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BACKGROUND: Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor disability disorder that also manifests characteristic abnormal thyroid hormone (TH) levels. AHDS is caused by inactivating mutations in monocarboxylate transporter 8 (MCT8), a specific TH plasma membrane transporter widely expressed in the central nervous system (CNS). MCT8 mutations cause impaired transport of TH across brain barriers, leading to insufficient neural TH supply. There is currently no successful therapy for the neurological symptoms. Earlier work has shown that intravenous (IV), but not intracerebroventricular adeno-associated virus serotype 9 (AAV9) -based gene therapy given to newborn Mct8 knockout (Mct8(−/y)) male mice increased triiodothyronine (T(3)) brain content and partially rescued TH-dependent gene expression, suggesting a promising approach to treat this neurological disorder. METHODS: The potential of IV delivery of AAV9 carrying human MCT8 was tested in the well-established Mct8(−/y)/Organic anion-transporting polypeptide 1c1 (Oatp1c1)(−/ −) double knockout (dKO) mouse model of AHDS, which, unlike Mct8(−/y) mice, displays both neurological and TH phenotype. Further, as the condition is usually diagnosed during childhood, treatment was given intravenously to P30 mice and psychomotor tests were carried out blindly at P120–P140 after which tissues were collected and analyzed. RESULTS: Systemic IV delivery of AAV9-MCT8 at a juvenile stage led to improved locomotor and cognitive functions at P120–P140, which was accompanied by a near normalization of T(3) content and an increased response of positively regulated TH-dependent gene expression in different brain regions examined (thalamus, hippocampus, and parietal cortex). The effects on serum TH concentrations and peripheral tissues were less pronounced, showing only improvement in the serum T(3)/reverse T(3) (rT(3)) ratio and in liver deiodinase 1 expression. CONCLUSION: IV administration of AAV9, carrying the human MCT8, to juvenile dKO mice manifesting AHDS has long-term beneficial effects, predominantly on the CNS. This preclinical study indicates that this gene therapy has the potential to ameliorate the devastating neurological symptoms in patients with AHDS.

Background CLN8-Batten disease (CLN8 disease) is a rare neurodegenerative disorder characterized phenotypically by progressive deterioration of motor and cognitive abilities, visual symptoms, epileptic seizures, and premature death. Mutations in CLN8 results in characteristic Batten disease symptoms and brain-wide pathology including accumulation of lysosomal storage material, gliosis, and neurodegeneration. Recent investigations of other subforms of Batten disease (CLN1, CLN3, CLN6) have emphasized the influence of biological sex on disease and treatment outcomes; however, little is known about sex differences in the CLN8 subtype. To determine the impact of sex on CLN8 disease burden and progression, we utilized a Cln8mnd mouse model to measure the impact and progression of histopathological and behavioral outcomes between sexes. Results Several notable sex differences were observed in the presentation of brain pathology, including Cln8mnd female mice consistently presenting with greater GFAP+ astrocytosis and CD68+ microgliosis in the somatosensory cortex, ventral posteromedial/ventral posterolateral nuclei of the thalamus, striatum, and hippocampus when compared to Cln8mnd male mice. Furthermore, sex differences in motor-behavioral assessments revealed Cln8mnd female mice experience poorer motor performance and earlier death than their male counterparts. Cln8mnd mice treated with an AAV9-mediated gene therapy were also examined to assess sex differences on therapeutics outcomes, which revealed no appreciable differences between the sexes when responding to the therapy. Conclusions Taken together, our results provide further evidence of biologic sex as a modifier of Batten disease progression and outcome, thus warranting consideration when conducting investigations and monitoring therapeutic impact.

CLN6-Batten disease, a form of neuronal ceroid lipofuscinosis is a rare lysosomal storage disorder presenting with gradual declines in motor, visual, and cognitive abilities and early death by 12–15 years of age. We developed a self-complementary adeno-associated virus serotype 9 (scAAV9) vector expressing the human CLN6 gene under the control of a chicken β-actin (CB) hybrid promoter. Intrathecal delivery of scAAV9.CB.hCLN6 into the cerebrospinal fluid (CSF) of the lumbar spinal cord of 4-year-old non-human primates was safe, well tolerated, and led to efficient targeting throughout the brain and spinal cord. A single intracerebroventricular (i.c.v.) injection at post-natal day 1 in Cln6 mutant mice delivered scAAV9.CB.CLN6 directly into the CSF, and it prevented or drastically reduced all of the pathological hallmarks of Batten disease. Moreover, there were significant improvements in motor performance, learning and memory deficits, and survival in treated Cln6 mutant mice, extending survival from 15 months of age (untreated) to beyond 21 months of age (treated). Additionally, many parameters were similar to wild-type counterparts throughout the lifespan of the treated mice., Graphical Abstract, CLN6-Batten disease is a fatal lysosomal storage disorder that primarily affects children. Weimer and colleagues developed an AAV9 gene therapy that was well tolerated in mice and non-human primates. Delivery of this therapy into the brain of CLN6-Batten mice prevented most neurodegenerative symptoms, with treated mice living a full lifespan.

CLN8 disease is a rare form of neuronal ceroid lipofuscinosis caused by biallelic mutations in the CLN8 gene, which encodes a transmembrane endoplasmic reticulum protein involved in trafficking of lysosomal enzymes. CLN8 disease patients present with myoclonus, tonic-clonic seizures, and progressive declines in cognitive and motor function, with many cases resulting in premature death early in life. There are currently no treatments that can cure the disease or substantially slow disease progression. Using a mouse model of CLN8 disease, we tested the safety and efficacy of an intracerebroventricularly (i.c.v.) delivered self-complementary adeno-associated virus serotype 9 (scAAV9) gene therapy vector driving expression of human CLN8. A single neonatal injection was safe and well tolerated, resulting in robust transgene expression throughout the CNS from 4 to 24 months, reducing histopathological and behavioral hallmarks of the disease and restoring lifespan from 10 months in untreated animals to beyond 24 months of age in treated animals. While it is unclear whether some of these behavioral improvements relate to preserved visual function, improvements in learning/memory, or other central or peripheral benefits, these results demonstrate, by far, the most successful degree of rescue reported in an animal model of CLN8 disease, and they support further development of gene therapy for this disorder.

A lack of stratification methods in patients with amyotrophic lateral sclerosis (ALS) is likely implicated in therapeutic failures. Regional diversities and pathophysiological abnormalities in astrocytes from mice with SOD1 mutations (mSOD1-ALS) can now be explored in human patients using somatic cell reprogramming. Here, fibroblasts from four sporadic (sALS) and three mSOD1-ALS patients were transdifferentiated into induced astrocytes (iAstrocytes). ALS iAstrocytes were neurotoxic toward HB9-GFP mouse motor neurons (MNs) and exhibited subtype stratification through GFAP, CX43, Ki-67, miR-155 and miR-146a expression levels. Up- (two cases) and down-regulated (three cases) miR-146a values in iAstrocytes were recapitulated in their secretome, either free or as cargo in small extracellular vesicles (sEVs). We previously showed that the neuroprotective phenotype of depleted miR-146 mSOD1 cortical astrocytes was reverted by its mimic. Thus, we tested such modulation in the most miR-146a-depleted patient-iAstrocytes (one sALS and one mSOD1-ALS). The miR-146a mimic in ALS iAstrocytes counteracted their reactive/inflammatory profile and restored miR-146a levels in sEVs. A reduction in lysosomal activity and enhanced synaptic/axonal transport-related genes in NSC-34 MNs occurred after co-culture with miR-146a-modulated iAstrocytes. In summary, the regulation of miR-146a in depleted ALS astrocytes may be key in reestablishing their normal function and in restoring MN lysosomal/synaptic dynamic plasticity in disease sub-groups.

Sporadic amyotrophic lateral sclerosis (ALS) is a fatal disease with unknown etiology, characterized by a progressive loss of motor neurons leading to paralysis and death typically within 3–5 years of onset. Recently, there has been remarkable progress in understanding inherited forms of ALS in which well defined mutations are known to cause the disease. Rodent models in which the superoxide dismutase-1 (SOD1) mutation is overexpressed recapitulate hallmark signs of ALS in patients. Early anatomical changes in mouse models of fALS are seen in the neuromuscular junctions (NMJs) and lower motor neurons, and selective reduction of toxic mutant SOD1 in the spinal cord and muscle of these models has beneficial effects. Therefore, much of ALS research has focused on spinal motor neuron and NMJ aspects of the disease. Here we show that, in the SOD1G93Arat model of ALS, spinal motor neuron loss occurs presymptomatically and before degeneration of ventral root axons and denervation of NMJs. Although overt cell death of corticospinal motor neurons does not occur until disease endpoint, we wanted to establish whether the upper motor neuron might still play a critical role in disease progression. Surprisingly, the knockdown of mutant SOD1 in only the motor cortex of presymptomatic SOD1G93Arats through targeted delivery of AAV9–SOD1–shRNA resulted in a significant delay of disease onset, expansion of lifespan, enhanced survival of spinal motor neurons, and maintenance of NMJs. This datum suggests an early dysfunction and thus an important role of the upper motor neuron in this animal model of ALS and perhaps patients with the disease.

Oligodendrocytes have recently been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS). Here we show that, in vitro, mutant superoxide dismutase 1 (SOD1) mouse oligodendrocytes induce WT motor neuron (MN) hyperexcitability and death. Moreover, we efficiently derived human oligodendrocytes from a large number of controls and patients with sporadic and familial ALS, using two different reprogramming methods. All ALS oligodendrocyte lines induced MN death through conditioned medium (CM) and in coculture. CM-mediated MN death was associated with decreased lactate production and release, whereas toxicity in coculture was lactate-independent, demonstrating that MN survival is mediated not only by soluble factors. Remarkably, human SOD1 shRNA treatment resulted in MN rescue in both mouse and human cultures when knockdown was achieved in progenitor cells, whereas it was ineffective in differentiated oligodendrocytes. In fact, early SOD1 knockdown rescued lactate impairment and cell toxicity in all lines tested, with the exclusion of samples carrying chromosome 9 ORF 72 (C9orf72) repeat expansions. These did not respond to SOD1 knockdown nor did they show lactate release impairment. Our data indicate that SOD1 is directly or indirectly involved in ALS oligodendrocyte pathology and suggest that in this cell type, some damage might be irreversible. In addition, we demonstrate that patients with C9ORF72 represent an independent patient group that might not respond to the same treatment.

Astrocytes isolated from individuals with amyotrophic lateral sclerosis (ALS) are toxic to motor neurons (MNs) and play a non-cell autonomous role in disease pathogenesis. The mechanisms underlying the susceptibility of MNs to cell death remain unclear. Here we report that astrocytes derived from either mice bearing mutations in genes associated with ALS or human subjects with ALS reduce the expression of major histocompatibility complex class I (MHCI) molecules on MNs; reduced MHCI expression makes these MNs susceptible to astrocyte-induced cell death. Increasing MHCI expression on MNs increases survival and motor performance in a mouse model of ALS and protects MNs against astrocyte toxicity. Overexpression of a single MHCI molecule, HLA-F, protects human MNs from ALS astrocyte-mediated toxicity, whereas knockdown of its receptor, the killer cell immunoglobulin-like receptor KIR3DL2, on human astrocytes results in enhanced MN death. Thus, our data indicate that, in ALS, loss of MHCI expression on MNs renders them more vulnerable to astrocyte-mediated toxicity.

Spinal muscular atrophy (SMA) is the most frequent lethal genetic neurodegenerative disorder in infants. The disease is caused by low abundance of the survival of motor neuron (SMN) protein leading to motor neuron degeneration and progressive paralysis. We previously demonstrated that a single intravenous injection (IV) of self-complementary adeno-associated virus-9 carrying the human SMN cDNA (scAAV9-SMN) resulted in widespread transgene expression in spinal cord motor neurons in SMA mice as well as nonhuman primates and complete rescue of the disease phenotype in mice. Here, we evaluated the dosing and efficacy of scAAV9-SMN delivered directly to the cerebral spinal fluid (CSF) via single injection. We found widespread transgene expression throughout the spinal cord in mice and nonhuman primates when using a 10 times lower dose compared to the IV application. Interestingly, in nonhuman primates, lower doses than in mice can be used for similar motor neuron targeting efficiency. Moreover, the transduction efficacy is further improved when subjects are kept in the Trendelenburg position to facilitate spreading of the vector. We present a detailed analysis of transduction levels throughout the brain, brainstem, and spinal cord of nonhuman primates, providing new guidance for translation toward therapy for a wide range of neurodegenerative disorders.

Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (ALS) resulting in progressive motor neuron death through one or more acquired toxicities. Involvement of wild-type SOD1 has been linked to sporadic ALS, as misfolded SOD1 has been reported in affected tissues of sporadic patients and toxicity of astrocytes derived from sporadic ALS patients to motor neurons has been reported to be reduced by lowering the synthesis of SOD1. We now report slowed disease onset and progression in two mouse models following therapeutic delivery using a single peripheral injection of an adeno-associated virus serotype 9 (AAV9) encoding an shRNA to reduce the synthesis of ALS-causing human SOD1 mutants. Delivery to young mice that develop aggressive, fatal paralysis extended survival by delaying both disease onset and slowing progression. In a later-onset model, AAV9 delivery after onset markedly slowed disease progression and significantly extended survival. Moreover, AAV9 delivered intrathecally to nonhuman primates is demonstrated to yield robust SOD1 suppression in motor neurons and glia throughout the spinal cord and therefore, setting the stage for AAV9-mediated therapy in human clinical trials.