CORRECTION OF THE GENETIC DEFECT IN INDUCED PLURIPOTENT STEM CELL LINES THROUGH CHROMOSOME TRANSPLANTATION

Since the discovery of stem cell in 1961, the knowledge and applications of pluripotent stem cells have expanded significantly, opening remarkable opportunities for fundamental discoveries and biomedical advancements, such as regenerative medicine, disease modelling, and drug screening. A pivotal milestone in this field was the generation of induced pluripotent stem cells (iPSCs) by Yamanaka and Takahashi in 2006, through the use of four reprogramming factors (Oct4, Sox2, Klf4 and c-Myc). This finding enabled the generation of iPSCs from adult patient-specific cells, overcoming numerous ethical concerns by the use of embryonic stem cells (ESCs), while maintaining key advantages of ESCs (e.g. indefinite self-renewal and the ability to differentiate into various cell types). Thanks to these properties, iPSCs give a model that resemble better the human anatomy and complexity compared to animal models across various applications. The coupling of iPSCs with advanced gene-editing technologies, such as CRISPR/Cas9, represents one of the most promising strategies for gene correction in a wide range of genetic diseases. However, some genetic defects, especially those involving genomic structural abnormalities (e.g. large deletions, inversions, aneuploidies, or complex rearrangements) cannot be corrected using conventional gene therapy methods. A group of these disorders falls within the dystrophinopathies. Dystrophinopathies are X-linked disorders caused by mutations in the DMD gene (Xp21.2), which encodes the dystrophin protein. The dystrophin or DMD gene is one of the largest protein-coding genes in the human genome (79 exons) and gives rise to various isoforms. The full-length dystrophin protein (427 kDa) is expressed primarily in skeletal and cardiac muscles, where is part of the dystrophin-associated protein complex (DAPC) or dystrophin glycoprotein complex (DGC) and plays a structural role by linking the cytoskeleton to the extracellular matrix (ECM) to maintain the sarcolemma integrity. Dystrophin is also crucial for proper muscle contraction and relaxation cycle. Within dystrophinopathies, Duchenne (DMD) and Becker (BMD) muscular dystrophies are the most well-known forms. These conditions are characterized by progressive loss of respiratory and cardiac muscle strength, as well as nerve tissue damage. In my laboratory, we study therapeutic strategies for both DMD and BMD; however, during my PhD research, I focused specifically on the correction and functional validation of the milder form, BMD. This pathology has an estimated worldwide prevalence of approximately 1:19,000, and it leads to progressive muscle degeneration weakness, typically beginning in mid- to late adulthood or sometimes limited to the quadriceps muscles. Cardiomyopathy, present in 70% of cases, is the leading cause of death in affected individuals. Mutations in the DMD gene in BMD typically result in in-frame pattern, leading to the production of misfolded or abnormal truncated protein, or a reduced quantity of functional protein, thereby impairing muscle function. Due to the large size of the DMD gene and the prevalence of gross deletion associated with the disease, conventional gene therapy approaches face limitations for correcting these defects. To address the genetic defect in the dystrophin gene, my laboratory pioneered the genomic technique of chromosome transplantation (CT), in iPSCs. CT consists of the complete substitution of an endogenous defective chromosome with an exogenous normal one, resulting in a normal euploid karyotype and the full correction of the genetic defect, including large mutations. After performing CT, corrected clones were first analysed to confirm the presence of the WT DMD gene. In addition, we assessed genomic stability through karyotype analysis and verified the retention of pluripotency and differentiation potential by evaluating the expression of stemness markers and the ability to generate derivatives of the three germ layers. Subsequently, both uncorrected BMD iPSCs and CT-corrected iPSC clones were differentiated into cardiomyocytes (CMs), a cell type that expresses the full-length dystrophin isoform. The restoration of dystrophin was evaluated at a transcriptional, protein and functional levels by electro-functional assays using Ion Optix and Patch Clamp platforms. This study proves the feasibility of fully correcting the genetic defect in BMD and functionally confirming the rescue of dystrophin. It lays the foundation for applying this approach also to other X-linked genomic disorders characterized by large or complex mutations.