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Background: Xenobiotic exposures can extensively influence the expression and alternative splicing of drug-metabolizing enzymes, including cytochromes P450 (CYPs), though their transcriptome-wide impact on splicing remains underexplored. This study used a well-characterized splicing event in the Cyp2b2 gene to validate a sandwich-cultured primary rat hepatocyte model for studying global splicing in vitro. Using endpoint PCR, RNA sequencing, and bioinformatics tools (rSeqDiff, rMATs, IGV), we analyzed differential gene expression and splicing in CYP and nuclear receptor genes, as well as the entire transcriptome, to understand how xenobiotic exposures shape alternative splicing and activate xenosensors., Methods: Primary rat hepatocytes in sandwich culture were exposed to two methylenedioxybenzene (MDB) congeners and carbamazepine, with gene expression and splicing assessed. A 3D-clustergram integrating KEGG pathway analysis with differential gene expression provided distinct splicing landscapes for each xenobiotic, showing that splicing diversity does not always align with gene expression changes., Results: Endpoint PCR revealed a Cyp2b2v to wild-type Cyp2b2 splicing ratio near 1:1 (100%) under most conditions, while RNA-seq showed a stable baseline closer to 40%. C6-MDB reduced this ratio to ~ 50% by PCR and ~ 39% by RNA-seq, showing slight method-dependent variations yet consistent trends. In contrast, exon 6 skipping in Cyp1a1 occurred only with MDB exposure, implicating AHR activation. Xenobiotic treatments also induced alternative splicing in defensome and stress-responsive genes, including the phase II enzyme Gstm3, Albumin, Orm1, and Fxyd1, highlighting their roles in xenobiotic response modulation. Significant splicing changes in factors such as SRSF1, SRSF7, and METTL3 suggest a coordinated feedback loop involving epitranscriptomic modulation and cross-talk within SR protein networks, refining splice site selection, transcript stability, and cellular fate., Conclusions: This study demonstrates how xenobiotic structural features influence gene expression and splicing, revealing splicing patterns that expand our understanding of transcriptome diversity and function. By identifying regulatory mechanisms, including AHR activation, epitranscriptomic modulation, and crosstalk within SR protein networks, that shape adaptive responses to xenobiotic stress, this work offers insights into the splicing and transcriptional networks that maintain cellular homeostasis. These findings provide predictive biomarkers for toxic exposures and highlight the potential of splicing profiles as diagnostic tools for assessing the health impacts of chemical exposure., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

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., Competing Interests: The authors PI, NiK, and NoK all are co-inventors of the AtheroVax vaccine and share interest in commercial development of the vaccine. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Iversen, Kipshidze, Kipshidze, Dangas, Ramacciotti, Kakabadze and Fareed.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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., (© 2021. The Author(s).)

Competing Interests: We declare no competing interests.

Competing Interests: We declare no competing interests.

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.

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ewing's sarcoma treatment failures are associated with high mortality indicating a need for new therapeutic approaches. We used a k-mer counting approach to identify cancer-specific mRNA transcripts in 3 Ewing's Family Tumor (EFT) cell lines not found in the normal human transcriptome. Phosphorodiamidate morpholino oligomers targeting six EFT-specific transcripts were evaluated for cytotoxicity in TC-32 and CHLA-10 EFT lines and in HEK293 renal epithelial control cells. Average morpholino efficacy (EC 50 ) was 0.66 ± 0.13 in TC-32, 0.25 ± 0.14 in CHLA-10 and 3.07 ± 5.02 µM in HEK293 control cells (ANOVA p < 0.01). Synergy was observed for a cocktail of 12 morpholinos at low dose (0.3 µM) in TC-32 cells, but not in CHLA-10 cells. Paired synergy was also observed in both EFT cell lines when the PHGDH pre-mRNA transcript was targeted in combination with XAGE1B or CYP4F22 transcripts. Antagonism was observed when CCND1 was targeted with XAGE1B or CYP4F22, or when IGFBP-2 was targeted with CCND1 or RBM11. This transcriptome profiling approach is highly effective for cancer drug discovery, as it identified new EWS-specific target genes (e.g. CYP4F22, RBM11 and IGBP-2), and predicted effective antisense agents (EC 50 < 1 µM) that demonstrate both synergy and antagonism in combination therapy., Competing Interests: CONFLICTS OF INTEREST Author P.L. Iversen has a financial interest in LS Pharma, LLC. Authors J.D. Bushman and J.E Summerton have a financial interest in Onco-Tools, LLC. Authors A.J. Annalora, S.T. O’Neal, and C.B. Marcus have no financial conflicts of interest to report.

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., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Marburg virus (MARV) is an Ebola-like virus in the family Filovirdae that causes sporadic outbreaks of severe hemorrhagic fever with a case fatality rate as high as 90%. AVI-7288, a positively charged antisense phosphorodiamidate morpholino oligomer (PMOplus) targeting the viral nucleoprotein gene, was evaluated as a potential therapeutic intervention for MARV infection following delayed treatment of 1, 24, 48, and 96 h post-infection (PI) in a nonhuman primate lethal challenge model. A total of 30 cynomolgus macaques were divided into 5 groups of 6 and infected with 1,830 plaque forming units of MARV subcutaneously. AVI-7288 was administered by bolus infusion daily for 14 days at 15 mg/kg body weight. Survival was the primary endpoint of the study. While none (0 of 6) of the saline group survived, 83-100% of infected monkeys survived when treatment was initiated 1, 24, 48, or 96 h post-infection (PI). The antisense treatment also reduced serum viremia and inflammatory cytokines in all treatment groups compared to vehicle controls. The antibody immune response to virus was preserved and tissue viral antigen was cleared in AVI-7288 treated animals. These data show that AVI-7288 protects NHPs against an otherwise lethal MARV infection when treatment is initiated up to 96 h PI.

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.).

Unlabelled: Ebola viruses (EBOV) cause severe disease in humans and nonhuman primates with high mortality rates and continue to emerge in new geographic locations, including several countries in West Africa, the site of a large ongoing outbreak. Phosphorodiamidate morpholino oligomers (PMOs) are synthetic antisense molecules that are able to target mRNAs in a sequence-specific fashion and suppress translation through steric hindrance. We previously showed that the use of PMOs targeting a combination of VP35 and VP24 protected rhesus monkeys from lethal EBOV infection. Surprisingly, the present study revealed that a PMOplus compound targeting VP24 alone was sufficient to confer protection from lethal EBOV infection but that a PMOplus targeting VP35 alone resulted in no protection. This study further substantiates recent data demonstrating that VP24 may be a key virulence factor encoded by EBOV and suggests that VP24 is a promising target for the development of effective anti-EBOV countermeasures., Importance: Several West African countries are currently being ravaged by an outbreak of Ebola virus (EBOV) that has become a major epidemic affecting not only these African countries but also Europe and the United States. A better understanding of the mechanism of virulence of EBOV is important for the development of effective treatments, as no licensed treatments or vaccines for EBOV disease are currently available. This study of phosphorodiamidate morpholino oligomers (PMOs) targeting the mRNAs of two different EBOV proteins, alone and in combination, demonstrated that targeting a single protein was effective at conferring a significant survival benefit in an EBOV lethal primate model. Future development of PMOs with efficacy against EBOV will be simplified if only one PMO is required instead of a combination, particularly in terms of regulatory approval., (Copyright © 2015 Warren et al.)

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Ebola virus causes an acute hemorrhagic fever lethal in primates and rodents. The contribution of host immune factors to pathogenesis has yet to be determined and may reveal efficacious targets for potential treatment. In this study, we show that the interleukin (IL)-10 signaling pathway modulates Ebola pathogenesis. IL-10(-/-) mice and wild-type mice receiving antisense targeting IL-10 signaling via disrupting expression through aberrant splice altering were resistant to ebola virus infection. IL-10(-/-) mice exhibited reduced viral titers, pathology, and levels of IL-2, IL-6, keratinocyte-derived chemokine (KC), and macrophage inflammatory protein-1 α and increased interferon (IFN)-γ relative to infected wild-type mice. Furthermore, antibody depletion studies in IL-10(-/-) mice suggest a requirement for natural killer cells and IFN-γ for protection. Together, these data demonstrate that resistance to ebola infection is regulated by IL-10 and can be targeted in a prophylactic manner to protect against lethal hemorrhagic virus challenge.

Activated and regulatory T cells express the negative co-stimulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that binds B7 on antigen-presenting cells to mediate cellular responses. Single nucleotide polymorphisms in the CTLA-4 gene have been found to affect alternative splicing and are linked to autoimmune disease susceptibility or resistance. Increased expression of a soluble splice form (sCTLA-4), lacking the transmembrane domain encoded by exon 3, has been shown to accelerate autoimmune pathology. In contrast, an exon 2-deficient form lacking the B7 ligand binding domain (liCTLA-4), expressed by diabetes resistant mouse strains has been shown to be protective when expressed as a transgene in diabetes susceptible non-obese diabetic (NOD) mice. We sought to employ an antisense-targeted splice-switching approach to independently produce these CTLA-4 splice forms in NOD mouse T cells and observe their relative impact on spontaneous autoimmune diabetes susceptibility. In vitro antisense targeting of the splice acceptor site for exon 2 produced liCTLA-4 while targeting exon 3 produced the sCTLA-4 form in NOD T cells. The liCTLA-4 expressing T cells exhibited reduced activation, proliferation and increased adhesion to intercellular adhesion molecule-1 (ICAM-1) similar to treatment with agonist α-CTLA-4. Mice treated to produce liCTLA-4 at the time of elevated blood glucose levels exhibited a significant reduction in the incidence of insulitis and diabetes, whereas a marked increase in the incidence of both was observed in animals treated to produce sCTLA-4. These findings provide further support that alternative splice forms of CTLA-4 affects diabetes susceptibility in NOD mice and demonstrates the therapeutic utility of antisense mediated splice-switching for modulating immune responses.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays multiple roles in regulation of immune and inflammatory responses. The ability of certain AhR ligands to induce regulatory T cells (Tregs) has generated interest in developing AhR ligands for therapeutic treatment of immune-mediated diseases. To this end, we designed a screen for novel Treg-inducing compounds based on our understanding of the mechanisms of Treg induction by the well-characterized immunosuppressive AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We screened a ChemBridge small molecule library and identified 10-chloro-7H-benzimidazo[2,1-a]benzo[de]Iso-quinolin-7-one (10-Cl-BBQ) as a potent AhR ligand that was rapidly metabolized and not cytotoxic to proliferating T cells. Like TCDD,10-Cl-BBQ altered donor CD4(+) T cell differentiation during the early stages of a graft versus host (GVH) response resulting in expression of high levels of CD25, CTLA-4 and ICOS, as well as several genes associated with Treg function. The Treg phenotype required AhR expression in the donor CD4(+) T cells. Foxp3 was not expressed in the AhR-induced Tregs implicating AhR as an independent transcription factor for Treg induction. Structure-activity studies showed that unsubstituted BBQ as well as 4, 11-dichloro-BBQ were capable of inducing AhR-Tregs. Other substitutions reduced activation of AhR. Daily treatment with 10-Cl-BBQ during the GVH response prevented development of GVH disease in an AhR-dependent manner with no overt toxicity. Together, our data provide strong support for development of select BBQs that activate the AhR to induce Tregs for treatment of immune-mediated diseases.

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.

Phosphorodiamidate morpholino oligomers (PMO) are neutrally charged, sequence-specific antisense agents that interfere with targeted gene expression. PMO have been shown to be highly specific and potent therapies after cellular uptake, yet methods to detect PMO in tissue and inside the cell are limited. We offer in this report novel methods for the detection of cellular resident PMO using flow cytometry-fluorescence in situ hybridization (flow FISH) and a sandwich hybridization technique to quickly and sensitively quantify tissue resident PMO. These methods rely on oligonucleotide probes complementary to a PMO to specifically detect and quantify cell-associated and tissue resident PMO after in vitro or in vivo administration. Using the sandwich hybridization technique, we show that intranasally delivered PMO demonstrates zero-order clearance kinetics from the lung. Furthermore, PMO was detected in nonhematopoietic and hematopoietic cells of the lung regardless of influenza virus infection, although an increase in PMO uptake in infected hematopoietic cells was observed. Coincident measurement of target knock-down to cell-associated influenza A PMO concentration allowed for the calculation of an EC50.

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 β-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 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.

There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates.

Targeting bacterial essential genes using antisense phosphorodiamidate morpholino oligomers (PMOs) represents an important strategy in the development of novel antibacterial therapeutics. PMOs are neutral DNA analogues that inhibit gene expression in a sequence-specific manner. In this study, several cationic, membrane-penetrating peptides were conjugated to PMOs (PPMOs) that target 2 bacterial essential genes: acyl carrier protein (acpP) and gyrase A (gyrA). These were tested for their ability to inhibit growth of Bacillus anthracis, a gram-positive spore-forming bacterium and causative agent of anthrax. PPMOs targeted upstream of both target gene start codons and conjugated with the bacterium-permeating peptide (RFF)(3)R were found to be most effective in inhibiting bacterial growth in vitro. Both of the gene-targeted PPMOs protected macrophages from B. anthracis induced cell death. Subsequent, in vivo testing of the PPMOs resulted in increased survival of mice challenged with the virulent Ames strain of B. anthracis. Together, these studies suggest that PPMOs targeting essential genes have the potential of being used as antisense antibiotics to treat B. anthracis infections.

The p53 tumor suppressor gene encodes a transcription factor that is commonly mutated in cancer. Tumors arise when premalignant cells are unable to undergo p53-dependent apoptosis, cell cycle arrest or DNA repair. The p53-signaling pathway affects not only tumor development, but also the response of tumors to chemotherapeutic drugs. In this study, we use cell penetrating peptide conjugates of phosphorodiamidate morpholino oligomers (PPMOs) to inhibit p53 expression. We examine the functional properties of endogenous p53 isoforms that are produced upon PPMO-mediated inhibition of p53 translation and splicing, and report that loss of N-terminal or C-terminal sequences interferes with the transcriptional activity of p53. Importantly, we report that PPMO-mediated inhibition of p53 expression sensitizes human cancer cells with wild-type p53 to chemotherapeutic drugs.

CD4+CD25+regulatory T cells (T(reg)) impair anti-tumor and anti-viral immunity. As there are higher T(reg) levels in cancer patients compared with healthy individuals, there is considerable interest in eliminating them or altering their function as part of cancer or viral immunotherapy strategies. The scurfin transcriptional regulator encoded by the member of the forkhead winged helix protein family (FOXP3) is critical for maintaining the functions of T(reg). We hypothesized that targeting FOXP3 expression with a novel arginine-rich, cell-penetrating, peptide-conjugated phosphorodiamidate morpholino (PPMO) based antisense would eliminate T(reg) and enhance the induction of effector T-cell responses. We observed that the PPMO was taken up by activated T cells in vitro and could downregulate FOXP3 expression, which otherwise increases during antigen-specific T-cell activation. Generation of antigen-specific T cells in response to peptide stimulation was enhanced by pre-treatment of peripheral blood mononuclear cells with the FOXP3-targeted PPMO. In summary, modulation of T(reg) levels using the FOXP3 PPMO antisense-based genomic strategy has the potential to optimize immunotherapy strategies in cancer and viral immunotherapy.

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.

Currently, no vaccines or therapeutics are licensed to counter Ebola or Marburg viruses, highly pathogenic filoviruses that are causative agents of viral hemorrhagic fever. Here we show that administration of positively charged phosphorodiamidate morpholino oligomers (PMOplus), delivered by various dosing strategies initiated 30-60 min after infection, protects>60% of rhesus monkeys against lethal Zaire Ebola virus (ZEBOV) and 100% of cynomolgus monkeys against Lake Victoria Marburg virus (MARV) infection. PMOplus may be useful for treating these and other highly pathogenic viruses in humans.

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.

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.

Phosphorodiamidate morpholino oligomers (PMO), which have a neutral chemistry, are extensively being used as tools for selective inhibition of gene expression in cell culture models and are currently in human clinical trials. Unlike phosphorothioates (PS ODN) and other charged oligonucleotides, little is known about the uptake characteristics of neutral oligomers. The purpose of this study was to understand the kinetics of PMO transport in cells and correlate with antisense activity. In contrast to primary cells and some transformed cell lines which were uptake permissive, established cancer cell lines showed very poor uptake with an occasional diffuse intracellular pattern. Differential PMO uptake was also observed in immune cells, with dendritic cells and monocytes showing highest uptake compared to T and B cells. In addition, PMO localization was observed to be heterogeneous within a population of uptake permissive cells. Unassisted PMO delivery targeting specific genes was correlated with functional antisense efficacy in experiments showing correction of pre-mRNA missplicing and inhibition of target enzyme activity in cells in culture. PMO internalization in uptake-permissive cells was identified to be specific, saturable, and energy-dependent, suggesting a receptor mediated uptake mechanism. Understanding PMO transport should facilitate the design of more effective synthetic antisense oligomers as therapeutic agents.

Two types of phosphorodiamidate morpholino oligomers (PMOs) were tested for inhibition of growth of Salmonella enterica serovar Typhimurium. Both PMOs have the same 11-base sequence that is antisense to the region near the start codon of acpP, which is essential for lipid biosynthesis and viability. To the 3' end of each is attached the membrane-penetrating peptide (RXR)4XB (R, X, and B indicate arginine, 6-aminohexanoic acid, and beta-alanine, respectively). One peptide-PMO (AcpP PPMO) has no charge on the PMO moiety. The second PPMO has three cations (piperazine) attached to the phosphorodiamidate linkages (3+Pip-AcpP PPMO). A scrambled-sequence PPMO (Scr PPMO) was synthesized for each type of PMO. The MICs of AcpP PPMO, 3+Pip-AcpP PPMO, and either one of the Scr PPMOs were 1.25 microM (7 microg/ml), 0.156 microM (0.94 microg/ml), and >160 microM (>900 microg/ml), respectively. 3+Pip-AcpP PPMO at 1.25 or 2.5 microM significantly reduced the growth rates of pure cultures, whereas AcpP PPMO or either Scr PPMO had no effect. However, the viable cell count was significantly reduced at either concentration of 3+Pip-AcpP PPMO or AcpP PPMO, but not with either Scr PPMO. In other experiments, macrophages were infected intracellularly with S. enterica and treated with 3 microM 3+Pip-AcpP PPMO. Intracellular bacteria were reduced >99% with 3+Pip-AcpP PPMO, whereas intracellular bacteria increased 3 orders of magnitude in untreated or Scr PPMO-treated cultures. We conclude that either AcpP PPMO or 3+Pip-AcpP PPMO inhibited growth of S. enterica in pure culture and that 3+Pip-AcpP PPMO reduced intracellular viability of S. enterica in macrophages.

Contact dermatitis is the result of inflammatory responses mediated by hapten-specific activated CD8+ and CD4+ T cells. Activation-induced cell death (AICD) is a naturally occurring process regulating the resolution of T-cell responses through decreased expression of the antiapoptotic molecule cellular FLICE inhibitory protein (cFLIP). We show that targeting cFLIP expression in vitro and in vivo, with morpholino antisense applied systemically or topically in conjunction with antigen, sensitizes T cells to undergo "early" AICD resulting in tolerance. Analysis of antisense-treated CD8+ OT-1 splenocytes after co-culture with SIINFEKL-pulsed DCs showed apoptosis occurring in a dose-dependent manner with respect to cFLIP peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) concentration. A transplant acceptance model using male DO.11 donor cells and female BALB/c recipient mice showed that cFLIP antisense treatment could promote antigen tolerance. Hypersensitivity responses induced in mice by the epicutaneous application of the haptens FITC and oxazolone confirmed that topically applied cFLIP antisense could reduce inflammation. Treatment of the skin produced significant reduction in dermatitis and localized infiltration of lymphocytes. Moreover, the treatment was target- and antigen-specific, dose-dependent, and capable of inducing long-lived tolerance. These data suggest that the targeted expression of immune-regulating molecules is possible through the application of antisense to the skin.

The modulation of cellular processes by small molecule inhibitors, gene inactivation, or targeted knockdown strategies combined with phenotypic screens are powerful approaches to delineate complex cellular pathways and to identify key players involved in disease pathogenesis. Using chemical genetic screening, we tested a library of known phosphatase inhibitors and identified several compounds that protected Bacillus anthracis infected macrophages from cell death. The most potent compound was assayed against a panel of sixteen different phosphatases of which CD45 was found to be most sensitive to inhibition. Testing of a known CD45 inhibitor and antisense phosphorodiamidate morpholino oligomers targeting CD45 also protected B. anthracis-infected macrophages from cell death. However, reduced CD45 expression did not protect anthrax lethal toxin (LT) treated macrophages, suggesting that the pathogen and independently added LT may signal through distinct pathways. Subsequent, in vivo studies with both gene-targeted knockdown of CD45 and genetically engineered mice expressing reduced levels of CD45 resulted in protection of mice after infection with the virulent Ames B. anthracis. Intermediate levels of CD45 expression were critical for the protection, as mice expressing normal levels of CD45 or disrupted CD45 phosphatase activity or no CD45 all succumbed to this pathogen. Mechanism-based studies suggest that the protection provided by reduced CD45 levels results from regulated immune cell homeostasis that may diminish the impact of apoptosis during the infection. To date, this is the first report demonstrating that reduced levels of host phosphatase CD45 modulate anthrax pathogenesis.

Phosphorodiamidate morpholino oligomers (PMOs) are uncharged nucleic acid-like molecules designed to inactivate the expression of specific genes via the antisense-based steric hindrance of mRNA translation. PMOs have been successful at knocking out viral gene expression and replication in the case of acute viral infections in animal models and have been well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents that may be useful for combating filoviral infections. We have previously shown that mice treated with a PMO whose sequence is complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebola virus challenge. In the present study, we report on the abilities of two additional VP24-specific PMOs to reduce the cell-free translation of a VP24 reporter, to inhibit the in vitro replication of Ebola virus, and to protect mice against lethal challenge when the PMOs are delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess the enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of various lengths and compositions, positioning of conjugated peptides to either the 5' or the 3' terminus, and the conferring of charge modifications by the addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and mice during lethal Ebola virus challenge.

Alternative splicing of pre-mRNA leads to variation in the exons that form the mRNA, and provides eukaryotic organisms with an additional qualitative control of gene expression. Disruptions in the regulation of pre-mRNA splicing caused by heritable genomic mutations or quantitative shifts in the regulation of exon inclusion can lead to disease. Alternative exon inclusion (pre-mRNA splicing) produces different proteins with alternative activities, derived from the same pre-mRNA, and is utilized by the immune system to expand gene function. Recent advances in the delivery of splice switching oligomers to lymphoid cells, combined with the ability to manipulate mRNA splicing to either correct mis-splicing or to alter the balance of different splice forms, holds great promise for the development of new therapeutic strategies for the treatment of immune-related disease. Antisense-based targeted manipulation of various immune modulating molecules as therapeutic approaches are discussed in this review.

Measles virus (MeV) is a highly contagious human pathogen. Despite the success of measles vaccination programs, measles is still responsible for an estimated 245,000 deaths each year. There are currently no antiviral compounds available for the treatment of measles. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are antisense compounds that enter cells readily and can interfere with mRNA function by steric blocking. A panel of PPMO was designed to target various sequences of MeV RNA that are known to be important for viral replication. Five PPMO, targeting MeV genomic RNA or mRNA, inhibited the replication of MeV, in a dose-responsive and sequence-specific manner in cultured cells. One of the highly active PPMO (PPMO 454), targeting a conserved sequence in the translation start site of the mRNA coding for the nucleocapsid protein, inhibited multiple genotypes of MeV. This report provides evidence that PPMO treatment represents a promising approach for developing antiviral agents against measles and other paramyxoviruses.

The potency of antisense peptide-phosphorodiamidate morpholino oligomers (PPMOs) was improved by varying the peptide composition. An antisense phosphorodiamidate morpholino oligomer (PMO) complementary to the mRNA of the essential gene acpP (which encodes the acyl carrier protein required for lipid biosynthesis) in Escherichia coli was conjugated to the 5' ends of various cationic membrane-penetrating peptides. Each peptide had one of three repeating sequence motifs: C-N-N (motif 1), C-N (motif 2), or C-N-C (motif 3), where C is a cationic residue and N is a nonpolar residue. Variations in the cationic residues included arginine, lysine, and ornithine (O). Variations in the nonpolar residues included phenylalanine, valine, beta-alanine (B), and 6-aminohexanoic acid (X). The MICs of the PPMOs varied from 0.625 to >80 microM (about 3 to 480 microg/ml). Three of the most potent were the (RX)(6)B-, (RXR)(4)XB-, and (RFR)(4)XB-AcpP PMOs, which were further tested in mice infected with E. coli. The (RXR)(4)XB-AcpP PMO was the most potent of the three conjugates tested in mice. The administration of 30 microg (1.5 mg/kg of body weight) (RXR)(4)XB-AcpP PMO at 15 min postinfection reduced CFU/ml in blood by 10(2) to 10(3) within 2 to 12 h compared to the numbers in water-treated controls. All mice treated with 30 microg/dose of (RXR)(4)XB-AcpP PMO survived infection, whereas all water-treated mice died 12 h postinfection. The reduction in CFU/ml in blood was proportional to the dose of PPMO from 30 to 300 microg/ml. In summary, the C-N-C motif was more effective than the other two motifs, arginine was more effective than lysine or ornithine, phenylalanine was more effective than 6-aminohexanoic acid in vitro but not necessarily in vivo, and (RXR)(4)XB-AcpP PMO reduced bacterial infection and promoted survival at clinically relevant doses.

Background: Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder, is caused by protein-truncating mutations in the dystrophin gene. Absence of functional dystrophin renders muscle fibres more vulnerable to damage and necrosis. We report antisense oligomer (AO) induced exon skipping in the B6Ros.Cg-Dmd(mdx-4Cv)/J (4(CV)) mouse, a muscular dystrophy model arising from a nonsense mutation in dystrophin exon 53. Both exons 52 and 53 must be excised to remove the mutation and maintain the reading frame., Methods: A series of 2'-O-methyl modified oligomers on a phosphorothioate backbone (2OMeAOs) were designed and evaluated for the removal of each exon, and the most effective compounds were then combined to induce dual exon skipping in both myoblast cultures and in vivo. Exon skipping efficiency of 2OMeAOs and phosphorodiamidate morpholino oligomers (PMOs) was evaluated both in vitro and in vivo at the RNA and protein levels., Results: Compared to the original mdx mouse studies, induction of exon skipping from the 4(CV) dystrophin mRNA was far more challenging. PMO cocktails could restore synthesis of near-full length dystrophin protein in cultured 4(CV) myogenic cells and in vivo, after a single intramuscular injection., Conclusions: By-passing the protein-truncating mutation in the 4(CV) mouse model of muscular dystrophy could not be achieved with single oligomers targeting both exons and was only achieved after the application of AO cocktails to remove exons 52 and 53. As in previous studies, the stability and efficiency of PMOs proved superior to 2OMeAOs for consistent and sustained protein induction in vivo., ((c) 2008 John Wiley & Sons, Ltd.)

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.