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.