Viral delivery of a microRNA to Gba to the mouse central nervous system models neuronopathic Gaucher disease.

Pathological mutations in GBA, encoding lysosomal glucocerebrosidase (GCase), cause Gaucher disease (GD). GD is a multi-system disease with great phenotypic variation between individuals. It has been classified into type 1 with primarily peripheral involvement and types 2 and 3 with varying degrees of neurological involvement. GD is characterized by decreased GCase activity and subsequent accumulation of its lipid substrates, glucosylceramide and glucosylsphingosine. Current murine models of neuronopathic GD mostly replicate the severe aspects of the neurological symptoms developing rapid progression and early lethality, thus presenting a short window for therapeutic testing. In order to develop a model of chronic neuronopathic GD, we reduced GCase in the central nervous system (CNS) of a mild GD mouse model (GbaD409V/D409V) via intracerebroventricular administration of an adeno-associated virus encoding a microRNA to Gba (AAV-GFP-miR-Gba). GbaD409V/D409V mice have significantly reduced GCase activity and increased substrate accumulation in the CNS. Phenotypically, these mice partially recapitulate features of mild type 1 GD. Their neurological examination reveals cognitive impairment with normal motor features. Administration of AAV-GFP-miR-Gba into GbaD409V/D409V pups in the CNS caused progressive lipid substrate accumulation. Phenotypically, AAV1-GFP-miR-Gba-treated mice were indistinguishable from their littermates until 10 weeks of age, when they started developing progressive neurological impairments, including hyperactivity, abnormal gait, and head retroflexion. Importantly, these impairments can be prevented by simultaneous administration of a miR-resistant GBA, demonstrating that the pathological effects are specifically due to Gba mRNA reduction. This novel model of neuronopathic GD offers several advantages over current models including slower progression of neurological complications and an increased lifespan, which make it more amenable for therapeutic testing.

Gene Therapy Strategies to Restore ER Proteostasis in Disease.

Proteostasis alterations are proposed as a transversal hallmark of several pathological conditions, including metabolic disorders, mechanical injury, cardiac malfunction, neurodegeneration, and cancer. Strategies to improve proteostasis aim to reduce the accumulation of specific disease-related misfolded proteins or bolster the endogenous mechanisms to fold and degrade abnormal proteins. Endoplasmic reticulum (ER) stress is a common pathological signature of a variety of diseases, which engages the unfolded protein response (UPR) as a cellular reaction to mitigate ER stress. Pharmacological modulation of the UPR is challenging considering the physiological importance of the pathway in various organs. However, local targeting of ER stress responses in the affected tissue using gene therapy is emerging as a possible solution to overcome side effects. The delivery of ER chaperones or active UPR components using adeno-associated virus (AAV) has demonstrated outstanding beneficial effects in several disease models (e.g., neurodegenerative conditions, eye disorders, and metabolic diseases). Here, we discuss current efforts to design and optimize gene therapy strategies to improve ER proteostasis in different disease contexts.

TDP43 nuclear export and neurodegeneration in models of amyotrophic lateral sclerosis and frontotemporal dementia.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative disorders marked in most cases by the nuclear exclusion and cytoplasmic deposition of the RNA binding protein TDP43. We previously demonstrated that ALS-associated mutant TDP43 accumulates within the cytoplasm, and that TDP43 mislocalization predicts neurodegeneration. Here, we sought to prevent neurodegeneration in ALS/FTD models using selective inhibitor of nuclear export (SINE) compounds that target exportin-1 (XPO1). SINE compounds modestly extend cellular survival in neuronal ALS/FTD models and mitigate motor symptoms in an in vivo rat ALS model. At high doses, SINE compounds block nuclear egress of an XPO1 cargo reporter, but not at lower concentrations that were associated with neuroprotection. Neither SINE compounds nor leptomycin B, a separate XPO1 inhibitor, enhanced nuclear TDP43 levels, while depletion of XPO1 or other exportins had little effect on TDP43 localization, suggesting that no single exporter is necessary for TDP43 export. Supporting this hypothesis, we find overexpression of XPO1, XPO7 and NXF1 are each sufficient to promote nuclear TDP43 egress. Taken together, our results indicate that redundant pathways regulate TDP43 nuclear export, and that therapeutic prevention of cytoplasmic TDP43 accumulation in ALS/FTD may be enhanced by targeting several overlapping mechanisms.

Cre-dependent AAV vectors for highly targeted expression of disease-related proteins and neurodegeneration in the substantia nigra.

Recombinant adeno-associated virus (AAV) vectors are a popular genetic approach in neuroscience because they confer such efficient transgene expression in the brain and spinal cord. A number of studies have used AAV to express pathological disease-related proteins in the dopaminergic neurons of the substantia nigra in situ ( e.g., α-synuclein to model aspects of Parkinson's disease). The neuropathology and neurodegeneration of Parkinson's disease occur in a circumscribed pattern in the brain, and one of the most important goals of any gene transfer study is accurate, pinpoint targeting. By combining Cre recombinase-dependent AAVs in Cre-driver rats in which Cre is expressed only in the tyrosine hydroxylase neurons, we have achieved more highly targeted expression of several disease-relevant neuropathological proteins in the substantia nigra pars compacta than using constitutive expression AAV vectors. Alpha-synuclein, tau, transactive response DNA-binding protein of 43 kDa, or the control fluorescent protein yellow fluorescent protein was individually expressed to induce highly targeted, dopaminergic neuron-specific neurodegeneration models. The refined targeting foreshadows a next-generation disease modeling system for expressing neurodegenerative disease-related proteins in a disease-relevant manner. We foresee specific utilities of this in vivo AAV vector targeting of pathological proteins to a well-defined and well-demarcated cell population.-Grames, M. S., Dayton, R. D., Jackson, K. L., Richard, A. D., Lu, X., Klein, R. L. Cre-dependent AAV vectors for highly targeted expression of disease-related proteins and neurodegeneration in the substantia nigra.

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction.

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

p62 Pathology Model in the Rat Substantia Nigra with Filamentous Inclusions and Progressive Neurodegeneration.

One of the proteins most frequently found in neuropathological lesions is the ubiquitin binding protein p62 (sequestosome 1). Post-mortem analysis of p62 is a defining diagnostic marker in several neurodegenerative diseases including amyotrophic lateral sclerosis and inclusion body myositis. Since p62 functions in protein degradation pathways including autophagy, the build-up of p62-positive inclusions suggests defects in protein clearance. p62 was expressed unilaterally in the rat substantia nigra with an adeno-associated virus vector (AAV9) in order to study p62 neuropathology. Inclusions formed within neurons from several days to several weeks after gene transfer. By electron microscopy, the inclusions were found to contain packed 10 nm thick filaments, and mitochondria cristae structure was disrupted, resulting in the formation of empty spaces. In corollary cell culture transfections, p62 clearly impaired mitochondrial function. To probe for potential effects on macroautophagy, we co-expressed p62 with a double fluorescent tagged reporter for the autophagosome protein LC3 in the rat. p62 induced a dramatic and specific dissociation of the two tags. By 12 weeks, a rotational behavior phenotype manifested, consistent with a significant loss of dopaminergic neurons analyzed post-mortem. p62 overexpression resulted in a progressive and robust pathology model with neuronal inclusions and neurodegeneration. p62 gene transfer could be a novel methodological probe to disrupt mitochondrial function or autophagy in the brain and other tissues in vivo.

Corrigendum: Better Targeting, Better Efficiency for Wide-Scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B.

[This corrects the article on p. 116 in vol. 9, PMID: 27867348.].

Better Targeting, Better Efficiency for Wide-Scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B.

Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats.

Severe respiratory changes at end stage in a FUS-induced disease state in adult rats.

BACKGROUND: Fused in sarcoma (FUS) is an RNA-binding protein associated with the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. ALS manifests in patients as a progressive paralysis which leads to respiratory dysfunction and failure, the primary cause of death in ALS. We expressed human FUS in rats to determine if FUS would induce ALS relevant respiratory changes to serve as an early stage disease indicator. The FUS expression was initiated in adult rats by way of an intravenously administered adeno-associated virus vector serotype 9 (AAV9) providing an adult onset model. RESULTS: The rats developed progressive motor impairments observed as early as 2-3 weeks post gene transfer. Respiratory abnormalities manifested 4-7 weeks post gene transfer including increased respiratory frequency and decreased tidal volume. Rats with breathing abnormalities also had arterial blood acidosis. Similar detailed plethysmographic changes were found in adult rats injected with AAV9 TDP-43. FUS gene transfer to adult rats yielded a consistent pre-clinical model with relevant motor paralysis in the early to middle stages and respiratory dysfunction at the end stage. Both FUS and TDP-43 yielded a similar consistent disease state. CONCLUSIONS: This modeling method yields disease relevant motor and respiratory changes in adult rats. The reproducibility of the data supports the use of this method to study other disease related genes and their combinations as well as a platform for disease modifying interventional strategies.

AAV9 supports wide-scale transduction of the CNS and TDP-43 disease modeling in adult rats.

AAV9 has emerged as an efficient adeno-associated virus (AAV) serotype for gene transfer to the central nervous system. We have used this technique to study aspects of amyotrophic lateral sclerosis (ALS) by administering AAV encoding the ALS-related gene transactive response DNA binding protein of 43 kDa (TDP-43) to neonatal rats. However, inducing the expression in adult subjects would be preferable to mimic the adult onset of symptoms in ALS. We expressed either green fluorescent protein (GFP) or TDP-43 in adult rats after an intravenous (i.v.) route of administration to attempt wide-scale transduction of the spinal cord for disease modeling. In order to optimize the gene transfer, we made comparisons of efficiency by age, gender, and across several AAV serotypes (AAV1, AAV8, AAV9, and AAV10). The data indicate more efficient neuronal transduction in neonates, with little evidence of glial transduction at either age, no gender-related differences in transduction, and that AAV9 was efficient in adults relative to the other serotypes tested. Based on these data, AAV9 TDP-43 was expressed at three vector doses in adult female rats yielding highly consistent, dose-dependent motor deficits. AAV9 can be delivered i.v. to adult rats to achieve consistent pathophysiological changes and a relevant adult-onset system for disease modeling.

Initial gene vector dosing for studying symptomatology of amyotrophic lateral sclerosis in non-human primates.

BACKGROUND: Most amyotrophic lateral sclerosis (ALS) research has focused on mice, but there are distinct differences in the functional neuroanatomy of the corticospinal pathway in primates vs. rodents. A non-human primate model may be more sensitive and more predictive for therapeutic efficacy. METHODS: Rhesus macaques received recombinant adeno-associated virus (AAV9) encoding either the ALS-related pathological protein TDP-43 or a green fluorescent protein (GFP) control by intravenous administration. Motor function and electromyography were assessed over a nine-month expression interval followed by post-mortem analyses. RESULTS: Recombinant TDP-43 or GFP was stably expressed long term. Although the TDP-43 subjects did not manifest severe paralysis and atrophy, there were trends of a partial disease state in the TDP-43 subjects relative to the control. CONCLUSIONS: These data indicate that a higher gene vector dose will likely be necessary for more robust effects, yet augur that a relevant primate model is feasible.