Infantile-onset Pompe Disease (IOPD), caused by mutations in lysosomal acid alpha-glucosidase (Gaa), manifests rapidly progressive fatal cardiac and skeletal myopathy incompletely attenuated by synthetic GAA intravenous infusions. The currently available murine model does not fully simulate human IOPD, displaying skeletal myopathy with late-onset hypertrophic cardiomyopathy. Bearing a Cre-LoxP induced exonic disruption of the murine Gaa gene, this model is also not amenable to genome-editing based therapeutic approaches. We report the early onset of severe hypertrophic cardiomyopathy in a novel murine IOPD model generated utilizing CRISPR-Cas9 homology-directed recombination to harbor the orthologous Gaa mutation c.1826dupA (p.Y609*), which causes human IOPD. We demonstrate the dual sgRNA approach with a single-stranded oligonucleotide donor is highly specific for the Gaa c.1826 locus without genomic off-target effects or rearrangements. Cardiac and skeletal muscle were deficient in Gaa mRNA and enzymatic activity and accumulated high levels of glycogen. The mice demonstrated skeletal muscle weakness but did not experience early mortality. Altogether, these results demonstrate that the CRISPR-Cas9 generated Gaa c.1826dupA murine model recapitulates hypertrophic cardiomyopathy and skeletal muscle weakness of human IOPD, indicating its utility for evaluation of novel therapeutics. Generation of transgenic murine knock-in models of human disease once relied solely upon targeted insertion of the desired sequence via Cre-Lox recombination and embryonic stem cell implantation. Though this strategy has resulted in many successful murine model systems, it is labor-intensive, time-consuming, and expensive. The advent of genome editing via engineered nucleases, especially clustered regularly interspaced short palindromic repeats (CRISPR)-based systems has allowed for a potentially accurate, efficient, and relatively inexpensive alternative to the traditional method of transgenic knock-in model generation 1,2. As an intriguing example of disorders that may uniquely benefit from genome editing, inherited metabolic disorders (IMDs) are a diverse group of genetic diseases affecting the proper breakdown or synthesis of essential compounds such as carbohydrates, amino acids, or organic acids. Many of these disorders are caused by single gene defects that alter the expression and/or activity of critical metabolic enzymes. Given the monogenic nature of IMD pathogenesis, this class of genetic disorders is quickly becoming an area of high interest for CRISPR-mediated genome editing therapeutics 3-5. Pompe disease, caused by the deficiency of acid α-glucosidase (GAA; EC 3.2.1.20), is characterized by lysosomal accumulation of glycogen in body tissues, primarily cardiac and skeletal muscle. Muscle lysosomal glycogen storage results in muscle weakness varying in age of onset and severity according to residual GAA enzymatic
The goal of this study is to generate and characterize a knock-in model of Pompe disease (PD) a rare, progressive, fatal disorder primarily affecting the cardiac and musculoskeletal systems. While a murine model of PD exists, it bears a Cre/loxP induced exonic insertion of a neomycin cassette and does not completely recapitulate severe human PD -displaying nonfatal hypertrophic cardiomyopathy only late in its natural history. We therefore designed a CRISPR-Cas9 knock-in system targeting the Gaa gene to introduce the known pathogenic CRIM negative Gaa mutation c.1826insA (p.Y609*).Following optimization of our knock-in strategy in cultured murine myoblasts, we successfully generated a Gaa c1826insA mouse model using a dual sgRNA with ssODN donor template approach. Whole genome sequencing and analysis of the Gaa c1826insA murine model establishes that our system is highly specific for the Gaa c1826 target locus and does not induce any off-target mutations or genomic rearrangements. Next, we examined GAA mRNA transcript, protein expression and enzymatic activity levels in our PD knock-in mice. Gaa c1826insA mice display significantly reduced levels of GAA expression and enzymatic activity relative to wild-type mice.We performed echocardiography on Gaa c1826insA mice to assess cardiac structure and function.Gaa c1826insA mice exhibit early-onset, progressive cardiac hypertrophy as measured by significant increases in left ventricular wall thickness and mass index by 3 months of age. We also conducted functional testsgrip strength, inverted screen, gait analysison Gaa c1826insA mice every 3 months to assess overall motor performance. Gaa c1826insA mice display impaired motor strength and coordination relative to wild-type mice. Altogether, our results demonstrate that the Gaa c1826insA murine model recapitulates human infantile-onset Pompe disease and is better suited for evaluation of therapeutic strategies such as genome correction.
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