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

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

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

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

Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy.

OBJECTIVE: To report safety, pharmacokinetics, exon 53 skipping, and dystrophin expression in golodirsen-treated patients with Duchenne muscular dystrophy (DMD) amenable to exon 53 skipping. METHODS: Part 1 was a randomized, double-blind, placebo-controlled, 12-week dose titration of once-weekly golodirsen; part 2 is an ongoing, open-label evaluation. Safety and pharmacokinetics were primary and secondary objectives of part 1. Primary biological outcome measures of part 2 were blinded exon skipping and dystrophin protein production on muscle biopsies (baseline, week 48) evaluated, respectively, using reverse transcription PCR and Western blot and immunohistochemistry. RESULTS: Twelve patients were randomized to receive golodirsen (n = 8) or placebo (n = 4) in part 1. All from part 1 plus 13 additional patients received 30 mg/kg golodirsen in part 2. Safety findings were consistent with those previously observed in pediatric patients with DMD. Most of the study drug was excreted within 4 hours following administration. A significant increase in exon 53 skipping was associated with ∼16-fold increase over baseline in dystrophin protein expression at week 48, with a mean percent normal dystrophin protein standard of 1.019% (range, 0.09%-4.30%). Sarcolemmal localization of dystrophin was demonstrated by significantly increased dystrophin-positive fibers (week 48, p < 0.001) and a positive correlation (Spearman r = 0.663; p < 0.001) with dystrophin protein change from baseline, measured by Western blot and immunohistochemistry. CONCLUSION: Golodirsen was well-tolerated; muscle biopsies from golodirsen-treated patients showed increased exon 53 skipping, dystrophin production, and correct dystrophin sarcolemmal localization. CLINICALTRIALSGOV IDENTIFIER: NCT02310906. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that golodirsen is safe and Class IV evidence that it induces exon skipping and novel dystrophin as confirmed by 3 different assays.

Validation of a Muscle-Specific Tissue Image Analysis Tool for Quantitative Assessment of Dystrophin Staining in Frozen Muscle Biopsies.

CONTEXT.­: Duchenne muscular dystrophy is a rare, progressive, and fatal neuromuscular disease caused by dystrophin protein loss. Common investigational treatment approaches aim at increasing dystrophin expression in diseased muscle. Some clinical trials include assessments of novel dystrophin production as a surrogate biomarker of efficacy, which may predict a clinical benefit from treatment. OBJECTIVES.­: To establish an immunofluorescent scanning and digital image analysis workflow that provides an objective approach for staining intensity assessment of the immunofluorescence dystrophin labeling and determination of the percentage of biomarker-positive fibers in muscle cryosections. DESIGN.­: Optimal and repeatable digital image capture was achieved by a rigorously qualified fluorescent scanning process. After scanning qualification, the MuscleMap (Flagship Biosciences, Westminster, Colorado) algorithm was validated by comparing high-power microscopic field total and dystrophin-positive fiber counts obtained by trained pathologists to data derived by MuscleMap. Next, the algorithm was tested on whole-slide images of immunofluorescent-labeled muscle sections from Duchenne muscular dystrophy, Becker muscular dystrophy, and control patients. RESULTS.­: When used under the guidance of a trained pathologist, the digital image analysis tool met predefined validation criteria and demonstrated functional and statistical equivalence with manual assessment. This work is the first, to our knowledge, to qualify and validate immunofluorescent scanning and digital tissue image-analysis workflow, respectively, with the rigor required to support the clinical trial environments. CONCLUSIONS.­: MuscleMap enables analysis of all fibers within an entire muscle biopsy section and provides data on a fiber-by-fiber basis. This will allow future clinical trials to objectively investigate myofibers' dystrophin expression at a greater level of consistency and detail.

Long-term treatment with eteplirsen in nonambulatory patients with Duchenne muscular dystrophy.

This analysis aims to describe the outcomes of two nonambulatory patients with Duchenne muscular dystrophy (DMD) who participated in two clinical studies. The two consecutive trials of eteplirsen (studies 201 and 202) were conducted in patients with DMD (N = 12) and confirmed genetic mutations amenable to exon 51 skipping.In study 201, 12 patients were randomized to receive once-weekly, double-blind intravenous infusions of eteplirsen 30 or 50 mg/kg or placebo for 24 weeks; patients then received open-label eteplirsen during weeks 25 through 28. All 12 patients continued onto open-label extension study 202 and received long-term treatment with eteplirsen. We compared cardiac, pulmonary, and upper limb function and dystrophin production in the nonambulatory twin patients versus the 10 ambulatory patients through 240 combined treatment weeks.Ten study patients remained ambulatory through both studies, while the identical twin patients both experienced early, rapid loss of ambulation. The twin patients had greater disease severity at baseline (6-minute walk test [6MWT], 330 and 256 m) versus the other patients (n = 10; 6MWT range, 341-418 m). They maintained cardiac and upper limb function through combined week 240, with outcomes similar to those of the patients who remained ambulatory. Dystrophin production was confirmed following eteplirsen treatment.Despite the loss of ambulation, other markers of disease progression remained relatively stable in the eteplirsen-treated twin patients and were similar to those of the ambulatory patients.

A new model of reproductive aging: the decline in ovarian non-growing follicle number from birth to menopause.

BACKGROUND: The primary determinant of reproductive age in women is the number of ovarian non-growing (primordial, intermediate and primary) follicles (NGFs). To better characterize the decline in NGF number associated with aging, we have employed modern stereology techniques to determine NGF number in women from birth to menopause. METHODS: Normal human ovaries were collected from 122 women (aged 0-51 years) undergoing elective oophorectomy, organ donation or autopsy. After gross pathologic examination, systematic random sampling was utilized to obtain tissue for analysis by the fractionator/optical disector method. Models to describe the resulting decay curve were constructed and evaluated. RESULTS: NGF decay was best described by a simple power function: log (y) = ax(b) + c, where a, b and c are constants and y = NGF count at age x (R(2) = 0.84, Sums of Squares Error = 28.18 on 119 degrees of freedom). This model implies that follicles decay faster with increasing age. CONCLUSIONS: Unlike previous models of ovarian follicle depletion, our model predicts no sudden change in decay rate, but rather a constantly increasing rate. The model not only agrees well with observed ages of menopause in women, but also is more biologically plausible than previous models. Although the model represents a significant improvement compared with earlier attempts, a considerable percentage of the variation in NGF number between women cannot be explained by age alone.

Eteplirsen treatment for Duchenne muscular dystrophy: Exon skipping and dystrophin production.

OBJECTIVE: To describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen. METHODS: Clinical study 202 was an observational, open-label extension of the randomized, controlled study 201 assessing the safety and efficacy of eteplirsen in patients with DMD with a confirmed mutation in the DMD gene amenable to correction by skipping of exon 51. Patients received once-weekly IV doses of eteplirsen 30 or 50 mg/kg. Upper extremity muscle biopsy samples were collected at combined study week 180, blinded, and assessed for dystrophin-related content by Western blot, Bioquant software measurement of dystrophin-associated immunofluorescence intensity, and percent dystrophin-positive fibers (PDPF). Results were contrasted with matched untreated biopsies from patients with DMD. Reverse transcription PCR followed by Sanger sequencing of newly formed slice junctions was used to confirm the mechanism of action of eteplirsen. RESULTS: Reverse transcription PCR analysis and sequencing of the newly formed splice junction confirmed that 100% of treated patients displayed the expected skipped exon 51 sequence. In treated patients vs untreated controls, Western blot analysis of dystrophin content demonstrated an 11.6-fold increase (p = 0.007), and PDPF analysis demonstrated a 7.4-fold increase (p < 0.001). The PDPF findings were confirmed in a re-examination of the sample (15.5-fold increase, p < 0.001). Dystrophin immunofluorescence intensity was 2.4-fold greater in treated patients than in untreated controls (p < 0.001). CONCLUSION: Taken together, the 4 assays, each based on unique evaluation mechanisms, provided evidence of eteplirsen muscle cell penetration, exon skipping, and induction of novel dystrophin expression. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence of the muscle cell penetration, exon skipping, and induction of novel dystrophin expression by eteplirsen, as confirmed by 4 assays.

Toxicological Characterization of Exon Skipping Phosphorodiamidate Morpholino Oligomers (PMOs) in Non-human Primates.

BACKGROUND: Phosphorodiamidate morpholino oligomers (PMOs) are a class of exon skipping drugs including eteplirsen, which has shown considerable promise for treatment of the degenerative neuromuscular disease, Duchenne musculardystrophy (DMD). OBJECTIVE: Toxicity studies in non-human primates (NHPs) of 12 weeks duration with two new PMOs for DMD, SRP-4045 and SRP-4053, along with results from a chronic study in NHPs of 39 weeks duration for eteplirsen, are described here. METHODS: PMOs were administered once-weekly by bolus intravenous (IV) injections to male NHPs. Endpoints evaluated included plasma exposures, clinical observations, body weight/food consumption, eye exams, electrocardiograms, male reproductive hormones/endpoints, complement alternative pathway, clinical pathology, urinalysis, and macroscopic/light microscopic pathology. RESULTS: Findings in these studies were limited to the kidneys, with a common presentation of tubular basophilia, vacuolation, and/or minimal degeneration that was considered non-adverse. No necrosis, glomerular lesions, or effects on renal function tests such as serum creatinine or urea nitrogen were observed, suggesting that PMO-related kidney findings are not likely to develop into frank nephrotoxicity. There were no adverse effects on other potential target organs after repeated IV injections at the highest dose levels tested, 320 mg/kg. CONCLUSIONS: Nonclinical results in NHPs for these three PMOs, together with the excellent clinical safety established for eteplirsen to date, suggest that once-weekly IV administration of PMOs for lifetime durations at therapeutic doses will be well tolerated by patients with DMD.

Delayed Time-to-Treatment of an Antisense Morpholino Oligomer Is Effective against Lethal Marburg Virus Infection in Cynomolgus Macaques.

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.

A single phosphorodiamidate morpholino oligomer targeting VP24 protects rhesus monkeys against lethal Ebola virus infection.

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.

Neuronal and glial cell number in the hippocampus after experimental traumatic brain injury: analysis by stereological estimation.

Fluid percussion (FP) brain injury causes spatial memory dysfunction in rats regardless of injury location (midline vs. lateral). Standard histological analysis of the injured brains shows hippocampal neuronal loss after lateral, but not midline FP injury. We have used the optical volume fractionator (OVF) stereological procedure to quantify neuronal loss and glial proliferation within specific subregions of the hippocampus after midline or lateral FP injury. The OVF method is a design-based cell counting procedure, which combines cellular numerical density estimates (from the optical disector) with volume estimates (generated by point counting and the fractionator stereology method) to produce an estimate of the absolute cell number. Fifteen adult male Sprague-Dawley rats were randomly divided into 3 groups (n = 5/group): midline injury, lateral injury and naive. A single fluid percussion pulse was delivered to anesthetized rats in the injured groups. At 14 days post-injury, strict morphological criteria enabled the estimation of neurons, astrocytes, oligodendrocytes, and microglia in defined hippocampal subregions. The results confirm that hippocampal neurons are selectively vulnerable to brain injury, particularly observed as a significant loss in the hilus following both types of injury and in area CA3 after lateral injury. In contrast, the number of astrocytes and oligodendrocytes remains unaffected by brain injury, regardless of subregion. However, the significant increase in microglia number (bilaterally after midline and ipsilateral following lateral injury) suggests that underlying cellular processes continue weeks following injury. The implications of the observed cell population changes are discussed in relation to the reported cognitive deficits associated with both lateral and midline FP brain injury.

Changes in cell cycle parameters and cell number in the rat midbrain during organogenesis.

We employed 5-bromo-2'-deoxyuridine (BrdU) labeling to identify in vivo changes in the cell cycle patterns of the rat midbrain during the major period of midbrain organogenesis, gestational days (gd) 11 to 16. We also used quantitative stereology to determine changes in absolute cell numbers during these gestational time points. Between gd 12 and 16, the length of S-phase did not change significantly while the fraction of cycling cells decreased from 73 to 11%. The average cell cycle length was determined to be 15 h on gd 12 and 17 h on gd 16, the difference not being statistically significant. The cell number in the midbrain increased from 1.3E5 cells on gd 11 to 1.7E7 cells on gd 16. On gd 12 and gd 13, there was a significant negative correlation between litter position and midbrain cell number, the effect diminishing on later days of gestation. The combined use of quantitative stereology and flow cytometry to study brain development represents a novel application that allows for simultaneous evaluation of changes in cell proliferation kinetics and the resulting effect of those kinetic changes on embryonic midbrain development.

Effect of methylmercury on glutamate-cysteine ligase expression in the placenta and yolk sac during mouse development.

The placenta and the yolk sac play critical roles in fetal development, including protection from oxidative stress through the presence of detoxifying enzymes. Glutathione (GSH; gamma-glutamylcysteinylglycine), a crucial molecule in the maintenance of cellular redox status, plays a critical role in development, and it is also protective against methylmercury toxicity. Glutamate-cysteine ligase (GCL), the enzyme that catalyzes the rate-limiting step in GSH synthesis, is widely expressed in the mouse embryo and extraembryonic membranes throughout development. The aim of this study was to investigate the effect of low-level subchronic methylmercury exposure on GCL expression in the mouse placenta and yolk sac, after describing the basal developmental expression of the enzyme in these tissues. We found that basal mRNA expression levels increased dramatically in the placenta and the yolk sac at gd 18, whereas protein levels did not increase in parallel with the mRNA. We also found that methylmercury induced GCLc mRNA expression in the placenta at gd 18 in a dose-dependent manner, suggesting an important role for this enzyme in the response of the placenta to toxicants. These changes in expression may be useful as a biomarker of MeHg exposure during development.

Estimating human ovarian non-growing follicle number: the application of modern stereology techniques to an old problem.

BACKGROUND Previous published reports on the number of non-growing follicles (NGFs) in the human ovary have employed model-based methods for number estimates. These methods are time-intensive, and require correction factors and assumptions that ultimately limit their accuracy. Here, we describe the modification, application and validation of a modern fractionator/optical disector technique for the estimation of human ovarian NGF number. METHODS Forty-eight pairs of normal human ovaries were collected from women (age 8-51 years) undergoing elective bilateral oophorectomy, organ donation, or from autopsy. After gross pathologic examination, systematic random sampling was utilized to obtain tissue for analysis by the fractionator/optical disector method. The precision of individual NGF counts was determined by calculating the observed coefficient of error (OCE). Intra-observer variability and variation in NGF number between ovaries within a pair were also determined. RESULTS The mean OCE was 16.6% with larger variations observed at lower follicle counts. In recount experiments of the same ovary, NGF number estimates varied by 15-29%, except at very low follicle counts where variation was greater, but absolute differences were small. There was no significant difference in NGF number between ovaries within a pair (Wilcoxon signed rank test, P = 0.81). CONCLUSIONS Modern stereology methods provide an unbiased, efficient method for estimating NGF number in the human ovary. Both ovaries within a pair contain similar numbers of NGFs.

Compensatory paracrine mechanisms that define the urothelial response to injury in partial bladder outlet obstruction.

Diseases and conditions affecting the lower urinary tract are a leading cause of dysfunctional sexual health, incontinence, infection, and kidney failure. The growth, differentiation, and repair of the bladder's epithelial lining are regulated, in part, by fibroblast growth factor (FGF)-7 and -10 via a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the receptor for FGF-7 and -10 within the transitional epithelium (urothelium). The FGF-7 gene is located at the 15q15-q21.1 locus on chromosome 15 and four exons generate a 3.852-kb mRNA. Five duplicated FGF-7 gene sequences that localized to chromosome 9 were predicted not to generate functional protein products, thus validating the use of FGF-7-null mice as an experimental model. Recombinant FGF-7 and -10 induced proliferation of human urothelial cells in vitro and transitional epithelium of wild-type and FGF-7-null mice in vivo. To determine the extent that induction of urothelial cell proliferation during the bladder response to injury is dependent on FGF-7, an animal model of partial bladder outlet obstruction was developed. Unbiased stereology was used to measure the percentage of proliferating urothelial cells between obstructed groups of wild-type and FGF-7-null mice. The stereological analysis indicated that a statistical significant difference did not exist between the two groups, suggesting that FGF-7 is not essential for urothelial cell proliferation in response to partial outlet obstruction. In contrast, a significant increase in FGF-10 expression was observed in the obstructed FGF-7-null group, indicating that the compensatory pathway that functions in this model results in urothelial repair.