A Japanese research team has demonstrated that the use of antisense oligonucleotides (ASOs) with modified bases and sugars can enhance the effectiveness of targeting diseases in the central nervous system (CNS) for clinical purposes. The alteration, known as a 2′,4′-BNA/LNA with a 9-(aminoethoxy)phenoxazine (BNAP-AEO), enhances the ability of ASOs to strongly bind to complementary RNAs, effectively suppress genes, and minimize acute CNS toxicity. The study was conducted at the Tokyo Medical and Dental University (TMDU) and Osaka University, and the peer-reviewed article was published in Molecular Therapy—Nucleic Acids.

Optimizing ASOs for neurological diseases

In recent years, the FDA has approved a growing number of ASO drugs for clinical use. Nevertheless, the advancement of ASO drug development has been impeded by the negative consequences of ASOs, such as off-target effects, immunostimulatory effects, toxicities in high-exposure organs, and thrombocytopenia.

An effective strategy to counteract these adverse impacts has involved the utilization of “gapmer” ASOs, which consist of a central DNA segment surrounded by modified nucleotides. By utilizing sequence complementarity, gapmers engage in hybridization with transcripts, leading to the recruitment of ribonuclease H and the subsequent induction of target RNA degradation. 

Yet even gapmers aren’t immune to problems. In a Phase III trial for amyotrophic lateral sclerosis (ALS), the intrathecal administration of the ASO tofersen demonstrated a tendency to reduce the decline in clinical function. However, it also resulted in adverse events in the central nervous system, such as meningitis. Although the FDA has granted accelerated approval for tofersen, there is a need for the development of gapmer ASOs that have high silencing efficacy and low CNS toxicity for treating different CNS diseases.

Base- and sugar-modification reduce ASO toxicity in the CNS 

A novel chemical modification called 2′,4′-BNA/LNA with a 9-(aminoethoxy)phenoxazine (BNAP-AEO) was recently created by researchers from the labs of Satoshi Obika and Takanori Yokota. This modification involves both base- and sugar-modification. The 9-(aminoethoxy)phenoxazine, known as G-clamp, is a modified cytosine analog that forms additional hydrogen bonds with guanine. This modification enhances its capacity to form a double-stranded structure.

Prior studies have indicated that gapmer ASOs containing a G-clamp have demonstrated significant gene silencing in laboratory settings through transfection. In contrast, oligonucleotides containing BNAP-AEO have shown enhanced resistance to degradation by complementary RNAs compared to oligonucleotides with only a G-clamp, only 2′,4′-BNA/LNA modifications, or unmodified oligonucleotides lacking any chemical modifications in the sugar moiety, base, and nucleotide linkage. In addition, oligonucleotides containing BNAP-AEO showed enhanced resistance to exonuclease degradation.

Co-lead authors Taiki Matsubayashi, Kotaro Yoshioka, and Su Su Lei Mon conducted a study to assess the effectiveness of BNAP-AEOs as potential treatments for CNS diseases. The evaluation was done in a live organism. The scientists created different gapmer ASOs, such as BNAP-AEO or its derivative, that specifically targeted the metastasis associated with lung adenocarcinoma transcript-1 (Malat1) RNA. They then assessed the strength of their binding to complementary RNAs, their ability to silence genes in both laboratory and living organisms, and their potential for causing acute toxicity in the central nervous system.

The study uncovered three significant observations about the use of ASOs with BNAP-AEO. ASOs with BNAP-AEO modification demonstrated a higher affinity for complementary RNAs than ASOs without BNAP-AEO modification. Second, ASOs with BNAP-AEO modifications showed effective gene silencing effects on the target gene in both in vitro and in vivo studies. Finally, when the ASOs with BNAP-AEO modification were injected into mice via the ICV route, their acute CNS toxicity, as measured by open-field tests and scoring systems, was significantly lower than that of the ASOs without BNAP-AEO.

This study highlights the efficient gene-silencing effect and low acute CNS toxicity of ASOs containing BNAP-AEO, indicating the possibility of future therapeutic applications.

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