Nucleic acid therapeutics最新文献

Phosphorothioate (PS)-modified antisense oligonucleotide (ASO) drugs enter cells through endocytic pathways where a majority are entrapped within membrane-bound endosomes and lysosomes, representing a limiting step for antisense activity. While late endosomes have been identified as a major site for productive PS-ASO release, how lysosomes regulate PS-ASO activity beyond macromolecule degradation remains not fully understood. In this study, we reported that SID1 transmembrane family, member 2 (SIDT2), a lysosome transmembrane protein, can robustly regulate PS-ASO activity. We showed that SIDT2 is required for the proper colocalization between PS-ASO and lysosomes, suggesting an important role of SIDT2 in the entrapment of PS-ASOs in lysosomes. Mechanistically, we revealed that SIDT2 regulates lysosome cellular location. Lysosome location is largely determined by its movement along microtubules. Interestingly, we also observed an enrichment of proteins involved in microtubule function among SIDT2-binding proteins, suggesting that SIDT2 regulates lysosome location via its interaction with microtubule-related proteins. Overall, our data suggest that lysosome protein SIDT2 inhibits PS-ASO activity potentially through its interaction with microtubule-related proteins to place lysosomes at perinuclear regions, thus, facilitating PS-ASO's localization to lysosomes for degradation.

While rare, some gapmer phosphorothioate (PS) antisense oligonucleotides (ASOs) can induce a noncanonical TLR9-dependent innate immune response. In this study, we performed systematic analyses of the roles of PS ASO backbone chemistry, 2' modifications, and sequence in PS ASO induced TLR9 signaling. We found that each of these factors can contribute to altering PS ASO induced TLR9 signaling, and in some cases the effects are quite dramatic. We also found that the positioning (5' vs. 3') of a particular backbone or 2' modification within a PS ASO can affect its TLR9 signaling. Interestingly, medicinal chemical strategies that decrease TLR9 signaling for one sequence can have opposing effects on another sequence. Our results demonstrate that TLR9 signaling is highly PS ASO sequence dependent, the mechanism of which remains unknown. Despite this, we determined that placement of two mesyl phosphoramidate linkages within the PS ASO gap is the most promising strategy to mitigate PS ASO dependent TLR9 activation to enhance the therapeutic index and, therefore, further streamline PS ASO drug development.

Circular RNAs (circRNAs) constitute an abundant class of covalently closed noncoding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys, as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. In this study, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We found that, while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes. In summary, our results demonstrate that LNA-modified ASOs complementary to backsplice-junction sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.

Downregulation of genes involved in the secondary pathology of Duchenne muscular dystrophy, for example, inflammation, fibrosis, and adiposis, is an interesting approach to ameliorate degeneration of muscle and replacement by fibrotic and adiposis tissue. Small interfering RNAs (siRNAs) are able to downregulate target genes, however, delivery of siRNAs to skeletal muscle still remains a challenge. We investigated delivery of fully chemically modified, cholesterol-conjugated siRNAs targeting Alk4, a nontherapeutic target that is expressed highly in muscle. We observed that a single intravenous or intraperitoneal (IP) injection of 10 mg/kg resulted in significant downregulation of Alk4 mRNA expression in skeletal muscles in both wild-type and mdx mice. Treatment with multiple IP injections of 10 mg/kg led to an overall reduction of Alk4 expression, reaching significance in tibialis anterior (39.7% ± 6.2%), diaphragm (32.7% ± 5.8%), and liver (41.3% ± 29.9%) in mdx mice. Doubling of the siRNA dose did not further increase mRNA silencing in muscles of mdx mice. The chemically modified conjugated siRNAs used in this study are very promising for delivery to both nondystrophic and dystrophic muscles and could have major implications for treatment of muscular dystrophy pathology.

Despite wide recognition as a disease of pandemic proportions, effective treatments for nonalcoholic fatty liver disease (NAFLD) remain elusive. Most of the current clinical programs aim to reduce hepatic fat accumulation and, thus, prevent downstream inflammation and fibrosis. To date, this therapeutic approach has helped identify a potential disconnect between steatosis reduction and disease resolution. Mounting preclinical evidence indicates liver inflammation may play a major role in steatosis development and fibrosis but has not garnered the same clinical representation. This may be owing to deficiencies in standard therapeutic modalities that limit their application in NAFLD. RNA interference (RNAi) is an attractive approach to targeting liver inflammation owing to its clinical safety profile, target specificity, and limited biodistribution. In this study, we characterize a simple cholesterol-short-interfering RNA (siRNA) conjugate system targeting Tnf mRNA in liver macrophages for the treatment of NAFLD. First, we observed delivery and anti-inflammatory activity in an acute liver inflammation model. In a follow-up murine NAFLD model, we observed total prevention of nearly all hallmarks of this disease: steatosis, inflammation, and fibrosis. This simple conjugate siRNA delivery system may be the first to show RNAi activity in liver macrophages and provide evidence for a novel therapeutic approach to inflammation in NAFLD.

The nucleic acid therapeutics field has made tremendous progress in the past decades. Continuous advances in chemistry and design have led to many successful clinical applications, eliciting even more interest from researchers including both academic groups and drug development companies. Many preclinical studies in the field focus on improving the delivery of antisense oligonucleotide drugs (ONDs) and/or assessing their efficacy in target tissues, often neglecting the evaluation of toxicity, at least in early phases of development. A series of consensus recommendations regarding regulatory considerations and expectations have been generated by the Oligonucleotide Safety Working Group and the Japanese Research Working Group for the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S6 and Related Issues (WGS6) in several white papers. However, safety aspects should also be kept in sight in earlier phases while screening and designing OND to avoid subsequent failure in the development phase. Experts and members of the network "DARTER," a COST Action funded by the Cooperation in Science and Technology of the EU, have utilized their collective experience working with OND, as well as their insights into OND-mediated toxicities, to generate a series of consensus recommendations to assess OND toxicity in early stages of preclinical research. In the past few years, several publications have described predictive assays, which can be used to assess OND-mediated toxicity in vitro or ex vivo to filter out potential toxic candidates before moving to in vivo phases of preclinical development, that is, animal toxicity studies. These assays also have the potential to provide translational insight since they allow a safety evaluation in human in vitro systems. Yet, small preliminary in vivo studies should also be considered to complement this early assessment. In this study, we summarize the state of the art and provide guidelines and recommendations on the different tests available for these early stage preclinical assessments.

SHANK3 is a member of the SHANK family of scaffolding proteins that localize to the postsynaptic density of excitatory synapses. Mutations within the SHANK3 gene or SHANK3 haploinsufficiency is thought to be one of the major causes for Phelan-McDermid Syndrome (PMDS) that is characterized by a broad spectrum of autism-related behavioral alterations. Several approaches have already been proposed to elevate SHANK3 protein levels in PMDS patients like transcriptional activation or inhibition of SHANK3 degradation. We undertook a systematic screening approach and tested whether defined antisense oligonucleotides (ASOs) directed against the 3' untranslated region (3'-UTR) of the human SHANK3 mRNA are suitable to elevate SHANK3 protein levels. Using human induced pluripotent stem cells (hiPSCs) and hiPSCs-derived motoneurons from controls and PMDS patients we eventually identified two 18 nucleotide ASOs (ASO 4-5.2-4 and 4-5.2-6) that were able to increase SHANK3 protein levels in vitro by about 1.3- to 1.6-fold. These findings were confirmed by co-transfection of the identified ASOs with a GFP-SHANK3-3'-UTR construct in HEK293T cells using GFP protein expression as read-out. Based on these results we propose a novel approach to elevate SHANK3 protein concentrations by 3'-UTR specific ASOs. Further research is needed to test the suitability of SHANK3-specific ASOs as pharmacological compounds also in vivo.

Antisense oligonucleotides (ASOs) can modulate pre-mRNA splicing. This offers therapeutic opportunities for numerous genetic diseases, often in a mutation-specific and sometimes even individual-specific manner. Developing therapeutic ASOs for as few as even a single patient has been shown feasible with the development of Milasen for an individual with Batten disease. Efforts to develop individualized ASOs for patients with different genetic diseases are ongoing globally. The N = 1 Collaborative (N1C) is an umbrella organization dedicated to supporting the nascent field of individualized medicine. N1C recently organized a workshop to discuss and advance standards for the rigorous design and testing of splice-switching ASOs. In this study, we present guidelines resulting from that meeting and the key recommendations: (1) dissemination of standardized experimental designs, (2) use of standardized reference ASOs, and (3) a commitment to data sharing and exchange.