Despite the increasing rate of diagnosis enabled by next generation sequencing, the molecular basis underlying about half of leukodystrophies is unknown. Among recently discovered hereditary leukodystrophies, several pathogenic variants are located within genes that converge on a common pathway, thus it is possible that they share common underlying mechanisms.

HIKESHI Hypomyelinating Leukodystrophy (HHL) is a rare and devastating congenital disorder with infantile onset during the first year of life. Symptoms include poor growth, delayed motor milestones, central hypotonia and other psychomotor disabilities. HHL patients have an increased sensitivity to heat stress. Febrile illness often leads to an irreversible deterioration of the neurological condition, and may even lead to death. There is currently no curative treatment for HHL and the symptomatic treatments are focused on prevention of fever.

When a gene mutation causes a protein to be missing or faulty, gene therapy may be able to restore the normal function of that protein. This approach has been recently translated from basic research to the clinic, for the treatment of SMA, a devastating infantile neurological condition. Thus, a similar approach can be used to treat additional conditions, such as HLL and other leukodystrophies. However, in order to test this approach it is necessary to develop appropriate research models.

Animal models have been traditionally used for pre‐clinical research, but increasing evidence suggest that mice are different than humans, and therefore curing a mouse model in the lab often does not translate into the clinic. Therefore it is crucial to develop human‐based models that will better represent human physiology.

Here, we propose to use cutting‐edge techniques that include patient‐specific stem cells (termed iPSCs) and engineered Organ‐on‐Chip to generate a personalized Brain‐on‐Chip. This platform will be used to identify disease‐relevant phenotypes, which we will use for testing our gene therapy approach. Successful rescue of such phenotype will indicate that gene therapy should be tested in the clinic. The approach that we will apply in this research is focused on HHL, but it can be applied to additional leukodystrophies and other neurological conditions in the future.

The goal of this project is to further develop and optimize a gene therapy protocol for Pelizaeus‐ Merzbacher‐like disease 1 (PMLD1). This disorder is caused by mutations affecting an important protein in oligodendrocytes, connexin47, which forms most of their direct communication channels with other cells in the brain, also called gap junctions. Connexin47 is found only in oligodendrocytes but not in other brain cells. Since oligodendrocytes are responsible for forming the myelin in the brain, their impairment caused by loss of connexin47 leads to inadequate formation of myelin early on during development, and to further destruction and degeneration of as the disease progresses. Previous studies in cultured cells and in experimental mouse models of the disease confirmed that loss of connexin47 function specifically in oligodendrocytes causes the disease. Therefore, in previous project funded by ELA, we developed a gene therapy approach to deliver the connexin47 gene to oligodendrocytes of mice that lack gap junction channels in these cells. These animals represent a relevant model of the human disease and present many of the characteristic pathological changes. We used an adeno‐associated viral (AAV) vector AAV1/2 serotype based on previous work showing that it targets efficiently oligodendrocytes. We injected the vector into mouse brain to deliver the connexin47 gene at the age of 10 days. We confirmed selective expression in oligodendrocytes and improvement of the brain pathology. In further preliminary studies we also tested intravenously delivered AAV9 vector (currently used in clinical treatments for other diseases) with positive results, but the intravenous approach would not be useful for PMLD1 patients beyond infancy due to limited penetration into the brain.

Alexander disease is a very rare brain disease. People with Alexander disease experience damage to the white matter in their brains, and they lose a lot of brain tissue. It can be diagnosed when a person is very young, even before they turn 2 years old, but sometimes it is not diagnosed until later in childhood or even adulthood. The symptoms of Alexander disease include problems with mental development, seizures, and muscle stiffness, and it ultimately leads to death. There have been about 500 cases of Alexander disease reported around the world. Unfortunately, there is currently no cure for this disease.

The disease is caused by mutations in the gene glial fibrillary acidic protein (GFAP), which codes for an astrocyte‐specific cytoskeletal protein. Astrocytes are essential for healthy brain functioning because they control many neuronal activities and homeostatic mechanisms of the brain. However, scientists still do not fully understand how this mutant GFAP protein in astrocytes causes Alexander disease.

In this project, we will closely collaborate between two teams to investigate Alexander patient‐derived human stem cell models at the ultrastructural level. We will generate miniature brain‐like structures called organoids from patient‐derived stem cells, and apply novel cell culture protocols to include white matter‐like structures. By analysing the cells in these organoids at the ultrastructural level, we will gain more knowledge on how GFAP mutations can lead to astrocyte and white matter pathology in the disease. This knowledge is essential to find new ways to treat Alexander disease.

Hypomyelinating leukodystrophy 16, also known as HLD16, is a rare genetic disorder that affects the development of myelin, the protective covering around nerve fibers in the brain. HLD16 is caused by a mutation in the gene TMEM106B. We genetically engineered mice in a way that they have the same mutation in TMEM106B that is found in human patients. We plan to investigate these mice to understand the disease and how the mutation in this gene, whose function is yet unknown, leads to this rare disease. This will help to develop future therapies and understand the function of TMEM106B in myelination.

Krabbe disease (KD, or Globoid cell leukodystrophy) is a neurodegenerative disease caused by a deficiency of the lysosomal enzyme GALC. The early childhood form represents 85‐90% of cases and the onset of symptoms is between the 3rd and the 6th month after birth. During the advanced state, blindness and deafness show up; then a vegetative state arises which ends with death within the first 1‐2 years after birth. Unfortunately, for infants who have already developed symptoms of KD, there is no treatment that can change the course of the disease. Treatment, therefore, focuses on managing symptoms and providing supportive care.

Currently, the literature indicates that GALC‐deficiency correction might not be sufficient to cure the patients with KD, suggesting that not yet identified players could be implicated in KD pathogenesis. In this context, we identified some clear evidence pointing to the protein named GPR65 as a key factor in the pathogenesis of the disease.

Here, we propose a deep investigation of GPR65 and its evaluation as a druggable target in KD. In particular, we will evaluate GPR65 expression in patients with KD and test drugs to correct GPR65 deficiency in KD cell models. Moreover, we will explore the combination of a GPR65 mRNA‐based therapy with the GALC correction.

In case of a successful project outcome and in a forward‐looking vision, GPR65 mRNA‐based therapy might be used in combination with a main GALC‐deficiency correcting therapy, to help in achieving the complete KD phenotype rescue.