RT Journal Article
SR Electronic
T1 CRISPR-Cas9 generated Pompe knock-in murine model exhibits early-onset cardiomyopathy and impaired skeletal muscle function
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 780320
DO 10.1101/780320
A1 Jeffrey Y. Huang
A1 Shih-Hsin Kan
A1 Anthony D. Rangel
A1 Yunghang Chan
A1 Jeremy Davis-Turak
A1 Nancy D. Dalton
A1 Jon Neumann
A1 Raymond Y. Wang
YR 2019
UL http://biorxiv.org/content/early/2019/09/24/780320.abstract
AB 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 Gaac1826insA mouse model using a dual sgRNA with ssODN donor template approach. Whole genome sequencing and analysis of the Gaac1826insA murine model establishes that our system is highly specific for the Gaac1826 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. Gaac1826insA mice display significantly reduced levels of GAA expression and enzymatic activity relative to wild-type mice. We performed echocardiography on Gaac1826insA mice to assess cardiac structure and function. Gaac1826insA 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 tests – grip strength, inverted screen, gait analysis – on Gaac1826insA mice every 3 months to assess overall motor performance. Gaac1826insA mice display impaired motor strength and coordination relative to wild-type mice. Altogether, our results demonstrate that the Gaac1826insA murine model recapitulates human infantile-onset Pompe disease and is better suited for evaluation of therapeutic strategies such as genome correction.