Cerebellar Astrocyte Transduction as Gene Therapy for Megalencephalic Leukoencephalopathy

Abstract

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare genetic disorder belonging to the group of vacuolating leukodystrophies. It is characterized by megalencephaly, loss of motor functions, epilepsy, and mild mental decline. In brain biopsies of MLC patients, vacuoles were observed in myelin and in astrocytes surrounding blood vessels. There is no therapy for MLC patients, only supportive treatment. We show here a preclinical gene therapy approach for MLC using the Mlc1 knock-out mouse. An adeno-associated virus coding for human MLC1 under the control of the glial fibrillary acidic protein promoter was injected in the cerebellar subarachnoid space of Mlc1 knock-out and wild-type animals at 2 months of age, before the onset of the disease, as a preventive approach. We also tested a therapeutic strategy by injecting the animals at 5 months, once the histopathological abnormalities are starting, or at 15 months, when they have progressed to a more severe pathology. MLC1 expression in the cerebellum restored the adhesion molecule GlialCAM and the chloride channel ClC-2 localization in Bergmann glia, which both are mislocalized in Mlc1 knock-out model. More importantly, myelin vacuolation was extremely reduced in treated mice at all ages and correlated with the amount of expressed MLC1 in Bergmann glia, indicating not only the preventive potential of this strategy but also its therapeutic capacity. In summary, here we provide the first therapeutic approach for patients affected with MLC. This work may have also implications to treat other diseases affecting motor function such as ataxias.

Megalencephalic Leukoencephalopathy: Insights Into Pathophysiology and Perspectives for Therapy

“…A dose-escalation study demonstrated a correlation between MLC1 expression and the degree of correction of the white matter vacuolation in the cerebellum of treated KO mice (Sánchez et al, 2020). However, wild-type animals injected with MLC1 did not show any deleterious effect, contrary to what was reported in the MLC1 over-expressing transgenic animal (Sugio et al, 2017), reinforcing the safety of the strategy and indicating that only supra-physiological levels of MLC1 may be toxic.…”

“…Moreover, particularly for lumbar puncture, it can be performed in an outpatient setting, as is much less invasive than intracranial administration. In the MLC mouse model, the best results were obtained by delivering the vector into the CSF by occipital subarachnoid administration, achieving a high percentage of transduction of the cerebellum without damaging the brain parenchyma, allowing for a more localized and efficient action and avoiding non-specific effects due to systemic expression (Sánchez et al, 2020). By this route, we efficiently achieved transduction in most of the cerebellar parenchyma, with the highest efficacy in the Bergmann glia, where MLC1 is highly expressed (Figure 4).…”

“…This is possible by combining a particular AAV serotype with the control of the transgene expression by recombinant promoters that recruit specific host cell-derived transcription factors (Lawlor et al, 2009). To design a gene therapy strategy for MLC, and taking into account that MLC1 is exclusively expressed by astrocytes in the brain, we choose an AAVrh10 vector (Sánchez et al, 2020), as it seems to transduce a larger number of glial cells in the adult brain, compared to AAV9 that showed a higher and specific tropism for neurons (Petrosyan et al, 2014). AAVrh10 was used in combination with a glial-derived fibrillary acidic protein recombinant promoter (GFAP), with high selectivity for astrocytes in the mature mouse brain (Brenner et al, 1994).…”

Emerging Concepts in Vector Development for Glial Gene Therapy: Implications for Leukodystrophies

“…MLC1 is exclusively expressed in gray and white matter astrocytes including cerebellar Bergmann glia in the CNS. In a recently published murine MLC1 knockout model that mirrors the spongiform phenotype of the human disease, a single astrocyte targeted AAV.rh10- GFAP-MLC1 gene replacement prevented vacuolization when administered before disease onset at 5 months, and even at 15 months provided near complete remission of vacuolization ( Sánchez et al, 2020 ). While likely due to the advanced age at infusion, the authors observed relatively poor transduction in the CNS following IT or ICV delivery.…”

Canavan Disease as a Model for Gene Therapy-Mediated Myelin Repair

“…These genetically rare myelin diseases are often characterized in early childhood with several clinical presenting forms, namely hypotonia, ataxia, and seizures (Traeger and Rapin, 1998;Barkovich, 2005; van der Knaap and Bugiani, 2017). Some symptoms have also been observed of advancing into more severe stages such as hydrocephaly and megalencephaly (Matalon et al, 1988;Bugiani et al, 2010;Desai and Grimm, 2013;Sánchez et al, 2020). Consequently, many diagnosed patients can progressively develop significant debilitation and cognitive impairment, thus negatively affecting their quality of life and contributing to emotional and financial burdens on their families.…”