Uptake Of Antisense Oligonucleotides Research Articles

A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides.

Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular uptake of ASOs remains critically low, warranting improvement. Transient receptor potential canonical (TRPC) channels regulate Ca2+ influx and are activated upon stimulation by phospholipase C-generated diacylglycerol. Herein, we report that a novel TRPC3/C6/C7 activator, L687, can induce cellular ASO uptake. L687-induced ASO uptake was enhanced in a dose- and incubation-time-dependent manner. L687 enhanced the knockdown activity of various ASOs both in vitro and in vivo. Notably, suppression of TRPC3/C6 by specific siRNAs reduced ASO uptake in A549 cells. Application of BAPTA-AM, a Ca2+ chelator, and SKF96365, a TRPC3/C6 inhibitor, suppressed Ca2+ influx via TRPC3/C6, resulting in reduced ASO uptake, thereby suggesting that Ca2+ influx via TRPC3/C6 is critical for L687-mediated increased ASO uptake. L687 also induced dextran uptake, indicating that L687 increased endocytosis. Adding ASO to L687 resulted in endosome accumulation; however, the endosomal membrane disruptor UNC7938 facilitated endosomal escape and enhanced knockdown activity. We discovered a new function for TRPC activators regarding ASO trafficking in target cells. Our findings provide an opportunity to formulate an innovative drug delivery system for the therapeutic development of ASO.

Lipid nanoparticle delivery limits antisense oligonucleotide activity and cellular distribution in the brain after intracerebroventricular injection

Antisense oligonucleotide (ASO) therapeutics are being investigated for a broad range of neurological diseases. While ASOs have been effective in the clinic, improving productive ASO internalization into target cells remains a key area of focus in the field. Here, we investigated how the delivery of ASO-loaded lipid nanoparticles (LNPs) affects ASO activity, subcellular trafficking, and distribution in the brain. We show that ASO-LNPs increase ASO activity up to 100-fold in cultured primary brain cells as compared to non-encapsulated ASO. However, in contrast to the widespread ASO uptake and activity observed following free ASO delivery invivo, LNP-delivered ASOs did not downregulate mRNA levels throughout the brain after intracerebroventricular injection. This lack of activity was likely due to ASO accumulation in cells lining the ventricles and blood vessels. Furthermore, we reveal a formulation-dependent activation of the immune system post dosing, suggesting that LNP encapsulation cannot mask cellular ASO backbone-mediated toxicities. Together, these data provide insights into how LNP encapsulation affects ASO distribution as well as activity in the brain, and a foundation that enables future optimization of brain-targeting ASO-LNPs.

Abstract B019: ASO-mediated NRAS knockdown overcomes gilteritinib late resistance in FLT3-AML

Abstract The internal tandem duplication of the FLT3 receptor (FLT3-ITD) leads to constitutive receptor activation and is one of the most common mutations (~30%) in acute myeloid leukemia (AML), resulting in poor prognosis. Multiple FLT3 inhibitors (FLT3i) have been developed, including the potent FDA-approved FLT3i, gilteritinib. However, AML patients only respond to gilteritinib for approximately 6 months due to the emergence of drug resistance. Through an approach that integrated genomic, metabolomic, proteomic, and phosphoproteomic analyses with complementary genetic and pharmacologic screens, we demonstrated that gilteritinib resistance occurs in a stepwise manner with two distinct phases of early and late resistance (Joshi et al., Cancer Cell, 2021). While gilteritinib efficiently eliminates leukemia cells in the blood, residual cells survive within the bone marrow, where they are protected by extrinsic microenvironmental factors fostering early resistance. These lingering cells eventually develop intrinsic mutations in NRAS that allow them to resist gilteritinib treatment, leading to late resistance. Acquisition of mutations in the RAS pathway, particularly NRAS, are a common mode of late resistance (~11%) for many patients that relapse on FLT3i. Developing therapeutics to block NRAS activity remains a challenge with the lack of direct small-molecule inhibitors. Indirect approaches to inhibit mutant NRAS/MAPK pathway signaling have also not met much clinical success. Hence, therapeutic nucleic acid-based approaches, including antisense oligonucleotides (ASOs), may offer an alternative approach to target NRAS-driven cancers. We evaluated the effectiveness of a novel ASO targeting NRAS mRNA to restore gilteritinib sensitivity in resistant cultures in vitro and in primary AML patient samples ex vivo. We first assessed the potency of the NRAS ASO in both gilteritinib-sensitive and late resistant MOLM-14 AML cell lines (N=9) that expressed wild-type or mutant NRAS (G12S or G12D) via free uptake of ASO. NRAS expression was evaluated by qPCR after 48 hours. ASO treatment down-regulated mutant NRAS mRNA expression in a dose-dependent manner with an IC50 of < 1 µM and decreased NRAS protein levels. No changes in NRAS mRNA or protein levels were observed with non-targeting ASO control. Decreased NRAS mRNA expression restored sensitivity to gilteritinib in late-resistant cultures, leading to decreased cell viability and increased apoptosis. A similar trend was observed in primary AML cells from patients (N=2) that relapsed on gilteritinib, where reduction of approximately 50% of mutant NRAS mRNA robustly sensitized patient cells to gilteritinib. In contrast, the combination of gilteritinib and the MEK inhibitor trametinib was not as effective in either cell cultures or patient samples. Our results suggest that ASO-mediated knockdown of NRAS may circumvent the development of resistance and improve the durability of FLT3i. Citation Format: Sunil K. Joshi, Janét Pittsenbarger, Xiaolin Luo, Hadi Maazi, Alexey Revenko, Brian J. Druker, Elie Traer. ASO-mediated NRAS knockdown overcomes gilteritinib late resistance in FLT3-AML [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B019.

GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells

Antisense oligonucleotides (ASOs) are short synthetic nucleic acids that recognize and bind to complementary RNA to modulate gene expression. It is well established that single-stranded, phosphorothioate-modified ASOs enter cells independent of carrier molecules, primarily via endocytic pathways, but that only a small portion of internalized ASO is released into the cytosol and/or nucleus, rendering the majority of ASO inaccessible to the targeted RNA. Identifying pathways that can increase the available ASO pool is valuable as a research tool and therapeutically. Here, we conducted a functional genomic screen for ASO activity by engineering GFP splice reporter cellsand applying genome-wide CRISPR gene activation. Thescreen can identify factors that enhance ASO splice modulationactivity. Characterization of hit genes uncovered GOLGA8, a largely uncharacterized protein, as a novel positive regulator enhancing ASO activity by ∼2-fold. Bulk ASO uptake is 2- to5-fold higher in GOLGA8-overexpressing cells where GOLGA8 and ASOs are observed in the same intracellular compartments.We find GOLGA8 is highly localized to the trans-Golgi and readily detectable at the plasma membrane. Interestingly, overexpression of GOLGA8 increased activity for both splice modulation and RNase H1-dependent ASOs. Taken together, these results support a novel role for GOLGA8 in productive ASO uptake.

Synthesis of multivalent fatty acid-conjugated antisense oligonucleotides: Cell internalization, physical properties, and in vitro and in vivo activities

Herein, we describe the design and synthesis of multi-conjugatable fatty acid monomer phosphoramidites and their conjugation to antisense oligonucleotides (ASOs). Multivalent long-chain fatty acid conjugation improved the cellular uptake of ASOs but decreased in vitro activity due to alterations in physical properties and cellular localization. In addition, multivalently fatty acid-conjugated ASOs showed different organ specificity compared with that of unconjugated ASO in in vivo experiment. Although optimization of the linker structure between the fatty acid moiety and the ASO may be required, divalent long-chain fatty acid conjugation provides a new approach to increase endocytosis, thereby potentially improving the activity of therapeutic ASOs.

Epidermal growth factor receptor mediates the basolateral uptake of phosphorothioate-modified antisense oligonucleotides in the kidney

Antisense oligonucleotides (ASOs) are highly biologically stable and specific therapeutic molecules that interfere with mRNA transcription and as a result effectively reduce the expression of a protein of interest. ASOs have low drug clearance and are retained in tissues. This is particularly evident in the kidneys where they accumulate in the renal proximal tubules. Receptor-mediated endocytosis (RME) plays a role in ASO uptake, however the membrane receptors facilitating the process and the full mechanism behind these transport and subsequent renal retention is still poorly understood. In the present study we employ a proximal tubule-on-a-chip (PTOC) that recapitulates the highly polarized nature of the renal epithelium and can discriminate between basal and apical uptake processes. The PTOC was used to determine the impact of cellular polarization in ASO uptake in the kidney and elucidate which receptors predominantly facilitated uptake. In the PTOC the uptake occurred predominantly from the basolateral side and was extensively inhibited using the selective epidermal growth factor receptor (EGFR) antagonist cetuximab. These results demonstrate that ASO uptake in a physiologically relevant model is predominately mediated by the EGFR and takes place from the basolateral side of the proximal tubules. In comparison to the 2D culture, PTOC could differentiate between distinct ASO uptake routes mediated by either Megalin or EGFR, which proper membrane expression is highly dependent on cellular polarity. Our results highlight the limitations of renal 2D models and demonstrate how an organ on a chip model can fill the physiological gap and is a powerful tool to study ASO, which have intricate transport mechanisms that ultimately impact both their efficacy and safety. • Continuously perfused proximal tubule-on-a-chip model recreates a highly polarized renal epithelium. • Uptake of antisense-oligonucleotides occurs from the basolateral side of the renal proximal tubules. • EGFR drives antisense-oligonucleotide uptake in our proximal tubule-on-a-chip model, an effect not observed in 2D culture.

Use of Glycine to Augment Exon Skipping and Cell Therapies for Duchenne Muscular Dystrophy.

Antisense oligonucleotide (AO)-based exon-skipping and cell therapies are the main therapeutic approaches for Duchenne muscular dystrophy (DMD). Insufficient systemic delivery leading to low therapeutic efficacy limits the former; low transplantation efficiency hampers the latter. Here we describe how glycine can address these issues by augmenting satellite proliferation and muscle regeneration, resulting in enhanced AO uptake in regenerating myofibers and cell transplantation efficiency in dystrophic mice. The dual functionality of glycine demonstrated in AO-based exon-skipping and cell therapies presents a simple and efficient method to augment AO potency and cell transplantation efficacy in DMD and other muscle diseases.

In vivo restoration of dystrophin expression in mdx mice using intra-muscular and intra-arterial injections of hydrogel microsphere carriers of exon skipping antisense oligonucleotides

Duchenne muscular dystrophy (DMD) is a genetic disease caused by a mutation in the X-linked Dytrophin gene preventing the expression of the functional protein. Exon skipping therapy using antisense oligonucleotides (AONs) is a promising therapeutic strategy for DMD. While benefits of AON therapy have been demonstrated, some challenges remain before this strategy can be applied more comprehensively to DMD patients. These include instability of AONs due to low nuclease resistance and poor tissue uptake. Delivery systems have been examined to improve the availability and stability of oligonucleotide drugs, including polymeric carriers. Previously, we showed the potential of a hydrogel-based polymeric carrier in the form of injectable PEG-fibrinogen (PF) microspheres for delivery of chemically modified 2′-O-methyl phosphorothioate (2OMePs) AONs. The PF microspheres proved to be cytocompatible and provided sustained release of the AONs for several weeks, causing increased cellular uptake in mdx dystrophic mouse cells. Here, we further investigated this delivery strategy by examining in vivo efficacy of this approach. The 2OMePS/PEI polyplexes loaded in PF microspheres were delivered by intramuscular (IM) or intra-femoral (IF) injections. We examined the carrier biodegradation profiles, AON uptake efficiency, dystrophin restoration, and muscle histopathology. Both administration routes enhanced dystrophin restoration and improved the histopathology of the mdx mice muscles. The IF administration of the microspheres improved the efficacy of the 2OMePS AONs over the IM administration. This was demonstrated by a higher exon skipping percentage and a smaller percentage of centered nucleus fibers (CNF) found in H&E-stained muscles. The restoration of dystrophin expression found for both IM and IF treatments revealed a reduced dystrophic phenotype of the treated muscles. The study concludes that injectable PF microspheres can be used as a carrier system to improve the overall therapeutic outcomes of exon skipping-based therapy for treating DMD.

Intracellular Absolute Quantification of Oligonucleotide Therapeutics by NanoSIMS.

Antisense oligonucleotide (ASO)-based therapeutics hold great potential for the treatment of a variety of diseases. Therefore, a better understanding of cellular delivery, uptake, and trafficking mechanisms of ASOs is highly important for early-stage drug discovery. In particular, understanding the biodistribution and quantifying the abundance of ASOs at the subcellular level are needed to fully characterize their activity. Here, we used a combination of electron microscopy and NanoSIMS to assess the subcellular concentrations of a 34S-labeled GalNAc-ASO and a naked ASO in the organelles of primary human hepatocytes. We first cross-validated the method by including a 127I-labeled ASO, finding that the absolute concentration of the lysosomal ASO using two independent labeling strategies gave matching results, demonstrating the strength of our approach. This work also describes the preparation of external standards for absolute quantification by NanoSIMS. For both the 34S and 127I approaches used for our quantification methodology, we established the limit of detection (5 and 2 μM, respectively) and the lower limit of quantification (14 and 5 μM, respectively).

Antisense oligonucleotide activity in tumour cells is influenced by intracellular LBPA distribution and extracellular vesicle recycling

Next generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad application. Here we compare intracellular traffic of anti KRAS antisense oligonucleotide (AZD4785) in tumour cell lines PC9 and LK2, with good and poor productive uptake, respectively. We find that the majority of AZD4785 is rapidly delivered to CD63+late endosomes (LE) in both cell lines. Importantly, lysobisphosphatidic acid (LBPA) that triggers ASO LE escape is presented in CD63+LE in PC9 but not in LK2 cells. Moreover, both cell lines recycle AZD4785 in extracellular vesicles (EVs); however, AZD4785 quantification by advanced mass spectrometry and proteomic analysis reveals that LK2 recycles more AZD4785 and RNA-binding proteins. Finally, stimulating LBPA intracellular production or blocking EV recycling enhances AZD4785 activity in LK2 but not in PC9 cells thus offering a possible strategy to enhance ASO potency in tumour cells with poor productive uptake of ASOs.

Evaluation of Phosphorus and Non-Phosphorus Neutral Oligonucleotide Backbones for Enhancing Therapeutic Index of Gapmer Antisense Oligonucleotides.

The phosphorothioate (PS) linkage in an essential component of therapeutic oligonucleotides. PS in the DNA region of gapmer antisense oligonucleotides (ASOs) supports RNaseH1 activity and enhances nuclease stability. PS also promotes binding to plasma, cell surface, and intracellular proteins, which facilitates tissue distribution, cellular uptake, and endosomal escape of PS ASOs. We recently showed that site-specific replacement of PS in the DNA gap with methoxylpropyl phosphonate (MOP) linkages can enhance the therapeutic index of gapmer ASOs. In this article, we explored 18 phosphorus- and non-phosphorus-based neutral backbone modifications to determine the structure-activity relationship of neutral linkages for enhancing therapeutic index. Replacing MOP with other alkyl phosphonate and phosphotriester linkages enhanced therapeutic index, but these linkages were susceptible to chemical degradation during oligonucleotide deprotection from solid supports following synthesis. Replacing MOP with non-phosphorus linkages resulted in improved chemical stability, but these linkages were introduced into ASOs as nucleotide dimers, which limits their versatility. Overall, linkages such as isopropyl and isobutyl phosphonates and O-isopropyl and O-tetrahydrofuranosyl phosphotriesters, formacetal, and C3-amide showed improved activity in mice relative to MOP. Our data suggest that site-specific incorporation of any neutral backbone linkage can improve therapeutic index, but the size, hydrophobicity, and RNA-binding affinity of the linkage influence ASO activity.

O3 Ex vivo normothermic machine perfusion facilitates gymnotic delivery of RNA interference therapeutics in donor kidneys

Abstract Introduction Normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for delivery of novel therapeutics to optimize organ quality. This includes RNA interference (RNAi) therapeutics e.g. antisense oligonucleotides (ASO) that block detrimental microRNAs. The intracellular kinetics of RNAi therapeutics are crucial for their pharmacological effect, however, it remains poorly understood. NMP provides an ideal platform to investigate this further. Method During NMP, human kidneys (n = 12) were treated for 6 hours with a fluorescently-labelled ASO designed to block microRNA-24-3p activity. Biopsies were taken at 0, 2, 4, and 6 hours. Kidney sections were stained with antibodies against early endosomes (Rab5), late endosomes (Rab7), RNA-induced silencing complexes (GW182) and lysosomes (LAMP2). Confocal microscopy images were obtained and co-localisation quantified using Hugyens™ software following batch deconvolution. The global transcriptomic impact of ASO therapy was also assessed using RNA sequencing. Result Following 2 hours of NMP, ASO was primarily found in tubular epithelial cells. Co-localisation studies revealed ASO uptake via endocytosis and endosomal sorting occurring during NMP. This was followed by cytoplasmic escape and co-localisation of ASO with GW182 proteins. This pattern of co-localisation was not seen in scrambled sequence or cold perfusion controls. RNAseq analysis revealed a decrease in inflammatory pathways and upregulation of microRNA-24-3p targets. Discussion This is the first study to demonstrate NMP facilitates gymnotic ASO delivery directly into the RISC, whereby, it blocks microRNA-mediated mRNA silencing and increases bioavailability of protective targets. This study highlights the capacity of NMP to re-programme gene expression in donor kidneys using RNAi therapeutics. Take-home Message Ex vivo normothermic machine perfusion of donor kidneys provides a unique window of opportunity prior to transplantation when we can deliver therapies to improve the quality of the organ. Novel genetic therapies designed to protect kidneys against ischemia reperfusion injury could potentially increase organ utilisation and improve post-transplant outcomes for the many patients on the kidney transplant waiting list.

Glucagon Like Peptide 1 Receptor Agonists for Targeted Delivery of Antisense Oligonucleotides to Pancreatic Beta Cell.

The extra hepatic delivery of antisense oligonucleotides (ASOs) remains a challenge and hampers the widespread application of this powerful class of therapeutic agents. In that regard, pancreatic beta cells are a particularly attractive but challenging cell type because of their pivotal role in diabetes and the fact that they are refractory to uptake of unconjugated ASOs. To circumvent this, we have expanded our understanding of the structure activity relationship of ASOs conjugated to Glucagon Like Peptide 1 Receptor (GLP1R) agonist peptide ligands. We demonstrate the key role of the linker chemistry and its optimization to design maleimide based conjugates with improved in vivo efficacy. In addition, truncation studies and scoping of a diverse set of GLP1R agonists proved fruitful to identify additional targeting ligands efficacious in vivo including native hGLP1(7-36)NH2. Variation of the carrier peptide also shed some light on the dramatic impact of subtle sequence differences on the corresponding ASO conjugate performance in vivo, an area which clearly warrant further investigations. We have confirmed the remarkable potential of GLP1R agonist conjugation for the delivery of ASOs to pancreatic beta cell by effectively knocking down islet amyloid polypeptide (IAPP) mRNA, a potential proapoptotic target, in mice.

High-resolution visualization and quantification of nucleic acid-based therapeutics in cells and tissues using Nanoscale secondary ion mass spectrometry (NanoSIMS).

Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial—both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2′-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.

Targeted Delivery of Antisense Oligonucleotides Using Neurotensin Peptides.

Despite recent advances, targeted delivery of therapeutic oligonucleotide to extra-hepatic tissues continues to be a challenging endeavor and efficient ligand-receptor systems need to be identified. To determine the feasibility of using neurotensin to improve the productive uptake of antisense oligonucleotides (ASO), we synthesized neurotensin-ASO conjugates and evaluated their cellular uptake and activity in cells and in mice. We performed a comprehensive structure-activity relationship study of the conjugates and determined the influence of ASO charge, ASO length, peptide charge, linker chemistry and ligand identity on receptor binding and internalization. We identified a modified neurotensin peptide capable of improving the cellular uptake and activity of gapmer ASOs in sortilin expressing cells (sixfold) and in spinal cord in mice (twofold). Neurotensin conjugation also improved the potency of morpholino ASO designed to correct splicing of survival motor neuron pre-mRNA in the cortex and striatum after intracerebroventricular injection. Neurotensin-mediated targeted delivery represents a possible approach for enhancing the potency of ASOs with diverse nucleic acid modifications.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

A 50‐nm‐Sized Micellar Assembly of Thermoresponsive Polymer‐Antisense Oligonucleotide Conjugates for Enhanced Gene Knockdown in Lung Cancer by Intratracheal Administration

AbstractFor intratracheal delivery of antisense oligonucleotides (ASOs) to lung cancer, a nano‐sized micellar assembly is fabricated from an ASO conjugate with thermoresponsive poly(2‐n‐propyl‐2‐oxazoline) (POX). POX‐ASO conjugate is prepared via a copper‐free click conjugation between azide‐terminal POX and dibenzocyclooctyne‐terminal ASO. Through the thermoresponsive transition of the POX segment from hydrophilic to hydrophobic at temperatures above a critical solution temperature, a 50‐nm‐sized micellar assembly with a narrow size distribution is successfully fabricated when the hydrophobicity of POX segment is strengthened by increasing the molecular weight to 30 kDa. The micellar assembly, termed “ASOball”, elicits more efficient cellular uptake of ASO and significantly higher gene knockdown, compared with non‐conjugated ASO, with negligible cytotoxicity in cultured human lung cancer cells. When the ASOballs are intratracheally administrated into an orthotopic lung cancer model mouse, they show enhanced retention in the tumor tissue 24 h post‐administration, compared with non‐conjugated ASO. Ultimately, the ASOballs significantly reduce the expression level of target long noncoding RNA (lncRNA) by ≈55% at a dose of 15 µg per mouse in a sequence‐specific manner 72 h post‐administration, whereas no significant knockdown effect is observed for non‐conjugated ASO. These results demonstrate the strong potential of ASOballs for intratracheal ASO delivery to lung cancers.

Advanced Fluorescence Imaging to Distinguish Between Intracellular Fractions of Antisense Oligonucleotides.

Antisense oligonucleotides (AON) have been intensively studied as tools in molecular cell biology and as novel therapeutics in various diseases over the past two decades. Especially cellular uptake and endosomal release of AONs are topics of interest, as these are crucial steps in reaching the subcellular AON target sites and achieving biological activity. We used cell-penetrating peptides (CPPs) to enhance uptake and endosomal release of AONs, and monitored these two processes and the subsequent fate of the AONs by advanced fluorescence microscopy in living cells. In this chapter, we discuss the use of automated time-lapse confocal laser scanning microscopy (CLSM) to follow AON uptake and trafficking in time, fluorescence lifetime imaging microscopy (FLIM) to distinguish between free and AON-bound fluorophore, and fluorescence correlation spectroscopy (FCS) to measure subcellular AON concentrations and molecular associations. Additionally, we expand on the analysis of these microscopy data.

A novel and translational role for autophagy in antisense oligonucleotide trafficking and activity.

Endocytosis is a mechanism by which cells sense their environment and internalize various nutrients, growth factors and signaling molecules. This process initiates at the plasma membrane, converges with autophagy, and terminates at the lysosome. It is well-established that cellular uptake of antisense oligonucleotides (ASOs) proceeds through the endocytic pathway; however, only a small fraction escapes endosomal trafficking while the majority are rendered inactive in the lysosome. Since these pathways converge and share common molecular machinery, it is unclear if autophagy-related trafficking participates in ASO uptake or whether modulation of autophagy affects ASO activity and localization. To address these questions, we investigated the effects of autophagy modulation on ASO activity in cells and mice. We found that enhancing autophagy through small-molecule mTOR inhibition, serum-starvation/fasting, and ketogenic diet, increased ASO-mediated target reduction in vitro and in vivo. Additionally, autophagy activation enhanced the localization of ASOs into autophagosomes without altering intracellular concentrations or trafficking to other compartments. These results support a novel role for autophagy and the autophagosome as a previously unidentified compartment that participates in and contributes to enhanced ASO activity. Further, we demonstrate non-chemical methods to enhance autophagy and subsequent ASO activity using translatable approaches such as fasting or ketogenic diet.

Dendrimer-Enabled Therapeutic Antisense Delivery Systems as Innovation in Medicine.

Antisense oligonucleotide (AON)-based therapies concern the treatment for genetic disorders or infections such as a range of neurodegenerative and neuromuscular diseases and have shown benefits in animal models and patients. Nevertheless, successes in the clinic are still strongly limited by unfavorable biodistribution and poor cellular uptake of AONs. Dendrimer macromolecules are synthetically accessible and consist of a core with repeated iterations (named branches) surrounding this core, and on the periphery functional groups which can be modified for ligand attachment. The generations of these branched nanoparticles are based on the number of branches emanating from the core with layered architectures. Dendrimers show promise in several biomedical applications based on their tunable surface modifications allowing the adjustment of their in vivo behavior related to biocompatibility and pharmacokinetic parameters. Dendrimers can be used as nanocarriers of various types of drugs including AONs or nanodrugs. As nanocarriers, polycationic dendrimers can complex multiple negatively charged DNA oligonucleotides on their surface and form stable complexes to promote internalization into the cells based on a good cell membrane affinity. These nanocarriers complexing antisense oligonucleotides must be stable enough to reach the cellular target, but with adequate in vivo global clearance, and have good pharmacokinetic (PK) and pharmacodynamic (PD) profiles. This Review was designed to analyze the development of AONs carried by polycationic and polyanionic (few example) dendrimers. This Review strongly supports the idea that dendrimers, with adequate modulation of their terminal groups, could be used to carry AONs in cells.