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Targeting ASTROcytes with cutting-edge EDITING technologies to treat the Alexander disease

Periodic Reporting for period 1 - ASTRO-EDITING (Targeting ASTROcytes with cutting-edge EDITING technologies to treat the Alexander disease)

Reporting period: 2020-07-01 to 2022-06-30

Alexander disease (AxD) is an autosomal dominant neurodegenerative disorder caused by mutations in the gene encoding for the glial fibrillary acidic protein (GFAP), the major intermediate filament protein in astrocytes. In AxD patients, GFAP mutations are mainly clustered in hotspots affecting arginines of structural helical coiled-coil rod domains that play a crucial role in filament assembly. Mutated GFAP protein oligomerizes and accumulates within Rosenthal fibers, leading to astrocytic dysfunctions and altered development and homeostasis of affected brain tissues. AxD patients typically present with seizures, megalencephaly, spasticity, or developmental delays. Cerebral white matter abnormalities define this disorder as a demyelinating leukodystrophy. Antisense oligonucleotide (ASO)-mediated downregulation of total GFAP content showed therapeutic benefits in AxD animal models. Still, the high ASO doses required to achieve an acceptable level of GFAP knock-down pose some safety concerns in the chronic treatment of pediatric patients. Currently, this orphan disease (estimated incidence about 1 in 1 million births) lacks a cure.
The ASTRO-EDITING project aim to assess the efficacy of novel and definitive base/gene editing strategies targeting Gfap gene or Gfap hotspot mutations to recover pathological phenotypes in astrocytes. The project was developed in accordance with the following specific objectives:
Objective 1: selection of the best-performing editing system targeting the Gfap hotspot mutations or Gfap gene in an in vitro AxD model.
Objective 2: in vivo proof-of-concept studies on the efficiency and efficacy of the selected editing approach upon its AAV-mediated delivery in the central nervous system (CNS) of neonatal AxD mice.
ASTRO-EDITING provided in vitro and in vivo proof-of-concept data on the efficiency and efficacy of a novel AAV-based gene editing strategy that reduces GFAP content and pathological hallmarks in astrocytes. Additionally, the researcher's competences and knowledge in the gene therapy and neuroscience fields were reinforced thanks to the expertise of the host laboratory, the international environment of OSR, and scientific collaborations. The researcher created a new research niche, published scientific reviews, and improved his organizational, management and interpersonal skills, thus acquiring independence as experienced researcher in the scientific community.
A first accomplishment of the project was the generation of murine cell lines engineered to stably express mutated GFAP proteins fused to fluorescent mCherry. The combination of these in vitro AxD models and FACS analyses could represent an easy-to-use platform for high-throughput screening of compounds (e.g. drugs, RNAi molecules, CRISPR/Cas9 systems) inducing GFAP downregulation and therapeutic benefits in AxD patients. In ASTRO-EDITING, these engineered cell lines were used to develop CRISPR/Cas9-based strategies targeting the Gfap hotspot mutations or Gfap gene. Single guide RNAs (sgRNAs) recognized by Cas9 nucleases were designed and selected to induce the silencing of alleles harbouring Gfap hotspot mutations (R76H and R236H). Additionally, adenine base editors were identified to convert the CAC mutation in the normal CGC codon, thus correcting the R76H Gfap mutated allele. These allele-specific strategies required Cas9 variants with relatively large size, making difficult the in vivo delivery of CRISPR/Cas9 system into the brain by using adeno-associated vectors (AAVs). Therefore, a strategy targeting both WT and mutated Gfap alleles was optimized by using the SaCas9 variant, which better fits in a single AAV vector because of its smaller size. The SaCas9/sgRNA system targeting both the WT and mutated Gfap alleles induced a robust downregulation of GFAP-mCherry expression, without relevant genotoxic events.
Selection of an AAV serotype with higher tropism for astrocytes, and optimization of a protocol for intracerebral injection of viral vectors were assessed to maximize the delivery of CRISPR/Cas9 system in disease-bearing cells. In an AxD mouse model carrying the R76H Gfap mutation, intracerebral injection of a therapeutic AAV vector (carrying the SaCas9/sgRNA targeting both the WT and mutated Gfap alleles) led to reduced expression of GFAP protein and decreased accumulation of Rosenthal fibers in white matter areas; thus, recovering the disease hallmarks in the astrocytes of the central nervous system.
Overall, ASTRO-EDITING offers preclinical data on an innovative and definitive gene therapy strategy to treat AxD patients. The results were disseminated to an academic and scientific audience through participation to conferences and the publication in open access journals of scientific reviews in the fields of gene therapy and disease modelling of leukodystrophies. Outreach activities with patient’s association and interviews on national radio were performed to increase the awareness in general public on rare genetic disorders and to diffuse knowledge on gene therapy and editing technologies.
We envisage that the promising results achieved in ASTRO-EDITING will pave the way for future studies aimed at addressing the safety and efficacy of a novel and definitive gene therapy for the treatment of AxD patients. The technologies and platforms developed in ASTRO-EDITING could be applied for the future progress of gene editing approaches to treat other neurological and psychiatric disorders characterized by primary astrocyte degeneration or dysfunctional/maladaptive astrogliosis. ASTRO-EDITING addressed the European issue of Horizon 2020, in the social challenge “Health, Demographic Change and Wellbeing” endeavours for the call “Better health and care, economic growth and sustainable health disease”, subject SC1-BMC-04-2018 “Rare Disease European Joint Programme Cofund”.
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