CRISPR-Cas9 Knock-In of T513M and G41S Mutations in the Murine β–Galactosyl-Ceramidase Gene Re-capitulates Early-Onset and Adult-Onset Forms of Krabbe Disease

Abstract

Krabbe Disease (KD) is a lysosomal storage disorder characterized by the genetic deficiency of the lysosomal enzyme β-galactosyl-ceramidase (GALC). Deficit or a reduction in the activity of the GALC enzyme has been correlated with the progressive accumulation of the sphingolipid metabolite psychosine, which leads to local disruption in lipid raft architecture, diffuse demyelination, astrogliosis, and globoid cell formation. The twitcher mouse, the most used animal model, has a nonsense mutation, which limits the study of how different mutations impact the processing and activity of GALC enzyme. To partially address this, we generated two new transgenic mouse models carrying point mutations frequently found in infantile and adult forms of KD. Using CRISPR-Cas9 gene editing, point mutations T513M (infantile) and G41S (adult) were introduced in the murine GALC gene and stable founders were generated. We show that GALCT513M/T513M mice are short lived, have the greatest decrease in GALC activity, have sharp increases of psychosine, and rapidly progress into a severe and lethal neurological phenotype. In contrast, GALCG41S/G41S mice have normal lifespan, modest decreases of GALC, and minimal psychosine accumulation, but develop adult mild inflammatory demyelination and slight declines in coordination, motor skills, and memory. These two novel transgenic lines offer the possibility to study the mechanisms by which two distinct GALC mutations affect the trafficking of mutated GALC and modify phenotypic manifestations in early- vs adult-onset KD.

Brain Targeted AAV1-GALC Gene Therapy Reduces Psychosine and Extends Lifespan in a Mouse Model of Krabbe Disease

“…However, the same position on human GALC is an aspartic acid, which has a longer carbon chain backbone, and therefore may alter the hydrogen bond formation, the GALC−saposin A interaction, and GALC activity. Additionally, expression of human GALC may trigger a host immune response that neutralizes the function of therapeutic protein in mouse models with endogenous mutant GALC expression such as the Twi-5J mouse [ 51 ] and the CRISPR-Cas9 human mutation knock-in models [ 52 ]. Taken together, these examples explain the tendency toward using murine GALC for AAV gene therapy studies in mouse models.…”

Preclinical studies in Krabbe disease: A model for the investigation of novel combination therapies for lysosomal storage diseases

“…Since the G41S mice model adult-onset KD, they do not present pathological abnormalities until ∼PND200 and, even then, display much milder disease signs and pathology than TWI. 21 …”

“… 16 , 17 , 18 , 19 , 20 Psychosine accumulation has been confirmed in KD patients, the Twitcher (TWI) mouse model of KD, new gene edited mouse KD models, and the canine model of KD. 11 , 14 , 15 , 21 Miyatake and Suzuki first proposed what is now known as the psychosine hypothesis in 1972 to address the relatively low levels of galactosylceramides and high levels of psychosine. This hypothesis suggests that, because of the metabolic block preventing the normal breakdown of galactosylceramides into galactose and ceramides, galactosylceramides are instead deacylated into psychosine, which cannot be catabolized due to GALC deficiency.…”

Globoid Cell Leukodystrophy (Krabbe Disease): An Update

“…A natural authentic model for human KD, is the twitcher mouse, which have mutations in their GALC gene, 21 , 22 associated with the deregulation of several proteins. 23 GalSph is increased in the kidneys, liver, spleen and highly accumulates in the brains of these animals.…”

“…In contrast, another variant, ie, GALC G41S/G41S mice have normal lifespan, modest decreases of GALC, and minimal psychosine accumulation. 22 …”