Abstract
Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of neuromuscular disease such as Duchenne muscular dystrophy has been an advance for the ...splice-switching field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. To overcome limitations of existing technologies, we engineered chimeric stereopure oligonucleotides with phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones. We demonstrate that these chimeric stereopure oligonucleotides have markedly improved pharmacology and efficacy compared with PS-modified oligonucleotides, preventing premature death and improving median survival from 49 days to at least 280 days in a dystrophic mouse model with an aggressive phenotype. These data demonstrate that chemical optimization alone can profoundly impact oligonucleotide pharmacology and highlight the potential for continued innovation around the oligonucleotide backbone. More specifically, we conclude that chimeric stereopure oligonucleotides are a promising splice-switching modality with potential for the treatment of neuromuscular and other genetic diseases impacting difficult to reach tissues such as the skeletal muscle and heart.
After nearly 40 years of development, oligonucleotide therapeutics are nearing meaningful clinical productivity. One of the key advantages of oligonucleotide drugs is that their delivery and potency ...are derived primarily from the chemical structure of the oligonucleotide whereas their target is defined by the base sequence. Thus, as oligonucleotides with a particular chemical design show appropriate distribution and safety profiles for clinical gene silencing in a particular tissue, this will open the door to the rapid development of additional drugs targeting other disease-associated genes in the same tissue. To achieve clinical productivity, the chemical architecture of the oligonucleotide needs to be optimized with a combination of sugar, backbone, nucleobase, and 3'- and 5'-terminal modifications. A portfolio of chemistries can be used to confer drug-like properties onto the oligonucleotide as a whole, with minor chemical changes often translating into major improvements in clinical efficacy. One outstanding challenge in oligonucleotide chemical development is the optimization of chemical architectures to ensure long-term safety. There are multiple designs that enable effective targeting of the liver, but a second challenge is to develop architectures that enable robust clinical efficacy in additional tissues.
MicroRNAs are being considered as a novel type of bio-markers and potential therapeutic targets for various diseases. Diverse chemical tools are being developed for the detection or regulation of ...microRNAs with bio-medical implications. Chemical probes have been developed for use in combination with in situ signal amplification strategies to realize sensitive detection of microRNAs of low abundance. Regulation of microRNAs aberrantly expressed in tumours represents a new approach to cancer chemotherapy. Synthetic oligonucleotides including antisense oligonucleotides and microRNA mimics have been successfully delivered into cells or tissues to inhibit or enhance the function of specific endogenous microRNAs. Small-molecule modifiers of microRNAs that modify the expression or function of endogenous microRNAs are emerging not only as useful probes to explore microRNA-involved regulatory networks, but also as potential therapeutic reagents. In this tutorial review, we discuss the strategies developed by chemists in recent years for microRNA detection and regulation, with a focus on the potential of these chemical tools in microRNA-related biomedical applications.
Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or ...incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2′Omethyl phosphorothioate (2′OMe), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knockout mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2′OMe as shown by competitive inhibition and colocalization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2′OMe. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.
Abstract
RNA plays a central role in the expression of all genes. Because any sequence within RNA can be recognized by complementary base pairing, synthetic oligonucleotides and oligonucleotide ...mimics offer a general strategy for controlling processes that affect disease. The two primary antisense approaches for regulating expression through recognition of cellular RNAs are single-stranded antisense oligonucleotides and duplex RNAs. This review will discuss the chemical modifications and molecular mechanisms that make synthetic nucleic acid drugs possible. Lessons learned from recent clinical trials will be summarized. Ongoing clinical trials are likely to decisively test the adequacy of our current generation of antisense nucleic acid technologies and highlight areas where more basic research is needed.
Splice-switching antisense oligonucleotides (ASOs) could be used to treat a subset of individuals with genetic diseases
, but the systematic identification of such individuals remains a challenge. ...Here we performed whole-genome sequencing analyses to characterize genetic variation in 235 individuals (from 209 families) with ataxia-telangiectasia, a severely debilitating and life-threatening recessive genetic disorder
, yielding a complete molecular diagnosis in almost all individuals. We developed a predictive taxonomy to assess the amenability of each individual to splice-switching ASO intervention; 9% and 6% of the individuals had variants that were 'probably' or 'possibly' amenable to ASO splice modulation, respectively. Most amenable variants were in deep intronic regions that are inaccessible to exon-targeted sequencing. We developed ASOs that successfully rescued mis-splicing and ATM cellular signalling in patient fibroblasts for two recurrent variants. In a pilot clinical study, one of these ASOs was used to treat a child who had been diagnosed with ataxia-telangiectasia soon after birth, and showed good tolerability without serious adverse events for three years. Our study provides a framework for the prospective identification of individuals with genetic diseases who might benefit from a therapeutic approach involving splice-switching ASOs.
Antisense oligonucleotides (ASOs) usually contain a fully phosphorothioate (PS) backbone, which possibly interact with many genes and proteins under intracellular conditions. G3139 is an ASO that ...targets Bcl-2 mRNA and induces cell apoptosis. Here, we report a kind of cytidinyl-lipid combined with a cationic lipid (DNCA/CLD, molar ration, 28:3, named mix), which may interact with oligonucleotides via H-bond formation, pi-stacking and electrostatic interaction, accompanied by low zeta potentials. The IC50 value of G3139 delivered by mix-lipid reduced from above 20 μM to 0.158 μM for MCF-7/ADR, and exhibited stronger antiproliferation upon other cancer cell lines. In addition, PS modification in the 3′-half of G3139 (especially at positions 13-16) enhanced serum stability, target specificity and anticancer activity. Also, a locked nucleic acid (LNA) gapmer G3139 (LNA-G3139) showed superior antiproliferation (78.5%) and Bcl-2 mRNA suppression effects (85.5%) at 200 nM, mainly due to its high complementary RNA affinity. More apoptosis-associated targets were identified, and a lower level of non-specific protein binding (HSA) revealed that both antisense and aptamer mechanisms might simultaneously exist. A combination of a new delivery system and chemical modifications, such as in LNA-G3139, may have potential clinical application prospects in the future.
Outlined description: Encapsulation and delivery by cytidinyl-lipid/cationic lipid (DNCA/CLD), ASOs phosphrothiolated in 3′-half region (such as site 13-16) significantly enhance target specificity and serum stability, while its LNA-gapmer (LNA-G3139) shows great mRNA affinity, inhibition of multiple targets expression (Bcl-2, ATAD3A and AARS), and antitumour ability in vitro and in vivo. Display omitted
Translationally controlled tumor protein (TCTP) has been implicated in a plethora of important cellular processes related to cell growth, cell cycle progression, malignant transformation and ...inhibition of apoptosis. Therefore, TCTP is now recognized as a potential therapeutic target in several cancers including prostate, breast and lung cancers. We previously showed that TCTP is overexpressed in castration-resistant prostate cancer (CRPC), and it has been implicated resistance to treatment. Recently, we developed TCTP antisense oligonucleotides (ASOs) to inhibit TCTP expression. However, the intracellular delivery and silencing activity of these oligonucleotides remains a challenge, and depend on the use of transfection agents and delivery systems. Here we show that lipid-modified ASO (LASOs) has improved penetration and efficiency in inhibiting TCTP expression in the absence of additional transfection agents, both in vitro and in vivo. Transfection with TCTP-LASO led to rapid and prolonged internalization via macropinocytosis, TCTP downregulation and significant decreased cell viability. We also show that lipid-modification led to delayed tumor progression in CRPC xenografts models, with no significant toxic effects observed.
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Single-stranded oligonucleotides have been explored as a therapeutic modality for more than 20 years. Only during the last 5 years have single-stranded oligonucleotides become a modality of choice in ...the fields of precision medicine and targeted therapeutics. Recently, there have been a number of development efforts involving this modality that have led to treatments for genetic diseases that were once untreatable. This review highlights key applications of single-stranded oligonucleotides that function in a sequence-dependent manner when applied to modulate precursor (pre-)mRNA splicing, gene expression, and immune pathways. These applications have been used to address diseases that range from neurological to muscular to metabolic, as well as to develop vaccines. The wide range of applications denotes the versatility of single-stranded oligonucleotides as a robust therapeutic platform. The focus of this review is centered on approved single-stranded oligonucleotide therapies and the evolution of oligonucleotide therapeutics into novel applications currently in clinical development.
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Single-stranded oligonucleotide therapeutics represent a platform of targeted and selective medicines that address severe genetic diseases that were once untreatable. Continued advances in chemistry, targeted delivery, and new applications have paved the way for a future in which ASO therapeutics become a common modality to treat genetic diseases and beyond.
Abstract
Antisense oligonucleotides (ASOs) modulate cellular target gene expression through direct binding to complementary RNA. Advances in ASO chemistry have led to the development of ...phosphorothioate (PS) ASOs with constrained-ethyl modifications (cEt). These next-generation cEt-ASOs can enter cells without transfection reagents. Factors involved in intracellular uptake and trafficking of cEt-ASOs leading to successful target knockdown are highly complex and not yet fully understood. AZD4785 is a potent and selective therapeutic KRAS cEt-ASO currently under clinical development for the treatment of cancer. Therefore, we used this to investigate mechanisms of cEt-ASO trafficking across a panel of cancer cells. We found that the extent of ASO-mediated KRAS mRNA knockdown varied significantly between cells and that this did not correlate with bulk levels of intracellular accumulation. We showed that in cells with good productive uptake, distribution of ASO was perinuclear and in those with poor productive uptake distribution was peripheral. Furthermore, ASO rapidly trafficked to the late endosome/lysosome in poor productive uptake cells compared to those with more robust knockdown. An siRNA screen identified several factors mechanistically involved in productive ASO uptake, including the endosomal GTPase Rab5C. This work provides novel insights into the trafficking of cEt-ASOs and mechanisms that may determine their cellular fate.
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