Steven F. DowdyRyan L. SettenXian-Shu CuiSatish G. Jadhav...
8页
查看更多>>摘要：RNA therapeutics, including siRNAs, antisense oligonucleotides, and other oligonucleotides, have great potential to selectively treat a multitude of human diseases, from cancer to COVID to Parkinson's disease. RNA therapeutic activity is mechanistically driven by Watson–Crick base pairing to the target gene RNA without the requirement of prior knowledge of the protein structure, function, or cellular location. However, before widespread use of RNA therapeutics becomes a reality, we must overcome a billion years of evolutionary defenses designed to keep invading RNAs from entering cells. Unlike small-molecule therapeutics that are designed to passively diffuse across the cell membrane, macromolecular RNA therapeutics are too large, too charged, and/or too hydrophilic to passively diffuse across the cellular membrane and are instead taken up into cells by endocytosis. However, similar to the cell membrane, endosomes comprise a lipid bilayer that entraps 99% or more of RNA therapeutics, even in semipermissive tissues such as the liver, central nervous system, and muscle. Consequently, before RNA therapeutics can achieve their ultimate clinical potential to treat widespread human disease, the rate-limiting delivery problem of endosomal escape must be solved in a clinically acceptable manner.
Lise Lolle HolmThomas K. DoktorUlrika S.S. PetersenMaría Bueno...
13页
查看更多>>摘要：e report two new 6-pyruvoyl-tetrahydropterin synthase splicing variants identified through genomic se-quencing and transcript analysis in a patient with tetrahydrobiopterin deficiency, presenting with hyperphe-nylalaninemia and monoamine neurotransmitter deficiency. Variant c.243+3A>G causes exon 4 skipping. Thedeep-intronic c.164–672C>T variant creates a potential 5￠splice site that leads to the inclusion of four over-lapping pseudoexons, corresponding to exonizations of an antisense short interspersed nuclear elementAluSqrepeat 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 predominantpseudoexon is nearly identical to a disease causing activated pseudoexon in theF8gene, with the same 3￠and 5￠splice sites. Splice switching antisense oligonucleotides (SSOs) were designed to hybridize with splice sitesand/or predicted binding sites for regulatory splice factors. Different SSOs corrected the aberrant pseudoexoninclusion, both in minigenes and in fibroblasts from patients carrying the new variant c.164–672C>T or thepreviously described c.164–716A>T. With SSO treatment PTPS protein was recovered, illustrating the thera-peutic potential of the approach, for patients with different pseudoexon activating variants in the region. Inaddition, the natural presence of pseudoexons in the wild type context suggests the possibility of applying theantisense strategy in patients with hypomorphicPTSvariants with the purpose of upregulating their expressionto increase overall protein and activity.
Thushara W. MadanayakeEric A. WelshLancia N.F. DarvilleJohn M. Koomen...
10页
查看更多>>摘要：We report a novel method to inhibit epidermal growth factor receptor (EGFR) signaling using custom morpholino antisense oligonucleotides (ASOs) to drive expression of dominant negative mRNA isoforms of EGFR by ASO-induced exon skipping within the transmembrane (16) or tyrosine kinase domains (18 and 21). In vivo ASO formulations induced >95% exon skipping in several models of nonsmall cell lung cancer (NSCLC) and were comparable in efficacy to erlotinib in reducing colony formation, cell viability, and migration in EGFR mutant NSCLC (PC9). However, unlike erlotinib, ASOs maintained their efficacy in both erlotinib-resistant subclones (PC9-GR) and wild-type overexpressing EGFR models (H292), in which erlotinib had no significant effect. The most dramatic ASO-induced phenotype resulted from targeting the EGFR kinase domain directly, which resulted in maximal inhibition of phosphorylation of EGFR, Akt, and Erk in both PC9 and PC9GR cells. Phosphoproteomic mass spectrometry confirmed highly congruent impacts of exon 16-, 18-, and 21-directed ASOs compared with erlotinib on PC9 genome-wide cell signaling. Furthermore, EGFR-directed ASOs had no impact in EGFR-independent NSCLC models, confirming an EGFR-specific therapeutic mechanism. Further exploration of synergy of ASOs with existing tyrosine kinase inhibitors may offer novel clinical models to improve EGFR-targeted therapies for both mutant and wild-type NSCLC patients.
Lingdi ZhangXue-hai LiangCheryl Li De HoyosMichael Migawa...
11页
查看更多>>摘要：Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2′-O-methyl (2′-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2′-methoxyethyl, 2′ constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2′-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5′ end of the gap or in the 3′-wing of a Gap 2′-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2′-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.
Valeriia S. DrozdAhmed A. EldeebDmitry M. KolpashchikovDaria D. Nedorezova...
9页
查看更多>>摘要：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.
查看更多>>摘要：In this study, the efficiency of RNA interference of small interfering RNAs (siRNAs) bearing 5′-O-methyl-2′-deoxythymidine (X) and 5′-amino-2′, 5′-dideoxythymidine (Z) at the 5′-end of the sense strand and the antisense strand of siRNA was investigated in HeLa cells stably expressing enhanced green fluorescent protein. The results indicated that when one strand of siRNA was modified with X or Z and the other was unmodified, the X or Z modification was predominant in the process of strand selection and the unmodified strand was selected as a guide strand. When both strands are modified with X or Z, the modified antisense strand with X or Z will be selected as a guide strand with a certain probability. The resulting mature RNA-induced silencing complex exerted reduced, but still moderate silencing activity remained. These results suggest that the modification of the sense strand with X or Z eliminates the off-target effects caused by the sense strand without affecting the silencing efficiency of the siRNA.
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Mary Ann Liebert, Inc