CRISPR-Cas9 generated Pompe knock-in murine model exhibits early-onset hypertrophic cardiomyopathy and skeletal muscle weakness

Abstract

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

CRISPR-Mediated Generation and Characterization of a Gaa Homozygous c.1935C>A (p.D645E) Pompe Disease Knock-in Mouse Model Recapitulates Human Infantile Onset-Pompe Disease

“…A significant divergence of the model from human c.1935C>A IOPD is the lack of infantile mortality in KI mice. This KI mouse, along with the Gaa Em1826dupA KI mouse strain previously generated in our laboratory[15] and other previously published Gaa KO models [24–26], all demonstrate null or nearly-zero GAA enzyme activity. Nevertheless, no neonatal mortality has been observed in any model, while neonatal death is the inevitable clinical outcome in untreated IOPD patients[27, 28].…”

“…Gaa c.1935 gRNA-2 demonstrated higher on-target editing (26.7±10.7%) and HDR efficiency (5.4±3.4%) than gRNA-1 (on-target editing: 13.2±3.7%; HDR efficiency: 3.8±0.6%) (Table 1). Given our prior success in generating Gaa Em1826dupA KI cell and transgenic mouse lines using a dual overlapping gRNA strategy [15], we applied this same method towards the generation of Gaa c.1935C>A KI C2C12 cells and Gaa Em1935C>A mice.…”

“…In vitro testing of Gaa c.1935 guide RNAs pX459-Gaa c.1935 gRNA expression vectors and donor ssODNs were transfected into murine C2C12 myoblast cells (ATCC CRL-1772) using the Neon TM Transfection System (ThermoFisher Scientific) as previously described [15]. In short, 3×10 5 cells were mixed with 4.5 μg gRNA expression vector(s) and 450 nM ssODN (Table 1), then and electroporated using the following parameters: pulse voltage, 1650V; pulse width: 10 ms; pulse number: 3.…”

Gene Therapy Developments for Pompe Disease

“…Finally, the availability of CRISPR tools to generate novel mouse models that better recapitulate human disease phenotypes could help accelerate the design of the next generation of PD therapies. Huang et al recently reported the generation of a novel murine IOPD model utilizing dual sgRNA CRISPR-Cas9 homology-directed recombination to harbor the orthologous Gaa mutation c.1826dupA (p.Y609 *) [ 161 ]. This model effectively recapitulated the patient-specific genotype, which results in hypertrophic cardiomyopathy and skeletal muscle weakness, hallmarks of human IOPD.…”

Preclinical Research in Glycogen Storage Diseases: A Comprehensive Review of Current Animal Models

“…Finally, the Gaa c.1826dupA mouse model was generated using the novel CRISPR-Cas9 homology-directed recombination to harbor the orthologous Gaa mutation c.1826dupA (p.Y609*), which causes human IOPD [ 245 ]. These mice showed a major decrease in GAA activity (>95% in heart, diaphragm and gastrocnemius), massive increases in glycogen content (185 and 28 fold increases in the heart and gastrocnemius, respectively), autophagy impairment, decreased forelimb muscle strength, significant reduction in body mass and abnormal cardiac function [ 245 ].…”