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Heterozygous mutations in the FOXG1 gene manifest as FOXG1 syndrome, a severe neurodevelopmental disorder characterized by structural brain anomalies, including agenesis of the corpus callosum, hippocampal reduction, and myelination delays. Despite the well-defined genetic basis of FOXG1 syndrome, therapeutic interventions targeting the underlying cause of the disorder are nonexistent. In this study, we explore the therapeutic potential of adeno-associated virus 9 (AAV9)-mediated delivery of the FOXG1 gene. Remarkably, intracerebroventricular injection of AAV9-FOXG1 to Foxg1 heterozygous mouse model at the postnatal stage rescues a wide range of brain pathologies. This includes the amelioration of corpus callosum deficiencies, the restoration of dentate gyrus morphology in the hippocampus, the normalization of oligodendrocyte lineage cell numbers, and the rectification of myelination anomalies. Our findings highlight the efficacy of AAV9-based gene therapy as a viable treatment strategy for FOXG1 syndrome and potentially other neurodevelopmental disorders with similar brain malformations, asserting its therapeutic relevance in postnatal stages.

Anc80L65 is a synthetic, ancestral adeno-associated virus that has high tropism toward retinal photoreceptors after subretinal injection in mice and non-human primates. We characterized, for the first time, the post-intravitreal cell-specific transduction profile of Anc80L65 compared with AAV9. Here we use Anc80L65 and AAV9 to intravitreally deliver a copy of the gene encoding GFP into WT C57Bl/6J mice. GFP expression was driven by one of two clinically relevant promoters, chicken β actin (CB) or truncated MECP2 (P546). After qualitative assessment of relative GFP expression, we found Anc80L65 and AAV9 to have similar transduction profiles. Through the development of a novel method for quantifying GFP-positive retinal cells, we found Anc80L65 to have higher tropism in Müller glia and AAV9 to have higher tropism in horizontal cells. In addition, we found P546 to promote GFP expression at a more moderate level compared with the high levels seen under the CB promoter. Finally, for the first time, we characterized Anc80L65 cross-reactivity in human sera; 83% of patients with AAV2 pre-existing antibodies were found to be seropositive for Anc80L65. This study demonstrates the expanded therapeutic applications of Anc80L65 to treat retinal disease and provides the first insights to Anc80L65 pre-existing immunity in humans.

Thirty genes are involved in the biosynthesis and modification of glycosylphosphatidylinositol (GPI)-anchored proteins, and defects in these genes cause inherited GPI deficiency (IGD). PIGA is X-linked and involved in the first step of GPI biosynthesis, and only males are affected by variations in this gene. The main symptoms of IGD are neurological abnormalities, such as developmental delay and seizures. There is no effective treatment at present. We crossed Nestin-Cre mice with Piga-floxed mice to generate CNS-specific Piga knockout (KO) mice. Hemizygous KO male mice died by P10 with severely defective growth. Heterozygous Piga KO female mice are mosaic for Piga expression and showed severe defects in growth and myelination and died by P25. Using these mouse models, we evaluated the effect of gene replacement therapy with adeno-associated virus (AAV). It expressed efficacy within 6 days, and the survival of male mice was extended to up to 3 weeks, whereas 40% of female mice survived for approximately 1 year and the growth defect was improved. However, liver cancer developed in all three treated female mice at 1 year of age, which was probably caused by the AAV vector bearing a strong CAG promoter.

Batten disease is a family of rare, fatal, neuropediatric diseases presenting with memory/learning decline, blindness, and loss of motor function. Recently, we reported the use of an AAV9-mediated gene therapy that prevents disease progression in a mouse model of CLN6-Batten disease (Cln6nclf), restoring lifespans in treated animals. Despite the success of our viral-mediated gene therapy, the dosing strategy was optimized for delivery to the brain parenchyma and may limit the therapeutic potential to other disease-relevant tissues, such as the eye. Here, we examine whether cerebrospinal fluid (CSF) delivery of scAAV9.CB.CLN6 is sufficient to ameliorate visual deficits in Cln6nclf mice. We show that intracerebroventricular (i.c.v.) delivery of scAAV9.CB.CLN6 completely prevents hallmark Batten disease pathology in the visual processing centers of the brain, preserving neurons of the superior colliculus, thalamus, and cerebral cortex. Importantly, i.c.v.-delivered scAAV9.CB.CLN6 also expresses in many cells throughout the central retina, preserving many photoreceptors typically lost in Cln6nclf mice. Lastly, scAAV9.CB.CLN6 treatment partially preserved visual acuity in Cln6nclf mice as measured by optokinetic response. Taken together, we report the first instance of CSF-delivered viral gene reaching and rescuing pathology in both the brain parenchyma and retinal neurons, thereby partially slowing visual deterioration.

Of familial amyotrophic lateral sclerosis (fALS) cases, 20% are caused by mutations in the gene encoding human cytosolic Cu/Zn superoxide dismutase (hSOD1). Efficient translation of the therapeutic potential of RNAi for the treatment of SOD1-ALS patients requires the development of vectors that are free of significant off-target effects and with reliable biomarkers to discern sufficient target engagement and correct dosing. Using adeno-associated virus serotype 9 to deliver RNAi against hSOD1 in the SOD1G93A mouse model, we found that intrathecal injection of the therapeutic vector via the cisterna magna delayed onset of disease, decreased motor neuron death at end stage by up to 88%, and prolonged the median survival of SOD1G93A mice by up to 42%. To our knowledge, this is the first report to demonstrate no significant off-target effects linked to hSOD1 silencing, providing further confidence in the specificity of this approach. We also report the measurement of cerebrospinal fluid (CSF) hSOD1 protein levels as a biomarker of effective dosing and efficacy of hSOD1 knockdown. Together, these data provide further confidence in the safety of the clinical therapeutic vector. The CSF biomarker will be a useful measure of biological activity for translation into human clinical trials. : Development of off-target effect-free AAV9-shRNA-mediated SOD1 silencing in an ALS mouse model, with reliable biomarkers to discern sufficient target engagement and correct dosing. Keywords: ALS, AAV9, shRNA, SOD1, clinical vector, off-target effects, cisterna magna, neurodegeneration, motor neuron, biomarker