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

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.

Glycogen storage disease type II, also called Pompe disease (PD; OMIM#232300), is an autosomal recessive disorder resulting from malfunction of lysosomal acid α-glucosidase (GAA; EC 3.2.10.20) caused by mutations in the GAA gene (OMIM#606800). GAA deficiency leads to reduced glycogen degradation and accumulation of intra-lysosomal glycogen with pronounced glycogen storage in cardiac and skeletal muscle. Increased glycogen storage in myocytes, brain, and spinal cord anterior horn neurons results in muscle weakness, which varies in age of onset and severity according to the level of residual GAA enzymatic activity 1 . PD presents as a spectrum of phenotypes, typically classified into infantile-onset form (IOPD) and late-onset form (LOPD) based on the time of disease onset [2][3][4] . Patients with severe IOPD have neonatal onset and a rapidly progressive disease with prominent cardiomyopathy, general muscle weakness and hypotonia, respiratory problems and drastically reduced life expectancy. Patients with LOPD have a more slowly progressive proximal skeletal expression vectors and their respective single-stranded donor oligonucleotides (ssODN) were electroporated into C2C12 mouse myoblasts to assess in vitro on-target editing and HDR efficiency. 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). Following puromycin-resistant selection, we were able to successfully isolate and expand Gaa c.1935C>A KI C2C12 clonal cells electroporated with Gaa c.1935 gRNA-1 and/ or gRNA-2 and their respective donor ssODN (Table 1; Fig. 1A). Sanger sequence results confirmed that the Gaa c.1935C>A KI mutation along with a Gaa c.1920C>T silent protospacer adjacent motif (PAM) mutation were successfully introduced into the clonal line (Fig. 1B).
In comparison to Gaa wt cells, Gaa c.1935C>A KI cells displayed increased PAS staining, indicating the accumulation of glycogen (Fig. 1C). Furthermore, GAA enzymatic activity was almost abolished in Gaa c.1935C>A KI cells relative to Gaa wt cells; less than 2.3% of WT GAA activity was detected in the KI cell line (Fig. 1D). Taken together, these results demonstrate that our Gaa c.1935C>A KI C2C12 cell line exhibits a molecular and biochemical phenotype observed in human PD and can be utilized as an in vitro model for further study.
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). Comparing gRNA-3 with gRNA-1 by in silico analysis, gRNA-3 had higher predicted on-target efficiency (0.5487 [gRNA-3] vs 0.3905 [gRNA-1]) by CRISPick 13 as well as lower predicted off-targets by GT-Scan 14 . We then applied the dual overlap- Table 1. Gaa c.1935 guide RNA on-target activity and HDR efficiency. The target sequence, PAM motifs and donor templates used for testing of Gaa c. 1935 guide RNAs in C2C12 mouse mybloasts are outlined. Gaa c.1935 locus for each gRNA target sequence is highlighted in yellow. Desired Gaa c. 1935 KI mutation (red), silent PAM site (either green or gold, corresponding to gRNA) and gRNA seed region (black) mutations are bolded and underlined in the donor template sequence. Total on-target Cas9 nuclease activity and HDR efficiency for each Gaa c.1935 guide RNA condition is displayed as the average of two independent experiments.   www.nature.com/scientificreports/ generation (Fig. 2E). Subsequently, mice harboring the c.1935C>A Gaa variant were backcrossed 10 generations to the C57BL/6NJ background before KI mice were characterized. As the generation of our KI mice involved CRISPR endonuclease-mediated mutation introduction, we followed the International Committee on Standardized Genetic Nomenclature for Mice 17 and named the KI transgenic mice as Gaa em1935C>A .
Gaa em1935C>A KI mice have severe GAA enzymatic deficiency and glycogen storage in cardiac, skeletal muscle, and brain tissue. The missense Gaa c.1935C>A mutation in exon 14 of the Gaa gene leads to an amino acid substitution; therefore, we did not expect any nonsense-mediated decay in Gaa c.1935C>A mRNA transcripts. The comparative ΔC t between mouse Gaa and housekeeping gene Gapdh acquired by RT-PCR among WT, HET, and KI groups are almost identical, indicating the Gaa c.1935C>A mutation does not affect Gaa mRNA levels (Fig. 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. For comparative purposes, we acquired Gaa homozygous knock-out (KO) (B6;129-Gaa tm1Rabn /J; exon 6 knock-out) 18 mouse tissues from Jackson Laboratory (Bar Harbor, ME). Compared to tissue from WT or HET animals, tissue from KI (Gaa em1935C>A ) and KO (Gaa tm1Rabn /J) animals had significantly decreased GAA enzymatic activity (about 1% of WT levels) (Fig. 3B).
Compared to the unaffected WT or HET groups, KI and KO mice had abnormally elevated lysosomal glycogen storage in heart, diaphragm, and gastrocnemius muscle tissue. Interestingly, increased glycogen storage in The average C t value from WT samples were further utilized to normalize with other groups. No significant difference in Gaa mRNA transcript expression was detected among WT, HET, and KI samples. (B) GAA enzyme activity in heart, diaphragm, and gastrocnemius muscle tissues and brain homogenate from WT (n = 5; black bars), HET (n = 5; striped bars), KI (Gaa em1935C>A ; n = 4; white bars), and KO (Gaa tm1Rabn ; n = 3; grey bars) mice was measured using a fluorometric 4-MU α-d-glucopyranoside assay and normalized to the amount of sample protein. (C) Glycogen level was measured in the same tissues used for analysis in (B) using a colorimetric assay. KO mice displayed significantly elevated glycogen levels relative to WT and HET mice in all tissues assayed. However, KI mice showed a significant elevation of glycogen levels in muscle tissues, but no significant elevation in brain. The amount of glycogen was normalized to the amount of sample protein. Data were generated from at least three independent experiments and shown as mean ± SD. All comparisons were analyzed using one-way ANOVA with the Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns: not significant. www.nature.com/scientificreports/ whole-brain homogenates was observed in KO mice, but not in KI mice, which had a slight, but not statistically significant, increase in glycogen load (Fig. 3C).
Gaa em1935C>A KI mice show increased muscle glycogen content and elevated LAMP1 marker in brain regions. PAS staining is routinely used to demonstrate abnormal carbohydrate accumulation in muscle tissue 19 . PAS staining was performed in different muscle tissues (heart, diaphragm, and gastrocnemius) from 3-month-old KI mice. Scattered red to magenta PAS staining particles representing the accumulation of glycogen were observed in all three muscle tissue types in the KI mice, but not in WT animals (Fig. 4A). PAS staining with diastase (PAS-D), an enzyme that digests only glycogen, was also applied to consecutive slides to confirm that the particles consisted of glycogen. A decrease in red/magenta signal confirms that excessive accumulation products in tissues comprised only glycogen ( Supplementary Fig. 1). The lysosomal associated membrane protein-1 (LAMP1) is commonly used as a biomarker for lysosomal storage. LAMP1 staining in the brain sections from 3-month-old WT and KI mice were examined in three representative areas of the brain (frontal cortex, hippocampus, and cerebellum), demonstrating markedly increased LAMP1 immunoreactivity in KI neuronal cell bodies compared to WT controls (Fig. 4B).
In summary, histopathology showed that the KI mice display early pathological glycogen accumulation in muscle tissues, which is analogous to muscle pathology in IOPD patients. In addition, the KI mice display a more pronounced lysosomal burden in the brain areas as early as 3-months of age compared to WT animals.
Gaa em1935C>A KI mice have impaired skeletal muscle autophagy. Excessive autophagic buildup is well-documented in PD patients and in PD mice 20,21 and may be a potential mechanism of PD pathogenesis. Microtubule-associated protein light chain 3 (LC3B) is a protein component of autophagosomes, which are quickly degraded under normal physiological conditions and are hardly detectable. Cleavage of LC3B at the carboxy terminus immediately following synthesis yields the cytosolic, non-autophagosome bound LC3B-I form. LC3B-I is converted to autophagosome-bound LC3B-II via conjugation to phosphatidylethanolamine when autophagic processes are activated. Following autophagosome-lysosome fusion, LC3B-II is then hydrolyzed back to LC3B-I via ATG5 22 .
To examine autophagic status of the Gaa em1935C>A KI mice, western blotting for LC3B was performed using tissue homogenate ( Fig. 5A and Supplementary Fig. 2). Both KI and KO models demonstrate elevated synthesis of LC3B-I in gastrocnemius, evidence of upregulated autophagy (Fig. 5B); further, autophagosomal LC3B-II is increased in KI heart, diaphragm, and gastrocnemius but not in brain (Fig. 5C). The ratio of LC3B-II:LC3B-I is increased (Fig. 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 . Gaa em1935C>A KI mice display left ventricular cardiac hypertrophy at 3 months of age. Neonatal-onset hypertrophic cardiomyopathy is a common clinical presentation in patients with IOPD. To explore the anatomical features and physiological function of hearts in the KI mice, echocardiography was performed on 3-month-old mice. M-mode images obtained by echocardiography were used to measure multiple parameters including wall thickness, internal diameter, and heart rate. Many additional functional parameters can be derived from these measurements to determine temporal left ventricular (LV) wall motion as an index for LV contractile patterns and chamber size (Fig. 6A).
Increases in interventricular septal diameter (IVSd), LV posterior wall diameter (LVPWd), and LV mass index (LVMI) were observed in KI mice, compared to WT and/or HET mice (Fig. 6B), indicating pronounced hypertrophic cardiomyopathy. Measurements of myocardial contraction showed a slight decrease in LV systolic internal www.nature.com/scientificreports/ diameter (LVIDs) in the KI mouse, but no significant difference in LV diastolic internal diameter (LVIDd) was observed among WT, HET, and KI mice (Fig. 6C). Increased fractional shortening indicative of cardiac contractile dysfunction was observed in KI mice (Fig. 6D). Echocardiographic data therefore indicates early hypertrophic cardiomyopathy phenotypes in 3-month-old Gaa em1935C>A KI mice. The data presented in Fig. 5 show no gender differences in these parameters ( Supplementary Fig. 3).
Reduced forelimb grip strength in Gaa em1935C>A KI mice. The forelimb grip strength test is commonly used to evaluate neuromuscular dysfunction in mice by measuring the deterioration of skeletal muscle. Peak tension force was recorded as the mice lost their grip on the force transducer bar and normalized to bodyweight for analysis by gender group. First, mouse body weight is known to differ between genders at 3 months of age 23 . The mean ± SD body weights of male and female mice in our study cohort were 28.73 ± 3.13 g and 21.76 ± 2.36 g, respectively. In each gender cohort, there was no significant difference in body weight across WT, HET, and KI mice (Fig. 7). In addition, at 3 months of age, the male Gaa em1935C>A KI mouse showed a significant reduction (~ 19%) in normalized peak tension force compared to WT mice, indicating decreased forelimb muscle strength in KI mice (Fig. 7). This reduction was observed only in male KI mice, but not in female KI mice.

Discussion
In populations of Southern Han ancestry, the GAA c.1935C>A (p.Asp645Glu) mutation represents 36%-80% of mutations 11,12,24 in IOPD patients. We have successfully applied CRISPR/Cas9 genome editing to install the Gaa c.1935C>A mutation in a mouse myoblast C2C12 cell line and create a novel Gaa em1935C>A KI mouse model; each of which represents a valuable resource for studying IOPD. The KI C2C12 line demonstrates severe GAA enzyme WT. Impaired autolysosomal formation (increased LC3B-II/LC3B-I ratio) is observed in KI skeletal muscle tissues. The ratio of LC3B-II and LC3B-I protein intensity was quantified by densitometric analysis of the western blots, and the ratio was further normalized with WT in each tissue assayed. Data were generated from at least three independent western blots and values are shown as mean ± SD. All comparisons were analyzed using one-way ANOVA with the Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. www.nature.com/scientificreports/ deficiency and glycogen accumulation; the KI mouse model successfully recapitulates molecular, biochemical, histologic, and phenotypic aspects of human IOPD. While no phenotypic differences were noted between GAA c.1935C>A HET and WT mice aside from the expected 50% reduction in HET GAA enzymatic activity, the homozygous KI mice demonstrated a significant, PD-like phenotype. KI mice had normal Gaa mRNA levels with significantly reduced level of GAA hydrolysis activity (about 1% of WT) in heart and skeletal muscle, as well as brain tissue. This aligns with observed levels of low GAA enzyme activity (0.08-0.82% of normal range for control) previously measured in homozygous GAA c.1935C>A patient fibroblasts 25 . Significant increases in glycogen storage were observed in KI mouse muscle tissues, consistent with the human GAA c.1935C>A IOPD phenotype. In addition, increased lysosomal burden, as indicated by LAMP1 immunostaining, was demonstrated in brain tissue from Gaa em1935C>A KI mice. Autophagic impairment was noted in skeletal muscle tissues, consistent with what is observed in human PD and other murine PD models 18 . Gaa em1935C>A mice developed hypertrophic cardiomyopathy at approximately two months of age, which becomes quite marked at three months of age. This muscle weakness phenotype may be due to a combination of sequelae from cardiomyopathy, impairment of lysosomal-autophagosomal fusion into autolysosomes, and catabolism of myofibril contractile proteins 22 . Studies are ongoing to assess the life span, natural history, and phenotypic progression of the model. 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 . Only one Gaa KO model on a DBA/2J background (homozygous Ltbp4 Δ36 alleles) is reported to have a shorter lifespan (but still not neonatal lethality) in male mice, compared to male Gaa KO mice on the C57BL/6;129 background 27 . The DBA/2J genetic background may exacerbate the severity of respiratory muscle weakness caused by Gaa KO deletion, leading to earlier death than is observed in other KO models 27 . striped bars), and KI (Gaa .em1935C>A ; n = 10; white bars) mice were obtained from 3-month-old mice. Data are shown as mean ± SD. Heart rate (HR) was maintained greater than 500 bpm throughout measurements. All comparisons were analyzed using one-way ANOVA with the Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns: not significant. www.nature.com/scientificreports/ Genome editing represents a new approach to the treatment of PD, compared to traditional treatments like ERT or gene therapy. A mouse that both recapitulates clinical features of human disease and harbors orthologous pathologic gene variants serves as a valuable system for the development of innovative therapies and, most importantly, studies enabling eventual clinical trials in humans. As this model undergoes in-depth validation and studies of its clinical and immune response to standard intravenous rhGAA enzyme infusions, subsequent avenues for exploration include variant rhGAA enzyme infusions, gene therapy, and CRISPR-based genomic editing. The latter approach can be performed using CRISPR "prime editing", which is capable of targeting more than 90% of known pathogenic mutations, including the c.1935C>A transversion 30 . In addition, multiple tissues can be obtained or derived from our Gaa em1935C>A KI mouse to investigate the potential tissue-specific efficacy of genome correction-based therapeutics in vitro, before in vivo studies are attempted. With these advances, and high sequence conservation surrounding the mutation, the Gaa em1935C>A KI mouse represents an ideal candidate for the development of personalized therapeutics like prime editing that correct pathogenic variants, restore GAA enzyme activity and further improve functional phenotypes before translational application in the clinic.  Male KI mice demonstrate decreased normalized peak tension force consistent with skeletal muscle weakness. Forelimb peak tension force was measured using a grip strength meter and taken as the average of 9 trials over 3 days. Data are shown as mean ± SD. All comparisons were analyzed using one-way ANOVA with the Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant. 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. Forty-eight hours post-transfection, cellular genomic DNA was obtained for Sanger sequencing around the Gaa c.1935 target locus. SpCas9 nuclease activity and HDR KI efficiency were determined by Tracking of Indels by Decomposition (TIDE) 31 or Tracking of Insertion, Deletions, and Recombination events (TIDER) 32 analysis of DNA sequence electropherogram files.

Materials and methods
Generation of a Gaa c.1935C>A KI C2C12 cell line. Similar parameters to those described above were applied to transfect 4.5 μg of gRNA-1, gRNA-2 under the U6 promoter in the pX459 expression vector and 450 nM ssODN into 3 × 10 5 C2C12 myoblast cells. pCMV6-AC-GFP (OriGene) was used as a marker for transfection; electroporated cells were selected for successful pX459 transfection by adding 2.5 μg/mL puromycin dihydrochloride (Sigma-Aldrich) to the culture medium beginning 24 h after electroporation. Puromycin was supplied every 48 h until all pCMV6-AC-GFP-transfected cells were no longer viable. After puromycin-resistance screening, single cell clones were selected by standard serial dilution methods in 96-well plates in the presence of 2.5 μg/mL puromycin dihydrochloride. Sanger sequencing was used to confirm the genotype of each single cell clone.
Generation of Gaa em1935C>A KI mice. The generation of Gaa em1935C>A KI mice was performed at the University of California-Irvine Transgenic Mouse Core, and all study procedures were reviewed and approved under IACUC protocol #AUP16-63. Standard methods were applied to produce pronuclear stage C57BL/6NJ embryos 16 . In brief, 3 μM crRNA/tracrRNA/3xNLS-Cas9 protein and 10 ng/μL ssODN were injected into pronuclear stage C57BL/6NJ embryos ( Table 2). Surviving embryos were implanted into oviducts of 0.5dpc ICR pseudo-pregnant females.
Whole-genome sequencing and analysis. Whole genome sequencing (WGS) and analyses were performed on tail samples from G 0 wild type (Gaa wt ), G 0 founder #1 (Gaa c.1935Founder#1 ), and G 0 founder #2 (Gaa c.1935Founder#2 ) mice. In brief, WGS was performed and analyzed on an Illumina HiSeq X Ten Sequencer at 40-50× read depth (Fulgent Genetics) using TrueSeq DNA libraries created from 1 μg fragmented genomic DNA. WGS on-target and off-target analyses were performed on the OnRamp BioInformatics platform. Data were aligned to the Mouse genome (mm10) using BWA 33 . PCR artifacts were identified with the memtest utility from Sentieon 34 , and filtered out using samtools 35 . Alignments were de-duplicated and realigned around insertions and deletions using LocusCollector, Dedup, and Realigner from Sentieon. SNV calling was performed with GVCFtyper from Sentieon, using the mouse dbSNP 142 data (http:// hgdow nload. cse. ucsc. edu/ golde npath/ mm10/ datab ase/ snp142. txt. gz) as the known SNPs. Known SNPs and variants falling in un-located chromosomes were removed from analysis. For off-target analysis, we used SNVs that had a C>A transversion and any one of the four following criteria indicating an ectopic HDR event: a de novo N → A mutation 3 bases upstream, N→A mutation 6 bases upstream, N→C mutation 12 bases upstream, or N → T mutation 15 bases upstream. This search step was repeated for the reverse complement sequences. The fully processed BAM files (after Realigner) were used as input to the Manta structural variant caller 36 . For each of the non-wild-type (WT) samples, Manta somatic caller was applied with the C57BL6-WT sample as "normal" and the sample of interest as "tumor, " thereby subtracting the background structural variants in C57BL6-WT compared to mm10. Vcf (https:// vcfto ols. github. io) was used to annotate the output VCF files from Manta. Experimental animals. The G 2 mice were backcrossed 10 generations onto a C57BL/6NJ background before any characterization was performed. Mice received ad libitum Teklad Global 16% Protein Rodent Diet (Envigo, Indianapolis, IN) and water in temperature-controlled environment. Animal were housed in groups of 4 mice/cage, separated by gender except for mating trios, and provided with 14-h light and 10-h dark cycle. The use and care of animals in this study adhered to the guidelines of the NIH Guide for the Care and Use of Laboratory Animals, as utilized by the CHOC Children's Institutional Animal Care and Use Committee under CHOC IACUC protocol #160,902. In addition, all experiments in this study were carried out in compliance with ARRIVE guidelines (https:// arriv eguid elines. org), and all methods were performed in accordance with relevant guidelines and regulations.
Quantitative real-time PCR. Total RNA was extracted from tail tip or liver homogenate using a Directzol RNA miniprep kit (Zymo Research) and reverse-transcribed using an iScript™ cDNA Synthesis Kit (Bio-Rad). As per the manufacturer's instructions, both oligo(dT) and random hexamer primers were used to synthesize cDNA. The resulting cDNA was diluted tenfold, and a 2-μl aliquot was used in a 12-μl PCR reaction with SsoAdvanced Universal Probes Supermix (Bio-Rad) and specific TaqMan primer/probe assays for Gaa (Taqman www.nature.com/scientificreports/ assay #Mm00484581_m1) and Gapdh (TaqMan assay #Mm99999915_g1). PCR reactions were run in triplicate and quantified with Bio-Rad CFX96 Touch Real-Time PCR Detection. Gapdh was used as an internal reference gene, and relative quantification of Gaa gene expression was calculated using the comparative ΔC t method for the difference in C t values of Gaa and Gapdh in the given sample. ΔC t values were further normalized with the average of the C t value of wildtype samples.

Biochemical analyses.
For the GAA activity assay, phosphate-buffered saline (PBS)-flushed mouse tissues or C2C12 myoblast cell pellets were homogenized in CelLytic M cell lysis reagent (MilliporeSigma). Acidic α-glucosidase enzyme activity was assessed as previously described with minor modifications 15,37 . In brief, 10 µL tissue homogenate was mixed with 10 µL of 6 mM 4-methylumbelliferyl-α-d-glucopyranoside substrate (Mil-liporeSigma) in McIlvaine citrate/phosphate buffer (pH 4.3) and quenched with 180 µL glycine carbonate buffer (pH 10.5) after 1-h incubation at 37 °C in a 96-well plate. GAA activity reactions were run in triplicate, and fluorescence measurements were obtained using an Infinite M Plex spectrofluorophotometer (Tecan) at excitation and emission wavelengths of 360 nm and 450 nm, respectively. One GAA enzymatic activity unit was defined as 1 nmol converted substrate per hour. Protein concentration was estimated using a Pierce BCA assay kit (ThermoFisher), using bovine serum albumin as a standard. Specific activity was calculated as units of GAA enzymatic activity per mg of protein.
Tissue glycogen levels were measured using a glycogen assay kit (Sigma-Aldrich) according to the manufacturer's instructions. In brief, 10 µL tissue homogenate was incubated with hydrolysis enzyme reaction mixture in a final volume of 50 µL at room temperature for 30 min before adding 50 µL development enzyme reaction mixture for 30 min incubation at room temperature. Absorbance at 570 nm was measured using an Infinite M Plex spectrofluorophotometer (Tecan). A standard curve was generated using standard glycogen solution provided in the assay kit. Glycogen quantification assays were performed in duplicate, and an extra reaction without hydrolytic enzyme treatment was used for background correction of endogenous glucose levels in each sample. Tissue glycogen level is expressed as µg of glycogen per mg of protein.
Tissue harvesting, processing, and histological staining. Three-month-old mice were euthanized using CO 2 asphyxiation and transcardially perfused with PBS. Brains were dissected sagittally along the midline; left hemispheres were rapidly frozen and stored at − 80 °C for biochemical analysis, and right hemispheres were post-fixed at 4 °C in zinc formalin. Heart, diaphragm, and gastrocnemius muscle were also harvested. Half of the tissue samples for biochemical studies were rapidly frozen and the other half of tissues were post-fixed at 4 °C in zinc formalin.
Samples for histological staining were processed and embedded in paraffin blocks for sectioning at 4-μm thickness, and periodic acid-Schiff (PAS) staining (Sigma-Aldrich) was performed according to the manufacturer's instructions. EVOS M5000 imaging system (Invitrogen) was used to capture representative images at 20× objective magnification on RGB-mode illumination.
LAMP1 immunohistochemistry staining in paraffin-embedded brain sections from study animals were performed with anti-LAMP1 polyclonal antibody (Cat#24170, Abcam, Waltham, MA) using an ImmPACT DAB Substrate Kit with Peroxidase (Vector Laboratories, Burlingame, CA) following manufacturer's instructions. Paraffin sections were deparaffinized and endogenous peroxidase activity was quenched by immersion in 1.5% hydrogen peroxide followed by heat-induced epitope retrieval in sodium citrate buffer. The sections were subsequently incubated overnight at 4 °C with mouse anti-LAMP1 antibody (1:50) following secondary antibody amplification before visualizing with diaminobenzidine (DAB) as chromogen. Represented images were captured by Keyence BZ-X800 microscopes (Keyence American, Itasca, IL) at 20× objective magnification with the same parameters of exposure.
LC3B western blot analysis. Frozen mouse tissues were homogenized in CelLytic M cell lysis reagent (MilliporeSigma) and cOmplete protease inhibitors (Roche) was added to prevent protein degradation. Total protein concentration of the supernatants from centrifuged tissue lysates was determined by BCA protein assay (Pierce). Eight micrograms of total protein lysate were resolved on 4-15% Mini-PROTEAN TGX Stain-free gels (Bio-Rad) and transferred onto Immuno-Blot PVDF membranes (Bio-Rad). Membrane blots were blocked with EveryBlot blocking buffer (Bio-Rad) and probed with an anti-LC3B primary antibody (cat# L7543, Sigma) followed by an HRP-conjugated secondary antibody before applying ECL HRP substrate (Bio-Rad) for chemiluminescence. Stain-free gels and blots were imaged using the stain-free and chemiluminescence settings on the ChemiDoc™ MP imaging system (Bio-Rad). LC3B-I and LC3B-II protein levels were measured by densitometric analysis of western blots using Fuji software (ImageJ version 2.0) 38 . Signals were normalized to the amount of total protein as determined by densitometric analysis of stain-free gels. LC3B-II/LC3B-I ratio was normalized to WT for each organ.
Murine echocardiography. Transthoracic echocardiography (M-mode and 2-dimensional echocardiography) was performed using a Vevo 2100 high-resolution ultrasound system, with a linear transducer of 32-55 MHz (VisualSonics Inc.). Chest fur was removed by using depilatory cream one day prior to the procedure. Mice were kept warm on a heated platform (37 °C) and anesthetized with 5% isoflurane delivered via nose cone for 15 s, then maintained at 0.5% throughout the echocardiography examination. Small needle electrodes for simultaneous electrocardiography were inserted into one upper and one lower limb. Measurements of chamber dimensions and wall thickness were performed while heartbeats of the mice were greater than 500 beats per minute (bpm). Percentage fractional shortening (%FS) was used as an indicator of left ventricular systolic cardiac function and calculated as follows: %FS = (LVIDd -LVIDs)/LVIDd * 100. www.nature.com/scientificreports/ Forelimb grip strength assay. Forelimb grip strength was measured as previously described 39 . Following acclimatization (at least one hour prior to grip strength measurement), each mouse was weighed and placed on a forelimb pull bar attached to an isometric force transducer (Columbus Instruments, Columbus, OH, USA). The mouse was pulled away from the bar by its tail, and the force required was recorded by the force transducer. Over 3 consecutive days, each mouse performed 3 pulls per day for a total of 9 pulls per test session. Peak tension force (N) was calculated as the average of each subject's 9 pulls over the test session and normalized by body weight.
Statistical analysis. All graphs and statistical comparisons were generated using GraphPad Prism 9. Statistical analyses were performed using the two-tailed unpaired t-test or one-way ANOVA followed by Tukey's HSD test. All data are presented in this study as mean ± standard deviation (SD).

Data availability
The data that support the findings of this study are available from the corresponding author, RYW, upon reasonable request. WGS FASTA sequences were uploaded to the National Institutes of Health National Library of Medicine submission portal.