Nucleic acid therapeutics最新文献

BK polyomavirus, a virus that latently resides in tubular epithelial cells of human kidneys, represents a major clinical problem for immunocompromised patients undergoing kidney transplantation. No proven effective specific antiviral therapies other than reduction of immunosuppressants exist. We exploited splice-modulating antisense oligonucleotides (ASOs) to block expression of the viral "gatekeeper protein" T antigen to specifically inhibit viral replication. Candidate oligonucleotides were screened for inhibitory activity in BK virus-infected cells, resulting in up to 97% reduction of viral T antigen mRNA and virus-encapsulating protein. The most promising ASO candidates were validated in BK virus-infected human kidney epithelial cells, showing sequence-specific activity that was exacerbated by chemical modifications. Administration of the candidate oligonucleotide in mice revealed long-lasting uptake in proximal tubules of the kidney, where BK virus resides. A final optimization round yielded an oligonucleotide displaying superior activity. Studies in transformed cells revealed a discernible shift in T antigen splicing not observed with a scrambled control, indicating that targeting of the donor splice site was responsible for the observed effects. Here, we demonstrate proof-of-principle for a direct-acting, splice-modulating, universal ASO that distributes to sites where BK viral replication occurs, warranting further development of this novel therapeutic to combat BK virus replication in kidney transplant patients.

Amido-bridged nucleic acid (AmNA) and a 2'-O,4'-C-spirocyclopropylene bridged nucleic acid (scpBNA) are bridged nucleic acid analogs with high binding affinity toward complementary strands along with high nuclease resistance. AmNA and scpBNA have been developed to overcome phosphorothioate modified gapmer hepatotoxicity, while the mechanism of reducing hepatotoxicity still remains unknown. Here, we found that antisense oligonucleotides (ASOs) with combination of AmNA, scpBNA, and phosphodiester (PO) bonds could significantly reduce hepatotoxicity in mice. Histopathological findings of the periportal spaces of the liver were observed only in the locked nucleic acid and AmNA-scpBNA groups, but not in the AmNA-scpBNA-PO group. Furthermore, bioinformatics and histopathological analysis revealed that the reduced hepatotoxicity might be related to mitochondrial abnormalities, such as decreased expression levels of Atp5o and Sdhb genes. Taken together, the results of this study demonstrated that AmNA, scpBNA, and PO modification are able to reduce hepatotoxicity for improving the potential of ASOs.

A 2023 workshop brought together stakeholders involved in the development and safety assessment of oligonucleotide (ONT) therapeutics. The purpose was to discuss potential strategies and opportunities for enhancing developmental and reproductive toxicity (DART) assessment of ONTs. The workshop was timely, bringing together regulators, industry representatives, consultants, and contract research organization partners interested in the ongoing development of internationally harmonized guidance for nonclinical safety assessment of ONTs. Given DART's importance in nonclinical safety assessment and the unique attributes of ONTs, the forum discussed case studies, consensus approaches, and areas needing further development to optimize DART strategies. This report covers the workshop proceedings, highlighting methods to achieve a robust DART assessment for ONTs. It includes case studies that described strategies for dose level selection, dosing frequency, species selection, and alternative animal model approaches. Topics also cover surrogate ONT use, exposure of the placenta and embryo/fetal compartment, and weight of evidence approaches. A goal of these workshop proceedings is to describe example approaches to hopefully inform the DART strategy expectations in the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidance currently under development for nonclinical safety assessment of ONTs.

Nonclinical safety screening of small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand is typically carried out in rats at exaggerated exposures in a repeat-dose regimen. We have previously shown that at these suprapharmacological doses, hepatotoxicity observed with a subset of GalNAc-siRNAs is largely driven by undesired RNA-induced silencing complex (RISC)-mediated antisense strand seed-based off-target activity, similar to microRNA-like regulation. However, the RISC component requirements for off-target activity of siRNAs have not been evaluated. Here, we evaluate the roles of major RISC components, AGO and TNRC6 (or GW182) proteins, in driving on- and off-target activity of GalNAc-siRNAs in hepatocytes, in vitro and in vivo. We demonstrate that knocking down AGO2, but not AGO1 or AGO4, is protective against GalNAc-siRNA-driven off-target activity and hepatotoxicity. As expected, knocking down AGO2, but not AGO1 or AGO4, reduces the on-target activity of GalNAc-siRNA. Similarly, knocking down TNRC6 paralogs, TNRC6A or TNRC6B, but not TNRC6C, is protective against off-target activity and hepatotoxicity while having minimal impact on the on-target activity of GalNAc-siRNA. These data indicate that while AGO2 is the only RISC component required for the on-target activity of GalNAc-siRNAs, the undesired off-target activity and hepatotoxicity of a subset of GalNAc-siRNAs are mediated via the RISC composed predominantly of AGO2 and TNRC6 paralogs TNRC6A and/or TNRC6B.

Chemically modified short interfering RNAs (siRNAs) unequivocally represent a groundbreaking class of drugs. The deliberate chemical modification of the natural structure has been pivotal to their resounding success. Specific modifications at certain positions bolster their potency, safety, stability, and specificity. In clinical research, 2'-O-methyl and 2'-fluoro are the most used modifications. The effects of a wide range of chemical changes in fully modified siRNAs have not been thoroughly evaluated for tolerability. In this study, we utilized two sequences in a fully modified siRNA to systematically assess the tolerability of single nucleotide backbone and sugar modifications, including deoxyribonucleic acid, 2'-O-(2-methoxyethyl), locked nucleic acid, unlocked nucleic acid, mismatches, butane diol substitution, and butane diol insertion. We synthesized 522 siRNA variants and evaluated their efficacy in vitro. Our findings demonstrate that individual tolerability is significantly influenced by the modification's sequence, pattern, and position, with limited universal principles identifiable from this dataset. The efficacy results are probably driven by the thermodynamic balance defined by a combination of parameters. The framework presented here will serve as a reference dataset to facilitate the expansion of chemical diversity in therapeutic siRNAs.

The Oligonucleotide Nonclinical Working Group (WG) of the European Federation of Pharmaceutical Industries and Associations conducted an industry survey to understand current practices and regulatory expectations for genotoxicity and carcinogenicity assessment of oligonucleotide therapeutics (ONTs), along with historical genotoxicity testing results. The survey, involving 29 pharmaceutical and biotechnology companies, revealed a consistent absence of genotoxicity across a diverse range of oligonucleotide classes and chemistries, consistent with previous observations. Despite the lack of genotoxicity, companies continue to follow standard testing guidelines, with only limited divergence. The survey data support the view that well-established ONT modifications can be considered "precedented," in terms of negligible genotoxic risk. As such, further testing of new ONT candidates containing only precedented modifications is unwarranted, when defined criteria are met. Further, we propose a pathway for novel ONT chemical modifications to achieve precedented status. The survey results also indicate that alternative strategies for carcinogenicity assessment (e.g., single-species testing) can be accepted by regulatory agencies under certain circumstances. Overall, the survey findings underscore the need for a more tailored approach to the nonclinical safety assessment of ONTs, and the WG proposes development of supplementary questions for International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S2(R1) guidance to reflect this broad industry experience.

The recent Nobel Prizes awarded to Ambros and Ruvkun have refocused attention on microRNAs (miRNAs). The importance of miRNAs for basic science has always been clear, but the application to therapy has lagged behind. This delay has been made even more apparent by the accelerating pace of successful programs using duplex RNAs and antisense oligonucleotides to target mRNA. Why has progress been slow? A clear understanding of how miRNAs function in mammalian cells is obscured by the fact that miRNAs can exert their effects through multiple complex mechanisms. This gap in our knowledge has complicated progress in drug discovery. Better insights into the mechanism of miRNAs, more rigorous definitions of miRNAs, and more powerful tools for establishing the physical contacts necessary for miRNA action are now available. These advances lead to a central question for nucleic acid therapy-can miRNAs be productive targets for drug discovery and development?