Evaluating glial and neuronal GFAP, MCP-1 and NfL as blood biomarkers for onset and disease severity in cerebral adrenoleukodystrophy

With a combined incidence of 1:14,700, X-ALD is the most common monogenetically inherited leukodystrophy. The disease is caused by mutations of the peroxisomal very long-chain fatty acid transporter ABCD1 that normally imports very long-chain fatty acids into the peroxisome for degradation. Accordingly, loss of ABCD1 function results in accumulation of very long-chain fatty acids in the plasma and body fluids of affected patients. X-ALD shows a striking phenotypic heterogeneity with inflammatory cerebral X-ALD (CALD) being the most severe form. In order to be treatable by bone marrow transplantation or gene therapy, CALD has to be recognized in its earliest stages.

The ultimate goal of this research project is to identify an easily accessible blood biomarker indicative for onset and progression of CALD. If successful, the identified blood biomarker could provide valuable information for decisions on clinical interventions and could also be used as treatment efficacy marker in clinical trials targeting CALD.

Characterization of lysosomal dysfunction and targeted intervention in X-ALD

X-linked adrenoleukodystrophy (ALD) is a severe disease, ranging from the fatal cerebral inflammatory demyelinating form affecting boys (cALD) to the peripheral dying back axonopathy that affects adults (AMN). There are no effective therapies for AMN, and the cause of cALD is poorly understood.

The goal is to develop, validate, and use for treatment discovery, a new model of ALD. To accomplish this, the group will use the small genetic vertebrate model zebrafish, which has low costs and the ability to perform high-throughput screens that are unfeasible in other vertebrate systems. Zebrafish have the same genes as humans, including ABCD1, the gene responsible for ALD. The data shows that mutations in zebrafish ABCD1 cause ALD in zebrafish. The team is using the ALD zebrafish model to validate a new potential treatment for ALD, to understand this drug’s mechanism, and to understand the cause of cALD.

Development of cutting-edge editing technologies to treat Alexander disease

Alexander disease (AxD) is a rare autosomal dominant disorder caused by point mutations in the gene encoding for the glial fibrillary acidic protein (GFAP), the major intermediate filament protein in astrocytes. Accumulation of GFAP protein in Rosenthal fibers leads to astrocytic dysfunctions and altered development and homeostasis of affected brain tissues.

No cures are currently available for AxD patients. In the proposed pilot project, the team aim to develop a gene editing approach to specifically downregulate the expression of the mutated GFAP protein in affected AxD astrocytes. This project will collect solid proof-of-concept data for the future progress of a novel and definitive gene therapy strategy for the treatment of AxD patients. In addition, they expect to outline novel editing platforms that could be applied prospectively for disease modelling studies and therapeutic treatments of other leukodystrophies characterized by astrocyte degeneration or dysfunctional/maladaptive astrogliosis.

Evaluating the mechanisms of macrophage/microglia-mediated enzymatic cross-correction of human MLD neurons and glial cells in vitro

Hematopoietic stem cell gene therapy (HSC GT) is an experimental treatment based on the transplantation of autologous hematopoietic precursor cells genetically modified to express high levels of ARSA enzyme. This treatment benefits late infantile and early juvenile MLD children if treated in the pre-symptomatic/early symptomatic stage of the disease but is less effective in MLD children treated in the progressive phase of disease, who experience severe neurological deterioration. In order to recognize the reasons behind this different clinical outcome we need a better understanding of the therapeutic mechanisms of correction of MLD neural cells by the progeny of transplanted hematopoietic cells that engraft in the brain.

In this project the investigators aim to fill this gap of knowledge taking advantage of unique and clinically relevant in vitro human disease models, i.e. neural and blood cells derived from healthy donors, untreated and gene therapy treated MLD patients. We expect to clarify how and to which extent the ARSA enzyme is transported from metabolically competent donor blood cells to MLD neurons and glia. Also, they will provide hints on the contribution of complementary mechanisms of correction that could be exploited to enhance the benefit of HSC GT and broaden its accessibility to larger cohorts of MLD patients who presently not meet the inclusion criteria of these experimental treatments.

Why does regeneration fail in cerebral X-linked adrenoleukodystrophy?

X-linked adrenoleukodystrophy is a genetic disease affecting mostly young male patients. In its severest form, patients develop a fulminant inflammatory destruction of central nervous system white matter, areas where all descending nerve fiber tracts are contained. Untreated, patients suffering from this disease mostly die within months to few years. The only effective therapy for halting the progression is an early transplantation of hematopoietic stem cells. Though progression often can be stopped this way, any disability that patients had accumulated before treatment recovers very poorly. This is in harsh contrast to another inflammatory disease affecting the white matter, multiple sclerosis. Here, patients often recover very well after an inflammatory and demyelinating bout. The reasons for this difference are not yet understood.

In this project, the team strive to investigate the reasons underlying the apparent lack of regeneration in X-ALD. They will use autopsy tissue from human patients who died of X-ALD as a starting point and let their research be guided by a careful re-examination of pathologic changes in this tissue. In addition, they will use novel proteome and transcriptome techniques that allow a very detailed investigation of molecular alterations in the tissue. In this way the researchers strive to identify novel molecular and cellular pathways of the disease that could be manipulated in order to improve clinical recovery in X-ALD. Furthermore, in their novel detailed analysis of the human histopathology they hope to provide a valuable tool also for other researchers working in the field.

Closing the circle on integrated stress response treatment targets in vanishing white matter

Leukodystrophies are a major source of handicap at all ages, but children are affected most. The team studies leukodystrophies since 1987. Initially their research was focused on describing new leukodystrophies and finding the underlying gene defects. Much of their research efforts have focused on the new leukodystrophy vanishing white matter (VWM), also called childhood ataxia with CNS hypomyelination (CACH). The disease may occur at all ages, but mainly affects young children (2-6 years). Children with VWM experience progressive neurological handicap and die early, usually a few years after diagnosis. There is no cure for VWM, but patients benefit from treatments that prevent fever and head trauma, as these events trigger a fast worsening of the disease.

Several years ago, the team founds that the gene defect for VWM lies in an enzyme complex that is crucial for protein synthesis. Since then, they study how the disease works (disease mechanisms), most of all to find openings for treatment. The group founds that cells in the white matter of the brain do not develop into mature cells that can execute their normal function of myelination and white matter repair properly. The problem with maturing in functional cells can well explain the severe white matter damage that is observed in patients. Their recent studies have demonstrated that a basic stress pathway is abnormally activated in VWM white matter cells. They have evidence that abnormal activation of this stress pathway may contribute to disease.

In the proposed study they will test the effects of three FDA-approved inhibitors of this stress pathway in representative disease models. The proposed work has the potential to open up new treatment options fast. Their vision on treatment of VWM is that effective cure of this complex disease is not achieved with any single therapeutic modality. The team believes that the treatment should target the disease at multiple levels, including reduction of stress pathways, reduction of the toxicity of the diseased white matter, provision of patient derived cured/healthy white matter cells (stem cell transplantation) and possibly gene therapy.

Allelespecific siRNAs as therapeutic option for ADLD: in vitro preclinical validation on unique human experimental models

The adult onset autosomal dominant leukodystrophy or ADLD, is a genetic, fatal and incurable neurodegenerative disease. It is characterized by a loss of the so-called "white matter" of the central nervous system and is manifested by movement disorders and severe alterations of the autonomic nervous system.

The genetic cause is the presence of three copies, instead of the two normally present, of the gene that contains the instructions to produce the lamin B1 (LMNB1) protein, which belongs to a group of structural proteins (lamins) forming the nuclear membrane of the cell. In patients, Lamin B1 accumulates into the cells causing the neurodegeneration.

Through our project, we will provide the first therapeutic option for ADLD, developing a technique called "allele-specific silencing". By using small molecules of RNA called “siRNAs”, we will be able to "turn off" one of the three copies of the gene, restoring the physiological lamin B1 levels, and in turn avoiding the accumulation of the protein and the disease.
To validate our therapeutic strategy mediated by siRNAs, we will generate two innovative in vitro models based on induced pluripotent stem cells (iPSC) derived from ADLD patients that will allow an authentic "disease-in-a-dish" approach. The iPSCs are a highly versatile tool, since they can be used to recreate in the laboratory different types of cells normally difficult or impossible to obtain from a patient, such as those of the central nervous system. In these models we will test genetic products already developed in our lab to measure efficacy and potency on LMNB1 reduction and the absence of negative or dangerous effects. Our project is intended to pave the way towards a therapy for ADLD and also establish distinctive human ADLD-relevant models and therapeutic approaches that may have great importance for future studies non only on A

nanoERT Nanoparticle based Enzyme Replacement Therapy for the treatment of Krabbe disease: a preclinical study in the Twitcher Mouse

 Krabbe disease (KD, or Globoid cell leukodystrophy) is an autosomal recessive, neurodegenerative disease caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). It is a lethal metabolic disorder, with a frequency of about 1/100000 newborns. The early childhood form represents the 85-90% of cases and the onset of symptoms is between the 3rd and the 6th month after birth. First symptoms are dysphagia, nervousness, hypertonia; convulsions are often present as well. 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, the systemic administration (e.g. by intravenous injections) of GALC is not effective because of the presence of the blood brain barrier (BBB) that forbids the translocation of bulky proteins like GALC into the central nervous system. No cure is currently available for KD, and treatment is symptomatic and supportive only.

Our strategy to overcome this issue is to exploit active nanoparticles capable of transporting GALC across the BBB. Thanks to a previous pilot study supported of ELA, we demonstrated that with this approach it is possible to achieve GALC activity recovery in the brain of the mouse model of KD, and in cells from KD patients.

With this project, we will perform a complete pre-clinical testing in the KD murine model. An enzyme replacement therapy (ERT) protocol will be optimized based on our nanoparticles to deliver functional GALC via systemic administration into the mouse brain. We will test if this therapy can improve the pathophysiology in terms of: i. life span, ii. prevention/slow-down of neuropathological alterations, and iii. preservation of motor functions.

Given that the materials used in this study are already approved for clinical use, in case of successful project outcome, this research has the potential for a short/medium term clinical translation. Finally, we would like to point out that our methodological approach, here proposed to correct GALC deficiency, is potentially applicable to other lysosomal storage disorders with major brain involvement, such as the Metachromatic Leukodystrophy, by changing the cargo (i.e. the functional enzyme) transported by the nanoparticles.

Megalencephalic leukoencephalopathy with subcortical cysts is an astrocyte disease of the neurovascular unit

MLC is a rare form of leukodystrophy mainly linked to mutations in the MLC1 gene. Patients with this disease suffer from macrocephaly and motor and cognitive symptoms associated with a progressive myelin degeneration. Why the absence of MLC1 leads to these defects is an open question. MLC1 is a molecule produced by astrocytes, which are the major glial cells of the brain and is enriched at their interface with the blood vessels. Since astrocytes control vascular functions in the brain, we proposed that brain vascular pathological mechanims could be involved in MLC, a question that has never been adressed. Using a mouse model deficient for MLC1, we started to uncover a gliovascular pathology in MLC. Our results suggest that it might be the first pathological sign of MLC. We now propose to further explore these vascular alterations with the underlying idea that knowing their causative mechanisms could provide therapeutic options for MLC patients.

AAVmediated Gene Delivery for the Motor and Sensory Phenotype of Adrenomyeloneuropathy

Adrenomyeloneuropathy is a debilitating lifelong disorder that currently has not treatments available. As an inherited disorder, gene correction is clearly necessary to alter the trajectory of disease burden. We have a developed an approach to deliver a healthy copy of the faulty gene directly into brain and spinal cord.

We have tested this in mice with the disease and have made key insights into a cell type critical to this process: neurons in the brain and alongside the spinal cord. With this knowledge we are now improving gene delivery using new viral vectors.

Importantly we have also developed industry partnerships that can help with manufacturing and setting up future clinical trials. Beyond creating a much-needed treatment, we are also through our studies gaining a better understanding of the disease biology of adrenomyeloneuropathy.

Development of chimeric lysosomal enzymes with improved bioavailability to advance gene therapy strategies for globoid cell leukodystrophy

Globoid Cell Leukodystrophy (GLD) is a neurodegenerative lysosomal storage disease (LSD) due to the genetic deficiency of beta-galactosylceramidase (GALC). The rapid disease progression of the infantile forms and the severe neurodegeneration pose major issues for the development of effective treatments. Currently, GLD patients lack real therapeutic options.

The promising but still modest results obtained in pre-clinical models using innovative approaches (i.e. gene/cell therapies) highlight the difficulty of providing timely (before onset of symptoms), pervasive (to all affected tissues), and long-term (ideally for the whole life) therapeutically relevant levels of GALC enzyme in a safe manner. This difficulty relies in part on our imperfect understanding of the mechanisms of enzymatic correction in the different cell types that are targets (i.e brain cells) or effectors (the progeny of blood stem cells) in the context of gene/cell therapy approaches. This is a gap that we aim to fill with this study. The long-term goal of this study is to design therapeutic approaches based on solid mechanistic ground achieved using relevant GLD models. We hypothesise that the use of a GALC enzyme engineered to increase its secretion and capability to cross the blood-brain barrier may boost the efficacy of gene/cell therapy approaches in GLD, as it does in pre-clinical models of similar diseases. Taking advantage of our expertise in the study and treatment of GLD, and building upon the availability of novel reagents and tools, we will design chimeric GALC enzymes that will be tested for secretion/bioavailablity, safety, and modality of action in direct comparison with the unmodified enzyme in relevant cells types (i.e. hematopoietic stem/progenitor cells and differentiated progeny) and, ultimately, in GLD mice.

Successful completion of this project will increase mechanistic knowledge of disease correction in GLD, paving the way to novel gene/cell therapy strategies using modified enzymes to be applied as independent treatments and/or in combination to achieve global disease correction.

Brain organoids to evaluate a new therapeutic strategy for Adrenoleukodystrophy

X-linked adrenoleukodystrophy (ALD) is the most common leukodystrophy. All ALD patients have a mutation in ABCD1 and accumulate very long-chain fatty acid (VLCFA) in tissues, including brain and spinal cord. In adulthood, virtually all males and >80% of women develop chronically progressive myelopathy (adrenomyeloneuropathy) for which no

disease-modifying therapy is available. Hematopoietic stem cell transplantation (HSCT) and ex vivo autologous gene therapy are effective in treating cerebral ALD, but only in the early stages of brain inflammation. Unfortunately, HSCT-treated patients can still develop myelopathy in adulthood, because HSCT is only effective at halting the inflammatory component of the disease without addressing the underlying biochemical defect. This therapeutic gap highlights the need to develop effective treatments aimed at the normalization of VLCFA levels in the brain and spinal cord. Using skin cells from ALD patients we have demonstrated that saturated VLCFA induce cellular stress, with prolonged exposure resulting in cell death.

This effect is not observed with mono-unsaturated VLCFA. We identified small-molecules that activate an alternative metabolic route that converts saturated to mono-unsaturated VLCFA.

Treatment of ALD cells completely corrects VLCFA levels. Treatment of the ALD mouse with these molecules added to their food results in a reduction in adrenals, spinal cord and brain. Unfortunately, are these small molecules not specific enough and cause side-effects. We are currently searching for more specific small-molecules. When found, these small-molecules must be tested in an ALD disease model. In this project we will develop a novel ALD disease model.

We already generated stem cells from control and ALD skin cells. These stem cells can be used to generate organoids, which is a miniaturized and simplified version of an organ. Interestingly, with the proper tools we can use these stem cells to generate brain organoids. The availability of control and ALD brain organoids would be a major step forwards in the development of a therapy for ALD and other leukodystrophies. Organoids will lead to a reduction in animal studies and they are a preclinical model that better reflects the disease.

Gene therapy for Megalencephalic leukoencephalopathy with subcortical cysts in two animal models of the disease.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare genetic disorder characterized by an abnormal head, loss of motor functions, epilepsy and mild mental decline. The disease is caused by mutations in two genes named MLC1 and GLIALCAM. There are no therapies for MLC patients, only palliative treatment. Interestingly, some patients with GLIALCAM mutations show a remitting phenotype, suggesting that it may be possible to improve the phenotype of MLC patients, even at later stages of the disease.

We present here a gene therapy preclinical therapeutic approach for MLC patients using two animal models of the disease, the Mlc1 and the GlialCAM knock-out mice. Therapeutic genes will be delivered by one-time treatment to the CNS, overcoming the difficulty of crossing the blood-brain barrier. We hope that the results of this project will be able to provide the first therapeutic tools for patients affected with MLC and may have also implications to treat other diseases affecting the myelin.

Pre-clinical testing of single and combined autophagy modulation by Lithium and Rapamycin in Globoid Cell Leukodystrophy.

Globoid Cell Leukodystrophy (GLD) is a rare, hereditary disorder (with a frequency of about 1/100000 newborns) triggered by a deficit of the lysosomal enzyme galactosylceramidase (GALC) and characterized by the accumulation of galactosylsphingosine (psychosine, PSY) in the nervous system. PSY is a cytotoxic sphingolipid, which leads to the widespread degeneration of oligodendrocytes and Schwann cells, causing demyelination. Little is known about the molecular mechanisms by which PSY imparts toxicity and there is currently no cure available for GLD. The early-infantile and most widespread form of this lysosomal storage disorder (LSD) is degenerative, rapidly progressive and lethal. Bone marrow transplantation is currently the only clinically applied method to treat GLD, but

gene therapy has yielded good results in experimental models. However, the recent literature strongly suggests that GALC-deficiency correction is not sufficient to completely rescue the GLD phenotype. Thus, supportive therapies specifically addressing secondary targets of the disease might be needed to improve the final therapeutic outcome. Autophagy, although much studied for many LSDs and neurodegenerative diseases (NDs), has been poorly

investigated in GLD. Autophagy dysregulation has been only recently demonstrated, by us and others, in two in vitro GLD cell models. Here, given our previous results and the wide data form literature showing autophagy activation as a promising therapeutic strategy for NDs and LSDs, we propose the pre-clinical testing of two autophagy activators in the naturally occurring murine model of GLD, the Twitcher mouse (TWI). Specifically, we aim to test in the TWI mouse the effects of Lithium, Rapamycin and of their combination, studying the rescue of a complete set of behavioural and biochemical parameters. Certainly, this study will be very useful to deeply understand the role of autophagy in the molecular pathogenesis of GLD. Furthermore, in case of positive results, therapy will be readily applicable to humans for clinical testing, thanks to the fact that both tested drugs are already available as pharmaceutical preparations. Therefore, in a forward-looking vision, Lithium and/or Rapamycin could be used in combination with a main GALC-deficiency correcting therapy (as gene therapy and/or enzyme replacement therapy) to help in the achievement of a complete GLD phenotype rescue.

Patient iPSC-based models for 4H leukodystrophy to identify complex neuron-glia dysfunctions.

4H syndrome, an inherited brain white matter disorder (leukodystrophy), leads to considerable clinical handicap, ranging from mild to severe. Most patients deteriorate over time. There is no treatment yet, and we do not understand what goes wrong in the brain. There are no good animal models for this disease, hampering research. We think that besides white matter and myelin, also the nerve cells (neurons, also called grey matter) and their processes (axons) are damaged from early disease stages on. In our project, we want to explore this grey matter involvement with an innovative approach.

We are able to make white and grey matter cells from 4H patient and control fibroblasts. Earlier studies showed that 4H patient cells show affected expression of genes involved in nerve cell development. As nerve cells provide signals to white matter cells and myelination, neuronal defects could underlie abnormalities found in grey and white matter areas in 4H patient brains. We want to study co-cultures of both grey and white matter cells derived from patient cells to know how they influence each other. In the future, this might be a good model for drug screening to treat 4H.

Towards preclinical proof-of-concept for HBSL gene therapy

Hypomyelination with Brain stem and Spinal cord involvement and Leg spasticity (HBSL) is a leukodystrophy caused by defective cytoplasmic Aspartyl-tRNA synthetase (DARS). This enzyme is involved in building proteins, a fundamental biological process in bacteria and man alike. HBSL is caused by autosomal recessive mutations in the DARS gene and all point mutations identified result in neurological disease. HBSL is a potentially fatal spectrum disorder with no treatment and unclear etiology. In a pioneering effort our team at the University of New South Wales has generated the first mouse model of HBSL by introducing the same mutations causing HBSL in patients into the mouse Dars gene. Accurate animal models are the prerequisite to study the disease mechanisms and to develop and test treatments.

Moreover, we have already identified that DARS expression in the brain of both mouse and man is enriched in neurons with far less expression in glia. This suggests that neurons might be the cells that a first line HBSL therapy should ideally target. In the first aim of this research proposal, we plan to engineer more mouse models that genetically mimic HBSL mutations and characterize them in order to model different forms of severity of HBSL. The second aim will be to develop a gene therapy platform for expression of a healthy copy of the DARS gene in mice. These experiments will help to identify the optimal route of delivery and the best timing for intervention. In the third aim we will perform a proof-of-concept gene therapy in the most relevant mouse model using optimized paramemters for DARS gene therapy identified in the previous aims. This project will yield an accurate animal model of HBSL that will be instrumental for preclinical testing of gene therapy or other treatment avenues. We propose that this study will generate data with high clinical relevance as our preclinical endpoints and gene therapy platform could easily be adapted for the treatment of other leukodystrophies caused by abnormal protein translation.

Preclinical studies specifically testing therapies (gene, cell, enzyme or pharmacologic therapies) for leukodystrophies.

Metachromatic Leukodystrophy (MLD) is a rare demyelinating disease, due to arylsulfatase A (ARSA) deficiency, an enzyme involved in the catabolism of sulfatides, the main component of the myelin sheath. This deficiency leads to progressive demyelination of central nervous system and peripheral neuropathy. The most frequent form of the disease is the late infantile form of the disease which is characterized by a rapid progression of the disease, especially after the first onset of symptom. Ex vivo gene therapy develop by the group of Alessandra Biffi, and based on engraftment of lentiviral transduced hematopoietic stem cells has been shown to be effective for presymptomatic forms of MLD but not effective in early symptomatic patients, probably due to the rapid evolution and the time needed for the engrafment. On the other hand, enzyme replacement was shown as potentially effective but needed a chronic delivery. In the group, we previously proposed a gene therapy approach for MLD based on intracerebral delivery of an AAVrh.10 encoding ARSA.

We had established proof of concept in mouse model of the disease and scale up study in non-human primates (NHP) leading us to propose a clinical trial which included 4 patients from 2013 to 2016. Despite ARSA expression in the brain and detection in cerebrospinal fluid (CSF) we failed to obtained any therapeutical benefits in these patients. Our point is that expressing ARSA in the CNS is fundamental to rapidly stop the disease progression, however, it could be essential also to bring back in patient healthy microglial cells. This project aim at establishing the proof of concept of rapid, sustained and important ARSA expression in the whole CNS (brain, spinal cord) and potentially also in the peripheral nerve. To achieve this aim, we propose to use a novel serotype of AAV: AAVPHP.eB that has been engineered to efficiently cross the blood brain barrier (BBB) after IV delivery. The project will be divided in two main parts, first a rapid study in the MLD mouse model both for pre and post symptomatic treatment, in order to evaluate efficiency of AAVPHP.eB-ARSA vector for biodistribution, ARSA expression, sulfatides correction, Purkinje cell prevention and behavioral testing. In a second phase, we want to test this vector in NHP thought several routes of delivery: intravenous (IV), intrathecal (IT) and intracerebroventricular (ICV), especially by potential coupling of these routes of administration to optimize CNS targeting. This with the final aim to propose a clinical trial for symptomatic MLD patients.

Selective galactosylceramidase ablation to study the pathogenesis of Krabbe leukodystrophy.

Krabbe leukodystrophy (KD) is a progressive and fatal neurologic lysosomal storage disorder that usually affects infants and causes death before two to three years of age. Hematopoietic Stem Cell Therapy extends the long-term survival with improved quality of life for KD patients, but it is not a cure. Using our newly developed KD animal model, we will identify which brain cell(s) needs to be efficiently cured with therapy.