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Allan-Herndon-Dudley syndrome (AHDS) is a severe X-linked intellectual and psychomotor disability disorder accompanied by abnormal thyroid hormone (TH) levels. AHDS is caused by inactivating mutations in the monocarboxylate transporter 8 (MCT8), a specific TH transporter widely expressed in the central nervous system. MCT8 gene 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. AAV9-based gene therapy is a promising approach to treat monogenic neurological disorders. Here, the potential of this approach was tested in the well-established double knockout (dKO) Mct8-/y;Oatp1c1-/- mouse model of AHDS, which displays disease-relevant neurological and TH phenotypes. Systemic intravenous delivery of AAV9-MCT8 at a juvenile stage led to improved locomotor and cognitive function, as well as rescue of T3-brain content and T3-related gene expression. This preclinical study indicates that this gene therapy may improve the neurological symptoms of AHDS patients.

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.

In May of 2019, the adeno-associated virus (AAV)-based gene therapy onasemnogene abeparvovec-xioi (Zolgensma) became the second Food and Drug Administration (FDA)-approved gene therapy with designated use for infants diagnosed with spinal muscular atrophy (SMA). The decision came nearly 10 years after results of the first preclinical models were initially reported. While the journey was an arduous one, the approval was an indication of the remarkable success of the first in-human clinical trials. According to the traditional classification system of autosomal recessive SMA, of which there are multiple types with phenotypic variability, SMA type 1 is the most common and most severe and represents 45% of the SMA patient population. Children with SMA type 1 cannot lift their heads without assistance and do not live past their second birthday. With Zolgensma, the first treated children with SMA type 1 have reached 5 years of age and some of them achieved the ability to sit unassisted or even walk. In this article, we review the work that led to FDA approval with emphasis on the development of preclinical and clinical studies.

NEDAMSS is a newly discovered neurodevelopmental disorder with regression, abnormal movements, loss of speech and seizures. The disease is caused by heterozygous mutations in the Interferon Regulatory Factor 2 Binding Protein-Like (IRF2BPL) gene. IRF2BPL is a ubiquitously expressed intron-less transcriptional regulator. The function of the protein is currently unknown, but the observed disease phenotype indicates a role in development and maintenance of the nervous system. To study this disease, we used patient skin-derived fibroblasts and reprogrammed them into induced neuronal progenitor cells (iNPCs). The iNPCs were then differentiated into astrocytes. Moreover, we also used a direct conversion method to generate neurons directly from the skin fibroblasts. Using these methods and performing molecular and functional analyses of the different cell types, we identified a novel disease mechanism not previously described. While IRF2BPL is localized in the nucleus in healthy individuals, we found that several patient cell lines displayed mis-localization in the form of aggregates in the cytoplasm. Moreover, the patient astrocytes also showed abnormal mitochondrial ATP-linked respiration levels and caused neuronal death in co-culture assays. Based on these observations, we tested treatment with CuATSM, a small molecule drug currently in clinical trials for ALS. We found that CuATSM successfully rescued neuronal survival and reduced ATP-linked respiration to normal levels in NEDAMSS astrocytes. We also established multiple AAV based gene therapy tools to modulate the expression of the protein and restore its normal function in vitro and potentially in vivo. In summary, we identified a novel disease mechanism involved in NEDAMSS disease and found promising therapeutic strategies that might be able to slow or halt the progression of the disease.

Noise-induced hearing loss (NIHL) is one of the most prevalent disabilities for which effective therapeutic treatment is currently lacking. Auditory injury caused by excessive or constant noise exposure damages the sensory elements of the ear, leading to metabolic or mechanical damage to hair cells and subsequent degeneration of spiral ganglion neurons (SGNs). To improve hearing in the NIHL patient population, maintenance and regeneration of inner hair cell-SGN synapses is critical. Although effective compounds for the treatment of NIHL have been suggested, challenges in delivery of the compounds to the inner hair cells (IHCs) hinder translation of these therapies to a clinical setting. Current routes of administration are ineffective in reaching IHCs or are invasive and may damage the cochlea. In this study, we examined less invasive delivery routes, via intrathecal (cerebrospinal fluid delivery) or intravenous injection, to administer adeno-associated virus serotype 9 (AAV9) expressing green fluorescent protein (GFP) to target IHCs and SGNs. To induce auditory injury, mice were exposed to 100 dB sound pressure level (SPL) octave band noise for 2 hours. Mice were then injected at various time points post noise injury to determine if noise exposure influenced targeting efficiency. Three weeks post injection, cochleae were processed and analyzed for GFP expression using immunofluorescence. Our results demonstrate that intravenous delivery was not successful in targeting of IHCs or SGNs at any time point measured. However, intrathecally injected AAV9 was highly effective in targeting cells in both healthy and noise-damaged cochleae at multiple time points post-noise exposure. We conclude that gene therapy techniques can be utilized to efficiently deliver therapeutic transgenes to the cochlea using cerebrospinal fluid as a delivery route. These findings determine a novel route of delivery and open a new avenue of therapeutic treatment for auditory injury and cochlear disorders.

Batten Disease is a fatal, lysosomal storage disorder characterized by cognitive and motor deficits, vision impairments, and seizures. Loss of vision is a hallmark of 10 of the 13 Batten Disease subtypes. Our group has pioneered AAV9 gene therapy treatments achieving widespread transduction of the brain and spinal cord. Two clinical trials are currently ongoing at Nationwide Children’s Hospital delivering this vector via cerebrospinal fluid (CSF) to the brain and spinal cord for treatment of the lethal neurodegenerative aspects of Batten Disease. However, AAV9 transduction of the retina after CSF delivery is limited and there is a critical need for a solution that prevents vision loss and further improves quality of life for Batten Disease patients. Similar to most genetic ocular diseases, photoreceptor degeneration is the most commonly cited pathology in patients. However, recent studies suggest that in some subtypes of Batten Disease, expression must be rescued also within the deeper layers of the retina that are difficult to reach with therapeutic vectors (inner nuclear layer, INL). We performed single cell RNA sequencing of mice and non-human primate retinas in collaboration with Dr. Fischer (OSU) and concluded that Batten Disease vision specific gene therapy needs to target a wide range of cells, including the INL, which is a major challenge for translation of a vision specific therapy to the clinic. We have recently discovered that administration of neuraminidase (NA), a sialidase enzyme, prior to or in combination with AAV9.GFP, drastically increases transduction throughout the murine C57Bl/6 retina including the INL and all the way through to the photoreceptor layer. Our preliminary data indicates GFP expression in almost all, if not all, retinal cell types using this method. Importantly, we have confirmed successful targeting of INL bipolar cells, a notoriously difficult cell-type to transduce, and up to 40% increase in Muller glia transduction. Preliminary histological examination indicates no damage or alterations in retinal integrity. While additional testing in large animal models is required and scheduled to occur in a WT pig model in Autumn 2020, this remarkable discovery suggests that it may now be possible to target every cell-type of the retina with a single AAV vector. This is especially important in regard to treatment of Batten Disease but has many additional implications. Current retinal therapies that require a more invasive subretinal delivery to photoreceptors could now opt for the safer intravitreal delivery strategy. In addition, this strategy would be highly useful in the field of optogenetics, where investigators continue to struggle to find an efficient way to express light-sensitive opsins in cells of the INL to restore sight in individuals that have already lost their photoreceptors.

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 (ICV)-delivered self-complementary AAV9 (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 brain and spinal cord from 4 to 24 months, reducing histopathological and behavioral hallmarks of the disease and completely restoring lifespan from 10 months in untreated animals to beyond 24 months of age in treated animals. These results demonstrate, by far, the most successful rescue reported in an animal model of CLN8 disease, and supports gene therapy as a promising therapeutic strategy for this disorder.

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.

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.