Delivery Of Oligonucleotides Research Articles

STING Expression in Peripheral Sensory Neurons Underlies Virus Recognition and Pain

Pain is an early and common symptom of viral infections, and yet we have a limited understanding about the mechanisms through which viruses induce pain. Peripheral sensory neurons are well-studied for their role in pain, and are emerging as new actors in pathogen recognition and defense. It has been reported that peripheral sensory neurons express several proteins associated with virus recognition, including the stimulator of interferon genes (STING). Although STING and interferon signaling has been recently proposed as a critical regulator of physiological pain and a promising new target for treating chronic pain, whether and how STING in peripheral sensory neurons participate in virus recognition, defense and pain is still completely unknown. Here, we report that intradermal delivery of viral oligonucleotides induced pain responses in mice via the expression of STING and the transient receptor potential vanilloid subtype 1 (TRPV1) in sensory neurons. In particular, we found that intradermal injections in mouse hindpaw of double-stranded DNA oligonucleotides derived from the herpes simplex virus (HSV-60) induced immediate spontaneous pain responses (hindpaw lifting, shacking and licking), as well as transitory mechanical pain hypersensitivities lasting up to 4 h. Remarkably, mice treated with the TRPV1 antagonist AMG9810 or resiniferatoxin, an ultrapotent TRPV1 agonist leading to the peripheral denervation, abrogated both HSV-60 induced pain responses. STING expression is enriched in TRPV1+ peripheral sensory neurons, and similar abrogation of HSV-60 induced pain responses was observed in conditional knockout mice lacking the expression of STING uniquely in peripheral sensory neurons. In support of these data, we also found that STING agonists directly can activate sensory neurons in vitro calcium imaging analysis and elicited pain responses in wild-type, but not TRPV1 knockout mice. Thus, our initial findings provide a previously undiscovered mechanism by which viruses are recognized by peripheral sensory neurons and induce pain. Grant support from NINDS R21 NS121946.

Nucleic acid and oligonucleotide delivery for activating innate immunity in cancer immunotherapy.

A group of nucleic acids and oligonucleotides play various roles in the innate immune system. They can stimulate pattern recognition receptors to activate innate immune cells, encode immunostimulatory proteins or peptides, or silence specific genes to block negative regulators of immune cells. Given the limitations of current cancer immunotherapy, there has been increasing interest in harnessing innate immune responses by nucleic acids and oligonucleotides. The poor biopharmaceutical properties of nucleic acids and oligonucleotides make it critical to use carriers that can protect them in circulation, retain them in the tumor microenvironment, and bring them to intracellular targets. Therefore, various gene carriers have been repurposed to deliver nucleic acids and oligonucleotides for cancer immunotherapy and improve their safety and activity. Here, we review recent studies that employed carriers to enhance the functions of nucleic acids and oligonucleotides and overall immune responses to cancer, and discuss remaining challenges and future opportunities in the development of nucleic acid-based immunotherapeutics.

Polyethylenimine/cGAMP Nanocomplexes for STING-Mediated Cancer Immunotherapy: Formulation and Characterization Using Orthogonal Techniques

Cyclic GMP-AMP (cGAMP) has lately been extensively investigated in cancer immunotherapy due its activation of the innate immunity stimulation of interferon genes (STING) pathway within antigen presenting cells (APC) leading to an increase in tumor specific CD8+ T cells. As negatively charged dinucleotides are prone to enzymatic degradation before being taken up by APC, there is a need for an appropriate carrier. Therefore, polyethylenimine (PEI), a gold standard for oligonucleotide delivery, was selected. Molecular weight, type of PEI and N/P ratio between PEI/cGAMP were investigated in terms of toxicity, efficacy and physicochemical properties of the nanocomplexes (NCs) such as size, zeta potential and shape. Due to lack of nano-medicine regulations and the need for a case-by case assessment, here we examine these parameters by several orthogonal methods, such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) and online asymmetric flow field flow fractionation (AF4) connected to DLS. N/P ratio of 2/1 ratio using linear PEI 25 kDa resulted in larger, positively charged particles of elongated shape, which were shown to have the best toxicity/efficacy ratio among different PEIs and ratios tested.

MicroRNA antagonist therapy during normothermic machine perfusion of donor kidneys

Normothermic machine perfusion (NMP) is a novel clinical approach to overcome the limitations of traditional hypothermic organ preservation. NMP can be used to assess and recondition organs prior to transplant and is the subject of clinical trials in solid organ transplantation. In addition, NMP provides an opportunity to deliver therapeutic agents directly to the organ, thus avoiding many limitations associated with systemic treatment of the recipient. We report the delivery of oligonucleotide-based therapy to human kidneys during NMP, in this case to target microRNA function (antagomir). An antagomir targeting mir-24-3p localized to the endothelium and proximal tubular epithelium. Endosomal uptake during NMP conditions facilitated antagomir co-localization with proteins involved in the RNA-induced silencing complex (RISC) and demonstrated engagement of the miRNA target. This pattern of uptake was not seen during cold perfusion. Targeting mir-24-3p action increased expression of genes controlled by this microRNA, including heme oxygenase-1 and sphingosine-1-phosphate receptor 1. The expression of genes not under the control of mir-24-3p was unchanged, indicating specificity of the antagomir effect. In summary, this is the first report of ex vivo gymnotic delivery of oligonucleotide to the human kidney and demonstrates that NMP provides the platform to bind and block detrimental microRNAs in donor kidneys prior to transplantation.

Difficulties translating antisense-mediated activation of Frataxin expression from cell culture to mice

ABSTRACT Friedreich’s ataxia (FA) is an inherited neurodegenerative disorder caused by decreased expression of frataxin (FXN) protein. Previous studies have shown that antisense oligonucleotides (ASOs) and single-stranded silencing RNAs can be used to increase expression of frataxin in cultured patient-derived cells. In this study, we investigate the potential for oligonucleotides to increase frataxin expression in a mouse model for FA. After confirming successful in vivo delivery of oligonucleotides using a benchmark gapmer targeting the nuclear noncoding RNA Malat1, we tested anti-FXN oligonucleotides designed to function by various mechanisms. None of these strategies yielded enhanced expression of FXN in the model mice. Our inability to translate activation of FXN expression from cell culture to mice may be due to inadequate potency of our compounds or differences in the molecular mechanisms governing FXN gene repression and activation in FA model mice.

An orally available, brain penetrant, small molecule lowers huntingtin levels by enhancing pseudoexon inclusion

Huntington’s Disease (HD) is a progressive neurodegenerative disorder caused by CAG trinucleotide repeat expansions in exon 1 of the huntingtin (HTT) gene. The mutant HTT (mHTT) protein causes neuronal dysfunction, causing progressive motor, cognitive and behavioral abnormalities. Current treatments for HD only alleviate symptoms, but cerebral spinal fluid (CSF) or central nervous system (CNS) delivery of antisense oligonucleotides (ASOs) or virus vectors expressing RNA-induced silencing (RNAi) moieties designed to induce mHTT mRNA lowering have progressed to clinical trials. Here, we present an alternative disease modifying therapy the orally available, brain penetrant small molecule branaplam. By promoting inclusion of a pseudoexon in the primary transcript, branaplam lowers mHTT protein levels in HD patient cells, in an HD mouse model and in blood samples from Spinal Muscular Atrophy (SMA) Type I patients dosed orally for SMA (NCT02268552). Our work paves the way for evaluating branaplam’s utility as an HD therapy, leveraging small molecule splicing modulators to reduce expression of dominant disease genes by driving pseudoexon inclusion.

Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides

The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.

Delivery of therapeutic oligonucleotides targeting Dectin-1 using quantized complexes

Delivery of therapeutic oligonucleotides targeting Dectin-1 using quantized complexes

NanoSIMS Imaging Reveals the Impact of Ligand-ASO Conjugate Stability on ASO Subcellular Distribution.

The delivery of antisense oligonucleotides (ASOs) to specific cell types via targeted endocytosis is challenging due to the low cell surface expression of target receptors and inefficient escape of ASOs from the endosomal pathway. Conjugating ASOs to glucagon-like peptide 1 (GLP1) leads to efficient target knockdown, specifically in pancreatic β-cells. It is presumed that ASOs dissociate from GLP1 intracellularly to enable an ASO interaction with its target RNA. It is unknown where or when this happens following GLP1-ASO binding to GLP1R and endocytosis. Here, we use correlative nanoscale secondary ion mass spectroscopy (NanoSIMS) and transmission electron microscopy to explore GLP1-ASO subcellular trafficking in GLP1R overexpressing HEK293 cells. We isotopically label both eGLP1 and ASO, which do not affect the eGLP1-ASO conjugate function. We found that the eGLP1 peptide and ASO are not detected at the same level in the same endosomes, within 30 min of GLP1R-HEK293 cell exposure to eGLP1-ASO. When we utilized different linker chemistry to stabilize the GLP1-ASO conjugate, we observed more ASO located with GLP1 compared to cell incubation with the less stable conjugate. Overall, our work suggests that the ASO separates from GLP1 relatively early in the endocytic pathway, and that linker chemistry might impact the GLP1-ASO function.

Insulin-Degrading Enzyme Is a Non Proteasomal Target of Carfilzomib and Affects the 20S Proteasome Inhibition by the Drug.

Carfilzomib is a last generation proteasome inhibitor (PI) with proven clinical efficacy in the treatment of relapsed/refractory multiple myeloma. This drug is considered to be extremely specific in inhibiting the chymotrypsin-like activity of the 20S proteasome, encoded by the β5 subunit, overcoming some bortezomib limitations, the first PI approved for multiple myeloma therapy which is however burdened by a significant toxicity profile, due also to its off-target effects. Here, molecular approaches coupled with molecular docking studies have been used to unveil that the Insulin-Degrading Enzyme, a ubiquitous and highly conserved Zn2+ peptidase, often found to associate with proteasome in cell-based models, is targeted by carfilzomib in vitro. The drug behaves as a modulator of IDE activity, displaying an inhibitory effect over 10-fold lower than for the 20S. Notably, the interaction of IDE with the 20S enhances in vitro the inhibitory power of carfilzomib on proteasome, so that the IDE-20S complex is an even better target of carfilzomib than the 20S alone. Furthermore, IDE gene silencing after delivery of antisense oligonucleotides (siRNA) significantly reduced carfilzomib cytotoxicity in rMC1 cells, a validated model of Muller glia, suggesting that, in cells, the inhibitory activity of this drug on cell proliferation is somewhat linked to IDE and, possibly, also to its interaction with proteasome.

Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver.

Systemically delivered lipid nanoparticles are preferentially taken up by hepatocytes. This hinders the development of effective, non-viral means of editing genes in tissues other than the liver. Here we show that lipid-nanoparticle-mediated gene editing in the lung and spleen of adult mice can be enhanced by reducing Cas9-mediated insertions and deletions in hepatocytes via oligonucleotides disrupting the secondary structure of single-guide RNAs (sgRNAs) and also via their combination with short interfering RNA (siRNA) targeting Cas9 messenger RNA (mRNA). In SpCas9 mice with acute lung inflammation, the systemic delivery of an oligonucleotide inhibiting an sgRNA targeting the intercellular adhesion molecule 2 (ICAM-2), followed by the delivery of the sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes and increased that in lung endothelial cells. In wild-type mice, the lipid-nanoparticle-mediated delivery of an inhibitory oligonucleotide, followed by the delivery of Cas9-degrading siRNA and then by Cas9 mRNA and sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes but not in splenic endothelial cells. Inhibitory oligonucleotides and siRNAs could be used to modulate the cell-type specificity of Cas9 therapies.

Delivery of Oligonucleotides: Efficiency with Lipid Conjugation and Clinical Outcome.

Oligonucleotides have shifted drug discovery into a new paradigm due to their ability to silence the genes and inhibit protein translation. Importantly, they can drug the un-druggable targets from the conventional small-molecule perspective. Unfortunately, poor cellular permeability and susceptibility to nuclease degradation remain as major hurdles for the development of oligonucleotide therapeutic agents. Studies of safe and effective delivery technique with lipid bioconjugates gains attention to resolve these issues. Our review article summarizes the physicochemical effect of well-studied hydrophobic moieties to enhance the cellular entry of oligonucleotides. The structural impacts of fatty acids, cholesterol, tocopherol, and squalene on cellular internalization and membrane penetration in vitro and in vivo were discussed first. The crucial assays for delivery evaluation within this section were analyzed sequentially. Next, we provided a few successful examples of lipid-conjugated oligonucleotides advanced into clinical studies for treating patients with different medical backgrounds. Finally, we pinpointed current limitations and outlooks in this research field along with opportunities to explore new modifications and efficacy studies.

Delivery of antisense oligonucleotides for splice-correction of androgen receptor pre-mRNA in castration-resistant prostate cancer models using cell-penetrating peptides.

BackgroundCell‐penetrating peptides (CPPs) are a promising approach for delivering antisense oligonucleotides (AONs) as they form nanosized complexes through noncovalent interactions that show efficient cellular uptake. Previously, we have designed an AON system to correct splicing of the androgen receptor (AR) pre‐mRNA, thereby preventing the generation of the splice variant AR‐V7 mRNA. AON‐mediated knockdown of AR‐V7 resulted in inhibition of androgen‐independent cell proliferation. In this study, we evaluated the CPP‐mediated delivery of this AON into castration‐resistant prostate cancer cell line models 22Rv1, DuCaP (dura mater cancer of the prostate), and VCaP (vertebral cancer of the prostate).MethodsNanoparticles (polyplexes) of AONs and CPPs were formed through rapid mixing. The impact of the peptide carrier, the formulation parameters, and cell incubation conditions on cellular uptake of fluorescently labeled AONs were assessed through flow cytometry. The cytotoxic activity of these formulations was measured using the CellTiter‐Glo cell viability assay. The effectivity of CPP‐mediated delivery of the splice‐correcting AON‐intronic splicing enhancer (ISE) targeting the ISE in the castration‐resistant prostate cancer (CRPC)‐derived 22Rv1, DuCaP, and VCaP cells was determined by measuring levels of AR‐V7 mRNA normalized to those of the human heterochromatin protein 1 binding protein 3 (HP1BP3). Western blot analysis was used to confirm AR‐V7 downregulation at a protein level. The cellular distribution of fluorescently labeled AON delivered by a CPP or a transfection reagent was determined through confocal laser scanning microscopy.ResultsThe amphipathic and stearylated CPP PepFect 14 (PF14) showed higher uptake efficiency than arginine‐rich CPPs. Through adjustment of formulation parameters, concentration and incubation time, an optimal balance between carrier‐associated toxicity and delivery efficiency was found with a formulation consisting of an amino/phosphate ratio of 3, 0.35 μM AON concentration and 30 min incubation time of the cells with polyplexes. Cellular delivery of AON‐ISE directed against AR pre‐mRNA achieved significant downregulation of AR‐V7 by 50%, 37%, and 59% for 22Rv1, DuCaP, and VCaP cells, respectively, and reduced androgen‐independent cell proliferation of DuCaP and VCaP cells.ConclusionsThis proof‐of‐principle study constitutes the basis for further development of CPP‐mediated delivery of AONs for targeted therapy in prostate cancer.

Nanomedicine, a valuable tool for skeletal muscle disorders: Challenges, promises, and limitations.

Muscular dystrophies are a group of rare genetic disorders characterized by progressive muscle weakness, which, in the most severe forms, leads to the patient's death due to cardiorespiratory problems. There is still no cure available for these diseases and significant effort is being placed into developing new strategies to either correct the genetic defect or to compensate muscle loss by stimulating skeletal muscle regeneration. However, the vast anatomical extension of the target tissue poses great challenges to these goals, highlighting the need for complementary strategies. Nanomedicine is an actively evolving field that merges nanotechnologies with biomedical and pharmaceutical sciences. It holds great potential in regenerative medicine, both in supporting tissue engineering and regeneration, and in optimizing drug and oligonucleotide delivery and gene therapy strategies. In this review, we will summarize the state‐of‐the‐art in the field of nanomedicine applied to skeletal muscle regeneration. We will discuss the recent work toward the development of nanopatterned scaffolds for tissue engineering, the efforts in the synthesis of organic and inorganic nanoparticles for gene therapy and drug delivery applications, as well as their use as immune modulators. Although nanomedicine holds great promise for muscle and other degenerative diseases, many challenges still need to be systematically addressed to assure a smooth transition from the bench to the bedside.This article is categorized under:Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement

Cross-Linkable Polyion Complex Micelles from Polypept(o)ide-Based ABC-Triblock Copolymers for siRNA Delivery.

ABC-type triblock copolymers are a rising platform especially for oligonucleotide delivery as they offer an additional functionality besides the anyhow needed functions of shielding and complexation. The authors present a polypept(o)ide-based triblock copolymer synthesized by amine-initiated ring-opening polymerization (ROP) of N-carboxyanhydrides (NCAs), comprising a shielding block A of polysarcosine (pSar), a poly(S-ethylsulfonyl-l-cystein) (pCys(SO2 Et)) block B for bioreversible and chemo-selective cross-linking and a poly(l-lysine) (pLys) block C for complexation to construct polyion complex (PIC) micelles as vehicle for small interfering RNA (siRNA) delivery. The self-assembly behavior of ABC-type triblocks is investigated to derive correlations between block lengths of the polymer and PIC micelle structure, showing an enormous effect of the β-sheet forming pCys(SO2 Et) block. Moreover, the block enables the introduction of disulfide cross-links by reaction with multifunctional thiols to increase stability against dilution. The right content of the additional block leads to well-defined cross-linked 50-60nm PIC micelles purified from production impurities and determinable siRNA loading. These PIC micelles can deliver functional siRNA into Neuro2A and KB cells evaluated by cellular uptake and specific gene knockdown assays.

Quantitative Measurement of Cytosolic and Nuclear Penetration of Oligonucleotide Therapeutics.

A major obstacle in the development of effective oligonucleotide therapeutics is a lack of understanding about their cytosolic and nuclear penetration. To address this problem, we have applied the chloroalkane penetration assay (CAPA) to oligonucleotide therapeutics. CAPA was used to quantitate cytosolic delivery of antisense oligonucleotides (ASOs) and siRNAs and to explore the effects of a wide variety of commonly used chemical modifications and their patterning. We evaluated potential artifacts by exploring the effects of serum, comparing activity data and CAPA data, and assessing the impact of the chloroalkane tag and its linker chemistry. We also used viral transduction to expand CAPA to the nuclear compartment in epithelial and neuronal cell lines. Using this enhanced method, we measured a 48-h time course of nuclear penetration for a panel of chemically diverse modified RNAs. Moving forward, CAPA will be a useful tool for deconvoluting the complex processes of endosomal uptake, escape into the cytosol, and subcellular trafficking of oligonucleotide therapeutics in therapeutically relevant cell types.

Cyclic peptides nanospheres: A ‘2-in-1′ self-assembled delivery system for targeting nucleus and cytoplasm

Vascular endothelial growth factor (VEGF) is considered as one of the vital growth factors for angiogenesis, which is primarily responsible for the progress and maintenance of new vascular network in tumor. Numerous studies report that inhibition of VEGF-induced angiogenesis is a potent technique for cancer suppression. Recently, RNA interference, especially small interfering RNA (siRNA) signified a promising approach to suppress the gene expression. However, the clinical implementation of biological macromolecules such as siRNA is significantly limited because of stability and bioavailability issues. Herein, self-assembled peptide nanospheres have been generated from L,L-cyclic peptides using hydrophobic (Trp), positively charged (Arg) and cysteine (Cys) amino acid residues and demonstrated as vehicles for intracellular delivery of VEGF siRNA and VEGF antisense oligonucleotide. Formation of peptide nanostructures is confirmed by HR-TEM, AFM, SEM and DLS analysis. Possible mechanism of self-assembly of the cyclic peptides and their binding with macromolecules are demonstrated by in-silico analysis. Gel electrophoresis reveals that the newly generated peptide based organic materials exhibit strong binding affinity toward siRNAs / antisense oligonucleotides (ASOs) at optimum concentration. Flow cytometry and confocal microscopy results confirm the efficiency of the new biomaterials toward the intracellular delivery of fluorescent labeled siRNA / ASOs. Furthermore, VEGF expression evaluated by western blot and RT-PCR upon the delivery of functional VEGF siRNA/ASOs suggests that very low concentrations of VEGF siRNA/ASOs cause significant gene knockdown at protein and mRNA levels, respectively.

Enhancement of Gene Knockdown on CD22-Expressing Cells by Chemically Modified Glycan Ligand-siRNA Conjugates.

Extrahepatic targeted delivery of oligonucleotides, such as small interfering RNA (siRNA) and antisense oligonucleotides (ASOs), is an attractive technology for the development of nucleic acid-based medicines. To target CD22-expressing B cells, several drug platforms have shown promise, including antibodies, antibody-drug conjugates, and nanoparticles, but to date CD22-targeted delivery of oligonucleotide therapeutics has not been reported. Here we report the uptake and enhancement of siRNA gene expression knockdown in CD22-expressing B cells using a chemically stabilized and modified CD22 glycan ligand-conjugated siRNA. This finding has the potential to broaden the use of siRNA technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of siRNAs to B cell lymphomas.

Nanocarriers for Delivery of Oligonucleotides to the CNS.

Nanoparticles with oligonucleotides bound to the outside or incorporated into the matrix can be used for gene editing or to modulate gene expression in the CNS. These nanocarriers are usually optimised for transfection of neurons or glia. They can also facilitate transcytosis across the brain endothelium to circumvent the blood-brain barrier. This review examines the different formulations of nanocarriers and their oligonucleotide cargoes, in relation to their ability to enter the brain and modulate gene expression or disease. The size of the nanocarrier is critical in determining the rate of clearance from the plasma as well as the intracellular routes of endothelial transcytosis. The surface charge is important in determining how it interacts with the endothelium and the target cell. The structure of the oligonucleotide affects its stability and rate of degradation, while the chemical formulation of the nanocarrier primarily controls the location and rate of cargo release. Due to the major anatomical differences between humans and animal models of disease, successful gene therapy with oligonucleotides in humans has required intrathecal injection. In animal models, some progress has been made with intraventricular or intravenous injection of oligonucleotides on nanocarriers. However, getting significant amounts of nanocarriers across the blood-brain barrier in humans will likely require targeting endothelial solute carriers or vesicular transport systems.

Delivery of Antisense Oligonucleotides to the Mouse Brain by Intracerebroventricular Injections.

The use of antisense oligonucleotides (AONs) is a promising therapeutic strategy for central nervous system disorders. However, the delivery of AONs to the central nervous system is challenging because their size does not allow them to diffuse over the blood-brain barrier (BBB) when injected systemically. The BBB can be bypassed by administering directly into the brain. Here we describe a method to perform single and repeated intracerebroventricular injections into the lateral ventricle of the mouse brain.