Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare and often devastating neurometabolic disorder characterized by impaired synthesis of dopamine, serotonin, and norepinephrine. Affected children present with severe developmental delays, motor dysfunction, autonomic instability, and behavioral symptoms, and current treatments remain largely supportive. Current gene therapy trials for AADC deficiency have demonstrated the safety and tolerability of bilateral AAV2-hAADC infusion into the putamen or midbrain. These approaches have restored dopamine synthesis in the basal ganglia and the limbic systems. However, they have not affected serotonin levels, likely due to the lack of transduction of serotonergic neurons by inadequate coverage of relevant brainstem nuclei. Restoring serotonergic signaling may be critical for improving cognitive and behavioral outcomes, which are still not fully addressed by current dopamine-focused therapies. To address this limitation, we evaluated the safety of a multi-target, magnetic resonance (MR)-guided convection-enhanced delivery strategy in nonhuman primates, administering AAV2-AADC into serotonergic and noradrenergic nuclei in addition to current dopaminergic targets. This broader targeting approach aims to enhance serotonin modulation and restore neuromodulatory balance, with potential benefits for cognitive and behavioral function in children with AADC deficiency undergoing gene therapy.

Adeno-associated virus serotype 2 (AAV2) remains one of the most common vectors for CNS gene delivery. Yet, its long-term intraparenchymal trafficking and the relationship between capsid persistence and transgene expression remain poorly understood. In this study, we tracked the spatial and temporal patterns of AAV2 following direct striatal infusion in rats, analyzing tissue at 3 days, 3 weeks, 12 weeks, 30 weeks, and 67 weeks after injection. Using immunofluorescence for the human aromatic L-amino acid decarboxylase (AADC) transgene and an epitope detecting intact AAV2 capsids (A20), we mapped capsid localization, clearance, and axonal transport over more than a year. Following direct striatal infusion, AAV2 capsids rapidly localized to striatal neurons, with early accumulation in the substantia nigra pars reticulata (SNpr) detectable by 3 days post-infusion, but without evidence of nigral neuron transduction. Transgene expression increased over time and peaked locally at 12 weeks, with a delayed yet robust AADC signal in striatonigral terminals by 30 weeks. By 67 weeks, capsid signal was minimal while AADC expression remained stable. These findings clarify the long-term dynamics of AAV2 distribution, capsid persistence, and axonal transport in the adult brain, informing our understanding of vector behavior and durability following intraparenchymal AAV2 gene delivery.

CRISPR-mediated genome editing of the central nervous system (CNS) has the potential to revolutionize the treatment of neurological disorders, including neurodegenerative disorders such as Huntington's disease (HD). However, the development of CRISPR therapeutics for the CNS has been hindered by challenges associated with delivery, specifically the lack of a clinically compatible, non-viral delivery technology facilitating genome editing of neurons in vivo . For most indications, two key obstacles must be overcome before therapeutic genome editing of the brain is feasible: non-toxic intracellular delivery of CRISPR cargo into neurons and establishment of strategies enabling targeted brain regions to be edited efficiently. While viral vectors have shown promise in pre-clinical models, non-viral approaches present distinct advantages: ease of manufacture as well as the transient presence of CRISPR machinery, which tempers risks of genotoxicity and immunogenicity. Peptide-enabled ribonucleoprotein (RNP) delivery of CRISPR (PERC) has emerged as a promising non-viral delivery strategy for CRISPR enzymes with initial use in primary human immune cells. In this study, we report the development of Neuro-PERC, a streamlined and optimized approach for in vivo editing of mammalian neurons. Administration of Neuro-PERC reagents via convection-enhanced delivery (CED) mediated efficient and well-tolerated neuronal genome editing. Neuro-PERC enabled robust neuronal editing in the brain of both small and large animal reporter models, and increased survival in a severe murine model of Huntington's disease. These results establish CED-administered Neuro-PERC as a candidate delivery technology to hasten clinical translation of CRISPR-based therapies for diseases of the CNS. Summary:Neuro-PERC, a peptide-mediated CRISPR enzyme delivery technology, enables efficient in vivo mammalian neuronal editing in the brain of mice and pigs, extending survival in a murine model of Huntington's disease when administered via convection-enhanced delivery (CED).

This study explores the potential of adeno-associated virus serotype 9 (AAV9) to deliver therapeutic genes directly into the memory circuit throughout the olfactory bulb (OB), a critical memory and sensory processing region. Using convection-enhanced delivery (CED) of AAV9 encoding green fluorescent protein (GFP), we mapped the extensive neural connectivity from the OB to key memory-related brain regions, including the entorhinal cortex (EC) and hippocampus. Our findings reveal significant transduction of neural pathways and underscore the potential of targeting the OB connectome for therapeutic interventions in progressive neurodegenerative disorders such as Alzheimer’s disease or mild cognitive impairment. Targeting the OB connectome will pave the way for new therapeutic strategies to preserve neuronal function and slow the progression, offering a promising avenue beyond symptomatic relief to address the underlying mechanisms of the disease.

Alcohol use disorder (AUD) exacts enormous personal, social and economic costs globally. Return to alcohol use in treatment-seeking patients with AUD is common, engendered by a cycle of repeated abstinence-relapse episodes even with use of currently available pharmacotherapies. Repeated ethanol use induces dopaminergic signaling neuroadaptations in ventral tegmental area (VTA) neurons of the mesolimbic reward pathway, and sustained dysfunction of reward circuitry is associated with return to drinking behavior. We tested this hypothesis by infusing adeno-associated virus serotype 2 vector encoding human glial-derived neurotrophic factor (AAV2-hGDNF), a growth factor that enhances dopaminergic neuron function, into the VTA of four male rhesus monkeys, with another four receiving vehicle, following induction of chronic alcohol drinking. GDNF expression ablated the return to alcohol drinking behavior over a 12-month period of repeated abstinence–alcohol reintroduction challenges. This behavioral change was accompanied by neurophysiological modulations to dopamine signaling in the nucleus accumbens that countered the hypodopaminergic signaling state associated with chronic alcohol use, indicative of a therapeutic modulation of limbic circuits countering the effects of alcohol. These preclinical findings suggest gene therapy targeting relapse prevention may be a potential therapeutic strategy for AUD. In a preclinical study, the delivery of an AAV-based gene therapy encoding GDNF in the brain prevented the return to alcohol use behaviors in a non-human primate model.

Lysosomal storage disorders (LSDs) constitute a large group of rare, multisystemic, progressive, inherited disorders of metabolism. The aberrant metabolic processes often lead to the cellular accumulation of incompletely metabolized macromolecules or their metabolic byproducts. Most of the patients affected by LSD can experience a variety of neurological presentations including, but not limited to, psychiatric complications, seizures, and/or developmental delays. The onset of symptoms can range from birth to adulthood, and disease severity can vary. Since there is significant overlap in the symptomatology of LSDs, diagnosis is typically confirmed through biochemical and molecular assays. There are currently no approved cures for any LSDs; however, in most cases, treatment of symptoms can lead to better outcomes and improvements in quality of life. The use of hematopoietic stem cell transplantation, enzyme replacement or substrate reduction therapy, and viral vector gene transfer is the subject of many ongoing and completed clinical trials. In this mini review, we provide an overview of LSDs with neurological manifestations, describe the current endeavors in alleviating peripheral symptoms and discuss effective therapeutics strategies.

Gene therapy shows great promise for the treatment of neurological disorders, and accessing cerebrospinal fluid (CSF) from the cerebellomedullary cistern through the posterior atlanto-occipital membrane has become a common route of delivery in preclinical studies. Unlike direct brain parenchymal infusions, CSF delivery offers broader coverage to the central and peripheral nervous system. This prospectively increases its translational value, more specially to treat global brain dysfunctions in which the pathology is disseminated throughout the brain and not focalized in one specific brain structure. Also, from the practical point of view, this approach offers a more reliable method for neurological gene replacement in infants, whose immature cranial suture preclude the use of skull-mounted devices. Here we describe a consistent, precise, and safe method for CSF injection.