NEWS

Update on ASTRO-EDITING

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 off-target events.

In perspective of in vivo studies, Dr. Meneghini characterized the progression of the pathology in an AxD mouse model carrying the R76H mutation. Progressive accumulation of GFAP protein and increased formation of Rosenthal fibers were observed in the white and grey matter regions within the first 3 months of age, suggesting that an early treatment of AxD mice could prevent disease onset and progression. Selection of an AAV serotype with higher tropism for astrocytes, and optimization of a protocol for intracerebral injection of viral vectors in neonatal mice were performed to maximize the delivery of CRISPR/Cas9 system in disease-bearing cells of brain areas affected by AxD pathology. Injection of a therapeutic AAV vector (carrying the SaCas9 nuclease and the sgRNA targeting both the WT and mutated Gfap alleles) in the cerebrospinal fluid of AxD pups resulted in: (i) relevant transduction of astrocytes; (ii) reduced expression of GFAP protein; (iii) decreased accumulation of Rosenthal fibers in white matter areas affected by AxD pathology in 3-month old mice.

ASTRO-EDITING offers preliminary data on an innovative and definitive gene therapy strategy to treat AxD patients. 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.