CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease

Abstract

Pompe disease, an autosomal recessive disorder caused by deficient lysosomal acid α-glucosidase (GAA), is characterized by accumulation of intra-lysosomal glycogen in skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant, the most frequent GAA pathogenic mutation in people of Southern Han Chinese ancestry, causes infantile-onset Pompe disease (IOPD), presenting neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, respiratory failure, and infantile mortality. We applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a Gaaem1935C>A knock-in mouse model and a myoblast cell line carrying the Gaa c.1935C>A mutation. Herein we describe the molecular, biochemical, histological, physiological, and behavioral characterization of 3-month-old homozygous Gaaem1935C>A mice. Homozygous Gaaem1935C>A knock-in mice exhibited normal Gaa mRNA expression levels relative to wild-type mice, had near-abolished GAA enzymatic activity, markedly increased tissue glycogen storage, and concomitantly impaired autophagy. Three-month-old mice demonstrated skeletal muscle weakness and hypertrophic cardiomyopathy but no premature mortality. The Gaaem1935C>A knock-in mouse model recapitulates multiple salient aspects of human IOPD caused by the GAA c.1935C>A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes.

Treatment of infantile-onset Pompe disease in a rat model with muscle-directed AAV gene therapy

“…Given the quick progression of the disease, Pompe rats showed a marked reduction of mean and maximum lifespan, with no PD rats surviving over 8 months of age. Therefore, PD rats showed the lowest survival rate achieved in comparison with any existing PD mouse model [ [13] , [14] , [15] , [16] , 20 , 21 , [33] , [34] , [35] , [36] , [37] , [38] , [39] , [40] ]. To date, seven different PD mouse models have been generated [ 15 , [33] , [34] , [35] , [36] , [37] , 39 , 40 ].…”

“…Therefore, PD rats showed the lowest survival rate achieved in comparison with any existing PD mouse model [ [13] , [14] , [15] , [16] , 20 , 21 , [33] , [34] , [35] , [36] , [37] , [38] , [39] , [40] ]. To date, seven different PD mouse models have been generated [ 15 , [33] , [34] , [35] , [36] , [37] , 39 , 40 ]. All of them accumulated glycogen in the heart and skeletal muscles but showed variable disease severity.…”

“…The Gaa KO DBA2/J mice showed early-onset spinal cord and respiratory defects but no striking differences in muscle disease and strength compared to 6 neo /6 neo mice [ 15 ]. Two knock-in PD mouse models exhibited skeletal and cardiac muscle pathology but did not display premature mortality [ 39 , 40 ]. Importantly, one advantage of rat models is their close physiological similarity to humans [ 63 , 64 ].…”

“…Here, we have engineered AAVMYO3 vectors expressing GAA under the control of muscle-targeted promoter/transcriptional cis -regulatory element (CRE) [ 32 ] to treat IOPD. To overcome the limitations of the existing mouse models of PD, which do not fully develop the critical features of human IOPD [ [13] , [14] , [15] , [16] , 20 , 21 , [33] , [34] , [35] , [36] , [37] , [38] , [39] , [40] ], a new PD rat model was developed. This newly generated PD rat model that suffered from devastating alterations characteristic of IOPD patients was used to test the efficacy of the gene therapy.…”

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“…Technology: In a study by Kan et al was aimed to create a mouse model that recapitulates the human form of the disease caused by a specific mutation (c.1935C>A) in the GAA gene [1]. Researchers used CRISPR-Cas9 technology to introduce the c.1935C>A mutation into the mouse genome.…”