AVI-7537 for Ebola: Seeking the Next Version Patrick L. Iversen, Ph.D. Department of Environmental & Molecular Toxicology, Oregon State University, Corvallis, Oregon and LS Pharma, Lebanon, Oregon, USA.
Published: October 28, 2018 Citation: Iversen P.L. (2018) AVI-7537 for Ebola: Seeking the Next Version. Science Publishing Group Journal 1(2). Corresponding Author: Patrick L. Iversen [email protected]
Abstract Objective:
Our goal is the discovery and development of innovative ebolavirus countermeasures.
Methods:
Antisense phosphorodiamidate morpholino oligomers (PMO) targeting Ebola VP24 (AVI-7537) expression was selected based on efficacy in mouse, guinea pig, and nonhuman primate lethal challenge models. AVI-7537 was evaluated in both single ascending dose and multiple ascending dose studies that included pharmacokinetic studies.
Results:
Support for continued development of AVI-7537 was terminated in 2013 due to the federal budget sequester. Reduced enthusiasm for relatively viral strain specific and expensive therapeutic approaches including AVI-7288 and AVI-7537 led to discovery of innovative human host genes including RIG-I, IL-10, and CCL2.
Conclusion:
AVI-7537 is effective and well-tolerated with a human equivalent dose of 11 mg/kg 14 daily iv injection. Broader spectrum treatment options led to investigations into targeting critical host genes capable of mitigating the severity of viral pathology. We are building on studies that identified candidate host genes for a new generation of antisense therapeutics for infectious disease.
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Introduction
Filoviruses consist of Marburgvirus, Cuevavirus, and Ebolavirus genera, each emerging as a spectrum of distinct isolates from regions with high biodiversity (Pigott et al. 2014). Ebolavirus transmitted as an aerosol is controversial but is a bioterror threat with qualitative differences from infection acquired from skin penetration (Mekibib B and Arien KK 2016). In addition, viral reservoirs in immune privileged sites including brain, eye, and testes (Mate et al. 2015) can evade existing therapeutic management strategies. Our goal is the discovery and development of innovative countermeasures in addition to the emerging bounty of agents in development.
This review provides an update of AVI-7537, an antisense agent directed at Ebola VP24, in late stage development for the treatment of Ebola Zaire infections. The context for the termination of AVI-7537 points to practical influences of competition, cost, and a changing landscape in the ebolavirus therapeutic space. Response to the changing landscape revealed more innovative approaches to the therapeutic management of ebolavirus disease via antisense directed at specific host genes.
AVI-7537 for Treatment of Ebola Virus Infections
Our initial strategy involved exploring the antiviral capacity of Phosphorodiamidate Morpholino Oligomers (PMOs). The procedure involved preparing 5 or more oligomers targeting a specific virus as a means of identifying optimal viral targets necessary for multi-log reductions in viral growth. We started with the small single-stranded positive sense RNA viruses. Successful studies began in cell culture with caliciviruses (Stein et al. 2001; Bok et al. 2008) followed by treatment of an outbreak in kittens (Smith et al. 2008). Inhibition of picornaviruses included coxackievirus b3 (Yuan et al. 2006), foot and mouth disease virus (Vagnozzi et al. 2007), and polio and enterovirus (Stone et al. 2008). Inhibition of flaviviruses included dengue (Kinney et al. 2005; Holden et al. 2006; Stein et al. 2008), and West Nile Virus (Deas et al. 2005; Deas et al. 2007). In addition, we explored inhibition of Venezuelan Equine Encephalitis (Paessler et al. 2008), hepatitis E virus (Yuchen et al. 2016), and SARS (Newman et al. 2004; Newman et al. 2005).
The more complex single-stranded negative sense RNA genome viruses were then investigated. Successful studies involved the paramyxovirus respiratory syncytial virus (Lai et al. 2008), and the orthomyxivirus influenza A (Ge et al. 2006; Lupfer et al. 2008; Gabriel et al. 2008). Ultimately, we were able to identify effective inhibitors of DNA viruses including the Kaposi’s sarcoma-associated herpesvirus (Zhang et al. 2008) and herpes simplex virus 1 (Moerdyk-Schauwecker et al. 2009). The assembly of effective viral targets assembled into a target database supported a rapid response therapeutic program for emerging infectious disease and confidence to initiate a filovirus program targeting Ebola and Marburg viruses (FIGURE 1).
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PMO candidates were designed to bind to Ebola Zaire L, VP24, VP30, GP, VP40, VP35, NP, and flanking genome targets. A head-to-head evaluation in which mice were treated with 0.5 mg PMO i.p. just prior to mouse adapted Ebola viral challenge. The survival ratio for untreated controls was 0.06 ± 0.09 over 19 different experiments with 10 mice in each experiment. The survival ratio for a scrambled control PMO was 0.08 ± 0.12 (20 different experiments) confirming no significant survival benefit and no difference from an untreated control (Iversen et al. 2012). The leader (0.10 ± 0.14 survival ratio), NP (0.20 ± 0.00), VP40 (0.45 ± 0.07), GP (0.15 ± 0.13) and VP30 (0.35 ± 0.29) provided less survival benefit in the mouse model and were not selected as potential candidates. Viral targets including VP35 (0.79 ± 0.30) and VP24 (0.69 ± 0.29) provided significant survival benefit and were selected as candidates as well as L (0.35 ± 0.29) because its role as the viral polymerase.
A PMO cocktail administered as 500 µg/mouse (~25 mg/kg) of VP24, VP35 and L protected mice completely (100 percent) from lethal challenge with mouse adapted Ebola virus (Warfield et al. 2006). These encouraging observations led to evaluation in nonhuman primates. Rhesus macaques were treated (a combination of subcutaneous, i.p., and intramuscular routes) with a PMO targeting VP35 (n = 4) and the VP35+VP24+L cocktail (n = 4) compared to untreated monkeys (n = 3). All VP35 and untreated monkeys died between 7 and 9 days of viral challenge but 3 of 4 (75%) of those treated with the cocktail survived to day 14 post challenge (2 of 4 or 50% survived to the end of the study) when their viral titer in plasma was no longer detected.
The nonhuman data were promising but the relatively large dose focused efforts on enhancing the potency of the PMO core chemistry. A collection of peptide conjugated and piperazine modifications to the PMO linkage targeting Ebola virus VP24 were evaluated in the mouse adapted lethal challenge model (Swenson et al. 2009). Impressive potency benefit was observed
FIGURE 1. Update AVI-7537 for Ebola Virus Infection
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for a PMO conjugated with (RX)8B as a 5 µg/mouse (~0.25 mg/kg) dose provided 100 % survival compared to 15 to 20 % survival with unconjugated VP24 PMO at 50 µg/mouse (~2.5 mg/kg). The piperazine linkage modifications (PMOplus) also enhanced potency with up to 70 % survival with a 50 µg/mouse dose. The encouraging potency improvements were then confirmed in the nonhuman primate lethal challenge model with a human viral isolate. The cumulative NHP survival with 350 mg/kg PMO was 25 % (3 of 12 treated) establishing a baseline for comparison. The PPMO maximum tolerated dose was 18 mg/kg which failed to provide survival benefit with 0 % survival (0 of 3 treated). The PMOplus cocktail of VP35 (AVI-7539) and VP24 (AVI-7537) at 40 mg/kg led to a survival benefit of 61 % (8 of 13 treated). This two PMOplus cocktail, AVI-6002, became the lead candidate.
Detailed studies investigating the efficacy of AVI-6002 for Ebola and AVI-6003 for Marburg revealed a 28-30 mg/kg dose would reliably lead to 60 and 100 percent survival, respectively (Warren et al. 2010). Survival benefit was dose dependent and treatment was initiated post exposure but prior to symptoms. The minimum PMO component required for protection and attention to gender as a possible variable compared the benefit of AVI-7539 with AVI-7537 and the cocktail, AVI-6002 at 40 mg/kg i.v. (Warren et al. 2015). We observed 62.5 % (5 of 8) survival in the AVI-6002 cocktail but 75 % (6 of 8) with the single component AVI-7537 (targeting VP24) and 0% (0 of 8) with the single component AVI-7539 (targeting VP35). Survival was observed to day 42 post infection and AVI-7537 reduced viral titer to undetectable levels by day 9. The surprising observation established AVI-7537 as the lead candidate and may have identified a difference between mouse and nonhuman primate models and possible greater role of interferon inhibition in the mouse (Bradfute and Bavari 2011). Further, VP24 may play a greater role in the viral life cycle (Ramanan et al. 2011; Mateo et al. 2011; Watt et al. 2011).
We then explored the duration post infection the PMO could continue to provide survival benefit. The federal budget sequester caused the termination of the AVI-7537 program in 2013 but AVI-7288 continued for treatment of Marburg virus, also a filovirus. A delay in time-to-treatment study was conducted in the nonhuman lethal challenge model for Marburg virus and involved AVI-7288. Survival in NHP challenged with Marburg virus to 41 days post-infection was 83% (5 of 6 at 1 hour post challenge), 83% (5 of 6 at 24 hours post challenge), 100% (6 of 6 at 48 hours post challenge), and 83% (5 of 6 at 96 hours post challenge) when treated with AVI-7288 (targeting NP) compared to 0% (0 of 6) in the untreated group. Deep sequencing of the viral genomes in non-survivors did not reveal emergence of resistance to the sequence of AVI-7288. Deep sequencing of viral genome sequences from NHP treated with AVI-7537 did not reveal evidence of emerging sequence deviation suggestive of minimal emerging resistance to the antisense therapeutic (Khiabanian et al. 2014).
Human clinical trials in healthy volunteers involved single ascending dose (SAD) studies for both AVI-6002 and AVI-6003 (Heald et al. 2014). The PMOplus compounds were administered intravenously (i.v.) at 0.005, 0.05, 0.5, 1.5, 3, and 4.5 mg/kg/sequence into 30 healthy male and female subjects were found safe and well tolerated. The mean peak plasma concentration and area under the plasma concentration over time curve (AUC) increased linearly in proportion to the dose administered. The estimated plasma half-life was 2 to 5 hours and total clearance was constant over all dose levels. Finally, 40 healthy volunteers were treated i.v. daily for 14 days
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with 1, 4, 8, 12, or 16 mg/kg with AVI-7288 in a multiple ascending dose clinical trial (Heald et al. 2015). Comparison of the linear AUC in nonhuman primates and humans allowed estimation of a protective human dose of 9.6 mg/kg and Monte Carlo simulations supported a dose of 11 mg/kg.
Ebola Lessons Learned
The 2014-2016 outbreak exceeded the combined total number of cases reported in the 20 outbreaks since the 1970s with 28,652 Ebola cases of which 11,325 died. The 1976 outbreak in Zaire caused 280 deaths and a case fatality rate of 88 percent (280 deaths/318 cases). Subsequent sporadic outbreaks (22 in total) in Eastern and Central African nations had CFR ranging from 25 to 90 percent. Hence, the CFR in the 2014-2016 epidemic was 40 percent. Given the number of cases in the 2014-2016 epidemic, the weight averaged CFR for Ebola Zaire is near 40 percent. It is possible that the unprecedented international response provided a substantial reduction from initial CFR observations of 88 percent.
Improving readiness in at-risk countries and remaining prepared for Ebola and other health threats is essential as “More people died because of Ebola than from Ebola.” The epidemic interrupted responses to malaria, tuberculosis, vaccine-preventable diseases, and other conditions. It is harder to estimate the number of cases that did not develop due to improvements in surveillance, contact tracing, diagnostic testing, community engagement, infection prevention and control, border health, emergency management, and vaccine evaluation in response to the 2014 outbreak.
A significant reduction in Ebola viral disease mortality has been achieved with basic supportive therapies (Weppelmann et al. 2016). Patients are frequently dehydrated and respond to intravenous or oral fluids with electrolytes (Bah et al. 2015; Zhong et al. 2014). The overall mortality early in the 2014 outbreak was 53.5 percent but quarantine and hospitalization reduced this to nearly 25 percent (TABLE 1). The likely reduction in mortality was due to prevention of vascular volume depletion and replacement of electrolytes that reduced complications from hypovolemic shock.
Accelerated development of Ebola vaccines began September 4-5, 2014 marked by the World Health Organization (WHO) review of the most promising vaccine candidates and consider the ethics of evaluating these vaccines during the epidemic. The CDC initiated the STRIVE (NCT02378753) vaccine trial in Sierra Leone in October and the WHO led a ring vaccination trial in Guinea. Two vaccine candidates: a replication-deficient recombinant chimpanzee adenovirus type-3 vectored vaccine (ChAd3-EBOV; licensed to GSK) and a replication-competent, recombinant vesicular stomatitis virus vectored vaccine (rVSV-ZEBOV; licensed to Merck). A heterologous boost vaccination with a modified vaccinia Ankara with EBOV glycoprotein (MVA-EBOV) was added to the ChAd3-EBOV regimen to ensure a durable protection. The logistic demands of the ChAd3-EBOV/MVA-EBOV prime and boost regimen with two different agents led to selection of a single dose rVSV-ZEBOV vaccine at 2 x 107 pfu/mL for the trial.
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TABLE 1. Progress in Management of Ebola Virus Infections
Treatment Therapeutic Agents Effectiveness Status
Supportive Care Hydration/electrolytes Antibiotics/co-Infection therapy
RNA-based TKM-Ebola (siRNA L + VP35) AVI-7537 (PMOplus-VP35)
NHP efficacy NHP efficacy
Phase II terminated Discontinued
Vaccination began April 9, 2015 in seven enrollment sites in Sierra Leone and was completed on December 12, 2015. A total of 8,826 patients were enrolled and 8,016 were vaccinated. While 64 participants had illnesses investigated as suspected Ebola, none were confirmed as Ebola suggestive of efficacy. No serious adverse events related to vaccination were reported. Efficacy could not be measured because the health care workers that were vaccinated were not associated with Ebola transmission during this period of the epidemic (they were likely well trained and wore PPE when in contact with infected patients). A rVSV-ZEBOV ring vaccine in a trial conducted by the WHO in Guinea vaccinated 2119 people with no cases of Ebola disease within a period of 10-21 days after the vaccination. In comparison, 16 cases were identified within the same time frame among 2041 people that did not receive the vaccine immediately. The vaccine has been reported to be 100 percent effective. The vaccine, most recently deployed on June 21, 2018 for the outbreak that began in May 2018 in the Democratic Republic of the Congo, represents a prudent medical measure. The vaccine is not yet approved but represents
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a new tool in the management of Ebola outbreaks and epidemics. Vaccines continue to be developed for Ebola by GSK (the ChAd3-EBOV), Merck, and Janssen and a new self-amplifying RNA vaccine technology has demonstrated efficacy in a guinea pig lethal challenge model (Lundstrom 2018). Now that significant challenges in logistics and ethics of clinical trials in outbreak/epidemic settings have been overcome, progress for these vaccines is likely to be enhanced.
Passive immunization with convalescent serum, humanized monoclonal antibodies (mAb), and mAb cocktails represent a promising approach to managing the Ebola infected patient. Initially, polyclonal antibodies recovered from convalescent patients provided evidence of therapeutic potential (Olinger et al. 2012; Dye et al. 2012; Gutfraind and Meyers 2015; Kreil 2015). mAb were capable of preventing progression of a lethal Ebola virus infection in nonhuman primates (Qiu and Kobinger 2014) focusing attention on finding optimal mAb. The experimental ZMapp cocktail of three mAb targeting different epitopes of the viral GP protein that were produced in tobacco leaves was administered to several patients during the 2014 outbreak (Hampton 2014; Goodman 2014; Qui et al. 2014). ZMapp treated patients conditions improved albeit in the context of aggressive rehydration, electrolyte balancing, and other measures such that the efficacy was not established (Lyon et al. 2015). A suggested dose of 10 g mAb per patient may limit broad deployment of mAb therapy.
The search for small molecule therapeutics for Ebola include re-tasking approved drugs suggesting antimalarial (amodiaquine and chloraquine), antibiotics (erythromycin), estrogen receptor agonists (estradiol and toremifene), and serotonin uptake inhibitors (fluoxetine and paroxetine) (Madrid et al. 2014). Extending the spectrum of use of Favipiravir, an influenza agent in development that demonstrated efficacy against Ebola (Oestereich et al. 2014). Type-I interferons, interferon α and β, are under consideration as supplementation to other therapies for Ebola (Subramanian et al. 2008). In addition, therapeutics are in development with focus on Ebola include a nucleoside analog, BCX4430 (Warren et al. 2014). A prodrug of an adenosine analog, GS-5734, is effective against Ebola (Warren et al. 2016).
A female nurse involved in the humanitarian effort in Sierra Leone became infected and then recovered from Ebola virus. She had undetectable virus RNA in peripheral blood when she returned to her home in Scotland in January 2015. In October 2015, she was hospitalized with acute meningitis, and found to have Ebola virus in cerebrospinal fluid (CSF) when she was treated with 150 mg daily infusion of GS-5734. The Ebola virus RNA was undetectable after 14 days of treatment. The encouraging observation marks significant progress in the discovery of a small molecule therapeutics for emerging RNA viral infections.
RNA based therapeutics include TKM-Ebola and AVI-7537. TKM-Ebola is a lipid nanoparticle containing two siRNA targeting viral L and VP35 transcripts. Most patients experienced transient inflammatory response (6 to 24 hours post dose) and one severe cytokine release syndrome was diagnosed in human pharmacokinetic trials. Compassionate use of TKM-Ebola in the two patients in the United States led to a phase II study in Guinea during the 2014 outbreak. The phase II trial of TKM-Ebola was discontinued as it became clear an efficacy endpoint could not be achieved.
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Changing Development Landscape.
The Ebola therapeutic effort was initiated in a context of severe unmet medical need. The probability of surviving infection was about 12 percent, no therapeutics were in advanced stages and no effective vaccine was available. Outbreaks were infrequent shifting attention to potential laboratory accidents. The challenges to development are significant including demonstrating efficacy in human clinical trials. Consider the cost of drug development of $1,000 million and the cost of a course of therapy at $20,000, sales will need to exceed 50,000 to break even for costs already expended. This number exceeds the number of reported cases since the initial Ebola outbreak of 1976. The potential for return on investment for companies considering development of Ebola treatment projects are uncertain and unlikely to satisfy investors.
What is the price of a human life? Estimating the value of an Ebola therapeutic is challenging but creating a “straw man” using the gross domestic product (GDP) of the US divided by the number of people is ~$80,000/person. The product of GDP per person and the case fatality ratio (0.88) provides a starting place for the value proposition, ~$70,000 for a single course of therapy (FIGURE 2). The reduction in case fatality ratio through improved natural history studies and benefits of quarantine procedures, hydration therapy, and early treatment of co-infection were lessons learned from the 2014 outbreak (TABLE 1). In addition, advanced development of effective vaccines and multiple therapeutics in the development pipeline have improved the probability of survival dramatically. These successes alter the value proposition to below $10,000 per course of therapy (~$80,000/person * 0.2 CFR = ~$16,000).
FIGURE 2. Illustrative estimate of therapeutic value and improved survival probability. The inset graph of the relationship between estimated survival probability on the abscissa versus cost of a course of therapy (GDP/person * CFR). The larger graph indicates year versus improving survival. The horizontal lines indicate possible return on investment break point.
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The synthesis of an oligomer by solid phase synthesis for large-scale manufacture is feasible but the cost of goods remains relatively high. The cost per gram of a PMOplus agent like AVI-7537 is not reported but will not likely fall below $400/gram and a rough estimate of $1000/gram is a conservative estimate. Given the human dose estimate of fourteen daily doses of ~1 gram per day (11 mg/kg * 80 kg individual) is 14 grams for a course of therapy (Heald et al. 2015). The $1000/gram cost of goods means the cost would be ~$14,000 for a course of therapy. This does not include development costs, operational costs or a profit thus limiting the incentive to take such a therapeutic to commercial availability. However, incentives such as the Rare Tropical Disease waiver or indirect costs provided by grants and contracts provide significant interest for small companies. The federal budget sequester in 2013 terminated AVI-7537 support (FIGURE 2).
Given the changing context in options for management of Ebola infections and the limited return on investment to public companies, the company focused efforts on the emerging treatment for Duchenne Muscular Dystrophy, eteplirsen. While the 30 mg/kg dose is larger and ~60 grams of compound per year are greater dose obligations, the rare disease market offers a high price point.
Targeting Host Genes- Broad Spectrum Therapeutics for Infectious Disease
Viral and bacterial infections are an intrusion of xenobiotic nucleic acid information into a host. A direct result of the intrusion is the synthesis of xenobiotic proteins that ultimately interact with an array of host proteins and pathways. Insufficient immune responses die to senescence or naivety often result in viral pathogenesis (Panum 1940). Conversely, immune mediated cell destruction can lead to severe, possibly fatal pathology (Nathanson and Ahmed 2007). The immune response is not a digital system reflected in two states, on or off. The premise for our studies is that qualitative gene-specific and transcript-specific immune responses can be manipulated with antisense oligomers to improve responses to infection.
Antisense inhibition of ICAM-1 in patients with mild to moderate ulcerative colitis provided benefit (Van Deventer, Tami, Wedel. 2004). Inhibition of alpha 4 integrin provided significant benefit in a mouse model of multiple sclerosis (Meyers et al. 2005). We found lymphocytic choriomeningitis virus (LCMV) produces a hemorrhagic-like disease in the FVB mouse strain (Schnell et al. 2012). Depletion of T-cells in the mice prevented LCMV mortality and antisense inhibition of IL-17 cytokine expression provided significant protection. Inhibition of cFLIP in skin desensitizes responses to contact dermatitis (Mourich et al. 2009). Oligomer induced exon-skipping in CD45 pre-mRNA provided survival benefit from Anthrax infection in mice (Panchal et al. 2009) and in CTLA-4 pre-mRNA prevented autoimmune diabetes in non-obese diabetic mice (Mourich et al. 2014). Our hypothesis became antisense manipulation of specific immune response genes could improve survival from Ebola lethal challenge.
A total of 79 genes (248 PPMO) were evaluated with 34 targeting the innate immune response, 9 targeted genes that may be considered either innate or adaptive immune response, 30 targeting the adaptive immune response, 7 targeting programmed cell death, 1 scramble control, 3 irrelevant target controls and 1 positive control, VP35. The probability that a single PPMO
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designed to inhibit translation of a new gene was expected to be near 90 percent and two PPMOs were considered necessary to interfere with pre-mRNA splicing to provide 90 percent confidence in efficacy. The project utilized 1 translation suppressing and 2 splice-switching oligomers (when possible) for each gene to be evaluated for a combined 99 percent probability for success.
The experimental design involved injection of 50ug/mouse by the intraperitioneal (ip) route four hours prior to infection. The mice were challenged with 1000 pfu of mouse adapted Ebola Zaire. Each treatment group involved 10 mice (of mixed gender) and the endpoint was survival to day 14 post infection. The study utilized an established model in which a positive control PPMO targeting the VP35 viral gene was evaluated. All studies were conducted in BSL4 containment at USAMRIID laboratories in Fredrick MD.
TABLE 2. Summary of Observations by Cellular Localization of the Product of the Target Gene.
The percent survival in mice treated with PPMO targeting 34 innate immune response genes were distributed over 61 treatment groups with average survival 41 percent (249/608) a significant improvement over 12.2 percent (4/49) survival in mice treated with scramble control PPMO. Survival of mice treated with PPMO targeting 30 adaptive immune response genes in 47 treatment groups led to 44.5 percent (209/470) also improved over scramble controls but not different from innate immune response targets. The survival in groups of innate or adaptive immune responses is not free from bias due to our allocation of genes to one group or the other and we did not seek to identify random representations from either innate or adaptive immune responses so interpretation is limited.
Comparing the translation suppression design with oligomer induced exon skipping did not reveal a global superior strategy. We evaluated 74 PPMO in 109 treatment groups and observed
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29.4 percent survival (319/1087). We evaluated 127 PPMO in 196 treatment groups with induced exon skipping and observed 30.5 percent survival (597/1959). Further, exon-skipping induced by targeting the splice acceptor (SA) was not different from targeting the splice donor (SD), 31 percent (518/1669) for SA versus 27 percent (79/290) for the SD. We did not attempt to skip exons in frame so most were likely to produce out of frame transcripts and loss of the transcript by nonsense mediated decay.
The most effective PPMO for each gene target was examined with respect to the subcellular location of the target protein. A summary of these data are provided in TABLE 2. Receptor ligands were the best targets in general with an average of 46.2% survival. Four genes (IL10, IL12a, HMGB1 and CSF1) out of the 15 genes in this group provided impressive survival benefit. Four genes (MyD88, NOD1, RAGE and RIG1) out of the 9 (44%) adaptor genes targeted provided significant survival benefit. One transcription factor gene (IRF5) provided significant survival benefit of the five evaluated. The cellular effector gene, p53, is an apoptotic regulator and inhibition resulted in 80% survival. These data indicate multiple host gene targets were identified from the in vivo screening method. Further, the probability of finding an effective target appears to favor genes that are: 1) transcribed in response to infection, 2) proteins with relatively short half-lives, 3) proteins that interact with multiple cellular signal transduction pathways, and 4) proteins that are expressed at relatively low abundance.
TABLE 3. Host Target Genes Associated with Benefit in the Lethal Challenge Mouse Model
Gene Survival Benefit PPMO/Gene Genes/Pathway
RIG-I 100 1/3 1/1
IL-10 100 3/3 5/9
CCL2 100 3/3 1/1
NOD1 90 2/2 1/1
HMGB1 90 3/3 1/1
CD160 90 3/4 1/1
CSF-1 80 4/4 1/1
IL-10 rec α 80 3/3 5/9
DEC205 80 3/3 1/1
IRF5 80 2/2 1/4
An integrated evaluation for genes identified from these studies is provided in TABLE 3. Survival benefit for a gene is confirmed when multiple oligomers targeting that same gene are active (indicated as PPMO/Gene). Confidence in the observations is enhanced when multiple elements in a signal transduction pathway are identified (indicated as Genes/Pathway). One pathway of interest is the IL-10 signal transduction cascade which includes IL-10 receptor alpha, SOCS3, TNF-α and IL-6. This represents a compelling argument that IL-10 signaling is critical for filovirus induced pathogenesis (Panchal et al. 2014).
This project teaches a great deal about the power of the antisense approach to manipulation of host gene expression for the management of infection induced pathogenesis. First, the
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approach to the design of the oligomer permits selection of host genes with very high probability of success with of 2 - 3 oligomers per gene. Second, selection of genes favors those in which transcription is induced upon infection, the protein is low abundance, is involved in multiple protein:protein interactions and has a relatively short half-life. These criteria provide guidance for a productive interface with bioinformatics collaborators to identify meaningful candidate agents. Third, the PMO approach allows evaluation of genes, such as CD160, expressed in low abundance NK T-cells. Information from low abundance cell types tends to be under represented in RNA expression arrays, RNAseq, or protein expression profiling. Activity in these low abundance cell populations tends to confirm the effective distribution of the oligomers throughout the body. Fourth, the utilization of an intact animal as a screening tool allows for the discovery of the role of genes like CSF-1, which is likely to exert its effect on stimulating the replication of myeloid derived dendritic cells. Use of mice to screen for active genes relied on the platform technology for the feasibility of a limited number of agents per gene, reliable pharmacokinetic profile resulting in consistent dose and duration of action, minimal off target activity, and very low potential for toxicity.
The ongoing efforts in the discovery of antisense therapeutics for Ebola and other emerging infections disease involves deeper investigations into these identified host gene targets. Emphasis on aspects of the infection that may benefit most from therapeutic intervention. One aspect is identifying targets that provide benefit several days after infection, a setting based on viral diagnosis and possible onset of symptoms. A second characteristic will be that host targets that are compatible with responses to vaccines, possibly even providing enhancement of the vaccine response.
Discussion
Antisense therapeutics began with discovery of novel antiviral agents. Paul Zamecnik, the pioneer of antisense technology explored antisense oligodeoxynucleotides to block infection from Rous Sarcoma Virus (RSV) (Zamecnik and Stephenson 1978). They progressed to the human T-cell lymphotropic virus type III (HTLV-III) (Zamecnik et al. 1986) and then to the human immunodeficiency virus (HIV) (Goodchild et al. 1988) establishing a foundation for antisense antivirals. Indeed, my first antisense target was HIV-tat with a poly-L-lysine conjugate to a 3’-morpholino modified oligodeoxyribonucleotides (Stephenson and Iversen 1989).
Ebola provided an aggressive viral infection and a rigorous challenge for an antisense approach. The project leading to AVI-7537 for Ebola and AVI-7288 for Marburg was successful in providing survival benefit in mouse, hamster, and nonhuman primate lethal challenge models (Warren et al. 2010; Iversen et al. 2012; Warren et al. 2015). Further, GLP toxicology studies permitted evaluation in human trials that revealed the compounds are well tolerated and PK observations established a human dose estimate of 11 mg/kg to be administered daily for 14 consecutive days (Heald et al. 2014; Heald et al. 2015). However, limitations associated with the iv route of administration, cost of manufacture and single viral strain specificity diminished enthusiasm for the final effort to gain approval under the animal rule.
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The large outbreak of 2014 in West Africa forced improved Ebola infection management strategies including emerging vaccines, competing therapeutics, and potential repurposing of approved drugs. A favorable reduction in case fatality and growing availability of effective management led to attrition of drugs in development. Therapeutics are constantly challenged by emerging knowledge, protocols, and competing therapeutics forcing re-evaluation of their relevance. An acknowledged risk during drug development is newly approved therapeutics that displace standards of care. Vitravene (Fomiversen) was the first antisense drug to gain FDA approval in 1998 (Stein and Castanotto 2017). It was an intraocular injection developed to treat cytomegalovirus retinitis in immunocompromised patients with AIDS (Katzung 2006). The drug was withdrawn because the development of highly active anti-retroviral therapy (HAART) reduced the number of CMV cases (Bubela and McCabe 2006). Indeed, only one of 23.9 new molecular entities (NME) developed in preclinical stages becomes an approved drug (Bunnage 2011). AVI-7537, a NEM, experienced the attrition the majority of NME experience during drug development.
Market trends in the anti-infective space favor broad-spectrum agents. While medical practitioners tend to favor narrow spectrum agents for their precision, broad-spectrum agents represent a hedge against risk and a larger field of use. Rational drug design based on human genome and proteome screening hold the potential to reduce attrition of NME and risk to drug developers. Targeting human genes to treat Ebola infections can limit viral resistance while expanding spectrum of use. Our studies in the Ebola mouse lethal challenge model identified multiple targets for lead compound discovery and development. These observations may provide a useful starting point for small molecule discovery but also a detailed search for oligonucleotide-induced exon skipping strategies.
The observation of significant, 59% (41/70), survival benefit provided by inhibiting the expression of IL-10 by inducing skipping of exon 4 (ANOVA p < 0.001) is similar to survival in the IL-10 knockout mouse of 70% (7/10). We observed survival benefit by blocking multiple downstream elements of the IL-10 signaling pathway including: (i) 57% (17/30) survival inhibiting IL-10 receptor α by inducing exclusion of exon 2, (ii) 50% (5/10) survival inhibiting the IL-10 receptor associated JAK2 by excluding exon 8, (iii) 70% (7/10) survival inhibiting the translation start site of STAT1, and (iv) 90% (9/10) inhibiting SOCS3 by excluding exon 2. Nearly all category A and B pathogens induce the IL-10 pathway (Couper et al. 2008) and L-10 has been reported to support viral clearance (Brooks et al. 2006). It appears inhibiting the IL-10 pathway enhances the cytotoxic T-cell lymphocytic response shifting immune response from a less efficient B-cell response. These studies support consideration of IL-10 pathway inhibitors as vaccine adjuvants as well as anti-infective therapeutics.
The future of host gene directed antisense for the management of emerging infectious disease is promising. Such agents will not be limited to specific viral strains, are insensitive to many viral mutations thus limiting resistance, and are likely to find utility in non-infective diseases.
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Conclusions
AVI-7537 is an effective Ebola antiviral that has completed most of the key regulatory requirements under the animal rule: (i) Efficacy in a nonhuman primate Ebola lethal challenge model that is a surrogate for human infection, (ii) PK studies enable estimation of a human equivalent dose 0f 11 mg/kg, and (iii) it is well tolerated in multiple dose healthy human trials. However, development of AVI-7537 has been discontinued. Alternative approaches to Ebola therapeutics targeting human genes now presents a feasible and improved path to therapeutic discovery.
Acknowledgements
I wish to acknowledge the contributions of USAMRIID investigators: Sina Bavari, Ricka Panchal, Dana Swenson, Kelly Warfield, Travis Warren, and Lisa Welch. I also wish to acknowledge the contributions of individuals at AVI BioPharma (now Sarepta Therapeutics) including: Jay Charleston, Laura Hauck, Alison Heald, Dan Mourich, Dwight Weller, and Mike Wong. The author wishes to thank support from the Daugherty Foundation.
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