Efficient Delivery Of Oligonucleotides Research Articles

Fluoropolymer Coated DNA Nanoclews for Volumetric Visualization of Oligonucleotides Delivery and Near Infrared Light Activated Anti-Angiogenic Oncotherapy.

The potential of microRNA regulation in oncotherapy is limited by the lack of delivery vehicles. Herein, it is shown that fluoropolymer coated DNA nanoclews (FNCs) provide outstanding ability to deliver oligonucleotide through circulation and realize near infrared (NIR) light activated angiogenesis suppression to abrogate tumors. Oligonucleotides are loaded in DNA nanoclews through sequence specific bindings and then a fluorinated zwitterionic polymer is coated onto the surface of nanoclews. Further incorporating quantum dots in the polymer coating endows the vectors with NIR-IIb (1500-1700nm) fluorescence and NIR light triggered release ability. The FNC vector can deliver oligonucleotides to cancer cells systemically and realize on-demand cytosolic release of the cargo with high transfection efficiency. Taking advantage of the NIR-IIb emission, the whole delivery process of FNCs is visualized volumetrically in vivo with a NIR light sheet microscope. Loaded by FNCs, an oligonucleotide can effectively silence the target miRNA when activated with NIR light, and inhibit angiogenesis inside tumor, leading to complete ablation of cancer. These findings suggest FNCs can be used as an efficient oligonucleotide delivery platform to modulate the expression of endogenous microRNA in gene therapy of cancer.

Therapeutic Oligonucleotides: An Outlook on Chemical Strategies to Improve Endosomal Trafficking.

The potential of oligonucleotide therapeutics is undeniable as more than 15 drugs have been approved to treat various diseases in the liver, central nervous system (CNS), and muscles. However, achieving effective delivery of oligonucleotide therapeutics to specific tissues still remains a major challenge, limiting their widespread use. Chemical modifications play a crucial role to overcome biological barriers to enable efficient oligonucleotide delivery to the tissues/cells of interest. They provide oligonucleotide metabolic stability and confer favourable pharmacokinetic/pharmacodynamic properties. This review focuses on the various chemical approaches implicated in mitigating the delivery problem of oligonucleotides and their limitations. It highlights the importance of linkers in designing oligonucleotide conjugates and discusses their potential role in escaping the endosomal barrier, a bottleneck in the development of oligonucleotide therapeutics.

Conjugation Approaches for Peptide-Mediated Delivery of Oligonucleotides Therapeutics

Oligonucleotide-based agents are versatile biomolecules that modulate gene expression. The last decade has seen the emergence of oligonucleotide-based tools for biochemical investigations. Importantly, several oligonucleotide-based drugs and vaccines are currently used for various therapeutic applications ranging from anti-inflammatory and anti-viral agents to those used in cardiovascular, ophthalmic, and neuro-muscular disorders. Despite a broad range of applications, achieving efficient oligonucleotide delivery remains a major limitation. A possible solution is to conjugate cell-penetrating peptides with oligonucleotides. This review provides an overview of chemical strategies used to synthesise peptide–oligonucleotide conjugates. The merits and liabilities of these strategies are discussed in the context of synthetic efficiency, and bio-reversible and -irreversible linkages.

Advances in oligonucleotide drug delivery.

Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technological advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches — including chemical modification, bioconjugation and the use of nanocarriers — which aim to address the delivery challenge.

Natural polyphenol assisted delivery of single-strand oligonucleotides by cationic polymers

Single-strand oligonucleotides provide promising potential as new therapeutics towards various diseases. However, the efficient delivery of oligonucleotide therapeutics is still challenging due to their susceptibility to nuclease degradation and the lack of effective carriers for condensation. In this study, we reported the use of natural polyphenol to facilitate the condensation of single-strand oligonucleotides by cationic polymers. Green tea catechin complexed with single-strand oligonucleotides to form anionic nanoparticles, which were further coated by low molecular weight cationic polymers to increase their cell internalization. The resulting core-shell structured nanoparticles, so-called green nanoparticles (GNPs), showed improved cargo stability, and achieved high efficiency in the delivery of several types of single-strand oligonucleotides including antisense oligonucleotides, anti-miRNA, and DNAzyme. This study provides a facile strategy for the efficient delivery of single-strand oligonucleotides.

Polyethylenimine-based Formulations for Delivery of Oligonucleotides.

Polyethyleneimine (PEI) is well-known as a non-viral gene delivery vector, especially for oligonucleotide delivery. However, its clinical applications are significantly limited due to its high cationic charge, lack of specificity, and interaction with the proteins and nontarget cells in the biological fluids, resulting in high cytotoxicity, poor stability and low transfection efficiency for oligonucleotides transporting. It has been shown that the molecular weight (MW) of PEI, degree of branching, N/P ratio, buffer capacity, oligonucleotide structure, culture medium pH, serum, presence or absence of and method of preparation make a significant difference in the cytoxicity, stability, and transfection efficiency for the PEI-based oligonucleotides delivery systems. Ligands, hydrophobic, hydrophilic, and amphiphilic modification of PEI have been investigated to reduce the cytoxicity and improve the stability, the transfection efficiency, and therapeutic effect. Moreover, various intelligent modifications of PEI, such as pH-responsive (hydrazone bond) and redox sensitive linkers (disulfide bond) can control oligonucleotides release and have attracted much attention. In general, more efficient oligonucleotide delivery can be achieved by the introduction of modifications to PEI and by optimization of parameters of PEI or PEI-based formulations.

A Kinetic Model of Oligonucleotide-Brush Interactions for the Rational Design of Gene Delivery Vectors.

Polymer brushes are attractive candidates for the design of gene delivery vectors as they allow the systematic study of the impact of structural (type, size, and shape of nanomaterials core) and physicochemical parameters (cationic monomer chemistry, brush thickness, and grafting density) on transfection efficiency. However, relatively little is known of their interactions of oligonucleotides. To study such interactions, we use surface plasmon resonance and developed a kinetic model of brush binding and infiltration. We identify the striking impact that brush grafting density and thickness have on oligonucleotide kinetics of infiltration, binding affinity, and maximum loading. Surprisingly, double-stranded RNA molecules are found to load at significantly higher levels compared to DNA molecules of identical sequence (apart from uracils/thymines). Furthermore, analysis of the kinetics of adsorption of these oligonucleotides indicates that the stoichiometry of binding (ratio of amine versus phosphate residues) is close to parity for the uptake of 20 bp double-stranded RNA. Finally, nanoparticles were designed to be used as gene transfection vectors and to quantify if the brush grafting density and thickness significantly impact transfection efficiencies in a small interfering RNA knockdown assay. Therefore, this study demonstrates the rational design of polymer brush-based nanoparticle vectors for efficient delivery of oligonucleotides. The model developed will allow to uncover how the refinement of the physicochemical and structural properties of polymer brushes enable the tuning of RNA binding and allow the systematic study of cationic vectors efficiency for RNA delivery.

CLIP6-PNA-Peptide Conjugates: Non-Endosomal Delivery of Splice Switching Oligonucleotides.

Efficient delivery of oligonucleotides still remains a challenge in the field of oligonucleotide based therapy. Peptide nucleic acid (PNA), a DNA analogue that is typically synthesized by solid phase peptide chemistry, has been conjugated to a variety of cell penetrating peptides (CPP) as a means of improving its cellular uptake. These CPPs typically deliver their cargoes into cells by an endosomal-dependent mechanism resulting in lower bioavailability of the cargo. Herein, we designed and synthesized PNA-peptide conjugates as splice switching oligonucleotides (SSO) targeting the Mnk2 gene, a therapeutic target in cancer. In humans, the MKNK2 gene, is alternatively spliced, generating isoforms with opposite biological activities: Mnk2a and Mnk2b. It was found that the Mnk2a isoform is down-regulated in breast, lung, brain, and colon tumors and is a tumor suppressor, whereas MnK2b is oncogenic. We have designed and synthesized PNAs that were conjugated to either of the following peptides: a nuclear localization sequence (NLS) or a cytosol localizing internalization peptide (CLIP6). CLIP6-PNA demonstrates effective cellular uptake and exclusively employs a nonendosomal mechanism to cross the cellular membranes of glioblastoma cells (U87). Simple incubation of PNA-peptide conjugates in human glioblastoma cells up-regulates the Mnk2a isoform leading to cancer cell death.

Biodegradable lipid nanoparticles induce a prolonged RNA interference-mediated protein knockdown and show rapid hepatic clearance in mice and nonhuman primates

Lipid nanoparticles based on ionizable lipids have been clinically validated as a means of delivery for RNA interference (RNAi) therapeutics. The ideal properties of RNAi carriers are efficient delivery of oligonucleotides into target cells and rapid elimination after the function is performed. Here, we report that degradable lipid nanoparticles are effective carriers of small interfering RNA (siRNA) and have a high therapeutic index. The newly developed degradable lipid nanoparticles carrying siRNA showed potent gene-silencing activity in mouse hepatocytes (ED50≈0.02mg/kg siRNA). The ester bond in the lipid tail was hydrolyzed in the liver, resulting in rapid metabolism of the lipid. Toxicity assays showed that the degradable lipid was well-tolerated at siRNA doses of up to 16mg/kg in rats (over 800-fold higher than ED50). A single intravenous injection of siRNA targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) in cynomolgus monkeys resulted in more than 90% protein silencing, and a 50% decrease in plasma low-density lipoprotein (LDL) cholesterol, with a measurable reduction for 2 months. Moreover, quantification of lipids in liver biopsies revealed rapid hepatic clearance of the degradable lipid in nonhuman primates. These degradable lipid nanoparticles with a high therapeutic index hold promise for RNA-based treatments.

Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile.

Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride (“DQAsomes”) have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2′-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription–translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe the novel application of cationic bolasomes to deliver ASOs into bacteria. We also report the first successful in vitro antisense treatment to inhibit the growth of C. difficile.

Peptide-Based Multicomponent Oligonucleotide Delivery Systems: Optimisation of Poly-l-lysine Dendrons for Plasmid DNA Delivery

Gene therapy is a promising means to treat or prevent diseases either through gene silencing or expression. Some of the most effective delivery agents are polycationic dendrimers, which are highly branched constructs incorporating many positively charged groups. Two of the most effective dendrimers are polyethyleneimine (PEI) and poly(amidoamine) (PAMAM), which show high proficiency at overcoming barriers to oligonucleotide delivery. However, because of their abundance of cationic charge, they are associated with severe toxicity. We have therefore aimed to develop a low toxicity oligonucleotide delivery system, incorporating multiple components that have been selected and optimised to overcome the barriers to efficient oligonucleotide delivery. In this work we have focused on improving the toxicity, cellular uptake, and condensation of plasmid DNA (pDNA) through the fusion of synthetic poly-l-lysine (PLL) dendrons with the cell penetrating peptide TAT(48-60). A library of dendron structures, from 4+ to 16+ charge, and constructs containing six histidine residues, were synthesised. The effects of each modification on pDNA binding and condensation; cellular uptake and toxicity; and the size and zeta-potential of the complexes were assessed to identify the optimum dendron for incorporation into our systems. This work concluded that increasing the dendron charge from 4+ to 16+ significantly improved cellular uptake and pDNA condensation, with no effect on toxicity, while PLL dendrons with greater than 16+ charge could not be efficiently produced. In comparison, the incorporation of six histidines into these constructs had a variable effect on cellular uptake, and generated larger sized complexes, but did not affect toxicity.

Endosome Escape Strategies for Improving the Efficacy of Oligonucleotide Delivery Systems

Gene therapy requires safe and effective vectors to deliver genes to their target site of action. Non-viral gene delivery systems have attracted growing attention due to their low toxicity, low immunogenicity and ease of production compared to viral vectors. Most non-viral gene delivery systems enter cells via endocytic pathways, and their escape from endosomes is therefore crucial for successful transfection. Several reagents have been developed to promote endosomal escape, including peptides, polymers and lipids. Among these, endosome-disrupting peptides have been used in many studies, and have proven to be one of the most promising approaches to overcome endosomal entrapment and lysosomal degradation. This review provides an up-to-date summary of strategies for enhancing endosomal escape, with a focus on the modification of endosome-disrupting peptides to further increase the efficient delivery of oligonucleotides.

Synthesis and Studies on Cell-Penetrating Peptides

The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments.

Current status of delivery systems to improve target efficacy of oligonucleotides.

The tragic failure of gene therapy resulted in rolling back the research of gene-based medicine. Because of the poor delivery of gene-based medicines, such as antisense oligonucleotides, ribozyme, triplex, or gene both in vitro and in vivo, further development of gene-based medicines as therapeutic agents have stagnated. Although the delivery system plays a critical role in the overall efficacy of oligonucleotides, inappropriate target selection, improper evaluation methods and misinterpretation of results often caused the pessimistic view. Still, the decoding of the whole human genome has rekindled the enthusiastic development of delivery tools for gene-based medicine. We would like to focus on the newly developed delivery systems mainly for antisense oligonucleotides in this article. There are two ways to improve delivery efficacy of antisense oligonucleotides: One is the chemical modification of the antisense oligonucleotide backbone. The other way is by means of delivery vehicles, such as cationic liposomes, synthetic polymers, or non-viral vectors. We will review the current status of delivery vehicles both in vitro and in vivo. Delivery efficiency depends on the oligonucleotides' chemistry, length, size, net charge, cell/tissue type and administration route. It is difficult to deduce a common rule that affects delivery efficiency. Some cells like keratinocytes rapidly internalize oligonucleotides without a delivery system, which is contrary to common belief. Although we cannot extensively cover all reports, we will summarize several experiments with delivery system in vitro and in vivo. We will then address the possible factors promoting the efficient delivery of oligonucleotides.

Targeted oligonucleotide delivery in human lymphoma cell lines using a polyethyleneimine based immunopolyplex.

The efficacy of antisense gene therapy depends on efficient delivery of oligonucleotides into targeted cells. Although polyethyleneimine based polyplexes have been reported as good transfection reagents, they are inefficient in lymphoid cell transfection. We report the construction of an immunopolyplex, a targeted nonviral vector based on a polyplex backbone and its application for oligonucleotide transfer on human lymphoma cell lines. The salient characteristic of immunopolyplex lies in the possibility of easily replacing the targeting element (antibody), leaving the polyplex backbone intact. Furthermore, a study was made of the influence of endocytosis inhibitors on immunopolyplex activity. The capacity of the immunopolyplex for oligonucleotide transfer was studied in vitro using FITC-labeled oligonucleotides as fluorescent reporters, an anti-CD3 antibody as targeting element, and a CD3-positive cell line (Jurkat) as a target cell line, in the absence and presence of endocytosis inhibitors. A CD3-negative Jurkat-derived mutant cell line (J.RT3-T3.5) was used as control. A nine-fold increase in fluorescence in the CD3-positive above that in the CD3-negative cell line was observed, indicating that oligonucleotide transfer is mainly specific. Low fluorescence values were obtained in the presence of endocytosis inhibitor or with untargeted polyplexes. We conclude that the immunopolyplex is a good nonviral vector for specific oligonucleotide delivery. Abolition of immunopolyplex activity in the presence of endocytosis inhibitor suggests that targeted oligonucleotide transfer occurs through an endocytic pathway.

A pressure-mediated nonviral method for efficient arterial gene and oligonucleotide transfer.

In this study, we report a method of controlled pressure-mediated delivery of "naked" DNA that achieves efficient and safe arterial gene and oligonucleotide transfer. We demonstrated a pressure-dependent uptake of fluorescein-labeled (FITC) oligonucleotide (ODN) in rabbit carotid arteries with preexisting neointimal hyperplasia, using nondistending intravascular delivery pressures ranging from 0 to 760 mm Hg. At an infusion pressure of 50 mm Hg, 10.5+/-5% of neointimal cell nuclei were positive for nuclear uptake of FITC-ODN 4 days after transfection. With an infusion pressure of 760 mm Hg, the transfection efficiency increased to 84.2+/-5.3% of neointimal cells, and to 64.5+/-11.6 and 92.4+/-5.5% of medial and adventitial cells, respectively. Similar patterns of FITC-ODN uptake were seen in atherosclerotic injured arteries. We also investigated the pressure-mediated delivery of plasmid DNA. Transfection of a luciferase expression plasmid, using an infusion pressure of 760 mm Hg, yielded luciferase expression of 816.6+/-108.6 fg/mg protein in normal rabbit carotid arteries, as compared with 38.9+/-23.7 fg/mg protein at 100 mm Hg. Luciferase expression was significantly higher in pressure-transfected injured atherosclerotic arteries (5467.3+/-1047.6 fg/mg protein at 760 mm Hg). Transfection of beta-galactosidase indicated that significant transgene expression occurred in the neointima and media. These data indicate that this pressure-mediated transfection method yields efficient oligonucleotide delivery and enhances transduction with plasmid DNA in normal as well as injured nonatherosclerotic or atherosclerotic arteries.

Enhancement of Anti-Herpetic Activity of Antisense Phosphorothioate Oligonucleotides 5′ End Modified with Geraniol

We have previously shown that antisense phosphorothioate oligonucleotide (SON) targeted against immediate early (IE) pre-mRNA5 of the herpes simplex virus type I (HSV-I) possessed potent anti-herpetic activities in vitro system. However, anti-herpetic activities of SON were not still efficient enough. Lipophilic compounds have been often conjugated with antisense oligonucleotide to enhance the biological activity. In this study, we selected geraniol as a lipophilic compound and newly synthesized SON bearing 5' terminal geraniol (geranyl-SON) toward IE pre-mRNA 5 of the HSV-1 to enhance the anti-herpetic activity. Geraniol is a olefinic terpene alcohol which is found in many essential oils. It possesses lipophilic characteristic. It is thought to be absorbed in tissue. Geraniol enhanced the anti-herpetic activity of SON with less cytotoxicity in a sequence specific manner. Terminal modification with geraniol did not affect binding affinity with complimentary DNA. Cytoplasm distribution of geranyl-SON was confirmed by confocal microscope. While some of the geranyl-SON was seen in the nucleus, unmodified SON had a punctate distribution in the cytoplasm with little in the nucleus. These results suggested that geranyl modification enhances anti-herpetic activity by changing the subcellular distribution of the oligonucleotides. Consequently geraniol-modifica-tion could provide new means for the efficient delivery of oligo-nucleotides.

A new peptide vector for efficient delivery of oligonucleotides into mammalian cells.

The development of antisense and gene therapy has focused mainly on improving methods for oligonucleotide and gene delivery into cells. In the present work, we describe a potent new strategy for oligonucleotide delivery based on the use of a short peptide vector, termed MPG (27 residues), which contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen. The formation of peptide vector/oligonucleotide complexes was investigated by measuring changes in intrinsic tryptophan fluorescence of peptide and of mansyl-labelled oligonucleotides. MPG exhibits relatively high affinity for both single- and double-stranded DNA in a nanomolar range. Based on both intrinsic and extrinsic fluorescence titrations, it appears that the main binding between MPG and oligonucleotides occurs through electrostatic interactions, which involve the basic-residues of the peptide vector. Further peptide/peptide interactions also occur, leading to a higher MPG/oligonucleotide ratio (in the region of 20/1), which suggests that oligonucleotides are most likely coated with several molecules of MPG. Premixed complexes of peptide vector with single or double stranded oligonucleotides are delivered into cultured mammalian cells in less than 1 h with relatively high efficiency (90%). This new strategy of oligonucleotide delivery into cultured cells based on a peptide vector offers several advantages compared to other commonly used approaches of delivery including efficiency, stability and absence of cytotoxicity. The interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and crossing of the plasma membrane. The mechanism of cell delivery of oligonucleotides by MPG does not follow the endosomal pathway, which explains the rapid and efficient delivery of oligonucleotides in the nucleus. As such, we propose this peptide vector as a powerful tool for potential development in gene and antisense therapy.

Efficient oligonucleotide delivery using the HVJ-liposome method in the central nervous system.

We examined the efficiency and intracellular fate of oligodeoxy-nucleotides (ODN) in the central nervous system (CNS) after delivery with a hemagglutinating virus of Japan (HVJ)-liposome vector in vivo and in vitro. In primary cultured granular cells of the rat cerebellum, application of fluorescein isothiocyanate (FITC)-labeled ODN complexed with HVJ-liposomes in vitro resulted in strong fluorescence localized in nuclei that persisted for > or = 2 wk, in contrast to 3 days with ODN alone. In vivo ODN transfer was attempted by different approaches: infusions into the paraventricular nuclei of the hypothalamus and the lateral cerebroventricle. Injection of FITC-labeled ODN into the hypothalamus by the HVJ-liposome method produced a higher concentration and more persistent fluorescence than did injection of ODN alone. Administration of ODN into the lateral cerebroventricle with HVJ-liposomes yielded more conspicuous and prolonged fluorescence in the periventricular layer, predominantly in cell nuclei. Furthermore, the distribution of fluorescent cells was broader with the HVJ-liposome method. These results indicate that the HVJ-liposome method prolongs the half-life of ODN and concentrates them in cell nuclei. Thus it is an efficient method for ODN transfer and holds promise as a gene delivery method in the CNS.