AR-A IPSCS: GENERATION OF AN ADVANCED CELLULAR MODEL TO INVESTIGATE A NOVEL THERAPEUTIC APPROACH FOR SBMA

Spinal and Bulbar Muscular Atrophy (SBMA) is a rare, highly disabling, neurodegenerative disease that unfortunately still lacks an effective treatment to date. Patients affected by SBMA present muscular impairment, characterized by cramps and muscle twitching in the early stages and by difficulty in swallowing and walking in the later stages, which not rarely leads to the need for a wheelchair. In addition, other systemic symptoms are present, such as sensory neuropathy, androgen insensitivity, sexual dysfunction, gynecomastia, fatty liver disease, and insulin resistance, making SBMA a disease highly impacting on patients’ life quality. SBMA is caused by an expansion of a CAG triplet repeat present in the region encoding the first exon of the androgen receptor (AR) gene, which is located on the X chromosome. SBMA is not only an X-linked inherited disease but also is dependent on levels of testosterone (T) and its active metabolite 5α-dihydrotestosterone (DHT). Physiologically, AR binds DHT, which induces a conformational change in the protein, allowing its dimerization and its translocation into the nucleus, where it acts as a transcription factor. When the CAG expansion is present, the protein presents an elongated polyQ tract (ARpolyQ). When the ARpolyQ protein interacts with DHT, only partial translocation into the nucleus occurs leading to a transcriptional loss of function (LoF), while this interaction also unmasks the polyQ tract, leading to AR toxic gain of function (GoF) and aggregation in both cytoplasm and nucleus. Given these characteristics, an ideal therapeutic approach for SBMA should reduce the ARpolyQ aggregation while maintaining its transcriptional activity. Interestingly, the AR mRNA transcript presents more than one translational start codon: in particular, an AUG (AUG-II) downstream of the CAG repeat is present, which generates a shorter isoform of the receptor called AR-A. Preliminary data from our laboratory showed that AR-A has a positive effect on ARpolyQ aggregation, but there is still a gap in our understanding of its mechanism. With the purpose to characterize AR-A functions and investigate its potential therapeutical activity, this study aims to provide a novel model to deepen AR-A role through the generation of induced Pluripotent Stem Cells (iPSCs) expressing AR-A (AR-A iPSCs). Starting from an SBMA patient-derived iPSC line which expresses AR with a polyQ tract of 51Qs (AR-Q51 iPSCs), CRISPR/Cas9 gene editing technique was exploited to block the translation of the ARpolyQ and allowing AR-A expression only. After generation of the AR-A iPSC line, iPSCs were validated for pluripotency, checking both expression of pluripotency markers and AR-A iPSCs ability to differentiate to the 3 germ layers, and AR expression. Both AR-A and AR-Q51 iPSC lines were used to generate first neuronal precursor cells (AR-A NCPs and AR-Q51 NPCs, respectively) and subsequently motoneurons (AR-A MNs and AR-Q51 MNs, respectively), since these are among the primary affected cells in SBMA. This model allowed us to study the role of AR-A in MNs generation and function, and compare them to the pathological AR-Q51 MNs. Since SBMA primary affects both MNs and muscles, the AR-A iPSC line was also exploited to generate iPSC-derived myoprogenitors (MPs), to establish whether AR-A affects muscle generation and differentiation compared to AR-Q51. The main findings reveal that AR-A expression only is sufficient to allow both MNs and MPs correct differentiation. In addition, 3D differentiation of AR-A MNs was successfully performed, generating assembloids composed by AR-A MN spheres and either immortalized human control muscle cell spheres or iPSC-derived control MP spheres. Assembloids allowed us to study AR-A MNs behaviour in model which better mimics the complex interaction of MNs and muscles in the neuromuscular junction (NMJ). Finally, the generation of an established model of MNs or myoprogenitors expressing AR-A only represents a progress in SBMA research, since it could be exploited in future research to deepen AR-A molecular mechanisms in both SBMA and healthy models, and can be useful for drug screening with the aim of inducing AR-A expression, which could then be used to counteract ARpolyQ aggregation in SBMA patients.