Structural and functional comparisons of salivary α-glucosidases from the mosquito vectors Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus.

Mosquito vectors of medical importance both blood and sugar feed, and their saliva contains bioactive molecules that aid in both processes. Although it has been shown that the salivary glands of several mosquito species exhibit α-glucosidase activities, the specific enzymes responsible for sugar digestion remain understudied. We therefore expressed and purified three recombinant salivary α-glucosidases from the mosquito vectors Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus and compared their functions and structures. We found that all three enzymes were expressed in the salivary glands of their respective vectors and were secreted into the saliva. The proteins, as well as mosquito salivary gland extracts, exhibited α-glucosidase activity, and the recombinant enzymes displayed preference for sucrose compared to p-nitrophenyl-α-D-glucopyranoside. Finally, we solved the crystal structure of the Ae. aegypti α-glucosidase bound to two calcium ions at a 2.3 Ångstrom resolution. Molecular docking suggested that the Ae. aegypti α-glucosidase preferred di- or polysaccharides compared to monosaccharides, consistent with enzymatic activity assays. Comparing structural models between the three species revealed a high degree of similarity, suggesting similar functional properties. We conclude that the α-glucosidases studied herein are important enzymes for sugar digestion in three mosquito species.

Infantile-onset pompe disease: a case report emphasizing the role of genetic counseling and prenatal testing.

BACKGROUND: Pompe disease, classified as glycogen storage disease type II, arises from a deficiency in the acid alpha-glucosidase (GAA) enzyme, leading to glycogen accumulation in multiple tissues. The unique correlation between genotype and enzyme activity is a key feature. This case highlights an infantile-onset form, emphasizing genetic counseling and prenatal testing importance. CASE PRESENTATION: An 18-week-old infant with respiratory distress, cyanosis, and fever was admitted. Born healthy, her sibling died from Pompe disease. She presented with cardiomegaly, hypotonia, and absent reflexes. Diagnosis was confirmed by significantly reduced GAA activity. Despite treatment initiation, the patient succumbed to cardiac arrest. CONCLUSIONS: The case underscores genetic counseling's role, offering insights into prenatal testing advancements, antenatal diagnosis through echocardiography, and the significance of early intervention, particularly in infantile-onset Pompe disease. SYNOPSIS: Genetic risk assessment and prenatal testing are crucial for families with a history of Pompe disease to improve early diagnosis and management outcomes.

Muscle-specific, liver-detargeted adeno-associated virus gene therapy rescues Pompe phenotype in adult and neonate Gaa-/- mice.

Pompe disease (PD) is a neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency. Reduced GAA activity leads to pathological glycogen accumulation in cardiac and skeletal muscles responsible for severe heart impairment, respiratory defects, and muscle weakness. Enzyme replacement therapy with recombinant human GAA (rhGAA) is the standard-of-care treatment for PD, however, its efficacy is limited due to poor uptake in muscle and the development of an immune response. Multiple clinical trials are ongoing in PD with adeno-associated virus (AAV) vectors based on liver- and muscle-targeting. Current gene therapy approaches are limited by liver proliferation, poor muscle targeting, and the potential immune response to the hGAA transgene. To generate a treatment tailored to infantile-onset PD, we took advantage of a novel AAV capsid able to increase skeletal muscle targeting compared to AAV9 while reducing liver overload. When combined with a liver-muscle tandem promoter (LiMP), and despite the extensive liver-detargeting, this vector had a limited immune response to the hGAA transgene. This combination of capsid and promoter with improved muscle expression and specificity allowed for glycogen clearance in cardiac and skeletal muscles of Gaa-/- adult mice. In neonate Gaa-/- , complete rescue of glycogen content and muscle strength was observed 6 months after AAV vector injection. Our work highlights the importance of residual liver expression to control the immune response toward a potentially immunogenic transgene expressed in muscle. In conclusion, the demonstration of the efficacy of a muscle-specific AAV capsid-promoter combination for the full rescue of PD manifestation in both neonate and adult Gaa-/- provides a potential therapeutic avenue for the infantile-onset form of this devastating disease.

Description of clinical and genetic features of 122 patients included in the Spanish Pompe registry.

Pompe disease is a rare genetic disorder with an estimated prevalence of 1:60.000. The two main phenotypes are Infantile Onset Pompe Disease (IOPD) and Late Onset Pompe Disease (LOPD). There is no published data from Spain regarding the existing number of cases, regional distribution, clinical features or, access and response to the treatment. We created a registry to collect all these data from patients with Pompe in Spain. Here, we report the data of the 122 patients registered including nine IOPD and 113 LOPD patients. There was a high variability in how the diagnosis was obtained and how the follow-up was performed among different centres. Seven IOPD patients were still alive being all treated with enzymatic replacement therapy (ERT) at last visit. Ninety four of the 113 LOPD patients had muscle weakness of which 81 were receiving ERT. We observed a progressive decline in the results of muscle function tests during follow-up. Overall, the Spanish Pompe Registry is a valuable resource for understanding the demographics, patient's journey and clinical characteristics of patients in Spain. Our data supports the development of agreed guidelines to ensure that the care provided to the patients is standardized across the country.

A Comprehensive Update on Late-Onset Pompe Disease.

Pompe disease (PD) is an autosomal recessive disorder caused by mutations in the GAA gene that lead to a deficiency in the acid alpha-glucosidase enzyme. Two clinical presentations are usually considered, named infantile-onset Pompe disease (IOPD) and late-onset Pompe disease (LOPD), which differ in age of onset, organ involvement, and severity of disease. Assessment of acid alpha-glucosidase activity on a dried blood spot is the first-line screening test, which needs to be confirmed by genetic analysis in case of suspected deficiency. LOPD is a multi-system disease, thus requiring a multidisciplinary approach for efficacious management. Enzyme replacement therapy (ERT), which was introduced over 15 years ago, changes the natural progression of the disease. However, it has limitations, including a reduction in efficacy over time and heterogeneous therapeutic responses among patients. Novel therapeutic approaches, such as gene therapy, are currently under study. We provide a comprehensive review of diagnostic advances in LOPD and a critical discussion about the advantages and limitations of current and future treatments.

Genotype, phenotype and treatment outcomes of 17 Malaysian patients with infantile-onset Pompe disease and the identification of 3 novel GAA variants.

BACKGROUND: Pompe disease is a rare glycogen storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA), leading to glycogen deposition in multiple tissues. Infantile-onset Pompe disease (IOPD) patients present within the first year of life with profound hypotonia and hypertrophic cardiomyopathy. Treatment with enzyme replacement therapy (ERT) has significantly improved survival for this otherwise lethal disorder. This study aims to describe the clinical and molecular spectrum of Malaysian IOPD patients, and to analyze their long term treatment outcomes. METHODS: Seventeen patients diagnosed with IOPD between 2000 and 2020 were included in this retrospective cohort study. Clinical and biochemical data were collated and analyzed using descriptive statistics. GAA enzyme levels were performed on dried blood spots. Molecular analysis of the GAA gene was performed by polymerase chain reaction and Sanger sequencing. Structural modelling was used to predict the effect of the novel mutations on enzyme structure. RESULTS: Our cohort had a median age of presentation of 3 months and median age of diagnosis of 6 months. Presenting features were hypertrophic cardiomyopathy (100%), respiratory insufficiency (94%), hypotonia (88%), failure to thrive (82%), feeding difficulties (76%), and hepatomegaly (76%). Fourteen different mutations in the GAA gene were identified, with three novel mutations, c.1552-14_1552-1del, exons 2-3 deletion and exons 6-10 deletion. The most common mutation identified was c.1935C > A p.(D645E), with an allele frequency of 33%. Sixteen patients received ERT at the median age of 7 months. Overall survival was 29%. Mean age of death was 17.5 months. Our longest surviving patient has atypical IOPD and is currently 20 years old. CONCLUSIONS: This is the first study to analyze the genotype and phenotype of Malaysian IOPD patients, and has identified the c.1935C > A p.(D645E) as the most common mutation. The three novel mutations reported in this study expands the mutation spectrum for IOPD. Our low survival rate underscores the importance of early diagnosis and treatment in achieving better treatment outcomes.

Effects of enzyme replacement therapy on bone density in late onset Pompe disease.

Pompe disease is an autosomal recessive disorder caused by a deficiency of α-glucosidase, resulting in the accumulation of glycogen in smooth, cardiac, and skeletal muscles, leading to skeletal muscle dysfunction, proximal muscle weakness, and early respiratory insufficiency. Although many patients exhibit decreased bone mineral density (BMD) and increased fractures, there is currently no official protocol for surveillance and management of osteoporosis and osteopenia in late onset Pompe disease (LOPD). Enzyme replacement therapy (ERT) has therapeutic effects on muscle function; however, very few studies report on the effect of ERT on bone mineralization in LOPD patients. Our study included 15 Pompe patients from 25 to 76 years of age on ERT for variable durations. Progressive impact of ERT on BMD of the hips and spine, and the frequency of osteopenia or osteoporosis was studied using DEXA scanning, and correlations were made with age of initiation of ERT, duration of ERT and six-minute walk test. We found a significant positive correlation between the age of ERT initiation and age of the subject, with increases in the Z-scores for the femur and lumbar region. Females had a significantly higher risk for developing osteoporosis compared to males. These results highlight the significance of ERT on reducing progression of osteoporosis in LOPD patients.

AAV-mediated delivery of secreted acid α-glucosidase with enhanced uptake corrects neuromuscular pathology in Pompe mice.

Gene therapy is under advanced clinical development for several lysosomal storage disorders. Pompe disease, a debilitating neuromuscular illness affecting infants, children, and adults with different severity, is caused by a deficiency of lysosomal glycogen-degrading enzyme acid α-glucosidase (GAA). Here, we demonstrated that adeno-associated virus-mediated (AAV-mediated) systemic gene transfer reversed glycogen storage in all key therapeutic targets - skeletal and cardiac muscles, the diaphragm, and the central nervous system - in both young and severely affected old Gaa-knockout mice. Furthermore, the therapy reversed secondary cellular abnormalities in skeletal muscle, such as those in autophagy and mTORC1/AMPK signaling. We used an AAV9 vector encoding a chimeric human GAA protein with enhanced uptake and secretion to facilitate efficient spread of the expressed protein among multiple target tissues. These results lay the groundwork for a future clinical development strategy in Pompe disease.

Insight into broad substrate specificity and synergistic contribution of a fungal α-glucosidase in Chinese Nong-flavor daqu.

BACKGROUND: Chinese Nong-favor daqu, the presentative liquor starter of Baijiu, has been enriched with huge amounts of enzymes in degrading various biological macromolecules by openly man-made process for thousand years. According to previous metatranscriptomics analysis, plenty of α-glucosidases were identified to be active in NF daqu and played the key role in degrading starch under solid-state fermentation. However, none of α-glucosidases was characterized from NF daqu, and their actual functions in NF daqu were still unknown. RESULTS: An α-glucosidase (NFAg31A, GH31-1 subfamily), the second highest expressed α-glucosidases in starch degradation of NF daqu, was directly obtained by heterologous expression in Escherichia coli BL21 (DE3). NFAg31A exhibited the highest sequence identities of 65.8% with α-glucosidase II from Chaetomium thermophilum, indicating its origin of fungal species, and it showed some similar features with homologous α-glucosidase IIs, i.e., optimal activity at pH ~ 7.0 and litter higher temperature of 45 ℃, well stability at 41.3 ℃ and a broad pH range of pH 6.0 to pH 10.0, and preference on hydrolyzing Glc-α1,3-Glc. Besides this preference, NFAg31A showed comparable activities on Glc-α1,2-Glc and Glc-α1,4-Glc, and low activity on Glc-α1,6-Glc, indicating its broad specificities on α-glycosidic substrates. Additionally, its activity was not stimulated by any of those detected metal ions and chemicals, and could be largely inhibited by glucose under solid-state fermentation. Most importantly, it exhibited competent and synergistic effects with two characterized α-amylases of NF daqu on hydrolyzing starch, i.e., all of them could efficiently degrade starch and malto-saccharides, two α-amylases showed advantage in degrading starch and long-chain malto-saccharides, and NFAg31A played the competent role with α-amylases in degrading short-chain malto-saccharides and the irreplaceable contribution in hydrolyzing maltose into glucose, thus alleviating the product inhibitions of α-amylases. CONCLUSIONS: This study provides not only a suitable α-glucosidase in strengthening the quality of daqu, but also an efficient way to reveal roles of the complicated enzyme system in traditional solid-state fermentation. This study would further stimulate more enzyme mining from NF daqu, and promote their actual applications in solid-state fermentation of NF liquor brewing, as well as in other solid-state fermentation of starchy industry in the future.

Structural and functional analyses of Pcal_0917, an α-glucosidase from hyperthermophilic archaeon Pyrobaculum calidifontis.

Genome analysis of Pyrobaculum calidifontis revealed the presence of α-glucosidase (Pcal_0917) gene. Structural analysis affirmed the presence of signature sequences of Type II α-glucosidases in Pcal_0917. We have heterologously expressed the gene and produced recombinant Pcal_0917 in Escherichia coli. Biochemical characteristics of the recombinant enzyme resembled to that of Type I α-glucosidases, instead of Type II. Recombinant Pcal_0917 existed in a tetrameric form in solution and displayed highest activity at 95 °C and pH 6.0, independent of any metal ions. A short heat-treatment at 90 °C resulted in a 35 % increase in enzyme activity. A slight structural shift was observed by CD spectrometry at this temperature. Half-life of the enzyme was >7 h at 90 °C. Pcal_0917 exhibited apparent Vmax values of 1190 ± 5 and 3.9 ± 0.1 U/mg against p-nitrophenyl α-D-glucopyranoside and maltose, respectively. To the best of our knowledge, Pcal_0917 displayed the highest ever reported p-nitrophenyl α-D-glucopyranosidase activity among the characterized counterparts. Moreover, Pcal_0917 displayed transglycosylation activity in addition to α-glucosidase activity. Furthermore, in combination with α-amylase, Pcal_0917 was capable of producing glucose syrup from starch with >40 % glucose content. These properties make Pcal_0917 a potential candidate for starch hydrolyzing industry.

GAA deficiency disrupts distal airway cells in Pompe disease.

Pompe disease is an autosomal recessive glycogen storage disease caused by mutations in the gene that encodes acid alpha-glucosidase (GAA)-an enzyme responsible for hydrolyzing lysosomal glycogen. GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption. Glycogen accumulation in skeletal muscles, motor neurons, and airway smooth muscle cells is known to contribute to respiratory insufficiency in Pompe disease. However, the impact of GAA deficiency on the distal alveolar type 1 and type 2 cells (AT1 and AT2) has not been evaluated. AT1 cells rely on lysosomes for cellular homeostasis so that they can maintain a thin barrier for gas exchange, whereas AT2 cells depend on lysosome-like structures (lamellar bodies) for surfactant production. Using a mouse model of Pompe disease, the Gaa-/- mouse, we investigated the consequences of GAA deficiency on AT1 and AT2 cells using histology, pulmonary function and mechanics, and transcriptional analysis. Histological analysis revealed increased accumulation of lysosomal-associated membrane protein 1 (LAMP1) in the Gaa-/- mice lungs. Furthermore, ultrastructural examination showed extensive intracytoplasmic vacuoles enlargement and lamellar body engorgement. Respiratory dysfunction was confirmed using whole body plethysmography and forced oscillometry. Finally, transcriptomic analysis demonstrated dysregulation of surfactant proteins in AT2 cells, specifically reduced levels of surfactant protein D in the Gaa-/- mice. We conclude that GAA enzyme deficiency leads to glycogen accumulation in the distal airway cells that disrupts surfactant homeostasis and contributes to respiratory impairments in Pompe disease.NEW & NOTEWORTHY This research highlights the impact of Pompe disease on distal airway cells. Prior to this work, respiratory insufficiency in Pompe disease was classically attributed to pathology in respiratory muscles and motor neurons. Using the Pompe mouse model, we note significant pathology in alveolar type 1 and 2 cells with reductions in surfactant protein D and disrupted surfactant homeostasis. These novel findings highlight the potential contributions of alveolar pathology to respiratory insufficiency in Pompe disease.

Increased yield of 2-O-α-d-glucopyranosyl-l-ascorbic acid synthesis by α-glucosidase using rational design that regulating the ground state of enzyme and substrate complex.

BACKGROUND: α-Glucosidase (AG) is a bifunctional enzyme, it has a capacity to synthesize 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) from l-ascorbic acid (L-AA) and low-cost maltose under mild conditions, but it can also hydrolyze AA-2G, which leads to low synthesis efficiency of AA-2G. MAIN METHODS AND MAJOR RESULTS: This study introduces a rational molecular design strategy to regulate enzymatic reactions based on inhibiting the formation of ground state of enzyme-substrate complex. Y215 was analyzed as the key amino acid site affecting the affinity of AG to AA-2G and L-AA. For the purpose of reducing the hydrolysis efficiency of AA-2G, the mutant Y215W was obtained by analyzing the molecular docking binding energy and hydrogen bond formation between AG and the substrates. Compared with the wild-type, isothermal titration calorimetry (ITC) results showed that the equilibrium dissociation constant (KD ) of the mutant for AA-2G was doubled; the Michaelis constant (Km ) for AA-2G was reduced by 1.15 times; and the yield of synthetic AA-2G was increased by 39%. CONCLUSIONS AND IMPLICATIONS: Our work also provides a new reference strategy for the molecular modification of multifunctional enzymes and other enzymes in cascade reactions system.

Generation of two induced pluripotent stem cell lines (CHOCi002-A and CHOCi003-A) from Pompe disease patients with compound heterozygous mutations in the GAA gene.

Pompe disease is an autosomal recessive lysosomal storage disease caused by pathogenic variants in GAA, which encodes an enzyme integral to glycogen catabolism, acid α-glucosidase. Disease-relevant cell lines are necessary to evaluate the efficacy of genotype-specific therapies. Dermal fibroblasts from two patients presenting clinically with Pompe disease were reprogrammed to induced pluripotent stem cells using the Sendai viral method. One patient is compound heterozygous for the c.258dupC (p.N87QfsX9) frameshift mutation and the c.2227C>T (p.Q743X) nonsense mutation. The other patient harbors the c.-32-13T>G splice variant and the c.1826dupA (p.Y609X) frameshift mutation in compound heterozygosity.

[Analysis of lysosomal enzyme activity and genetic variants in a child with late-onset Pompe disease].

OBJECTIVE: To explore the clinical features, lysosomal enzymatic [acid α-glucosidase (GAA)] activities and genetic variants in a child with late-onset Pompe disease (LOPD). METHODS: Clinical data of a child who had presented at the Genetic Counseling Clinic of West China Second University Hospital in August 2020 was retrospectively analyzed. Blood samples were collected from the patient and her parents for the isolation of leukocytes and lymphocytes as well as DNA extraction. The activity of lysosomal enzyme GAA in leukocytes and lymphocytes was analyzed with or without addition of inhibitor of GAA isozyme. Potential variants in genes associated with neuromuscular disorders were analyzed, in addition with conservation of the variant sites and protein structure. The remaining samples from 20 individuals undergoing peripheral blood lymphocyte chromosomal karyotyping were mixed and used as the normal reference for the enzymatic activities. RESULTS: The child, a 9-year-old female, had featured delayed language and motor development from 2 years and 11 months. Physical examination revealed unstable walking, difficulty in going upstairs and obvious scoliosis. Her serum creatine kinase was significantly increased, along with abnormal electromyography, whilst no abnormality was found by cardiac ultrasound. Genetic testing revealed that she has harbored compound heterozygous variants of the GAA gene, namely c.1996dupG (p.A666Gfs*71) (maternal) and c.701C>T (p.T234M) (paternal). Based on the guidelines from the American College of Medical Genetics and Genomics, the c.1996dupG (p.A666Gfs*71) was rated as pathogenic (PVS1+PM2_Supporting+PM3), whilst the c.701C>T (p.T234M) was rated as likely pathogenic (PM1+PM2_Supporting+PM3+PM5+PP3). The GAA in the leukocytes from the patient, her father and mother were respectively 76.1%, 91.3% and 95.6% of the normal value without the inhibitor, and 70.8%, 112.9% and 128.2% of the normal value with the inhibitor, whilst the activity of GAA in their leukocytes had decreased by 6 ~ 9 times after adding the inhibitor. GAA in lymphocytes of the patient, her father and mother were 68.3%, 59.0% and 59.5% of the normal value without the inhibitor, and 41.0%, 89.5% and 57.7% of the normal value with the inhibitor, the activity of GAA in lymphocytes has decreased by 2 ~ 5 times after adding the inhibitor. CONCLUSION: The child was diagnosed with LOPD due to the c.1996dupG and c.701C>T compound heterozygous variants of the GAA gene. The residual activity of GAA among LOPD patients can range widely and the changes may be atypical. The diagnosis of LOPD should not be based solely on the results of enzymatic activity but combined clinical manifestation, genetic testing and measurement of enzymatic activity.

Pompe disease ascertained through The Lantern Project, 2018-2021: Next-generation sequencing and enzymatic testing to overcome obstacles to diagnosis.

The Lantern Project is an ongoing complimentary diagnostic program for patients in the United States sponsored by Sanofi and implemented by PerkinElmer Genomics. It combines specific enzymatic, biomarker, and genetic testing to facilitate rapid, accurate laboratory diagnosis of Pompe disease and several other lysosomal storage diseases, and a multigene next-generation sequencing panel including Pompe disease, LGMD, and other neuromuscular disorders. This article reports data for Pompe disease collected from October 2018 through December 2021, including acid α-glucosidase (GAA) enzyme assay and GAA sequencing (standard or expedited for positive newborn screening [NBS] to rule out infantile-onset Pompe disease [IOPD]) and the Focused Neuromuscular Panel, which includes GAA. One hundred forty patients (12 received only GAA enzyme testing, 128 had GAA sequencing alone or in addition to enzyme assay) have been confirmed with Pompe disease in this project. Eight of the 140 had a variant of unknown significance, but GAA activity ≤2.10 µmol/L/h, thus were confirmed with Pompe disease. Three diagnosed patients 0-2 years old had cross-reactive immunologic material (CRIM)-negative GAA variants and thus IOPD. One additional infant with presumptive IOPD had a homozygous frameshift c.1846del, likely CRIM-negative; symptoms were not provided. Among the 128 patients with molecular results, the c.-32-13T>G splice variant was homozygous in 11, compound-heterozygous in 98, and absent in 19. Proximal muscle weakness (58 patients) was the most common sign reported at testing; elevated creatine kinase (29 patients) was the most common laboratory result. The most common symptom categories were muscular (73 patients), musculoskeletal (13 patients), and respiratory (23 patients). Clinical information was not available for 42 samples, and 17 infants had only "abnormal NBS" or "low GAA" reported. Cardiac symptoms in 7 included potentially age-related conditions in five c.-32-13T>G-compound-heterozygous adults (myocardial infarction, heart murmur/palpitations, congestive heart failure: 1 each; 2 with atrial fibrillation) and hypertrophic cardiomyopathy in 2 children (1 and 2 years old) with presumptive IOPD. One novel GAA variant was observed in a patient with enzyme activity 0.31 µmol/L/h: c.1853_1854ins49, a frameshift pathogenic variant. The Lantern Project demonstrates the combinatorial utility of enzyme assay, targeted single-gene testing, and a focused neuromuscular next-generation sequencing panel in diagnosing Pompe disease.

Degeneration of muscle spindles in a murine model of Pompe disease.

Pompe disease is a debilitating medical condition caused by a functional deficiency of lysosomal acid alpha-glucosidase (GAA). In addition to muscle weakness, people living with Pompe disease experience motor coordination deficits including an instable gait and posture. We reasoned that an impaired muscle spindle function might contribute to these deficiencies and therefore analyzed proprioception as well as muscle spindle structure and function in 4- and 8-month-old Gaa-/- mice. Gait analyses showed a reduced inter-limb and inter-paw coordination in Gaa-/- mice. Electrophysiological analyses of single-unit muscle spindle proprioceptive afferents revealed an impaired sensitivity of the dynamic and static component of the stretch response. Finally, a progressive degeneration of the sensory neuron and of the intrafusal fibers was detectable in Gaa-/- mice. We observed an increased abundance and size of lysosomes, a fragmentation of the inner and outer connective tissue capsule and a buildup of autophagic vacuoles in muscle spindles from 8-month-old Gaa-/- mice, indicating lysosomal defects and an impaired autophagocytosis. These results demonstrate a structural and functional degeneration of muscle spindles and an altered motor coordination in Gaa-/- mice. Similar changes could contribute to the impaired motor coordination in patients living with Pompe disease.

Immune transgene-dependent myocarditis in macaques after systemic administration of adeno-associated virus expressing human acid alpha-glucosidase.

Immune responses to human non-self transgenes can present challenges in preclinical studies of adeno-associated virus (AAV) gene therapy candidates in nonhuman primates. Although anti-transgene immune responses are usually mild and non-adverse, they can confound pharmacological readouts and complicate translation of results between species. We developed a gene therapy candidate for Pompe disease consisting of AAVhu68, a clade F AAV closely related to AAV9, that expresses an engineered human acid-alpha glucosidase (hGAA) tagged with an insulin-like growth factor 2 variant (vIGF2) peptide for enhanced cell uptake. Rhesus macaques were administered an intravenous dose of 1x1013 genome copies (GC)/kg, 5x1013 GC/kg, or 1 x 1014 GC/kg of AAVhu68.vIGF2.hGAA. Some unusually severe adaptive immune responses to hGAA presented, albeit with a high degree of variability between animals. Anti-hGAA responses ranged from absent to severe cytotoxic T-cell-mediated myocarditis with elevated troponin I levels. Cardiac toxicity was not dose dependent and affected five out of eleven animals. Upon further investigation, we identified an association between toxicity and a major histocompatibility complex class I haplotype (Mamu-A002.01) in three of these animals. An immunodominant peptide located in the C-terminal region of hGAA was subsequently identified via enzyme-linked immunospot epitope mapping. Another notable observation in this preclinical safety study cohort pertained to the achievement of robust and safe gene transfer upon intravenous administration of 5x1013 GC/kg in one animal with a low pre-existing neutralizing anti-capsid antibodies titer (1:20). Collectively, these findings may have significant implications for gene therapy inclusion criteria.

Phase I study of liver depot gene therapy in late-onset Pompe disease.

Gene therapy with an adeno-associated virus serotype 8 (AAV8) vector (AAV8-LSPhGAA) could eliminate the need for enzyme replacement therapy (ERT) by creating a liver depot for acid α-glucosidase (GAA) production. We report initial safety and bioactivity of the first dose (1.6 × 1012 vector genomes/kg) cohort (n = 3) in a 52-week open-label, single-dose, dose-escalation study (NCT03533673) in patients with late-onset Pompe disease (LOPD). Subjects discontinued biweekly ERT after week 26 based on the detection of elevated serum GAA activity and the absence of clinically significant declines per protocol. Prednisone (60 mg/day) was administered as immunoprophylaxis through week 4, followed by an 11-week taper. All subjects demonstrated sustained serum GAA activities from 101% to 235% of baseline trough activity 2 weeks following the preceding ERT dose. There were no treatment-related serious adverse events. No subject had anti-capsid T cell responses that decreased transgene expression. Muscle biopsy at week 24 revealed unchanged muscle glycogen content in two of three subjects. At week 52, muscle GAA activity for the cohort was significantly increased (p < 0.05). Overall, these initial data support the safety and bioactivity of AAV8-LSPhGAA, the safety of withdrawing ERT, successful immunoprophylaxis, and justify continued clinical development of AAV8-LSPhGAA therapy in Pompe disease.

Transcriptomic characterization of clinical skeletal muscle biopsy from late-onset Pompe patients.

Pompe disease is a rare lysosomal storage disorder arising from recessive mutations in the acid α-glucosidase gene and resulting in the accumulation of glycogen, particularly in the cardiac and skeletal muscle. The current standard of care is administration of enzyme replacement therapy in the form of alglucosidase alfa or the recently approved avalglucosidase alfa. In order to better understand the underlying cellular processes that are disrupted in Pompe disease, we conducted gene expression analysis on skeletal muscle biopsies obtained from late-onset Pompe disease patients (LOPD) prior to treatment and following six months of enzyme replacement with avalglucosidase alfa. The LOPD patients had a distinct transcriptomic signature as compared to control patient samples, largely characterized by perturbations in pathways involved in lysosomal function and energy metabolism. Although patients were highly heterogeneous, they collectively exhibited a strong trend towards attenuation of the dysregulated genes following just six months of treatment. Notably, the enzyme replacement therapy had a strong stabilizing effect on gene expression, with minimal worsening in genes that were initially dysregulated. Many of the cellular process that were altered in LOPD patients were also affected in the more clinically severe infantile-onset (IOPD) patients. Additionally, both LOPD and IOPD patients demonstrated enrichment across several inflammatory pathways, despite a lack of overt immune cell infiltration. This study provides further insight into Pompe disease biology and demonstrates the positive effects of avalglucosidase alfa treatment.