Safety and Biodistribution of Nanoligomers Targeting the SARS-CoV-2 Genome for the Treatment of COVID-19.

As the world braces to enter its fourth year of the coronavirus disease 2019 (COVID-19) pandemic, the need for accessible and effective antiviral therapeutics continues to be felt globally. The recent surge of Omicron variant cases has demonstrated that vaccination and prevention alone cannot quell the spread of highly transmissible variants. A safe and nontoxic therapeutic with an adaptable design to respond to the emergence of new variants is critical for transitioning to the treatment of COVID-19 as an endemic disease. Here, we present a novel compound, called SBCoV202, that specifically and tightly binds the translation initiation site of RNA-dependent RNA polymerase within the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome, inhibiting viral replication. SBCoV202 is a Nanoligomer, a molecule that includes peptide nucleic acid sequences capable of binding viral RNA with single-base-pair specificity to accurately target the viral genome. The compound has been shown to be safe and nontoxic in mice, with favorable biodistribution, and has shown efficacy against SARS-CoV-2 in vitro. Safety and biodistribution were assessed using three separate administration methods, namely, intranasal, intravenous, and intraperitoneal. Safety studies showed the Nanoligomer caused no outward distress, immunogenicity, or organ tissue damage, measured through observation of behavior and body weight, serum levels of cytokines, and histopathology of fixed tissue, respectively. SBCoV202 was evenly biodistributed throughout the body, with most tissues measuring Nanoligomer concentrations well above the compound KD of 3.37 nM. In addition to favorable availability to organs such as the lungs, lymph nodes, liver, and spleen, the compound circulated through the blood and was rapidly cleared through the renal and urinary systems. The favorable biodistribution and lack of immunogenicity and toxicity set Nanoligomers apart from other antisense therapies, while the adaptability of the nucleic acid sequence of Nanoligomers provides a defense against future emergence of drug resistance, making these molecules an attractive potential treatment for COVID-19.

Immunogenicity Assessment of Inotersen, a 2'-O-(2-Methoxyethyl) Antisense Oligonucleotide in Animals and Humans: Effect on Pharmacokinetics, Pharmacodynamics, and Safety.

Inotersen (TEGSEDI™) is a 2'-O-(2-methoxyethyl)-modified antisense oligonucleotide, intended for treating hereditary transthyretin (TTR) amyloidosis with polyneuropathy. The potential immunogenicity (IM) response to inotersen was evaluated in chronic nonclinical safety studies and the pivotal phase 2/3 clinical study. The evaluation was designed to assess the characteristics of antidrug antibodies (ADAs) and their effects on the pharmacokinetics, pharmacodynamics, clinical efficacy, and safety in animals and humans. No immunogenic response was observed after long-term treatment with inotersen in mice. In monkeys, the incidence rate of IM to inotersen appeared to be dose dependent, with 28.6%-50.0% of animals developing ADAs after 36 weeks of treatment. This was characterized as late onset (median onset of 185 days) with low titers (median titer of 8, or 400 if minimum required dilution of 50 is included). The overall incidence rate of patients who developed ADAs was 30% after 65 weeks of treatment with median onset of 203 days and median peak titer of 300. IM had minimal effect on plasma peak (Cmax) and total exposure (i.e. area under curve, AUC) of inotersen, but showed elevated plasma trough levels in both IM-positive animals and humans. However, ADAs had no effect on tissue exposure, TTR messenger RNA, or plasma TTR levels in the long-term monkey study. Similarly, IM showed no effect on plasma TTR levels in clinical studies. Thus, ADAs antibodies were binding antibodies, but not neutralizing antibodies. Finally, no association was observed between IM and toxicity findings (eg, platelet, complement activation, and histopathology findings) in the inotersen 9-month monkey study. In humans, no difference was observed in hematology, including platelets, kidney function tests, or incidence of adverse events between IM-positive and -negative patients. Overall, IM showed no effect on toxicity or safety of inotersen evaluated in both monkeys and humans. ClinicalTrials.gov Identifier: NCT01737398.

MTL-CEBPA, a Small Activating RNA Therapeutic Upregulating C/EBP-α, in Patients with Advanced Liver Cancer: A First-in-Human, Multicenter, Open-Label, Phase I Trial.

PURPOSE: Transcription factor C/EBP-α (CCAAT/enhancer-binding protein alpha) acts as a master regulator of hepatic and myeloid functions and multiple oncogenic processes. MTL-CEBPA is a first-in-class small activating RNA oligonucleotide drug that upregulates C/EBP-α. PATIENTS AND METHODS: We conducted a phase I, open-label, dose-escalation trial of MTL-CEBPA in adults with advanced hepatocellular carcinoma (HCC) with cirrhosis, or resulting from nonalcoholic steatohepatitis or with liver metastases. Patients received intravenous MTL-CEBPA once a week for 3 weeks followed by a rest period of 1 week per treatment cycle in the dose-escalation phase (3+3 design). RESULTS: Thirty-eight participants have been treated across six dose levels (28-160 mg/m2) and three dosing schedules. Thirty-four patients were evaluable for safety endpoints at 28 days. MTL-CEBPA treatment-related adverse events were not associated with dose, and no maximum dose was reached across the three schedules evaluated. Grade 3 treatment-related adverse events occurred in nine (24%) patients. In 24 patients with HCC evaluable for efficacy, an objective tumor response was achieved in one patient [4%; partial response (PR) for over 2 years] and stable disease (SD) in 12 (50%). After discontinuation of MTL-CEBPA, seven patients were treated with tyrosine kinase inhibitors (TKIs); three patients had a complete response with one further PR and two with SD. CONCLUSIONS: MTL-CEBPA is the first saRNA in clinical trials and demonstrates an acceptable safety profile and potential synergistic efficacy with TKIs in HCC. These encouraging phase I data validate targeting of C/EBP-α and have prompted MTL-CEBPA + sorafenib combination studies in HCC.

Comparison of the Class Effects of Antisense Oligonucleotides in CByB6F1-Tg(HRAS)2Jic and CD-1 Mice.

The 6-month Tg.rasH2 mouse carcinogenicity model provides an acceptable alternative to the 2-year carcinogenicity study in CD-1 mice. However, key questions related to the use of this model for testing antisense oligonucleotides (ASOs) include the similarity in the biologic response between mouse strains and the feasibility of using data from the CD-1 mouse to set doses and dose schedules for a Tg.rasH2 carcinogenicity study. To evaluate the potential strain differences, four distinct 2'- O-(2-methoxyethyl) ASOs were administered to CByB6F1 (wild type), Tg.rasH2 (hemizygous), and CD-1 mice. There were no meaningful differences in clinical signs, body weight, food consumption, or serum chemistry and hematology parameters. Histopathology evaluation indicated little to no difference in the spectrum or magnitude of changes present. The cytokine/chemokine response was also not appreciably different between the strains. This was consistent with the similarity in ASO concentration in the liver between the mouse strains tested. As the class effects of the ASOs were not meaningfully different between CD-1, CByB6F1, or Tg.rasH2 mice, data from nonclinical studies in CD-1 mice can be used for dose selection and expectation of effect in the Tg.rasH2 mouse.

Safety, pharmacokinetics and pharmacodynamics of the anti-hepcidin Spiegelmer lexaptepid pegol in healthy subjects.

BACKGROUND AND PURPOSE: Anaemia of chronic disease is characterized by impaired erythropoiesis due to functional iron deficiency, often caused by excessive hepcidin. Lexaptepid pegol, a pegylated structured l-oligoribonucleotide, binds and inactivates hepcidin. EXPERIMENTAL APPROACH: We conducted a placebo-controlled study on the safety, pharmacokinetics and pharmacodynamics of lexaptepid after single and repeated i.v. and s.c. administration to 64 healthy subjects at doses from 0.3 to 4.8 mg·kg(-1) . KEY RESULTS: After treatment with lexaptepid, serum iron concentration and transferrin increased dose-dependently. Iron increased from approximately 20 µmol·L(-1) at baseline by 67% at 8 h after i.v. infusion of 1.2 mg·kg(-1) lexaptepid. The pharmacokinetics showed dose-proportional increases in peak plasma concentrations and moderately over-proportional increases in systemic exposure. Lexaptepid had no effect on hepcidin production or anti-drug antibodies. Treatment with lexaptepid was generally safe and well tolerated, with mild and transient transaminase increases at doses ≥2.4 mg·kg(-1) and with local injection site reactions after s.c. but not after i.v. administration. CONCLUSIONS AND IMPLICATIONS: Lexaptepid pegol inhibited hepcidin and dose-dependently raised serum iron and transferrin saturation. The compound is being further developed to treat anaemia of chronic disease.

Effect of the antihepcidin Spiegelmer lexaptepid on inflammation-induced decrease in serum iron in humans.

Increased hepcidin production is key to the development of anemia of inflammation. We investigated whether lexaptepid, an antihepcidin l-oligoribonucleotide, prevents the decrease in serum iron during experimental human endotoxemia. This randomized, double-blind, placebo-controlled trial was carried out in 24 healthy males. At T = 0 hours, 2 ng/kg Escherichia coli lipopolysaccharide was intravenously administered, followed by an intravenous injection of 1.2 mg/kg lexaptepid or placebo at T = 0.5 hours. The lipopolysaccharide-induced inflammatory response was similar in subjects treated with lexaptepid or placebo regarding clinical and biochemical parameters. At T = 9 hours, serum iron had increased by 15.9 ± 9.8 µmol/L from baseline in lexaptepid-treated subjects compared with a decrease of 8.3 ± 9.0 µmol/L in controls (P < .0001). This study delivers proof of concept that lexaptepid achieves clinically relevant hepcidin inhibition enabling investigations in the treatment of anemia of inflammation. This trial was registered at www.clinicaltrial.gov as #NCT01522794.

Renal uptake and tolerability of a 2'-O-methoxyethyl modified antisense oligonucleotide (ISIS 113715) in monkey.

The primary target organ for uptake of systemically administered phosphorothioate oligonucleotides is the kidney cortex and the proximal tubular epithelium in particular. To determine the effect of oligonucleotide uptake on renal function, a detailed renal physiology study was performed in cynomolgus monkeys treated with 10-40 mg/kg/week ISIS 113715 for 4 weeks. The concentrations of oligonucleotide in the kidney cortex ranged from 1400 to 2600 µg/g. These concentrations were associated with histologic changes in proximal tubular epithelial cells that ranged from the appearance of cytoplasmic basophilic granules to atrophic and degenerative changes at higher concentrations. However, there were no renal functional abnormalities as determined by the typical measurements of blood urea nitrogen, serum creatinine, creatinine clearance, or urine specific gravity. Nor were there changes in glomerular filtration rate, or renal blood flow. Specific urinary markers of tubular epithelial cell damage, such as N-acetyl-glucosaminidase, and α-glutathione-s-transferase were not affected. Tubular function was further evaluated by monitoring the urinary excretion of amino acids, ß(2)-microglobulin, or glucose. Renal function was challenged by administering a glucose load and by examining concentrating ability after a 4-h water deprivation. Neither challenge produced any evidence of change in renal function. The only change observed was a low incidence of increased urine protein/creatinine ratio in monkeys treated with ≥40 mg/kg/week which was rapidly reversible. Collectively, these data indicate that ISIS 113715-uptake by the proximal tubular epithelium has little or no effect on renal function at concentrations of 2600 µg/g.

Synthesis and biological activity of phosphonoacetate- and thiophosphonoacetate-modified 2'-O-methyl oligoribonucleotides.

Chimeric 2'-O-methyl oligoribonucleotides (2'-OMe ORNs) containing internucleotide linkages which were modified with phosphonoacetate (PACE) or thiophosphonoacetate (thioPACE) were prepared by solid-phase synthesis. The modified 2'-OMe ORNs contained a central phosphate or phosphorothioate sequence with up to 4 PACE or thioPACE modifications, respectively, at either end of the ORN in a "gapmer" motif. Both PACE and thioPACE 2'-OMe ORNs formed stable duplexes with complementary RNA. The majority of these duplexes had higher thermal melting temperatures than an unmodified RNA:RNA duplex. The modified 2'-OMe ORNs were effective passenger strands with complementary, unmodified siRNAs, for inducing siRNA activity in a dual luciferase assay in the presence of a lipid transfecting agent. As single strands, thioPACE 2'-OMe ORNs were efficiently taken up by HeLa cells in the absence of a lipid transfecting agent. Furthermore, thioPACE modifications greatly improved the potency of a 2'-OMe phosphorothioate ORN as an inhibitor of microRNA-122 in Huh7 cells, without lipid transfection.

Synthesis and preliminary evaluation of pro-RNA 2'-O-masked with biolabile pivaloyloxymethyl groups in an RNA interference assay.

The cellular delivery of bioactive nucleic acid-based drugs such as small interfering RNA (siRNA) represents a major technical hurdle for their pharmaceutical application. Prodrug-like approaches provide an attractive concept to address the delivery problem. With the aim to prepare RNA-based prodrugs bearing biolabile protections which facilitate cellular uptake and are prone to be removed enzymatically inside cells in order to release functional RNA, we synthesized pro-RNA totally or partially masked in 2'-OH position with pivaloyloxymethyl (PivOM) groups. A suitable strategy has been developed to synthesize and to purify base-sensitive mixed 2'-OH/2'-O-PivOM oligoribonucleotides, and to include them in siRNA. In this strategy, the fluoride labile [(triisopropylsilyl)oxy]-benzyloxycarbonyl group (tboc) as nucleobase protection (for A and C), the TBS group as 2'-OH protection and the Q-linker to solid-support were compatible with the PivOM groups masking some 2'-OH. We have taken advantage of the specific stability of the PivOM group to apply selected acidic, basic, and fluoride ions treatment for the deprotection and release of pro-RNA. This kind of pro-siRNA was studied in a human cell culture-based RNAi assay and preliminary promising data are discussed.

Spiegelmer inhibition of CCL2/MCP-1 ameliorates lupus nephritis in MRL-(Fas)lpr mice.

The monocyte chemoattractant protein CCL2 is crucial for monocyte and T cell recruitment from the vascular to the extravascular compartment at sites of inflammation. CCL2 is expressed in human lupus nephritis and was shown to mediate experimental lupus; therefore, CCL2 antagonists may be beneficial for therapy. This study describes the l-enantiomeric RNA oligonucleotide mNOX-E36, a so-called Spiegelmer that binds murine CCL2 with high affinity and neutralizes its action in vitro and in vivo. The mirror image configuration of the Spiegelmer confers nuclease resistance and thus excellent biostability. mNOX-E36 does not induce type I IFN via Toll-like receptor-7 or cytosolic RNA receptors, as recently shown for certain synthetic D-RNA. Autoimmune-prone MRL(lpr/lpr) mice that were treated with a polyethylene glycol form of mNOX-E36 from weeks 14 to 24 of age showed prolonged survival associated with a robust improvement of lupus nephritis, peribronchial inflammation, and lupus-like inflammatory skin lesions. Thus, mNOX-E36-based inhibition of CCL2 represents a novel strategy for the treatment of autoimmune tissue injury, such as lupus nephritis.

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.

Delivery of 2-5A cargo into living cells using the Tat cell penetrating peptide: 2-5A-tat.

2',5'-Oligoadenylate tetramer (2-5A) has been chemically conjugated to short HIV-1 Tat peptides to provide 2-5A-tat chimeras. Two different convergent synthetic approaches have been employed to provide such 2-5A-tat bioconjugates. One involved generation of a bioconjugate through reaction of a cysteine terminated Tat peptide with a alpha-chloroacetyl derivative of 2-5A. The second synthetic strategy was based upon a cycloaddition reaction of an azide derivative of 2-5A with a Tat peptide bearing an alkyne function. Either bioconjugate of 2-5A-tat was able to activate human RNase L. The union of 2-5A and Tat peptide provided an RNase L-active chimeric nucleopeptide with the ability to be taken up by cells by virtue of the Tat peptide and to activate RNase L in intact cells. This strategy provides a valuable vehicle for the entry of the charged 2-5A molecule into cells and may provide a means for targeted destruction of HIV RNA in vivo.

Lack of pharmacokinetic interaction for ISIS 113715, a 2'-0-methoxyethyl modified antisense oligonucleotide targeting protein tyrosine phosphatase 1B messenger RNA, with oral antidiabetic compounds metformin, glipizide or rosiglitazone.

BACKGROUND: ISIS 113715 is a 20-mer phosphorothioate antisense oligonucleotide (ASO) that is complementary to the protein tyrosine phosphatase 1B (PTP-1B) messenger RNA and subsequently reduces translation of the PTP-1B protein, a negative regulator of insulin receptor. ISIS 113715 is currently being studied in early phase II clinical studies to determine its ability to improve or restore insulin receptor sensitivity in patients with type 2 diabetes mellitus. Future work will investigate the combination of ISIS 113715 with antidiabetic compounds. METHODS: In vitro ultrafiltration human plasma protein binding displacement studies and a phase I clinical study were used to characterise the potential for pharmacokinetic interaction of ISIS 113715 and three marketed oral antidiabetic agents. ISIS 113715 was co-incubated with glipizide and rosiglitazone in whole human plasma and tested for increased free drug concentrations. In a phase I clinical study, 23 healthy volunteers received a single oral dose of an antidiabetic compound (either metformin, glipizide or rosiglitazone) both alone and together with subcutaneous ISIS 113715 200 mg in a sequential crossover design. A comparative pharmacokinetic analysis was performed to determine if there were any effects that resulted from coadministration of ISIS 113715 with these antidiabetic compounds. RESULTS: In vitro human plasma protein binding displacement studies showed only minor effects on rosiglitazone and no effect on glipizide when co-incubated with ISIS 113715. The results of the phase I clinical study further indicate that there were no measurable changes in glipizide (5 mg), metformin (500 mg) or rosiglitazone (2 mg) exposure parameters, maximum plasma concentration and the area under the concentration-time curve, or pharmacokinetic parameter, elimination half-life when coadministered with ISIS 113715. Furthermore, there was no effect of ISIS 113715, administered in combination with metformin, on the urinary excretion of metformin. Conversely, there were no observed alterations in ISIS 113715 pharmacokinetics when administered in combination with any of the oral antidiabetic compounds. CONCLUSION: These data provide evidence that ISIS 113715 exhibits no clinically relevant pharmacokinetic interactions on the disposition and clearance of the oral antidiabetic drugs. The results of these studies support further study of ISIS 113715 in combination with antidiabetic compounds.

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.

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.

Delivery of oligoribonucleotides to human hepatoma cells using cationic lipid particles conjugated to ferric protoporphyrin IX (heme).

The receptor-ligand interaction between hepatocyte heme receptors and heme was evaluated as a basis for developing a targeted cationic lipid delivery reagent for nucleic acids. Heme (ferric protoporphyrin IX) was conjugated to the aminolipid dioleoyl phosphatidylethanolamine (DOPE) and used to form cationic lipid particles with dioleoyl trimethylammonium propane (DOTAP). These lipids particles (DDH) protect oligoribonucleotides from degradation in human serum and increase oligoribonucleotide uptake into 2.2.15 human hepatoma cells (to a level of 50-60 ng oligo/10(4) cells) when compared with the same lipid particles (DD) prepared identically without heme. The DDH heme level that was optimal for oligoribonucleotide delivery was also optimal for maximum expression of plasmid-encoded luciferase. The enhancing effect of heme was evident only at net particle negative charge. Fluorescence microscopy showed that DDH delivered oligoribonucleotides into both the 2.2.15 cell cytoplasm and nucleus. DDH may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, appropriate for use in such liver diseases as viral hepatitis, hepatoma, and hypercholesterolemia.