Several neurodevelopmental and neuropsychiatric disorders are characterized by intermittent episodes of pathological activity. Although genetic therapies offer the ability to modulate neuronal excitability, a limiting factor is that they do not discriminate between neurons involved in circuit pathologies and “healthy” surrounding or intermingled neurons. We describe a gene therapy strategy that down-regulates the excitability of overactive neurons in closed loop, which we tested in models of epilepsy. We used an immediate early gene promoter to drive the expression of Kv1.1 potassium channels specifically in hyperactive neurons, and only for as long as they exhibit abnormal activity. Neuronal excitability was reduced by seizure-related activity, leading to a persistent antiepileptic effect without interfering with normal behaviors. Activity-dependent gene therapy is a promising on-demand cell-autonomous treatment for brain circuit disorders.

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in SLC6A3, encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-μ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human SLC6A3 complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human SLC6A3 using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS.

Infantile parkinsonism-dystonia due to dopamine transporter deficiency syndrome (DTDS) is an ultrarare childhood movement disorder caused by biallelic loss-of-function mutations in the SLC6A3 gene. Advances in genomic analysis have revealed an evolving spectrum of SLC6A3-related neurological and neuropsychiatric disorders. Since the initial clinical and genetic characterisation of DTDS in 2009, there have been thirty-one published cases with a variety of protein-truncating variants (nonsense variants, splice-site changes, and deletions) and missense changes. Amino acid substitutions result in mutant proteins with impaired dopamine transporter function due to reduced transporter activity, impaired dopamine binding, reduced cell-surface expression, and aberrant posttranslational protein modification with impaired glycosylation. In this review, we provide an overview of the expanding clinical phenotype of DTDS and the precision therapies in development, including pharmacochaperones and gene therapy.

Neurodevelopmental disorders encompass a group of debilitating diseases presenting with motor and cognitive dysfunction, with variable age of onset and disease severity. Advances in genetic diagnostic tools have facilitated the identification of several monogenic chromatin remodeling diseases that cause Neurodevelopmental disorders. Chromatin remodelers play a key role in the neuro-epigenetic landscape and regulation of brain development; it is therefore not surprising that mutations, leading to loss of protein function, result in aberrant neurodevelopment. Heterozygous, usually de novo mutations in histone lysine methyltransferases have been described in patients leading to haploinsufficiency, dysregulated protein levels and impaired protein function. Studies in animal models and patient-derived cell lines, have highlighted the role of histone lysine methyltransferases in the regulation of cell self-renewal, cell fate specification and apoptosis. To date, in depth studies of histone lysine methyltransferases in oncology have provided strong evidence of histone lysine methyltransferase dysregulation as a determinant of cancer progression and drug resistance. As a result, histone lysine methyltransferases have become an important therapeutic target for the treatment of different cancer forms. Despite recent advances, we still lack knowledge about the role of histone lysine methyltransferases in neuronal development. This has hampered both the study and development of precision therapies for histone lysine methyltransferases-related Neurodevelopmental disorders. In this review, we will discuss the current knowledge of the role of histone lysine methyltransferases in neuronal development and disease progression. We will also discuss how RNA-based technologies using small-activating RNAs could potentially provide a novel therapeutic approach for the future treatment of histone lysine methyltransferase haploinsufficiency in these Neurodevelopmental disorders, and how they could be first tested in state-of-the-art patient-derived neuronal models.

Induced pluripotent stem cells (iPSCs) have great potential as physiological disease models for human disorders where access to primary cells is difficult, such as neurons. In recent years, many protocols have been developed for the generation of iPSCs and the differentiation into specialised cell subtypes of interest. More recently, these models have been modified to allow large-scale phenotyping and high-content screening of small molecule compounds in iPSC-derived neuronal cells. Here, we describe the automated seeding of day 11 ventral midbrain progenitor cells into 96-well plates, administration of compounds, automated staining for immunofluorescence, the acquisition of images on a high-content screening platform and workflows for image analysis.

The dopaminergic system has key roles in human physiology and is implicated in a broad range of neurological and neuropsychiatric conditions that are increasingly investigated using induced pluripotent stem cell-derived midbrain models. To determine similarities of such models to human systems, here we undertake single-cell and spatial profiling of first and second trimester fetal midbrain and compare it to in vitro midbrain models. Histological examination reveals that, by the second trimester, fetal midbrain tissue exhibits structural complexity comparable to that of adults. At the molecular level, single-cell profiling uncovers differences in cellular composition across models, with brain organoids most closely resembling late first trimester tissue — an observation supported by meta-integration of existing midbrain datasets. By reconstructing developmental trajectories of neuronal and astrocytic lineages, we map gene expression dynamics associated with maturation. Importantly, integration of spatial transcriptomics provides critical context for aligning organoid models, revealing that their spatial organization and intercellular signaling resemble the architecture and microenvironment of the second trimester midbrain. Ultimately, we leverage our findings to study Dopamine Transporter Deficiency Syndrome progression in patient-derived midbrain organoids, validating their relevance. Understanding the extent of human tissue recapitulation in midbrain laboratory models is essential to justify their use as biological proxies.