Objective
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, paralysis, and death within 5 ...years of diagnosis. About 10% of cases are inherited, of which 20% are due to mutations in the superoxide dismutase 1 (SOD1) gene. Riluzole, the only US Food and Drug Administration–approved ALS drug, prolongs survival by only a few months. Experiments in transgenic ALS mouse models have shown decreasing levels of mutant SOD1 protein as a potential therapeutic approach. We sought to develop an efficient adeno‐associated virus (AAV)‐mediated RNAi gene therapy for ALS.
Methods
A single‐stranded AAV9 vector encoding an artificial microRNA against human SOD1 was injected into the cerebral lateral ventricles of neonatal SOD1G93A mice, and impact on disease progression and survival was assessed.
Results
This therapy extended median survival by 50% and delayed hindlimb paralysis, with animals remaining ambulatory until the humane endpoint, which was due to rapid body weight loss. AAV9‐treated SOD1G93A mice showed reduction of mutant human SOD1 mRNA levels in upper and lower motor neurons and significant improvements in multiple parameters including the numbers of spinal motor neurons, diameter of ventral root axons, and extent of neuroinflammation in the SOD1G93A spinal cord. Mice also showed previously unexplored changes in pulmonary function, with AAV9‐treated SOD1G93A mice displaying a phenotype reminiscent of patient pathophysiology.
Interpretation
These studies clearly demonstrate that an AAV9‐delivered SOD1‐specific artificial microRNA is an effective and translatable therapeutic approach for ALS. Ann Neurol 2016;79:687–700
Recessive gene mutations underlie many developmental disorders and often lead to disabling neurological problems. Here, we report identification of a homozygous c.170G>A (p.Cys57Tyr or C57Y) mutation ...in the gene coding for protein disulfide isomerase A3 (PDIA3, also known as ERp57), an enzyme that catalyzes formation of disulfide bonds in the endoplasmic reticulum, to be associated with syndromic intellectual disability. Experiments in zebrafish embryos show that PDIA3C57Y expression is pathogenic and causes developmental defects such as axonal disorganization as well as skeletal abnormalities. Expression of PDIA3C57Y in the mouse hippocampus results in impaired synaptic plasticity and memory consolidation. Proteomic and functional analyses reveal that PDIA3C57Y expression leads to dysregulation of cell adhesion and actin cytoskeleton dynamics, associated with altered integrin biogenesis and reduced neuritogenesis. Biochemical studies show that PDIA3C57Y has decreased catalytic activity and forms disulfide‐crosslinked aggregates that abnormally interact with chaperones in the endoplasmic reticulum. Thus, rare disease gene variant can provide insight into how perturbations of neuronal proteostasis can affect the function of the nervous system.
Synopsis
Dysregulation of endoplasmic reticulum (ER) proteostasis is associated with various neurological problems. Here, study of patients with homozygous mutation in PDIA3 links disturbed proteostasis in intellectual disability directly to effects on neuronal connectivity and function.
A homozygous mutation disrupting a redox motif of protein disulfide isomerase A3 (PDIA3) is identified as a possible cause of syndromic intellectual disability.
Pathogenic features associated with mutant PDIA3 include reduced enzymatic activity, formation of protein aggregates, and abnormal interaction with ER chaperones.
Impaired ER proteostasis due to mutant PDIA3 expression results in altered biogenesis of secretory pathway cargoes including integrins, key adhesion molecules involved in synaptic function and plasticity.
Mutant PDIA3 alters neuronal morphogenesis and connectivity, impairing cognitive function such as memory consolidation.
Study of a rare intellectual disability‐linked variant in ER enzyme PDIA3 links cellular proteostasis defects directly to cytoskeleton and adhesion pathways required for neuronal morphogenesis and connectivity.
The highly invasive property of glioblastoma (GBM) cells and genetic heterogeneity are largely responsible for tumor recurrence after the current standard‐of‐care treatment and thus a direct cause of ...death. Previously, we have shown that intracranial interferon‐beta (IFN‐β) gene therapy by locally administered adeno‐associated viral vectors (AAV) successfully treats noninvasive orthotopic glioblastoma models. Here, we extend these findings by testing this approach in invasive human GBM xenograft and syngeneic mouse models. First, we show that a single intracranial injection of AAV encoding human IFN‐β eliminates invasive human GBM8 tumors and promotes long‐term survival. Next, we screened five AAV‐IFN‐β vectors with different promoters to drive safe expression of mouse IFN‐β in the brain in the context of syngeneic GL261 tumors. Two AAV‐IFN‐β vectors were excluded due to safety concerns, but therapeutic studies with the other three vectors showed extensive tumor cell death, activation of microglia surrounding the tumors, and a 56% increase in median survival of the animals treated with AAV/P2‐Int‐mIFN‐β vector. We also assessed the therapeutic effect of combining AAV‐IFN‐β therapy with temozolomide (TMZ). As TMZ affects DNA replication, an event that is crucial for second‐strand DNA synthesis of single‐stranded AAV vectors before active transcription, we tested two TMZ treatment regimens. Treatment with TMZ prior to AAV‐IFN‐β abrogated any benefit from the latter, while the reverse order of treatment doubled the median survival compared to controls. These studies demonstrate the therapeutic potential of intracranial AAV‐IFN‐β therapy in a highly migratory GBM model as well as in a syngeneic mouse model and that combination with TMZ is likely to enhance its antitumor potency.
Our study shows that species‐matched interferon‐beta (IFN‐β) gene therapy by locally administered adeno‐associated viral vectors (AAV) is an effective therapeutic approach for treating both highly invasive human glioblastoma and syngeneic mouse glioblastoma in mouse orthotopic models. We also show that combination treatment with AAV‐IFN‐β and temozolomide (TMZ) provides enhanced therapeutic benefit if AAV‐IFN‐β is administered prior to TMZ.
Neurological disorders – disorders of the brain, spine and associated nerves – are a leading contributor to global disease burden with a shockingly large associated economic cost. Various treatment ...approaches – pharmaceutical medication, device-based therapy, physiotherapy, surgical intervention, among others – have been explored to alleviate the resulting extent of human suffering. In recent years, gene therapy using viral vectors – encoding a therapeutic gene or inhibitory RNA into a “gutted” viral capsid and supplying it to the nervous system – has emerged as a clinically viable option for therapy of brain disorders. In this Review, we provide an overview of the current state and advances in the field of viral vector-mediated gene therapy for neurological disorders. Vector tools and delivery methods have evolved considerably over recent years, with the goal of providing greater and safer genetic access to the central nervous system. Better etiological understanding of brain disorders has concurrently led to identification of improved therapeutic targets. We focus on the vector technology, as well as preclinical and clinical progress made thus far for brain cancer and various neurodegenerative and neurometabolic disorders, and point out the challenges and limitations that accompany this new medical modality. Finally, we explore the directions that neurological gene therapy is likely to evolve towards in the future.
This article is part of the Special Issue entitled “Beyond small molecules for neurological disorders”.
•Provides an overview of viral vector-mediated gene therapy for brain disorders.•Greater, safer genetic access to the central nervous system by viral vector tools.•Focus on clinical progress for neurological disorders and brain tumors.•Outline of existing challenges and future directions for clinical gene therapy.
Objective
GM2 gangliosidosis is usually fatal by 5 years of age in its 2 major subtypes, Tay‐Sachs and Sandhoff disease. First reported in 1881, GM2 gangliosidosis has no effective treatment today, ...and children succumb to the disease after a protracted neurodegenerative course and semi‐vegetative state. This study seeks to further develop adeno‐associated virus (AAV) gene therapy for human translation.
Methods
Cats with Sandhoff disease were treated by intracranial injection of vectors expressing feline β‐N‐acetylhexosaminidase, the enzyme deficient in GM2 gangliosidosis.
Results
Hexosaminidase activity throughout the brain and spinal cord was above normal after treatment, with highest activities at the injection sites (thalamus and deep cerebellar nuclei). Ganglioside storage was reduced throughout the brain and spinal cord, with near complete clearance in many regions. While untreated cats with Sandhoff disease lived for 4.4 ± 0.6 months, AAV‐treated cats lived to 19.1 ± 8.6 months, and 3 of 9 cats lived >21 months. Correction of the central nervous system was so effective that significant increases in lifespan led to the emergence of otherwise subclinical peripheral disease, including megacolon, enlarged stomach and urinary bladder, soft tissue spinal cord compression, and patellar luxation. Throughout the gastrointestinal tract, neurons of the myenteric and submucosal plexuses developed profound pathology, demonstrating that the enteric nervous system was inadequately treated.
Interpretation
The vector formulation in the current study effectively treats neuropathology in feline Sandhoff disease, but whole‐body targeting will be an important consideration in next‐generation approaches. ANN NEUROL 2023;94:969–986
Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that ...supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington's disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders.
Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of β-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its ...substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB-corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB-corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB-corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses.
Sandhoff disease (SD) is caused by decreased function of the enzyme β‐N‐acetylhexosaminidase, resulting in accumulation of GM2 ganglioside in tissues. Neural tissue is primarily affected and ...individuals with the infantile form of the disease generally do not survive beyond 4 years of age. Current treatments address neurometabolic deficits to improve lifespan, however, this extended lifespan allows clinical disease to become manifest in other tissues, including the musculoskeletal system. The impact of SD on bone and joint tissues has yet to be fully determined. In a feline model of infantile SD, animals were treated by intracranial injection of adeno‐associated virus vectors to supply the central nervous system with corrective levels of hexosaminidase, resulting in a twofold to threefold increase in lifespan. As treated animals aged, signs of musculoskeletal disease were identified. The present study characterized bone and joint lesions from affected cats using micro‐computed tomography and histology. All affected cats had similar lesions, whether or not they were treated. SD cats displayed a significant reduction in metaphyseal trabecular bone and markedly abnormal size and shape of epiphyses. Abnormalities increased in severity with age and appear to be due to alteration in the function of chondrocytes within epiphyseal cartilage, particularly the articular‐epiphyseal complex. Older cats developed secondary osteoarthritic changes. The changes identified are similar to those seen in humans with mucopolysaccharidoses. Statement of clinical significance: the lesions identified will have significant implications on the quality of life of individuals whose lifespans are extended due to treatments for the primary neurological effects of SD.
Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer ...exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery.
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