a set of specific U1 antisense snRNAs (U1_asRNAs) and tested their efficacy in vitro, upon transient cotransfection with RPGR minigene reporter systems in HEK-293T, 661W, and PC-12 cell lines. We thus identified three chimeric U1_asRNAs that efficiently mediate E9a skipping, correcting the genetic defect. Unexpectedly, the U1-5'antisense construct, which exhibited the highest exon-skipping efficiency in PC-12 cells, induced E9a inclusion in HEK-293T and 661W cells, indicating caution in the choice of preclinical model systems when testing RNA splicing-correcting therapies. Our data provide a proof of principle for the application of U1_snRNA exon skipping-based approach to correct splicing defects in RPGR.

RNase H1-dependent phosphorothioate oligonucleotides (PS-ASOs) have been developed to treat various diseases through specific degradation of target RNAs. Although many factors or features of RNA and PS-ASOs have been demonstrated to affect antisense activity of PS-ASOs, little is known regarding the roles of RNase H1-associated proteins in PS-ASO performance. In this study, we report that two nucleolar proteins, NAT10 and DDX21, interact with RNase H1 and affect the potency and safety of PS-ASOs. The interactions of these two proteins with RNase H1 were determined using BioID proximity labeling in cells and confirmed biochemically. Reduction of NAT10 and DDX21 decreased PS-ASO activity in cells, and purified NAT10 and DDX21 proteins enhanced RNase H1 cleavage rates, indicating that these two proteins facilitate RNase H1 endoribonuclease activity. Consistently, reduction of these proteins increased the levels of R-loops, and impaired pre-rRNA processing. In addition, reduction of the two proteins increased the cytotoxicity of toxic PS-ASOs, and treatment of toxic PS-ASOs also altered the localization of these proteins. Together, this study shows for the first time that NAT10 and DDX21 interact with RNase H1 protein and enhance its enzymatic activity, contributing to the potency and safety of PS-ASOs.

Guanidine-bridged nucleic acid (GuNA) is a novel 2',4'-bridged nucleic acid/locked nucleic acid (2',4'-BNA/LNA) analog containing cations that exhibit strong affinity for target RNA and superior nuclease resistance. In this study, Malat1 antisense oligonucleotide (ASO) bearing GuNA was evaluated for target knockdown (KD) activity and tolerability. The GuNA ASO did not interfere with RNase H recruitment on the target RNA/ASO heteroduplex and did show potent target KD activity in a skeletal muscle-derived cell line equivalent to that of the LNA ASO under gymnotic conditions, whereas almost no KD activity was observed in a hepatocyte-derived cell line. The GuNA ASO exhibited potent KD activity in various tissues; the KD activity in the skeletal muscle was equivalent with that of the LNA ASO, but the KD activities in the liver and kidney were clearly lower compared with the LNA ASO. In addition, despite the higher accumulation of the GuNA ASO in the liver, levels of aspartate aminotransferase and alanine aminotransferase with the GuNA ASO administration were not elevated compared with those induced by the LNA ASO. Our data indicate that the GuNA ASO is tolerable and exhibits unique altered pharmacological activities in comparison with the LNA ASO in terms of the relative effect between liver and skeletal muscle.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by episodic heterotopic ossification. The median life span of people with this disorder is ∼40 years, and currently, there is no effective treatment available. More than 95% of cases are caused by a recurrent mutation (c.617G>A; R206H) of Activin A receptor, type I (ACVR1)/Activin receptor-like kinase-2 (ALK2), a bone morphogenetic protein type I receptor. The mutation renders ACVR1 responsive to activin A, which does not activate wild-type ACVR1. Ectopic activation of ACVR1^R206H by activin A induces heterotopic ossification. Since ACVR1^R206H is a hyperactive receptor, a promising therapeutic strategy is to decrease the activity of mutated ACVR1. To accomplish this goal, we developed locked nucleic acid (LNA) gapmers. These are short DNA oligonucleotides with LNA modification at both ends. They induce targeted mRNA degradation and specific knockdown of gene expression. We demonstrated that some of these gapmers efficiently knocked down ACVR1^R206H expression at RNA levels, while ACVR1^WT was mostly unaffected in human FOP fibroblasts. Also, the gapmers suppressed osteogenic differentiation induced by ACVR1^R206H and activin A. These gapmers may be promising drug candidates for FOP. This novel strategy will also pave the way for antisense-mediated therapy of other autosomal dominant disorders.

Known limitations of unfractionated heparin (UFH) have encouraged the evaluation of anticoagulant aptamers as alternatives to UFH in highly procoagulant settings such as cardiopulmonary bypass (CPB). Despite progress, these efforts have not been totally successful. We take a different approach and explore whether properties of an anticoagulant aptamer can complement UFH, rather than replace it, to address shortcomings with UFH use. Combining RNA aptamer 11F7t, which targets factor X/Xa, with UFH (or low molecular weight heparin) yields a significantly enhanced anticoagulant cocktail effective in normal and COVID-19 patient blood. This aptamer-UFH combination (1) supports continuous circulation of human blood through an ex vivo membrane oxygenation circuit, as is required for patients undergoing CPB and COVID-19 patients requiring extracorporeal membrane oxygenation, (2) allows for a reduced level of UFH to be employed, (3) more effectively limits thrombin generation compared to UFH alone, and (4) is rapidly reversed by the administration of protamine sulfate, the standard treatment for reversing UFH clinically following CPB. Thus, the combination of factor X/Xa aptamer and UFH has significantly improved anticoagulant properties compared to UFH alone and underscores the potential of RNA aptamers to improve medical management of acute care patients requiring potent yet rapidly reversible anticoagulation.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by the expansion of a CAG repeat in the ATXN3 gene. This mutation leads to a toxic gain of function of the ataxin-3 protein, resulting in neuronal dysfunction and atrophy of specific brain regions over time. As ataxin-3 is a dispensable protein in rodents, ataxin-3 knockdown by gene therapy may be a powerful approach for the treatment of SCA3. In this study, we tested the feasibility of an adeno-associated viral (AAV) vector carrying a previously described artificial microRNA against ATXN3 in a striatal mouse model of SCA3. Striatal injection of the AAV resulted in good distribution throughout the striatum, with strong dose-dependent ataxin-3 knockdown. The hallmark intracellular ataxin-3 inclusions were almost completely alleviated by the microRNA-induced ATXN3 knockdown. In addition, the striatal lesion of dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) in the SCA3 mice was rescued by ATXN3 knockdown, indicating functional rescue of neuronal signaling and health upon AAV treatment. Together, these data suggest that microRNA-induced ataxin-3 knockdown is a promising therapeutic strategy in the treatment of SCA3.

Antisense oligonucleotides (ASOs), a novel paradigm in modern therapeutics, modulate cellular gene expression by binding to complementary messenger RNA (mRNA) sequences. While advances in ASO medicinal chemistry have greatly improved the efficiency of cellular uptake, selective uptake by specific cell types has been difficult to achieve. For more efficient and selective uptake, ASOs are often conjugated with molecules with high binding affinity for transmembrane receptors. Triantennary N-acetyl-galactosamine conjugated phosphorothioate ASOs (GalNAc-PS-ASOs) were developed to enhance targeted ASO delivery into liver through the hepatocyte-specific asialoglycoprotein receptor (ASGR). We assessed the kinetics of uptake and subsequent intracellular distribution of AlexaFluor 488 (AF488)-labeled PS-ASOs and GalNAc-PS-ASOs in J774A.1 mouse macrophages and primary mouse or rat hepatocytes using simultaneous coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence (2PF) imaging. The CARS modality captured the dynamic lipid distributions and overall morphology of the cells; two-photon fluorescence (2PF) measured the time- and dose-dependent localization of ASOs delivered by a modified treatment of suspension cells. Our results show that in macrophages, the uptake rate of PS-ASOs did not significantly differ from that of GalNAc-PS-ASOs. However, in hepatocytes, GalNAc-PS-ASOs exhibited a peripheral uptake distribution compared to a polar uptake distribution observed in macrophages. The peripheral distribution correlated with a significantly larger amount of internalized GalNAc-PS-ASOs compared to the PS-ASOs. This work demonstrates the relevance of multimodal imaging for elucidating the uptake mechanism, accumulation, and fate of different ASOs in liver cells that can be used further in complex in vitro models and liver tissues to evaluate ASO distribution and activity.