Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive, loss-of-function mutations in FIG4, encoding a phosphoinositol(3,5)P2-phosphatase. CMT4J patients have both neuron loss and demyelination in the peripheral nervous system, with vacuolization indicative of endosome/lysosome trafficking defects. Although the disease is highly variable, the onset is often in childhood and FIG4 mutations can dramatically shorten life span. There is currently no treatment for CMT4J. Here, we present the results of preclinical studies testing a gene-therapy approach to restoring FIG4 expression. A mouse model of CMT4J, the Fig4–pale tremor (plt) allele, was dosed with a single-stranded adeno-associated virus serotype 9 (AAV9) to deliver a codon-optimized human FIG4 sequence. Untreated, Fig4^plt/plt mice have a median survival of approximately 5 weeks. When treated with the AAV9-FIG4 vector at P1 or P4, mice survived at least 1 year, with largely normal gross motor performance and little sign of neuropathy by neurophysiological or histopathological evaluation. When mice were treated at P7 or P11, life span was still significantly prolonged and peripheral nerve function was improved, but rescue was less complete. No unanticipated adverse effects were observed. Therefore, AAV9-mediated delivery of FIG4 is a well-tolerated and efficacious strategy in a mouse model of CMT4J.

Epileptic encephalopathies may arise from single gene variants. In recent years, next-generation sequencing technologies have enabled an explosion of gene identification in monogenic epilepsies. One such example is the epileptic encephalopathy SLC13A5 deficiency disorder, which is caused by loss of function pathogenic variants to the gene SLC13A5 that results in deficiency of the sodium/citrate cotransporter. Patients typically experience seizure onset within the first week of life and have developmental delay and intellectual disability. Current antiseizure medications may reduce seizure frequency, yet more targeted treatments are needed to address the epileptic and non-epileptic features of SLC13A5 deficiency disorder. Gene therapy may offer hope to these patients and better clinical outcomes than current available treatments. Here, we discuss SLC13A5 genetics, natural history, available treatments, potential outcomes and assessments, and considerations for translational medical research for an AAV9-based gene replacement therapy.

Intrathecal (IT) lumbar puncture delivery of recombinant adeno-associated virus serotype 9 (rAAV9) is a gene therapy approach being explored in preclinical studies and ongoing gene therapy clinical trials for neurological diseases. Few studies address IT rAAV9 vector distribution, tropism, and expression with respect to age of administration. Therefore, we IT delivered a rAAV9/GFP vector in mice at ages ranging from early postnatal development through adulthood (P10-P90). Tissues were assessed for transgene expression, cell tropism, and vector distribution. In the CNS, transduction was highest when delivered at post-natal day 10 (P10) and there was an age-dependent decline in transduction. We found higher transduction of astrocytes relative to neurons when rAAV9 was administered at younger ages and a switch to higher neuronal transduction with delivery at older timepoints. Biodistribution analysis of peripheral tissues showed that when delivered at P10, rAAV9 has the greatest distribution to the heart. Conversely, at P90 rAAV9 liver distribution was highest. As rAAV9 IT-delivered gene therapies continue to emerge for neurological diseases, careful consideration of the age of delivery should be taken in relation to the expected distribution and cell expression in animal models, and how this may translate to human studies.

ECHS1 Deficiency (ECHS1D) is a rare and devastating pediatric disease that currently has no defined treatments. This disorder results from missense loss-of-function mutations in the ECHS1 gene that result in severe developmental delays, encephalopathy, hypotonia, and early death. ECHS1 enzymatic activity is necessary for the beta-oxidation of fatty acids and the oxidation of branched-chain amino acids within the inner mitochondrial matrix. The pathogenesis of disease remains unknown, however it is hypothesized that disease is driven by an accumulation of toxic metabolites from impaired valine oxidation. To expand our knowledge on disease mechanisms, a novel mouse model of ECHS1D was generated that possesses a disease-associated knock-in (KI) allele and a knock-out (KO) allele. To investigate the behavioral phenotype, a battery of testing was performed at multiple time points, which included assessments of learning, motor function, endurance, sensory responses, and anxiety. Neurological abnormalities were assessed using wireless telemetry EEG recordings, pentylenetetrazol (PTZ) seizure induction, and immunohistochemistry. Metabolic perturbations were measured within the liver, serum, and brain using mass spectrometry and magnetic resonance spectroscopy. To test disease mechanisms, mice were subjected to disease pathway stressors and then survival, body weight gain, and epilepsy were assessed. Mice containing KI/KI or KI/KO alleles were viable with normal development and survival, and the presence of KI and KO alleles resulted in a significant reduction in ECHS1 protein. ECHS1D mice displayed reduced exercise capacity and pain sensation. EEG analysis revealed increased slow wave power that was associated with perturbations in sleep. ECHS1D mice had significantly increased epileptiform EEG discharges, and were sensitive to seizure induction, which resulted in death of 60% of ECHS1D mice. Under basal conditions, brain structure was grossly normal, although histological analysis revealed increased microglial activation in aged ECHS1D mice. Increased dietary valine only affected ECHS1D mice, which significantly exacerbated seizure susceptibility and resulted in death. Lastly, acute inflammatory challenge drove regression and early lethality in ECHS1D mice. In conclusion, we developed a novel model of ECHS1D that may be used to further knowledge on disease mechanisms and to develop therapeutics. Our data suggests altered metabolic signaling and inflammation may contribute to epilepsy in ECHS1D, and these alterations may be attributed to impaired valine metabolism.

ECHS1 deficiency (ECHS1D) is a rare and devastating neurometabolic disease that currently has no defined treatments. This disorder results from missense loss-of-function mutations in the ECHS1 gene that results in severe developmental delays, encephalopathy, hypotonia and early death. ECHS1 enzymatic activity is necessary for the beta-oxidation of fatty acids and the oxidation of branched-chain amino acids within the inner mitochondrial matrix. The pathogenesis of disease remains poorly understood. To expand our knowledge on disease mechanisms, we generated a novel mouse model of ECHS1D that possesses a disease-associated variant knocked-in (KI) the Echs1 allele and a knock-out (KO) of the other Echs1 allele. Neurological and metabolic abnormalities were assessed under basal conditions, and acute inflammation was tested as a potential disease driver. Mice containing KI/KI or KI/KO alleles were viable with normal development and survival, and the combined KI and KO alleles resulted in more than a 95% reduction of Echs1 protein levels. ECHS1D mice had significantly increased epileptiform EEG activity and were sensitive to seizure induction, which resulted in the death of 60% of ECHS1D mice. Power spectral analysis revealed ECHS1D mice had increased slow-wave EEG power that was associated with sleep dysfunction. Under basal conditions, energy status and mitochondrial function within the brain was unaffected, while aromatic amino acid content was increased. Markers of neuroinflammation were increased in ECHS1D mice in an age-dependent manner and acute inflammatory challenge resulted in failure to thrive and early lethality in ECHS1D mice. In conclusion, we developed a novel model of ECHS1D that can be used to study disease mechanisms and for therapeutic development.