Towards a therapy for Vanishing White Matter: on stem cells and viral vectors

Vanishing White Matter (VWM) is a severe leukodystrophy caused by pathogenic variants in either of the five genes encoding the subunits of eukaryotic initiation factor 2B (eIF2B). The age of onset is correlated to disease severity. Most patients have an onset in early childhood. They suffer from neurological decline and early death. This thesis aimed to further develop curative treatment options for VWM in the form of cell replacement therapy and gene therapies. To stimulate future endeavors into treatment development, we also created a stem cell-derived in vitro model to study VWM and potential therapies. Our findings are summarized below. Chapter 2 describes the effects of cell replacement therapy using human stem cell-derived glial progenitors in a VWM mouse model. Recovery following transplantation was assessed on a known hallmark of VWM pathology in the brain. We showed that human glial progenitor populations that were treated with retinoic acid and maintained in vitro until a particular window of development prior to grafting exerted therapeutic effects. Further evaluation by regression analysis showed that VWM mice were improved on both motor behavior and brain pathology. Interestingly, the transplanted cells showed differential migration and cell fate in the VWM brain as compared to healthy brain. Thus, additional studies on translating mouse studies into cell replacement therapy for VWM patients should consider the diseased brain microenvironment. In chapter 3, [redacted]. In chapter 4 we investigated whether gene editing could correct the point mutation in the Eif2b5 VWM mouse, using dual-AAV to administer intracerebroventricular CRISPR/Cas9 in neonatal mice. We noted rapid deterioration of health, resulting in premature death, from 5 weeks of age onwards. Next Generation Sequencing indicated a much higher occurrence of insertion/deletion mutations (indels) than successful correction of the Eif2b5 point mutation. The VWM histological analysis revealed acute necrosis of the brainstem, which explains the observed lethal phenotype. Involvement of the brainstem has been noted in VWM patients. In conclusion, indel formation was too pronounced for CRISPR/Cas9 to be beneficial as a gene therapy and instead worsened the phenotype. Newer gene editing technologies may be better suited as gene therapy for VWM because they do not generate indels. Protocols for the generation of iPSC-derived grey matter- and white matter-like astrocytes are described in chapter 5 to better model the specific involvement of white matter astrocytes in VWM. These protocols can be used for both mouse and human stem cells. We found that cultures of oligodendrocyte precursor cells (OPCs) show a phenotype; medium conditioned by mouse or human iPSC-induced VWM astrocytes leads to perturbed maturation of OPCs, while medium conditioned by wild-type iPSC-induced astrocytes converts them into healthy, myelin-producing oligodendrocytes. Interestingly, the maturation defect induced by the VWM astrocyte secretome, as well as decreased apoptosis, were only observed when white matter astrocytes were used and not in grey matter astrocyte cultures, revealing a selective vulnerability of white matter astrocytes for pathogenic VWM variants. RNA sequencing analysis indicated that some biological pathways were affected in both mouse and human iPSC-derived white matter VWM astrocytes, including extracellular space/matrix, (cell) development, and immune system. In addition, some pathways were exclusive for the human white matter-like astrocytes and related to neuronal and mitochondrial functioning. We thus present a human stem cell model that recapitulates white matter astrocyte dysfunction in VWM and that can promote therapeutic testing and drug screening. Lastly, the implications of these findings are discussed and future directions for the development of VWM treatments are considered