Off target toxicities and links with physicochemical properties of medicinal products, including antibiotics, oligonucleotides, lipid nanoparticles (with cationic and/or anionic charges). Data review suggests an emerging pattern.

Toxicology is an essential part of any drug development plan. Circumnavigating the risk of failure because of a toxicity issue can be a challenge, and failure in late development is extremely costly. To identify potential risks, it requires more than just understanding the biological target. The toxicologist needs to consider a compound's structure, it's physicochemical properties (including the impact of the overall formulation), as well as the biological target (e.g., receptor interactions). Understanding the impact of the physicochemical properties can be used to predict potential toxicities in advance by incorporating key endpoints in early screening strategies and/or used to compare toxicity profiles across lead candidates. This review discussed the risks of off-target and/or non-specific toxicities that may be associated with the physicochemical properties of compounds, especially those carrying dominant positive or negative charges, including amphiphilic small molecules, peptides, oligonucleotides and lipids/liposomes/lipid nanoparticles. The latter of which are being seen more and more in drug development, including the recent Covid pandemic, where mRNA and lipid nanoparticle technology is playing more of a role in vaccine development. The translation between non-clinical and clinical data is also considered, questioning how a physicochemical driven toxicity may be more universal across species, which means that such toxicity may be reassuringly translatable between species and as such, this information may also be considered as a support to the 3 R's, particularly in the early screening stages of a drug development plan.

RNA-based drug discovery for spinal muscular atrophy: a story of small molecules and antisense oligonucleotides.

INTRODUCTION: Spinal Muscular Atrophy (SMA), the second most prevalent autosomal genetic disease affecting infants, is caused by the lack of SMN1, which encodes a neuron functioning vital protein, SMN. Improving exon 7 splicing in the paralogous gene SMN2, also coding for SMN protein, increases protein production efficiency from SMN2 to overcome the genetic deficit in SMN1. Several molecular mechanisms have been investigated to improve SMN2 functional splicing. AREAS COVERED: This manuscript will cover two of the three mechanistically distinct available treatment options for SMA, both targeting the SMN2 splicing mechanism. The first therapeutic, nusinersen (Spinraza®, 2017), is an antisense oligonucleotide (ASO) targeting the splicing inhibitory sequence in the intron downstream of exon 7 from SMN2, thus increasing exon 7 inclusion. The second drug is a small molecule, risdiplam (Evrysdi®, 2021), that enhances the binding of splice factors and also promotes exon 7 inclusion. Both therapies, albeit through different mechanisms, increase full-length SMN protein expression. EXPERT OPINION: Nusinersen and risdiplam have directly helped SMA patients and families, but they also herald a sea change in drug development for genetic diseases. This piece aims to draw parallels between both development histories; this may help chart the course for future targeted agents.

Local and systemic tolerability of a 2'O-methoxyethyl antisense oligonucleotide targeting interleukin-4 receptor-α delivery by inhalation in mouse and monkey.

Antisense oligonucleotides (ASOs) bind and facilitate degradation of RNA and inhibit protein expression in pathways not easily targeted with small molecules or antibodies. Interleukin (IL)-4 and IL-13 potentiate signaling through the shared IL-4 receptor-α (IL-4Rα) subunit of their receptors. ASO targeting of IL-4Rα mRNA in a mouse model of asthma led to attenuation of airway hyperactivity, demonstrating potential benefit in asthma patients. This study focused on tolerability of inhaled IL-4Rα-targeting ASOs. Toxicity studies were performed with mouse- (ISIS 23189) and human-specific (ISIS 369645) sequences administered by inhalation. Four week (monkey) or 13 week (mouse) repeat doses at levels of up to 15 mg/kg/exposure (exp) and 50 mg/kg/exp, respectively, demonstrated dose-dependent effects limited to increases in macrophage size and number in lung and tracheobronchial lymph nodes. The changes were largely non-specific, reflecting adaptive responses that occur during active exposure and deposition of ASO and other material in the lung. Reversibility was observed at a rate consistent with the kinetics of tissue clearance of ASO. Systemic bioavailability was minimal, and no systemic toxicity was observed at exposure levels appreciably above pharmacological doses and doses proposed for clinical trials.

Delivery and biodistribution of siRNA for cancer therapy: challenges and future prospects.

RNAi-based approaches provide a promising therapeutic modality for the treatment of cancer. The inaccessibility of tumors in different cancer types necessitates the development of safe, specific and efficient systemic delivery systems to meet therapeutic need. The translation of siRNA-based cancer therapeutics to the clinic is hindered by several challenges associated with the cargo (siRNA) and the delivery system, including susceptibility to nucleases; insufficient circulation half-life due to phagocytosis by the reticuloendothelial system, transient and poor biodistribution in the tumor tissue; cellular uptake; inability to escape endosomes and release into the cytosolic compartment for an RNAi-mediated effect; microRNA-like unintended off-target effects; undesirable immune stimulation; and carrier-related toxicity. This review provides an overview of the pharmacokinetic and biodistribution challenges witnessed in the delivery of siRNA when administered systemically. It also describes the current delivery approaches using liposome-, polymer- and peptide-based delivery systems shown to elicit significant gene silencing and tumor growth regression in proof-of-concept studies. As part of future perspectives, delivery agents that showed significant efficacy in preclinical rodent models and clinical trials are also reviewed.

An amino acid-based amphoteric liposomal delivery system for systemic administration of siRNA.

We demonstrate a systematic and rational approach to create a library of natural and modified, dialkylated amino acids based upon arginine for development of an efficient small interfering RNA (siRNA) delivery system. These amino acids, designated DiLA2 compounds, in conjunction with other components, demonstrate unique properties for assembly into monodisperse, 100-nm small liposomal particles containing siRNA. We show that DiLA2-based liposomes undergo a pH-dependent phase transition to an inverted hexagonal phase facilitating efficient siRNA release from endosomes to the cytosol. Using an arginine-based DiLA2, cationic liposomes were prepared that provide high in vivo siRNA delivery efficiency and are well-tolerated in both cell and animal models. DiLA2-based liposomes demonstrate a linear dose-response with an ED50 of 0.1 mg/kg against liver-specific target genes in BALB/c mice.

RNAi-based therapeutics targeting survivin and PLK1 for treatment of bladder cancer.

Harnessing RNA interference (RNAi) to silence aberrant gene expression is an emerging approach in cancer therapy. Selective inhibition of an overexpressed gene via RNAi requires a highly efficacious, target-specific short interfering RNA (siRNA) and a safe and efficient delivery system. We have developed siRNA constructs (UsiRNA) that contain unlocked nucleobase analogs (UNA) targeting survivin and polo-like kinase-1 (PLK1) genes. UsiRNAs were encapsulated into dialkylated amino acid-based liposomes (DiLA(2)) containing a nor-arginine head group, cholesteryl hemisuccinate (CHEMS), cholesterol and 1, 2-dimyristoyl-phosphatidylethanolamine-polyethyleneglycol 2000 (DMPE-PEG2000). In an orthotopic bladder cancer mouse model, intravesical treatment with survivin or PLK1 UsiRNA in DiLA(2) liposomes at 1.0 and 0.5 mg/kg resulted in 90% and 70% inhibition of survivin or PLK1 mRNA, respectively. This correlated with a dose-dependent decrease in tumor volumes which was sustained over a 3-week period. Silencing of survivin and PLK1 mRNA was confirmed to be RNA-induced silencing complex mediated as specific cleavage products were detected in bladder tumors over the duration of the study. This report suggests that intravesical instillation of survivin or PLK1 UsiRNA can serve as a potential therapeutic modality for treatment of bladder cancer.

Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs.

siRNAs confer sequence specific and robust silencing of mRNA. By virtue of these properties, siRNAs have become therapeutic candidates for disease intervention. However, their use as therapeutic agents can be hampered by unintended off-target effects by either or both strands of the siRNA duplex. We report here that unlocked nucleobase analogs (UNAs) confer desirable properties to siRNAs. Addition of a single UNA at the 5'-terminus of the passenger strand blocks participation of the passenger strand in RISC-mediated target down-regulation with a concomitant increase in guide strand activity. Placement of a UNA in the seed region of the guide strand prevents miRNA-like off-target silencing without compromising siRNA activity. Most significantly, combined substitution of UNA at the 3'-termini of both strands, the addition of a UNA at the 5'-terminus of the passenger strand, and a single UNA in the seed region of the guide strand, reduced the global off-target events by more than 10-fold compared to unmodified siRNA. The reduction in off-target events was specific to UNA placement in the siRNA, with no apparent new off-target events. Taken together, these results indicate that when strategically placed, UNA substitutions have important implications for the design of safe and effective siRNA-based therapeutics.

A potential therapeutic for pandemic influenza using RNA interference.

RNA interference (RNAi) involves sequence-specific downregulation of target genes, leading to gene silencing in vitro and in vivo. Synthetic small interfering RNAs (siRNAs), formulated with appropriate delivery agents, can serve as effective tools for RNAi-based therapeutics. The potential of siRNA to provide antiviral activity has been studied extensively in many respiratory viruses, including influenza virus, wherein specific siRNAs target highly-conserved regions of influenza viral genome, leading to potent inhibition of viral RNA replication. Despite various delivery strategies, such as polycations and liposomes that have been employed to formulate siRNAs, effective delivery modalities are still needed. Although current strategies can provide significant biodistribution and delivery into lungs allowing gene silencing, complete protection and prolonged survival rates against multiple strains of influenza virus still remains a key challenge. Here, we describe methods and procedures pertaining to screening and selection of highly effective influenza-specific siRNAs in cell culture, in mice, and in the ferret model. This will be potentially useful to evaluate RNAi as a therapeutic modality for future clinical application.

Inhibition of human metapneumovirus replication by small interfering RNA.

BACKGROUND: Human metapneumovirus (hMPV) is a major respiratory viral pathogen in young children, elderly individuals and immunocompromised patients. Despite its major effects related to bronchiolitis, pneumonia and its potential role in recurrent wheezing episodes, there is still no commercial treatment or vaccine available against this paramyxovirus. METHODS: We tested a therapeutic strategy for hMPV that was based on RNA interference. RESULTS: An hMPV genome-wide search for small interfering RNAs (siRNAs) by computational analysis revealed 200 potentially effective 21-mer siRNAs. Initial screening with a luciferase assay identified 57 siRNAs of interest. Further evaluation of their inhibitory potential against the four hMPV subgroups by quantitative real-time reverse transcriptase PCR and plaque immunoassay identified two highly potent siRNAs with 50% inhibitory concentration (IC50) values in the subnanomolar range. siRNA45 targets the nucleoprotein messenger RNA (mRNA) and had IC50 values <0.078 nM against representative strains from the four hMPV subgroups, whereas siRNA60, which targets the phosphoprotein mRNA, had IC50 values between 0.090-<0.078 nM against the same panel of hMPV strains. Longer25/27-mer siRNAs known as Dicer substrates designed from the top two siRNA candidates were also evaluated and were at least as effective as their corresponding 21-mer siRNAs. Interestingly, the presence of one or two nucleotide mismatches in the target mRNA sequence of some hMPV subgroups did not always affect hMPV inhibition in vitro. CONCLUSIONS: We successfully identified two highly efficient siRNAs against hMPV targeting essential components of the hMPV replication complex.

Maternal and fetal distribution of a phosphorothioate oligonucleotide in rats after intravenous infusion.

BACKGROUND: Fetal uptake of an antisense oligonucleotide was evaluated after intravenous (i.v.) dosing of ISIS 2105, a 20-base phosphorothioate oligonucleotide, in timed-pregnant Sprague-Dawley rats. METHODS: To maximize the potential for fetal exposure, ISIS 2105 was administered as a 3-hr infusion at 6.6 mg/kg/hr with a total dose of 20 mg/kg, or as a continuous 7-day infusion at 0.35 mg/kg/hr with a total dose of 59 mg/kg. This dosing regime is higher than a patient would be expected to receive in the clinical use of oligonucleotides. Infusions were delivered through a jugular vein cannula by syringe pump on gestation day (GD) 19 (3-hr exposure) or by osmotic pumps implanted subcutaneously (s.c.) starting on GD 12 (7-day exposures). RESULTS: After a 3-hr infusion, maternal and fetal plasma concentrations of ISIS 2105 were >100 microg/ml and <0.07 microg/ml, respectively with a maternal fetal ratio of >1,000. Maternal regions of the placenta had twice the oligonucleotide concentration compared to fetal regions of the placenta (6 microg/g vs. 3 microg/g). After this acute exposure the concentrations in fetal kidney and liver were approximately 140- and 500-fold less than the maternal kidney and liver respectively. After 7-day infusion maternal plasma concentrations were 0.82 microg/ml and fetal concentrations were <0.22 microg/ml. By capillary gel electrophoresis (CGE) only the fetal liver consistently had quantifiable oligonucleotide concentrations (range=1.01-4.95 microg/g) compared to a mean concentration of 50.11+/-1.71 microg/g in the maternal liver a maternal to fetal ratio of approximately 10:50 after 7 days of infusion. CONCLUSIONS: There was a low level of transfer from dam to fetus, consistent with a slow equilibrium but the permeability of placenta to this 6 kDa polyanionic compound seemed to be limited even at supraclinical doses.