Development of intravenously administered synthetic RNA virus immunotherapy for the treatment of cancer.

The therapeutic effectiveness of oncolytic viruses (OVs) delivered intravenously is limited by the development of neutralizing antibody responses against the virus. To circumvent this limitation and to enable repeated systemic administration of OVs, here we develop Synthetic RNA viruses consisting of a viral RNA genome (vRNA) formulated within lipid nanoparticles. For two Synthetic RNA virus drug candidates, Seneca Valley virus (SVV) and Coxsackievirus A21, we demonstrate vRNA delivery and replication, virus assembly, spread and lysis of tumor cells leading to potent anti-tumor efficacy, even in the presence of OV neutralizing antibodies in the bloodstream. Synthetic-SVV replication in tumors promotes immune cell infiltration, remodeling of the tumor microenvironment, and enhances the activity of anti-PD-1 checkpoint inhibitor. In mouse and non-human primates, Synthetic-SVV is well tolerated reaching exposure well above the requirement for anti-tumor activity. Altogether, the Synthetic RNA virus platform provides an approach that enables repeat intravenous administration of viral immunotherapy.

Functional outcome measures in young, steroid-naïve boys with Duchenne muscular dystrophy.

The purpose of this study was to quantitate motor performance in 196 genetically confirmed steroid-naïve boys with Duchenne muscular dystrophy (DMD), to evaluate the test-retest reliability of measures of motor performance in young DMD boys, and to assess correlations among the different functional outcomes including timed tests. Boys aged 4-7 years were recruited in the FOR-DMD study, a comparative effectiveness study of different steroid regimens in DMD. Eligible boys had to be able to rise from the floor independently and to perform pulmonary function testing consistently. The boys were evaluated with standardized assessments at the screening and baseline visits at 32 sites in 5 countries (US, UK, Canada, Italy, Germany). Assessments included timed rise from floor, timed 10 m walk/run, six-minute walk distance, North Star Ambulatory Assessment (NSAA) and forced vital capacity (FVC). Mean age at baseline was 5.9 years (range 4.1-8.1 years). Test-retest reliability was high for functional assessments, regardless of time lag between assessments (up to 90 days) and for the majority of age groups. Correlations were strong among the functional measures and timed tests, less so with FVC. Physiotherapy measures are reliable in a young, steroid-naïve population and rise from floor velocity appears to be a sensitive measure of strength in this population.

ONCR-177, an Oncolytic HSV-1 Designed to Potently Activate Systemic Antitumor Immunity.

ONCR-177 is an engineered recombinant oncolytic herpes simplex virus (HSV) with complementary safety mechanisms, including tissue-specific miRNA attenuation and mutant UL37 to inhibit replication, neuropathic activity, and latency in normal cells. ONCR-177 is armed with five transgenes for IL12, FLT3LG (extracellular domain), CCL4, and antagonists to immune checkpoints PD-1 and CTLA-4. In vitro assays demonstrated that targeted miRNAs could efficiently suppress ONCR-177 replication and transgene expression, as could the HSV-1 standard-of-care therapy acyclovir. Although ONCR-177 was oncolytic across a panel of human cancer cell lines, including in the presence of type I IFN, replication was suppressed in human pluripotent stem cell-derived neurons, cardiomyocytes, and hepatocytes. Dendritic cells activated with ONCR-177 tumor lysates efficiently stimulated tumor antigen-specific CD8+ T-cell responses. In vivo, biodistribution analyses suggested that viral copy number and transgene expression peaked approximately 24 to 72 hours after injection and remained primarily within the injected tumor. Intratumoral administration of ONCR-177 mouse surrogate virus, mONCR-171, was efficacious across a panel of syngeneic bilateral mouse tumor models, resulting in partial or complete tumor regressions that translated into significant survival benefits and to the elicitation of a protective memory response. Antitumor effects correlated with local and distant intratumoral infiltration of several immune effector cell types, consistent with the proposed functions of the transgenes. The addition of systemic anti-PD-1 augmented the efficacy of mONCR-171, particularly for abscopal tumors. Based in part upon these preclinical results, ONCR-177 is being evaluated in patients with metastatic cancer (ONCR-177-101, NCT04348916).

Evaluation of the innate immune responses to influenza and live-attenuated influenza vaccine infection in primary differentiated human nasal epithelial cells.

The host innate immune response to influenza virus is a key determinant of pathogenic outcomes and long-term protective immune responses against subsequent exposures. Here, we present a direct contrast of the host responses in primary differentiated human nasal epithelial cell (hNEC) cultures following infection with either a seasonal H3N2 influenza virus (WT) or the antigenically-matched live-attenuated vaccine (LAIV) strain. Comparison of the transcriptional profiles obtained 24 and 36h post-infection showed that the magnitude of gene expression was greater in LAIV infected relative to that observed in WT infected hNEC cultures. Functional enrichment analysis revealed that the antiviral and inflammatory responses were largely driven by type III IFN induction in both WT and LAIV infected cells. However, the enrichment of biological pathways involved in the recruitment of mononuclear leukocytes, antigen-presenting cells, and T lymphocytes was uniquely observed in LAIV infected cells. These observations were reflective of the host innate immune responses observed in individuals acutely infected with influenza viruses. These findings indicate that cell-intrinsic type III IFN-mediated innate immune responses in the nasal epithelium are not only crucial for viral clearance and attenuation, but may also play an important role in the induction of protective immune responses with live-attenuated vaccines.

Glucocerebrosidase gene therapy prevents α-synucleinopathy of midbrain dopamine neurons.

Diminished lysosomal function can lead to abnormal cellular accumulation of specific proteins, including α-synuclein, contributing to disease pathogenesis of vulnerable neurons in Parkinson's disease (PD) and related α-synucleinopathies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase (GCase), and mutations in GBA1 are a prominent genetic risk factor for PD. Previous studies showed that in sporadic PD, and in normal aging, GCase brain activity is reduced and levels of corresponding glycolipid substrates are increased. The present study tested whether increasing GCase through AAV-GBA1 intra-cerebral gene delivery in two PD rodent models would reduce the accumulation of α-synuclein and protect midbrain dopamine neurons from α-synuclein-mediated neuronal damage. In the first model, transgenic mice overexpressing wildtype α-synuclein throughout the brain (ASO mice) were used, and in the second model, a rat model of selective dopamine neuron degeneration was induced by AAV-A53T mutant α-synuclein. In ASO mice, intra-cerebral AAV-GBA1 injections into several brain regions increased GCase activity and reduced the accumulation of α-synuclein in the substantia nigra and striatum. In rats, co-injection of AAV-GBA1 with AAV-A53T α-synuclein into the substantia nigra prevented α-synuclein-mediated degeneration of nigrostriatal dopamine neurons by 6 months. These neuroprotective effects were associated with altered protein expression of markers of autophagy. These experiments demonstrate, for the first time, the neuroprotective effects of increasing GCase against dopaminergic neuron degeneration, and support the development of therapeutics targeting GCase or other lysosomal genes to improve neuronal handling of α-synuclein.

Sustained Systemic Glucocerebrosidase Inhibition Induces Brain α-Synuclein Aggregation, Microglia and Complement C1q Activation in Mice.

AIMS: Loss-of-function mutations in GBA1, which cause the autosomal recessive lysosomal storage disease, Gaucher disease (GD), are also a key genetic risk factor for the α-synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase and reductions in this enzyme result in the accumulation of the glycolipid substrates glucosylceramide and glucosylsphingosine. Deficits in autophagy and lysosomal degradation pathways likely contribute to the pathological accumulation of α-synuclein in PD. In this report we used conduritol-ß-epoxide (CBE), a potent selective irreversible competitive inhibitor of glucocerebrosidase, to model reduced glucocerebrosidase activity in vivo, and tested whether sustained glucocerebrosidase inhibition in mice could induce neuropathological abnormalities including α-synucleinopathy, and neurodegeneration. RESULTS: Our data demonstrate that daily systemic CBE treatment over 28 days caused accumulation of insoluble α-synuclein aggregates in the substantia nigra, and altered levels of proteins involved in the autophagy lysosomal system. These neuropathological changes were paralleled by widespread neuroinflammation, upregulation of complement C1q, abnormalities in synaptic, axonal transport and cytoskeletal proteins, and neurodegeneration. INNOVATION: A reduction in brain GCase activity has been linked to sporadic PD and normal aging, and may contribute to the susceptibility of vulnerable neurons to degeneration. This report demonstrates that systemic reduction of GCase activity using chemical inhibition, leads to neuropathological changes in the brain reminiscent of α-synucleinopathy. CONCLUSIONS: These data reveal a link between reduced glucocerebrosidase and the development of α-synucleinopathy and pathophysiological abnormalities in mice, and support the development of GCase therapeutics to reduce α-synucleinopathy in PD and related disorders.

Supporting Practices to Adopt Registry-Based Care (SPARC): protocol for a randomized controlled trial.

BACKGROUND: Diabetes is predicted to increase in incidence by 42% from 1995 to 2025. Although most adults with diabetes seek care from primary care practices, adherence to treatment guidelines in these settings is not optimal. Many practices lack the infrastructure to monitor patient adherence to recommended treatment and are slow to implement changes critical for effective management of patients with chronic conditions. Supporting Practices to Adopt Registry-Based Care (SPARC) will evaluate effectiveness and sustainability of a low-cost intervention designed to support work process change in primary care practices and enhance focus on population-based care through implementation of a diabetes registry. METHODS: SPARC is a two-armed randomized controlled trial (RCT) of 30 primary care practices in the Virginia Ambulatory Care Outcomes Research Network (ACORN). Participating practices (including control groups) will be introduced to population health concepts and tools for work process redesign and registry adoption at a meeting of practice-level implementation champions. Practices randomized to the intervention will be assigned study peer mentors, receive a list of specific milestones, and have access to a physician informaticist. Peer mentors are clinicians who successfully implemented registries in their practices and will help champions in the intervention practices throughout the implementation process. During the first year, peer mentors will contact intervention practices monthly and visit them quarterly. Control group practices will not receive support or guidance for registry implementation. We will use a mixed-methods explanatory sequential design to guide collection of medical record, participant observation, and semistructured interview data in control and intervention practices at baseline, 12 months, and 24 months. We will use grounded theory and a template-guided approach using the Consolidated Framework for Implementation Research to analyze qualitative data on contextual factors related to registry adoption. We will assess intervention effectiveness by comparing changes in patient-level hemoglobin A1c scores from baseline to year 1 between intervention and control practices. DISCUSSION: Findings will enhance our understanding of how to leverage existing practice resources to improve diabetes care in primary care practices by implementing and using a registry. SPARC has the potential to validate the effectiveness of low-cost implementation strategies that target practice change in primary care. TRIAL REGISTRATION: NCT02318108.

Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven AAV9 neonatal intracerebroventricular injection.

Adeno-associated viral (AAV) gene transfer holds great promise for treating a wide-range of neurodegenerative disorders. The AAV9 serotype crosses the blood-brain barrier and shows enhanced transduction efficiency compared to other serotypes, thus offering advantageous targeting when global transgene expression is required. Neonatal intravenous or intracerebroventricular (i.c.v.) delivery of recombinant AAV9 (rAAV9) have recently proven effective for modeling and treating several rodent models of neurodegenerative disease, however, the technique is associated with variable cellular tropism, making tailored gene transfer a challenge. In the current study, we employ the human synapsin 1 (hSYN1) gene promoter to drive neuron-specific expression of green fluorescent protein (GFP) after neonatal i.c.v. injection of rAAV9 in mice. We observed widespread GFP expression in neurons throughout the brain, spinal cord, and peripheral nerves and ganglia at 6 weeks-of-age. Region-specific quantification of GFP expression showed high neuronal transduction rates in substantia nigra pars reticulata (43.9±5.4%), motor cortex (43.5±3.3%), hippocampus (43.1±2.7%), cerebellum (29.6±2.3%), cervical spinal cord (24.9±3.9%), and ventromedial striatum (16.9±4.3%), among others. We found that 14.6±2.2% of neuromuscular junctions innervating the gastrocnemius muscle displayed GFP immunoreactivity. GFP expression was identified in several neuronal sub-types, including nigral tyrosine hydroxylase (TH)-positive dopaminergic cells, striatal dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32)-positive neurons, and choline acetyltransferase (ChAT)-positive motor neurons. These results build on contemporary gene transfer techniques, demonstrating that the hSYN1 promoter can be used with rAAV9 to drive robust neuron-specific transgene expression throughout the nervous system.

ALS-associated peripherin spliced transcripts form distinct protein inclusions that are neuroprotective against oxidative stress.

Intracellular proteinaceous inclusions are well-documented hallmarks of the fatal motor neuron disorder amyotrophic lateral sclerosis (ALS). The pathological significance of these inclusions remains unknown. Peripherin, a type III intermediate filament protein, is upregulated in ALS and identified as a component within different types of ALS inclusions. The formation of these inclusions may be associated with abnormal peripherin splicing, whereby an increase in mRNA retaining introns 3 and 4 (Per-3,4) leads to the generation of an aggregation-prone isoform, Per-28. During the course of evaluating peripherin filament assembly in SW-13 cells, we identified that expression of both Per-3,4 and Per-28 transcripts formed inclusions with categorically distinct morphology: Per-3,4 was associated with cytoplasmic condensed/bundled filaments, small inclusions (<10µM), or large inclusions (≥10µM); while Per-28 was associated with punctate inclusions in the nucleus and/or cytoplasm. We found temporal and spatial changes in inclusion morphology between 12 and 48h post-transfected cells, which were accompanied by unique immunofluorescent and biochemical changes of other ALS-relevant proteins, including TDP-43 and ubiquitin. Despite mild cytotoxicity associated with peripherin transfection, Per-3,4 and Per-28 expression increased cell viability during H2O2-mediated oxidative stress in BE(2)-M17 neuroblastoma cells. Taken together, this study shows that ALS-associated peripherin isoforms form dynamic cytoplasmic and intranuclear inclusions, effect changes in local endogenous protein expression, and afford cytoprotection against oxidative stress. These findings may have important relevance to understanding the pathophysiological role of inclusions in ALS.

Progressive axonal transport and synaptic protein changes correlate with behavioral and neuropathological abnormalities in the heterozygous Q175 KI mouse model of Huntington's disease.

A long-term goal of modeling Huntington's disease (HD) is to recapitulate the cardinal features of the disease in mice that express both mutant and wild-type (WT) huntingtin (Htt), as HD commonly manifests as a heterozygous condition in humans, and loss of WT Htt is associated with loss-of-function. In a new heterozygous Q175 knock-in (KI) mouse model, we performed an extensive evaluation of motor and cognitive functional deficits, neuropathological and biochemical changes and levels of proteins involved in synaptic function, the cytoskeleton and axonal transport, at 1-16 months of age. Motor deficits were apparent at 6 months of age in Q175 KI mice and at that time, postmortem striatal gamma-aminobutyric acid (GABA) levels were elevated and mutant Htt inclusions were present throughout the brain. From 6 months of age, levels of proteins associated with synaptic function, including SNAP-25, Rab3A and PSD-95, and with axonal transport and microtubules, including KIF3A, dynein and dynactin, were altered in the striatum, motor cortex, prefrontal cortex and hippocampus of Q175 KI mice, compared with WT levels. At 12-16 months of age, Q175 KI mice displayed motor and cognitive deficits, which were paralleled at postmortem by striatal atrophy, cortical thinning, degeneration of medium spiny neurons, dense mutant Htt inclusion formation, decreased striatal dopamine levels and loss of striatal brain-derived neurotrophic factor (BDNF). Data from this study indicate that the heterozygous Q175 KI mouse represents a realistic model for HD and also provides new insights into the specific and progressive synaptic, cytoskeletal and axonal transport protein abnormalities that may accompany the disease.

The effects of age and focality on delay-execute prospective memory.

In everyday prospective remembering, individuals must often delay the execution of a retrieved intention until they are in the appropriate setting. These so-called 'delay-execute' tasks are particularly troublesome for older adults, who consistently demonstrate impaired performance in this kind of laboratory task. To better understand this effect, we investigated delay-execute prospective memory performance in younger and older adults. Specifically, we examined the strategies individuals used to maintain intentions over a delay period by analyzing response times to the ongoing task, both before and after the cue event. The results suggest that younger and older individuals perform the task similarly by rehearsing or reformulating the intention. Despite performing the task in a similar manner, older adults showed greater impairments in delay-execute prospective remembering.

Working memory and the strategic control of attention in older and younger adults.

OBJECTIVE: The objective of this study was to investigate the effects of aging on the strategic control of attention and the extent to which this relationship is mediated by working memory capacity (WMC). This study also sought to investigate boundary conditions wherein age differences in selectivity may occur. METHOD: Across 2 studies, the value-directed remembering task used by Castel and colleagues (Castel, A. D., Balota, D. A., & McCabe, D. P. (2009). Memory efficiency and the strategic control of attention at encoding: Impairments of value-directed remembering in Alzheimer's Disease. Neuropsychology, 23, 297-306) was modified to include value-directed forgetting. Study 2 incorporated valence as an additional task demand, and age differences were predicted in both studies due to increased demands of controlled processing. Automated operation span and Stroop span were included as working memory measures, and working memory was predicted to mediate performance. RESULTS: Results confirmed these predictions, as older adults were less efficient in maximizing selectivity scores when high demands were placed on selectivity processes, and working memory was found to mediate performance on this task. DISCUSSION: When list length was increased from previous studies and participants were required to actively forget negative-value words, older adults were not able to selectively encode high-value information to the same degree as younger adults. Furthermore, WMC appears to support the ability to selectively encode information.

Arenavirus evasion of host anti-viral responses.

The innate response to infection by an Old World arenavirus is initiated and mediated by extracellular and intracellular receptors, and effector molecules. In response, the invading virus has evolved to inhibit these responses and create the best environment possible for replication and spread. Here, we will discuss both the host's response to infection with data from human infection and lessons learned from animal models, as well as the multitude of ways the virus combats the resulting immune response. Finally, we will highlight recent work identifying TLR2 as an innate sensor for arenaviruses and how the TLR2-dependent response differs depending on the pathogenicity of the strain.

Pathogenic Old World arenaviruses inhibit TLR2/Mal-dependent proinflammatory cytokines in vitro.

Lymphocytic choriomeningitis virus (LCMV), the prototype arenavirus, and Lassa virus (LASV), the causative agent of Lassa fever (LF), have extensive strain diversity and significant variations in pathogenicity for humans and experimental animals. The WE strain of LCMV (LCMV-WE), but not the Armstrong (Arm) strain, induces a fatal LF-like disease in rhesus macaques. We also demonstrated that LASV infection of human macrophages and endothelial cells resulted in reduced levels of proinflammatory cytokines. Here we have shown that cells infected with LASV or with LCMV-WE suppressed Toll-like receptor 2 (TLR2)-dependent proinflammatory cytokine responses. The persisting isolate LCMV clone 13 (CL13) also failed to stimulate interleukin-6 (IL-6) in macrophages. In contrast, nonpathogenic Mopeia virus, which is a genetic relative of LASV and LCMV-Arm induced robust responses that were TLR2/Mal dependent, required virus replication, and were enhanced by CD14. Superinfection experiments demonstrated that the WE strain of LCMV inhibited the Arm-mediated IL-8 response during the early stage of infection. In cells transfected with the NF-κB-luciferase reporter, infection with LCMV-Arm resulted in the induction of NF-κB, but cells infected with LCMV-WE and CL13 did not. These results suggest that pathogenic arenaviruses suppress NF-κB-mediated proinflammatory cytokine responses in infected cells.