Alternative Splicing in the Nuclear Receptor Superfamily Expands Gene Function to Refine Endo-Xenobiotic Metabolism.

The human genome encodes 48 nuclear receptor (NR) genes, whose translated products transform chemical signals from endo-xenobiotics into pleotropic RNA transcriptional profiles that refine drug metabolism. This review describes the remarkable diversification of the 48 human NR genes, which are potentially processed into over 1000 distinct mRNA transcripts by alternative splicing (AS). The average human NR expresses ∼21 transcripts per gene and is associated with ∼7000 single nucleotide polymorphisms (SNPs). However, the rate of SNP accumulation does not appear to drive the AS process, highlighting the resilience of NR genes to mutation. Here we summarize the altered tissue distribution/function of well characterized NR splice variants associated with human disease. We also describe a cassette exon visualization pictograph methodology for illustrating the location of modular, cassette exons in genes, which can be skipped in-frame, to facilitate the study of their functional relevance to both drug metabolism and NR evolution. We find cassette exons associated with all of the functional domains of NR genes including the DNA and ligand binding domains. The matrix of inclusion or exclusion for functional domain-encoding cassette exons is extensive and capable of significant alterations in cellular phenotypes that modulate endo-xenobiotic metabolism. Exon inclusion options are differentially distributed across NR subfamilies, suggesting group-specific conservation of resilient functionalities. A deeper understanding of this transcriptional plasticity expands our understanding of how chemical signals are refined and mediated by NR genes. This expanded view of the NR transcriptome informs new models of chemical toxicity, disease diagnostics, and precision-based approaches to personalized medicine. SIGNIFICANCE STATEMENT: This review explores the impact of alternative splicing (AS) on the human nuclear receptor (NR) superfamily and highlights the dramatic expansion of more than 1000 potential transcript variants from 48 individual genes. Xenobiotics are increasingly recognized for their ability to perturb gene splicing events, and here we explore the differential sensitivity of NR genes to AS and chemical exposure. Using the cassette exon visualization pictograph methodology, we have documented the conservation of splice-sensitive, modular, cassette exon domains among the 48 human NR genes, and we discuss how their differential expression profiles may augment cellular resilience to oxidative stress and fine-tune adaptive, metabolic responses to endo-xenobiotic exposure.

Alternative splicing of the vitamin D receptor modulates target gene expression and promotes ligand-independent functions.

Alternative splicing modulates gene function by creating splice variants with alternate functions or non-coding RNA activity. Naturally occurring variants of nuclear receptor (NR) genes with dominant negative or gain-of-function phenotypes have been documented, but their cellular roles, regulation, and responsiveness to environmental stress or disease remain unevaluated. Informed by observations that class I androgen and estrogen receptor variants display ligand-independent signaling in human cancer tissues, we questioned whether the function of class II NRs, like the vitamin D receptor (VDR), would also respond to alternative splicing regulation. Artificial VDR constructs lacking exon 3 (Dex3-VDR), encoding part of the DNA binding domain (DBD), and exon 8 (Dex8-VDR), encoding part of the ligand binding domain (LBD), were transiently transfected into DU-145 cells and stably-integrated into Caco-2 cells to study their effect on gene expression and cell viability. Changes in VDR promoter signaling were monitored by the expression of target genes (e.g. CYP24A1, CYP3A4 and CYP3A5). Ligand-independent VDR signaling was observed in variants lacking exon 8, and a significant loss of gene suppressor function was documented for variants lacking exon 3. The gain-of-function behavior of the Dex8-VDR variant was recapitulated in vitro using antisense oligonucleotides (ASO) that induce the skipping of exon 8 in wild-type VDR. ASO targeting the splice acceptor site of exon 8 significantly stimulated ligand-independent VDR reporter activity and the induction of CYP24A1 above controls. These results demonstrate how alternative splicing can re-program NR gene function, highlighting novel mechanisms of toxicity and new opportunities for the use of splice-switching oligonucleotides (SSO) in precision medicine.

AVI-7288 for Marburg Virus in Nonhuman Primates and Humans.

BACKGROUND: AVI-7288 is a phosphorodiamidate morpholino oligomer with positive charges that targets the viral messenger RNA that encodes Marburg virus (MARV) nucleoprotein. Its safety in humans is undetermined. METHODS: We assessed the efficacy of AVI-7288 in a series of studies involving a lethal challenge with MARV in nonhuman primates. The safety of AVI-7288 was evaluated in a randomized, multiple-ascending-dose study in which 40 healthy humans (8 humans per dose group) received 14 once-daily infusions of AVI-7288 (1 mg, 4 mg, 8 mg, 12 mg, or 16 mg per kilogram of body weight) or placebo, in a 3:1 ratio. We estimated the protective dose in humans by comparing pharmacokinetic variables in infected nonhuman primates, uninfected nonhuman primates, and uninfected humans. RESULTS: Survival in infected nonhuman primates was dose-dependent, with survival rates of 0%, 30%, 59%, 87%, 100%, and 100% among monkeys treated with 0 mg, 3.75 mg, 7.5 mg, 15 mg, 20 mg, and 30 mg of AVI-7288 per kilogram, respectively (P<0.001 with the use of the log-rank test for the comparison of survival across groups). No safety concern was identified at doses up to 16 mg per kilogram per day in humans. No serious adverse events were reported. Drug exposure (the area under the curve) was dose-dependent in both nonhuman primates and humans; drug clearance was independent of dose but was higher in nonhuman primates than in humans. The protective dose in humans was initially estimated, on the basis of exposure, to be 9.6 mg per kilogram per day (95% confidence interval, 6.6 to 12.5) for 14 days. Monte Carlo simulations supported a dose of 11 mg per kilogram per day to match the geometric mean protective exposure in nonhuman primates. CONCLUSIONS: This study shows that, on the basis of efficacy in nonhuman primates and pharmacokinetic data in humans, AVI-7288 has potential as postexposure prophylaxis for MARV infection in humans. (Funded by the Department of Defense; ClinicalTrials.gov number, NCT01566877.).

Alternative Splicing in the Cytochrome P450 Superfamily Expands Protein Diversity to Augment Gene Function and Redirect Human Drug Metabolism.

The human genome encodes 57 cytochrome P450 genes, whose enzyme products metabolize hundreds of drugs, thousands of xenobiotics, and unknown numbers of endogenous compounds, including steroids, retinoids, and eicosanoids. Indeed, P450 genes are the first line of defense against daily environmental chemical challenges in a manner that parallels the immune system. Several National Institutes of Health databases, including PubMed, AceView, and Ensembl, were queried to establish a comprehensive analysis of the full human P450 transcriptome. This review describes a remarkable diversification of the 57 human P450 genes, which may be alternatively processed into nearly 1000 distinct mRNA transcripts to shape an individual's P450 proteome. Important P450 splice variants from families 1A, 1B, 2C, 2D, 3A, 4F, 19A, and 24A have now been documented, with some displaying alternative subcellular distribution or catalytic function directly linked to a disease pathology. The expansion of P450 transcript diversity involves tissue-specific splicing factors, transformation-sensitive alternate splicing, trans-splicing between gene transcripts, single-nucleotide polymorphisms, and epigenetic regulation of alternate splicing. Homeostatic regulation of variant P450 expression is influenced also by nuclear receptor signaling, suppression of nonsense-mediated decay or premature termination codons, mitochondrial dysfunction, or host infection. This review focuses on emergent aspects of the adaptive gene-splicing process, which when viewed through the lens of P450-nuclear receptor gene interactions, resembles a primitive immune-like system that can rapidly monitor, respond, and diversify to acclimate to fluctuations in endo-xenobiotic exposure. Insights gained from this review should aid future drug discovery and improve therapeutic management of personalized drug regimens.

Safety and pharmacokinetic profiles of phosphorodiamidate morpholino oligomers with activity against ebola virus and marburg virus: results of two single-ascending-dose studies.

Two identical single-ascending-dose studies evaluated the safety and pharmacokinetics (PK) of AVI-6002 and AVI-6003, two experimental combinations of phosphorodiamidate morpholino oligomers with positive charges (PMOplus) that target viral mRNA encoding Ebola virus and Marburg virus proteins, respectively. Both AVI-6002 and AVI-6003 were found to suppress disease in virus-infected nonhuman primates in previous studies. AVI-6002 (a combination of AVI-7537 and AVI-7539) or AVI-6003 (a combination of AVI-7287 and AVI-7288) were administered as sequential intravenous (i.v.) infusions of a 1:1 fixed dose ratio of the two subcomponents. In each study, 30 healthy male and female subjects between 18 and 50 years of age were enrolled in six-dose escalation cohorts of five subjects each and received a single i.v. infusion of active study drug (0.005, 0.05, 0.5, 1.5, 3, and 4.5 mg/kg per component) or placebo in a 4:1 ratio. Both AVI-6002 and AVI-6003 were safe and well tolerated at the doses studied. A maximum tolerated dose was not observed in either study. The four chemically similar PMOplus components exhibited generally similar PK profiles. The mean peak plasma concentration and area under the concentration-time curve values of the four components exhibited dose-proportional PK. The estimated plasma half-life of all four components was 2 to 5 h. The safety of the two combinations and the PK of the four components were similar, regardless of the target RNA sequence.

Lymphocytic choriomeningitis virus infection in FVB mouse produces hemorrhagic disease.

The viral family Arenaviridae includes a number of viruses that can cause hemorrhagic fever in humans. Arenavirus infection often involves multiple organs and can lead to capillary instability, impaired hemostasis, and death. Preclinical testing for development of antiviral or therapeutics is in part hampered due to a lack of an immunologically well-defined rodent model that exhibits similar acute hemorrhagic illness or sequelae compared to the human disease. We have identified the FVB mouse strain, which succumbs to a hemorrhagic fever-like illness when infected with lymphocytic choriomeningitis virus (LCMV). FVB mice infected with LCMV demonstrate high mortality associated with thrombocytopenia, hepatocellular and splenic necrosis, and cutaneous hemorrhage. Investigation of inflammatory mediators revealed increased IFN-γ, IL-6 and IL-17, along with increased chemokine production, at early times after LCMV infection, which suggests that a viral-induced host immune response is the cause of the pathology. Depletion of T cells at time of infection prevented mortality in all treated animals. Antisense-targeted reduction of IL-17 cytokine responsiveness provided significant protection from hemorrhagic pathology. F1 mice derived from FVB×C57BL/6 mating exhibit disease signs and mortality concomitant with the FVB challenged mice, extending this model to more widely available immunological tools. This report offers a novel animal model for arenavirus research and pre-clinical therapeutic testing.

Gene-silencing antisense oligomers inhibit acinetobacter growth in vitro and in vivo.

BACKGROUND: Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA/RNA analogues that silence expression of specific genes. We studied whether PPMOs targeted to essential genes in Acinetobacter lwoffii and Acinetobacter baumannii are active in vitro and in vivo. METHODS: PPMOs were evaluated in vitro using minimum inhibitory concentration (MIC) and viability assays, and in vivo using murine pulmonary infection models with intranasal PPMO treatment. RESULTS: MICs of PPMOs ranged from 0.1 to 64 µM (approximately 0.6-38 µg/mL). The most effective PPMO tested was (RXR)4-AcpP, which is targeted to acpP. (RXR)4-AcpP reduced viability of A. lwoffii and A. baumannii by >10(3) colony-forming units/mL at 5-8 times MIC. Mice treated with ≥0.25 mg/kg of (RXR)4-AcpP survived longer and had less inflammation and bacterial lung burden than mice treated with a scrambled-sequence PPMO or phosphate-buffered saline. Treatment could be delayed after infection and still increase survival. CONCLUSIONS: PPMOs targeted to essential genes of A. lwoffii and A. baumannii were bactericidal and had MICs in a clinically relevant range. (RXR)4-AcpP increased survival of mice infected with A. lwoffii or A. baumannii, even when initial treatment was delayed after infection. PPMOs could be a viable therapeutic approach in dealing with multidrug-resistant Acinetobacter species.

Arginine-rich cell-penetrating peptide dramatically enhances AMO-mediated ATM aberrant splicing correction and enables delivery to brain and cerebellum.

Antisense morpholino oligonucleotides (AMOs) can reprogram pre-mRNA splicing by complementary binding to a target site and regulating splice site selection, thereby offering a potential therapeutic tool for genetic disorders. However, the application of this technology into a clinical scenario has been limited by the low correction efficiency in vivo and inability of AMOs to efficiently cross the blood brain barrier and target brain cells when applied to neurogenetic disorders such as ataxia-telangiecatasia (A-T). We previously used AMOs to correct subtypes of ATM splicing mutations in A-T cells; AMOs restored up to 20% of the ATM protein and corrected the A-T cellular phenotype. In this study, we demonstrate that an arginine-rich cell-penetrating peptide, (RXRRBR)(2)XB, dramatically improved ATM splicing correction efficiency when conjugated with AMOs, and almost fully corrected aberrant splicing. The restored ATM protein was close to normal levels in cells with homozygous splicing mutations, and a gene dose effect was observed in cells with heterozygous mutations. A significant amount of the ATM protein was still detected 21 days after a single 5 µm treatment. Systemic administration of an fluorescein isothiocyanate-labeled (RXRRBR)(2)XB-AMO in mice showed efficient uptake in the brain. Fluorescence was evident in Purkinje cells after a single intravenous injection of 60 mg/kg. Furthermore, multiple injections significantly increased uptake in all areas of the brain, notably in cerebellum and Purkinje cells, and showed no apparent signs of toxicity. Taken together, these results highlight the therapeutic potential of (RXRRBR)(2)XB-AMOs in A-T and other neurogenetic disorders.

A novel therapeutic vaccine targeting the soluble TNFα receptor II to limit the progression of cardiovascular disease: AtheroVax™.

The burden of atherosclerotic cardiovascular disease contributes to a large proportion of morbidity and mortality, globally. Vaccination against atherosclerosis has been proposed for over 20 years targeting different mediators of atherothrombosis; however, these have not been adequately evaluated in human clinical trials to assess safety and efficacy. Inflammation is a driver of atherosclerosis, but inflammatory mediators are essential components of the immune response. Only pathogenic forms of sTNFR2 are acted upon while preserving the membrane-bound (wild-type) TNFR2 contributions to a non-pathogenic immune response. We hypothesize that the inhibition of sTNRF2 will be more specific and offer long-term treatment options. Here we describe pre-clinical findings of an sTNFR2-targeting peptide vaccine (AtheroVax™) in a mouse model. The multiple pathways to synthesis of the soluble TNFRII receptor (sTNFRII) were identified as sTNFRII(PC), sTNFRII(Δ7), and sTNFRII(Δ7,9). The sTNFRII(Δ7) peptide, NH2-DFALPVEKPLCLQR-COOH is specific to sTNFR2 based on an mRNA splice-variant in which exon 6 is joined to exon 8. The role of sTNFRII(Δ7) as a mediator of prolonged TNFα activity by preventing degradation and clearance was investigated. Inflammation is a critical driver of onset, progression and expansion of atherosclerosis. The TNFα ligand represents a driver of inflammation that is mediated by a splice variant of TNFR2, referred to as sTNFRII(Δ7). The multiple forms of TNFRII, both membrane bound and soluble, are associated with distinctly different phenotypes. sTNFRII(PC) and sTNFRII(Δ7) are not equivalent to etanercept because they lack a clearance mechanism. The unique peptide associated with sTNFRII(Δ7) contains a linear B-cell epitope with amino acids from both exon 6 and exon 8 supporting the vaccine design. Animal studies to evaluate the vaccine are ongoing, and results will be forthcoming. We describe a peptide vaccine targeting sTNFR2 in limiting the progression of atherosclerosis. A therapeutic vaccine limiting the progression of atherosclerosis will greatly contribute to the reduction in morbidity and mortality from cardiovascular disease. It is likely the vaccine will be used in combination with the current standards of care and lifestyle modifications.

Bacterial resistance to antisense peptide phosphorodiamidate morpholino oligomers.

Peptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF)(3)RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is ß-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential gene acpP (which encodes acyl carrier protein). The MIC of (RFF)(3)RXB-AcpP was 2.5 µM (14 µg/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF)(3)RXB-AcpP was 4 × 10(-7) mutations/cell division. A spontaneous (RFF)(3)RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)(3)RXB-AcpP was 40 µM (224 µg/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)(3)RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF)(3)RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF)(3)RXB-AcpP. Deletion of sbmA caused resistance to (RFF)(3)RXB-AcpP. We conclude that resistance to (RFF)(3)RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF)(3)R-XB PPMOs may be transported across the plasma membrane by SbmA.

The development of broad-spectrum antiviral medical countermeasures to treat viral hemorrhagic fevers caused by natural or weaponized virus infections.

The Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense (JPEO-CBRND) began development of a broad-spectrum antiviral countermeasure against deliberate use of high-consequence viral hemorrhagic fevers (VHFs) in 2016. The effort featured comprehensive preclinical research, including laboratory testing and rapid advancement of lead molecules into nonhuman primate (NHP) models of Ebola virus disease (EVD). Remdesivir (GS-5734, Veklury, Gilead Sciences) was the first small molecule therapeutic to successfully emerge from this effort. Remdesivir is an inhibitor of RNA-dependent RNA polymerase, a viral enzyme that is essential for viral replication. Its robust potency and broad-spectrum antiviral activity against certain RNA viruses including Ebola virus and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) led to its clinical evaluation in randomized, controlled trials (RCTs) in human patients during the 2018 EVD outbreak in the Democratic Republic of the Congo (DRC) and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic today. Remdesivir was recently approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19 requiring hospitalization. Substantial gaps remain in improving the outcomes of acute viral infections for patients afflicted with both EVD and COVID-19, including how to increase therapeutic breadth and strategies for the prevention and treatment of severe disease. Combination therapy that joins therapeutics with complimentary mechanisms of action appear promising, both preclinically and in RCTs. Importantly, significant programmatic challenges endure pertaining to a clear drug and biological product development pathway for therapeutics targeting biodefense and emerging pathogens when human efficacy studies are not ethical or feasible. For example, remdesivir's clinical development was facilitated by outbreaks of Ebola and SARS-CoV-2; as such, the development pathway employed for remdesivir is likely to be the exception rather than the rule. The current regulatory licensure pathway for therapeutics targeting rare, weaponizable VHF agents is likely to require use of FDA's established Animal Rule (21 CFR 314.600-650 for drugs; 21 CFR 601.90-95 for biologics). The FDA may grant marketing approval based on adequate and well-controlled animal efficacy studies when the results of those studies establish that the drug is safe and likely to produce clinical benefit in humans. In practical terms, this is anticipated to include a series of rigorous, well-documented, animal challenge studies, to include aerosol challenge, combined with human safety data. While small clinical studies against naturally occurring, high-consequence pathogens are typically performed where possible, approval for the therapeutics currently under development against biodefense pathogens will likely require the Animal Rule pathway utilizing studies in NHPs. We review the development of remdesivir as illustrative of the effort that will be needed to field future therapeutics against highly lethal, infectious agents.

Eastern equine encephalitis virus rapidly infects and disseminates in the brain and spinal cord of cynomolgus macaques following aerosol challenge.

Eastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state-of-the-art telemetry to measure critical physiological parameters. Here, we report the results of a comprehensive pathology study of NHP tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61-68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation into EEEV pathology in a NHP model and will provide significant insights into the evaluation of countermeasure.

Ebola virus persistence and disease recrudescence in the brains of antibody-treated nonhuman primate survivors.

Effective therapeutics have been developed against acute Ebola virus disease (EVD) in both humans and experimentally infected nonhuman primates. However, the risk of viral persistence and associated disease recrudescence in survivors receiving these therapeutics remains unclear. In contrast to rhesus macaques that survived Ebola virus (EBOV) exposure in the absence of treatment, we discovered that EBOV, despite being cleared from all other organs, persisted in the brain ventricular system of rhesus macaque survivors that had received monoclonal antibody (mAb) treatment. In mAb-treated macaque survivors, EBOV persisted in macrophages infiltrating the brain ventricular system, including the choroid plexuses. This macrophage infiltration was accompanied by severe tissue damage, including ventriculitis, choroid plexitis, and meningoencephalitis. Specifically, choroid plexus endothelium-derived EBOV infection led to viral persistence in the macaque brain ventricular system. This resulted in apoptosis of ependymal cells, which constitute the blood-cerebrospinal fluid barrier of the choroid plexuses. Fatal brain-confined recrudescence of EBOV infection manifested as severe inflammation, local pathology, and widespread infection of the ventricular system and adjacent neuropil in some of the mAb-treated macaque survivors. This study highlights organ-specific EBOV persistence and fatal recrudescent disease in rhesus macaque survivors after therapeutic treatment and has implications for the long-term follow-up of human survivors of EVD.

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer.

Antisense oligonucleotide-mediated exon skipping is able to correct out-of-frame mutations in Duchenne muscular dystrophy and restore truncated yet functional dystrophins. However, its application is limited by low potency and inefficiency in systemic delivery, especially failure to restore dystrophin in heart. Here, we conjugate a phosphorodiamidate morpholino oligomer with a designed cell-penetrating peptide (PPMO) targeting a mutated dystrophin exon. Systemic delivery of the novel PPMO restores dystrophin to almost normal levels in the cardiac and skeletal muscles in dystrophic mdx mouse. This leads to increase in muscle strength and prevents cardiac pump failure induced by dobutamine stress in vivo. Muscle pathology and function continue to improve during the 12-week course of biweekly treatment, with significant reduction in levels of serum creatine kinase. The high degree of potency of the oligomer in targeting all muscles and the lack of detectable toxicity and immune response support the feasibility of testing the novel oligomer in treating Duchenne muscular dystrophy patients.

Antisense phosphorodiamidate morpholino oligomers targeted to an essential gene inhibit Burkholderia cepacia complex.

BACKGROUND: Members of the Burkholderia cepacia complex (Bcc) cause considerable morbidity and mortality in patients with chronic granulomatous disease and cystic fibrosis. Many Bcc strains are antibiotic resistant, which requires the exploration of novel antimicrobial approaches, including antisense technologies such as phosphorodiamidate morpholino oligomers (PMOs). METHODS: Peptide-conjugated PMOs (PPMOs) were developed to target acpP, which encodes an acyl carrier protein (AcpP) that is thought to be essential for growth. Their antimicrobial activities were tested against different strains of Bcc in vitro and in infection models. RESULTS: PPMOs targeting acpP were bactericidal against clinical isolates of Bcc (>4 log reduction), whereas a PPMO with a scrambled base sequence (scrambled PPMO) had no effect on growth. Human neutrophils were infected with Burkholderia multivorans and treated with AcpP PPMO. AcpP PPMO augmented killing, compared with neutrophils alone and compared with neutrophils alone plus scrambled PPMO. Mice with chronic granulomatous disease that were infected with B. multivorans were treated with AcpP PPMO, scrambled PPMO, or water at 0, 3, and 6 h after infection. Compared with water-treated control mice, the AcpP PPMO-treated mice showed an approximately 80% reduction in the risk of dying by day 30 of the experiment and relatively little pathology. CONCLUSION: AcpP PPMO is active against Bcc infections in vitro and in vivo.

Antisense oligonucleotide development for the selective modulation of CYP3A5 in renal disease.

CYP3A5 is the primary CYP3A subfamily enzyme expressed in the human kidney and its aberrant expression may contribute to a broad spectrum of renal disorders. Pharmacogenetic studies have reported inconsistent linkages between CYP3A5 expression and hypertension, however, most investigators have considered CYP3A5*1 as active and CYP3A5*3 as an inactive allele. Observations of gender specific differences in CYP3A5*3/*3 protein expression suggest additional complexity in gene regulation that may underpin an environmentally responsive role for CYP3A5 in renal function. Reconciliation of the molecular mechanism driving conditional restoration of functional CYP3A5*3 expression from alternatively spliced transcripts, and validation of a morpholino-based approach for selectively suppressing renal CYP3A5 expression, is the focus of this work. Morpholinos targeting a cryptic splice acceptor created by the CYP3A5*3 mutation in intron 3 rescued functional CYP3A5 expression in vitro, and salt-sensitive cellular mechanisms regulating splicing and conditional expression of CYP3A5*3 transcripts are reported. The potential for a G-quadruplex (G4) in intron 3 to mediate restored splicing to exon 4 in CYP3A5*3 transcripts was also investigated. Finally, a proximal tubule microphysiological system (PT-MPS) was used to evaluate the safety profile of morpholinos in proximal tubule epithelial cells, highlighting their potential as a therapeutic platform for the treatment of renal disease.

Remdesivir is efficacious in rhesus monkeys exposed to aerosolized Ebola virus.

Efficacious therapeutics for Ebola virus disease are in great demand. Ebola virus infections mediated by mucosal exposure, and aerosolization in particular, present a novel challenge due to nontypical massive early infection of respiratory lymphoid tissues. We performed a randomized and blinded study to compare outcomes from vehicle-treated and remdesivir-treated rhesus monkeys in a lethal model of infection resulting from aerosolized Ebola virus exposure. Remdesivir treatment initiated 4 days after exposure was associated with a significant survival benefit, significant reduction in serum viral titer, and improvements in clinical pathology biomarker levels and lung histology compared to vehicle treatment. These observations indicate that remdesivir may have value in countering aerosol-induced Ebola virus disease.

Cationic phosphorodiamidate morpholino oligomers efficiently prevent growth of Escherichia coli in vitro and in vivo.

OBJECTIVES: Phosphorodiamidate morpholino oligomers (PMOs) are uncharged DNA analogues that can inhibit bacterial growth by a gene-specific, antisense mechanism. Attaching cationic peptides to PMOs enables efficient penetration through the Gram-negative outer membrane. We hypothesized that cationic groups attached directly to the PMO would obviate the need to attach peptides. METHODS: PMOs with identical 11-base sequence (AcpP) targeted to acpP (an essential gene) of Escherichia coli were synthesized with various numbers of either piperazine (Pip) or N-(6-guanidinohexanoyl)piperazine (Gux) coupled to the phosphorodiamidate linker. Peptide-PMO conjugates were made using the membrane-penetrating peptide (RXR)(4)XB (X is 6-aminohexanoic acid; B is beta-alanine). RESULTS: MICs (microM/mg/L) were measured using E. coli: 3 + Pip-AcpP, 160/653; 6 + Pip-AcpP, 160/673; 2 + Gux-AcpP, 20/88; 5 + Gux-AcpP, 10/49; 8 + Gux-AcpP, 10/56; 3 + Pip-AcpP-(RXR)(4)XB, 0.3/2; and 5 + Gux-AcpP-(RXR)(4)XB, 0.6/4. In cell-free protein synthesis reactions, all PMOs inhibited gene expression approximately the same. These results suggested that Pip-PMOs inefficiently penetrated the outer membrane. Indeed, the MICs of 3 + Pip-AcpP and 6 + Pip-AcpP were reduced to 0.6 and 2.5 microM (1.2 and 10.5 mg/L), respectively, using as indicator a strain with a 'leaky' outer membrane. In vivo, mice were infected intraperitoneally with E. coli. Intraperitoneal treatment with 50 mg/kg 3 + Pip-AcpP, 15 mg/kg 5 + Gux-AcpP or 0.5 mg/kg 3 + Pip-AcpP-(RXR)(4)XB, or subcutaneous treatment with 15 mg/kg 5 + Gux-AcpP or (RXR)(4)XB-AcpP reduced bacteria in blood and increased survival. CONCLUSIONS: Cationic PMOs inhibited bacterial growth in vitro and in vivo, and Gux-PMOs were more effective than Pip-PMOs. However, neither was as effective as the equivalent PMO-peptide conjugates. Subcutaneous treatment showed that 5 + Gux-AcpP or (RXR)(4)XB-AcpP entered the circulatory system, reduced infection and increased survival.

Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies.

Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)(4) (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)(4)-PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure-activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs.

Recent successes in therapeutics for Ebola virus disease: no time for complacency.

The PALM trial in the Democratic Republic of the Congo identified a statistically significant survival benefit for two monoclonal antibody-based therapeutics in the treatment of acute Ebola virus disease; however, substantial gaps remain in improving the outcomes of acute Ebola virus disease and for the survivors. Ongoing efforts are needed to develop more effective strategies, particularly for individuals with severe disease, for prevention and treatment of viral persistence in immune-privileged sites, for optimisation of post-exposure prophylaxis, and to increase therapeutic breadth. As antibody-based approaches are identified and advanced, promising small-molecule antivirals currently in clinical stage development should continue to be evaluated for filovirus diseases, with consideration of their added value in combination approaches with bundled supportive care, their penetration in tissues of interest, the absence of interaction with glycoprotein-based vaccines, and filoviral breadth.