The basic helix-loop-helix transcription factor TCF4 impacts brain architecture as well as neuronal morphology and differentiation.

Germline mutations in the basic helix-loop-helix transcription factor 4 (TCF4) cause the Pitt-Hopkins syndrome (PTHS), a developmental disorder with severe intellectual disability. Here, we report findings from a new mouse model with a central nervous system-specific truncation of Tcf4 leading to severe phenotypic abnormalities. Furthermore, it allows the study of a complete TCF4 knockout in adult mice, circumventing early postnatal lethality of previously published mouse models. Our data suggest that a TCF4 truncation results in an impaired hippocampal architecture affecting both the dentate gyrus as well as the cornu ammonis. In the cerebral cortex, loss of TCF4 generates a severe differentiation delay of neural precursors. Furthermore, neuronal morphology was critically affected with shortened apical dendrites and significantly increased branching of dendrites. Our data provide novel information about the role of Tcf4 in brain development and may help to understand the mechanisms leading to intellectual deficits observed in patients suffering from PTHS.

TCF4 (E2-2) harbors tumor suppressive functions in SHH medulloblastoma.

The TCF4 gene encodes for the basic helix-loop-helix transcription factor 4 (TCF4), which plays an important role in the development of the central nervous system (CNS). Haploinsufficiency of TCF4 was found to cause Pitt-Hopkins syndrome (PTHS), a severe neurodevelopmental disorder. Recently, the screening of a large cohort of medulloblastoma (MB), a highly aggressive embryonal brain tumor, revealed almost 20% of adult patients with MB of the Sonic hedgehog (SHH) subtype carrying somatic TCF4 mutations. Interestingly, many of these mutations have previously been detected as germline mutations in patients with PTHS. We show here that overexpression of wild-type TCF4 in vitro significantly suppresses cell proliferation in MB cells, whereas mutant TCF4 proteins do not to the same extent. Furthermore, RNA sequencing revealed significant upregulation of multiple well-known tumor suppressors upon expression of wild-type TCF4. In vivo, a prenatal knockout of Tcf4 in mice caused a significant increase in apoptosis accompanied by a decreased proliferation and failed migration of cerebellar granule neuron precursor cells (CGNP), which are thought to be the cells of origin for SHH MB. In contrast, postnatal in vitro and in vivo knockouts of Tcf4 with and without an additional constitutive activation of the SHH pathway led to significantly increased proliferation of CGNP or MB cells. Finally, publicly available data from human MB show that relatively low expression levels of TCF4 significantly correlate with a worse clinical outcome. These results not only point to time-specific roles of Tcf4 during cerebellar development but also suggest a functional linkage between TCF4 mutations and the formation of SHH MB, proposing that TCF4 acts as a tumor suppressor during postnatal stages of cerebellar development.

Possibilities and limitations of antisense oligonucleotide therapies for the treatment of monogenic disorders.

Antisense oligonucleotides (ASOs) are incredibly versatile molecules that can be designed to specifically target and modify RNA transcripts to slow down or halt rare genetic disease progression. They offer the potential to target groups of patients or can be tailored for individual cases. Nonetheless, not all genetic variants and disorders are amenable to ASO-based treatments, and hence, it is important to consider several factors before embarking on the drug development journey. Here, we discuss which genetic disorders have the potential to benefit from a specific type of ASO approach, based on the pathophysiology of the disease and pathogenic variant type, as well as those disorders that might not be suitable for ASO therapies. We further explore additional aspects, such as the target tissues, intervention time points, and potential clinical benefits, which need to be considered before developing a compound. Overall, we provide an overview of the current potentials and limitations of ASO-based therapeutics for the treatment of monogenic disorders.

Joining forces to develop individualized antisense oligonucleotides for patients with brain or eye diseases: the example of the Dutch Center for RNA Therapeutics.

Antisense oligonucleotides (ASOs) offer versatile tools to modify the processing and expression levels of gene transcripts. As such, they have a high therapeutic potential for rare genetic diseases, where applicability of each ASO ranges from thousands of patients worldwide to single individuals based on the prevalence of the causative pathogenic variant. It was shown that development of individualized ASOs was feasible within an academic setting, starting with Milasen for the treatment of a patient with CLN7 Batten's disease in the USA. Inspired by this, the Dutch Center for RNA Therapeutics (DCRT) was established by three academic medical centers in the Netherlands with a track record in ASO development for progressive, genetic neurodegenerative, neurodevelopmental, and retinal disorders. The goal of the DCRT is to bundle expertise and address national ethical, regulatory, and financial issues related to ASO treatment, and ultimately to develop individualized ASOs for eligible patients with genetic diseases affecting the central nervous system in an academic, not-for-profit setting. In this perspective, we describe the establishment of the DCRT in 2020 and the achievements so far, with a specific focus on lessons learned: the need for processes and procedures, the need for global collaboration, the need to raise awareness, and the fact that N-of-1 is N-of-a-few.

Joining forces to develop individualized antisense oligonucleotides for patients with brain or eye diseases: the example of the Dutch Center for RNA Therapeutics Many rare diseases have a genetic cause. Antisense oligonucleotides (ASOs) are short pieces of modified DNA that have therapeutic potential for some patients with rare diseases. However, often this is in a patient-specific setting, meaning individualized therapy development is required, which has little commercial opportunity for pharmaceutical companies. It was shown however that individualized ASOs can be developed by academics, starting with Milasen, which was developed for a unique DNA variant found in a child with Batten's disease in the USA. Following in the footstep of these academic pioneers we established the Dutch Center for RNA Therapeutics (DCRT), which aims to develop individualized ASOs for eligible patients with eye or brain diseases in a not-for-profit setting. Our goal is to bundle expertise and address national, ethical, regulatory and financial issues related to individualized ASO development. In this perspective review we outline the achievements since establishing the DCRT in 2020, with a focus on lessons learnt along the way: the need for processes and procedures, the need for global collaboration, the need to raise awareness and the fact that very often ASOs developed for a single person, could be applied also to a few other patients with the same DNA variants.

Organ-on-a-chip technologies to study neuromuscular disorders: possibilities, limitations, and future hopes.

Neuromuscular disorders are a heterogeneous group of diseases ranging from mild to devastating phenotypes depending on the disorder's origin. Pathophysiologies for many of these disorders are not fully understood and efficient therapies are urgently needed. Recent advances in the field of induced pluripotent stem cells and organ-on-a-chip technologies have brought enormous improvement in modeling neuromuscular diseases. Even complex units, like the neuromuscular junction, can now be built, enabling researchers to study each component of the motor unit by itself or interacting with others, allowing the identification of disease mechanisms. This article aims to introduce these new modeling systems to study neuromuscular disorders and the possibilities of organ-on-a-chip platforms to shed light on disease pathologies and their use for therapy development.