Targeting RNA: A Transformative Therapeutic Strategy.

The therapeutic pathways that modulate transcription mechanisms currently include gene knockdown and splicing modulation. However, additional mechanisms may come into play as more understanding of molecular biology and disease etiology emerge. Building on advances in chemistry and delivery technology, oligonucleotide therapeutics is emerging as an established, validated class of drugs that can modulate a multitude of genetic targets. These targets include over 10,000 proteins in the human genome that have hitherto been considered undruggable by small molecules and protein therapeutics. The approval of five oligonucleotides within the last 2 years elicited unprecedented excitement in the field. However, there are remaining challenges to overcome and significant room for future innovation to fully realize the potential of oligonucleotide therapeutics. In this review, we focus on the translational strategies encompassing preclinical evaluation and clinical development in the context of approved oligonucleotide therapeutics. Translational approaches with respect to pharmacology, pharmacokinetics, cardiac safety evaluation, and dose selection that are specific to this class of drugs are reviewed with examples. The mechanism of action, chemical evolution, and intracellular delivery of oligonucleotide therapies are only briefly reviewed to provide a general background for this class of drugs.

Biodistribution studies of polymeric nanoparticles for drug delivery in mice.

Abstract Duchenne muscular dystrophy (DMD) is a severe hereditary neuromuscular disorder caused by mutations in the dystrophin gene. Antisense-mediated targeted exon skipping has been shown to restore dystrophin expression both in DMD patients and in the mdx mouse, the murine model of DMD, but the ineffective delivery of these molecules limits their therapeutic use. We demonstrated that PMMA/N-isopropil-acrylamide (ZM2) nanoparticles (NPs), administered both intraperitoneally and orally, were able to deliver 2'OMePS antisense inducing various extents of dystrophin restoration in the mdx mice. Defining NP biodistribution is crucial to improve effects on target and dose regimens; thus, we performed in vivo studies of novel ZM4 NPs. ZM4 are conjugated with NIR fluorophores as optical probes suitable for studies on the Odyssey Imaging System. Our results indicate that NPs are widely distributed in all body muscles, including skeletal muscles and heart, suggesting that these vehicles are appropriate to deliver antisense oligonucleotides for targeting striated muscles in the DMD animal model, thus opening new horizons for Duchenne therapy.

Synergistic effect of chemical penetration enhancer and iontophoresis on transappendageal transport of oligodeoxynucleotides.

Gap junction protein connexin43 (Cx43) specific antisense oligodeoxynucleotides (AsODN) have been shown to improve a number of inflammatory conditions and may therefore offer a novel strategy for persistent pain management. However, for such molecules to be clinically effective, delivery challenges owing to the molecules' high molecular weight, negative charge and hydrophilicity have to be overcome. In this study, the effect of various chemical penetration enhancers and cathodal iontophoresis on transdermal delivery was evaluated. Initial skin permeation studies revealed only a slight increase in the passive flux of the model anionic drug sodium fluorescein using limonene/ethanol. Applying cathodal iontophoresis, the amount of the model drug permeated through untreated skin was tripled, while a combination of chemical and physical penetration enhancement resulted in a fourfold increase in the fluorescein amount permeated. However, even the synergistic effect of limonene/ethanol and iontophoresis was insufficient to achieve complete permeation of Cy3-labeled Cx43 AsODN across the entire skin thickness. Instead, molecules were trapped in the epidermis or permeated deeply into the hair follicles. These results suggest that the synergistic effect of chemical and physical penetration enhancement increases intradermal delivery of oligonucleotides but is insufficient to deliver such large molecules across intact skin.

Radiolabeling small RNA with technetium-99m for visualizing cellular delivery and mouse biodistribution.

To develop a noninvasive direct method for the in vivo tracking of small interfering RNA (siRNA) used in RNA interference, two 18-nucleotide oligoribonucleotides were radiolabeled with technetium-99m ((99m)Tc-RNA). The ability of (99m)Tc-RNA to track delivery was tested in cultured cells and living mice. The cellular delivery of (99m)Tc-RNAs could be quantified by gamma counting and could be visualized by microautoradiography. Radiolabeled RNAs can be efficiently delivered into cells by reaching up to 3x10(5) molecules of small RNAs per cell. Moreover, RNAs were internalized with homogeneous distribution throughout the cytoplasm and nucleus. In tumor-bearing mice, whole-body images and biodistribution studies showed that (99m)Tc-RNAs were delivered to almost all tissues after intravenous injection. The imaging of living animals allowed noninvasive and longitudinal monitoring of the in vivo delivery of these small RNAs. In conclusion, using (99m)Tc radiolabeling, the delivery of small RNAs could be measured quantitatively in cultured cells and could be noninvasively visualized in living animals using a gamma camera. The results of this study could open up a new approach for measuring the in vivo delivery of small RNAs that might further facilitate the development of siRNAs as targeted therapies.

Antisense delivery and protein knockdown within the intact central nervous system.

The ability to down regulate the expression of a specific protein within the intact central nervous system (CNS) is highly desirable from both a research and therapeutic perspective. Antisense has the potential to do this. However, problems of invasive antisense delivery methods and short half life of remain problematic. We overcome this by using Pluronic gel to provide a sustained delivery antisense oligodeoxynucleotides (ODN's) to the intact central nervous system and achieving rapid penetration throughout the spinal cord in 2-3 hours and significant knockdown of our target protein connexin 43 (Cx43) in 4-8 hours (recovering at 48-72 hours). Interestingly CY3-siRNA probes could not be detected penetrating the intact CNS and no knockdown the Cx43 was found. This approach with conventional ODNs could provide a faster and cheaper alternative to knockout mice in the investigation of the functions of specific proteins within the CNS and may also have therapeutic implications for drug discovery and development.

Biodistribution of 68Ga-labelled phosphodiester, phosphorothioate, and 2'-O-methyl phosphodiester oligonucleotides in normal rats.

Antisense oligonucleotides may hybridise with high selectivity to mRNA sequences allowing monitoring of gene expression or inhibition of the manifestation of altered genes inducing diseases. As part of the development of positron emission tomography methods, 17-mer antisense phosphodiester (PO), phosphorothioate (PS) and 2'-O-methyl phosphodiester (OMe) oligonucleotides specific for point mutationally activated human K-ras oncogene were labelled with 68Ga radionuclide via a chelator coupled to the probe. Hybridisation in solution and non-denaturing polyacrylamide gel electrophoresis (PAGE) with a subsequent exposure of the gels was performed to verify the hybridisation ability after labelling. The biodistribution was studied in male Sprague-Dawley rats by injecting 2MBq of 68Ga-oligonucleotides via the tail vein and measuring the organ radioactivity concentration after 20, 60 and 120 min or using whole-body autoradiography with 10 MBq 68Ga-oligonucleotide and 20 min incubation time. Control experiments were performed with 68GaCl3 and 68Ga-chelator complex. The results revealed that 68Ga-labelling did not change the hybridisation abilities of the oligonucleotides. The biodistribution pattern depended on the nature of the oligonucleotide backbone. Bone marrow, kidney, liver, spleen and urinary bladder were the five organs of highest uptake with each oligonucleotide. The PO accumulated highly in the liver, whereas high kidney uptake dominated the PS and OMe patterns. Intact PS and OMe were detected in plasma samples taken 20 and 60 min after injection. This study supplies a base for the further development of 68Ga-labelled oligonucleotides as pharmacokinetic tools and a potential future use for in vivo imaging of gene expression.

Delivery of antisense oligonucleotides to leukemia cells by RNA bacteriophage capsids.

BACKGROUND: Acute myelogenous leukemia (AML) is the most common type of acute leukemia in adults. Because conventional treatments are associated with substantial side effects, novel therapeutic strategies are required. Antisense oligodeoxynucleotides (ODNs) have shown promise as the basis of emerging therapies for fighting cancer, although in vivo application is hampered by high sensitivity to cellular nuclease degradation. Encapsidation of ODNs in a drug-delivery capsule would reduce such degradation, thereby increasing the potency of therapy. MS2 bacteriophage capsid proteins may be used as novel virus-like particles (VLPs) to deliver ODNs. Here we report an analysis of the uptake mechanism of this VLP system and preliminary examples of its use to deliver a number of ODNs, including some targeting p120 messenger RNAs, a biomarker overexpressed in myelogenous leukemia cells. METHODS: The ODNs were synthesized as covalent extensions to the translational repressor/assembly initiation signal (TR), a 19 nt stem-loop, of the RNA phage MS2. The VLPs were constructed by soaking ODN-TR directly into recombinant RNA-free capsid shells. Targeting of the encapsidated ODNs into cells was achieved by a receptor-mediated endocytosis pathway identified by immunofluorescence microscopy or by transmission electron microscopy with gold-labeled antibodies. RESULTS: After covalent decoration with transferrin on their surface, the VLPs containing ODNs demonstrated increased effectiveness in killing target leukemia cells expressing transferrin receptors, suggesting that this system is worthy of more extensive analysis. CONCLUSIONS: The findings suggest that RNA phage VLPs may be useful as a new nanomaterial for targeted delivery of antisense ODNs, or other macromolecular drug candidates.

Technology evaluation: ISIS-113715, Isis.

Isis is developing ISIS-113715, an antisense inhibitor of the PTP1B gene, for the potential treatment of type 2 diabetes and obesity. ISIS-113715 is undergoing phase II clinical trials.

Poly(L-lysine)-modified silica nanoparticles for the delivery of antisense oligonucleotides.

Silica nanoparticles were prepared in a microemulsion system, using polyoxyethylene nonylphenyl ether/cyclohexane/ammonium hydroxide. The surface charge of the particle was modified with PLL [poly(L-lysine)]. PAGE demonstrated the ability of PMS-NP (PLL-modified silica nanoparticles) to bind and protect antisense ODNs (oligonucleotides). The intracellular localization of FITC-labelled ODN was investigated by fluorescence microscopy. The results demonstrated that ODN could be delivered to cytoplasm. Flow-cytometry analysis showed a 20-fold enhancement of ODN delivered by PMS-NP compared with free ODN for a serum-free medium. Blocking efficacy of c- myc antisense ODN, delivered by PMS-NP, was examined in HNE1 and HeLa cell lines. Significant down-regulation of c- myc mRNA levels was observed in both the cell lines. However, the cellular uptake efficiency and antisense effects on target gene decreased in the presence of serum-containing medium. The analysis of the filtration assay showed that PMS-NP interacted with serum proteins. These results indicated that PMS-NP was a suitable delivery vector for antisense ODN, although its delivery efficiency decreased in the presence of a serum-containing medium.

Antisense technologies. Improvement through novel chemical modifications.

Antisense agents are valuable tools to inhibit the expression of a target gene in a sequence-specific manner, and may be used for functional genomics, target validation and therapeutic purposes. Three types of anti-mRNA strategies can be distinguished. Firstly, the use of single stranded antisense-oligonucleotides; secondly, the triggering of RNA cleavage through catalytically active oligonucleotides referred to as ribozymes; and thirdly, RNA interference induced by small interfering RNA molecules. Despite the seemingly simple idea to reduce translation by oligonucleotides complementary to an mRNA, several problems have to be overcome for successful application. Accessible sites of the target RNA for oligonucleotide binding have to be identified, antisense agents have to be protected against nucleolytic attack, and their cellular uptake and correct intracellular localization have to be achieved. Major disadvantages of commonly used phosphorothioate DNA oligonucleotides are their low affinity towards target RNA molecules and their toxic side-effects. Some of these problems have been solved in 'second generation' nucleotides with alkyl modifications at the 2' position of the ribose. In recent years valuable progress has been achieved through the development of novel chemically modified nucleotides with improved properties such as enhanced serum stability, higher target affinity and low toxicity. In addition, RNA-cleaving ribozymes and deoxyribozymes, and the use of 21-mer double-stranded RNA molecules for RNA interference applications in mammalian cells offer highly efficient strategies to suppress the expression of a specific gene.

2-5A antisense telomerase RNA therapy for intracranial malignant gliomas.

Malignant gliomas are the most common intracranial tumors and are considered incurable. Therefore, exploration of novel therapeutic modalities is essential. Telomerase is a ribonucleoprotein enzyme that is detected in the vast majority of malignant gliomas but not in normal brain tissues. We, therefore, hypothesized that telomerase inhibition could be a very promising approach for the targeted therapy of malignant gliomas. Thus, 2-5A (5'-phosphorylated 2'-5'-linked oligoadenylate)-linked antisense against human telomerase RNA component (2-5A-anti-hTER) was investigated for its antitumor effect on an intracranial malignant glioma model. 2-5A is a mediator of one pathway of IFN actions by activating RNase L, resulting in RNA degradation. By linking 2-5A to antisense, RNase L degrades the targeted RNA specifically and effectively. Prior to the experiments using intracranial tumor models in nude mice, we modified the in vitro and in vivo treatment modality of 2-5A-anti-hTER using a cationic liposome to enhance the effect of 2-5A-anti-hTER. Here we demonstrate that 2-5A-anti-hTER complexed with a cationic liposome reduced the viability of five malignant glioma cell lines to 20-43% within 4 days but did not influence the viability of cultured astrocytes lacking telomerase. Furthermore, treatment of intracranial malignant gliomas in nude mice with 2-5A-anti-hTER was therapeutically effective compared with the control (P < 0.01). These findings clearly suggest the therapeutic potentiality of 2-5A-anti-hTER as a novel approach for the treatment of intracranial malignant gliomas.

Distribution of a 20-mer phosphorothioate oligonucleotide, CGP69846A (ISIS 5132), into airway leukocytes and epithelial cells following intratracheal delivery to brown-norway rats.

PURPOSE: To evaluate the pulmonary distribution of CGP69846A (ISIS 5132), a phosphorothioate oligonucleotide, following intra-tracheal (i.t.) instillation into Brown-Norway rats. METHODS: The pharmacokinetic profile of [3H]-CGP69846A was investigated following i.t. instillation into both naive and inflamed airways of Brown-Norway rats. The cellular distribution was determined using autoradiography, immunohistochemistry and flow cytometry/fluorescence microscopy, in inflamed airways. RESULTS: CGP69846A displayed a dose-dependent lung retention following i.t. administration which was unaffected by local inflammation. Autoradiography and immunohistochemistry showed distribution to alveolar macrophages, eosinophils, bronchial and tracheal epithelium and alveolar cells. Studies with [FITC]-CGP69846A demonstrated a preferential association of oligonucleotide with leukocytes in bronchial lavage fluid of: macrophages > eosinophils = neutrophils >> lymphocytes. CONCLUSIONS: The dose-dependency of lung retention together with cell-specific uptake suggests that the lung can be used as a local target for antisense molecules with potentially minimal systemic effects. Furthermore, the preferential targeting of macrophages and the airway epithelium by oligonucleotides may represent rational cellular targets for antisense therapeutics.