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

Abstract

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.

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

“…5D), demonstrating impaired autophagosome-lysosome fusion, in skeletal muscles (diaphragm and gastrocnemius) but not cardiac muscle of the KI model. This is an observation similar to what has been observed in both Gaa em1826dupA KI and KO mouse models 15,21 .…”

“…A significant divergence of the model from human GAA 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 18,26,27 , 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 28,29 .…”

“…All donor ssODNs were designed with 50-bp homology arms flanking the target locus and synonymous mutations in the PAM and seed region (5 nt upstream of PAM) to prevent further Cas9 activity after successful HDR. were transfected into murine C2C12 myoblast cells (ATCC CRL-1772) using the Neon™ 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, 1650 V; pulse width: 10 ms; pulse number: 3.…”

“…Generation and characterization of Gaa em1935C>A transgenic mice. Given our prior success in generating Gaa em1826dupA KI cell and transgenic mouse lines using a bi-directional, dual overlapping gRNA strategy 15 , an additional gRNA-3 (5′-GGG CGT GCC CCT GGT CGG GG-3′) was introduced (Fig. 2A).…”

“…3A). GAA enzymatic activity was measured with artificial fluorometric 4-MU substrate as described previously 15 . The results were consistent with the other findings from this study, showing that the HET group had close to 50% of the level of enzymatic activity observed in the WT group in each muscle tissue and brain tissue sample tested, indicating that the one WT allele produced functional enzyme, but not the c.1935C>A allele.…”