Acid α-glucosidase (GAA) activity and glycogen content in muscle biopsy specimens of patients with Pompe disease: A systematic review.

Pompe disease is a rare genetic disorder characterized by a deficiency of acid α-glucosidase (GAA), leading to the accumulation of glycogen in various tissues, especially in skeletal muscles. The disease manifests as a large spectrum of phenotypes from infantile-onset Pompe disease (IOPD) to late-onset Pompe disease (LOPD), depending on the age of symptoms onset. Quantifying GAA activity and glycogen content in skeletal muscle provides important information about the disease severity. However, the distribution of GAA and glycogen levels in skeletal muscles from healthy individuals and those impacted by Pompe disease remains poorly understood, and there is currently no universally accepted standard assay for GAA activity measurement. This systematic literature review aims to provide an overview of the available information on GAA activity and glycogen content levels in skeletal muscle biopsies from patients with Pompe disease. A structured review of PubMed and Google Scholar literature (with the latter used to check that no additional publications were identified) was conducted to identify peer-reviewed publications on glycogen storage disease type II [MeSH term] + GAA, protein human (supplementary concept), Pompe, muscle; and muscle, acid alpha-glucosidase. A limit of English language was applied. Results were grouped by methodologies used to quantify GAA activity and glycogen content in skeletal muscle. The search and selection strategy were devised and carried out in line with Preferred Reporting of Items in Systematic Reviews and Meta-Analysis guidelines and documented using a flowchart. Bibliographies of papers included in the analysis were reviewed and applicable publications not already identified in the search were included. Of the 158 articles retrieved, 24 (comprising >100 muscle biopsies from >100 patients) were included in the analysis, with four different assays. Analysis revealed that patients with IOPD exhibited markedly lower GAA activity in skeletal muscles than those with LOPD, regardless of the measurement method employed. Additionally, patients with IOPD had notably higher glycogen content levels in skeletal muscles than those with LOPD. In general, however, it was difficult to fully characterize GAA activity because of the different methods used. The findings underscore the challenges in the interpretation and comparison of the results across studies because of the substantial methodological variations. There is a need to establish standardized reference ranges of GAA activity and glycogen content in healthy individuals and in Pompe disease patients based on globally standardized methods to improve comparability and reliability in assessing this rare disease.

Effects of gene replacement therapy with resamirigene bilparvovec (AT132) on skeletal muscle pathology in X-linked myotubular myopathy: results from a substudy of the ASPIRO open-label clinical trial.

BACKGROUND: X-linked myotubular myopathy (XLMTM) is a rare, life-threatening congenital muscle disease caused by mutations in the MTM1 gene that result in profound muscle weakness, significant respiratory insufficiency, and high infant mortality. There is no approved disease-modifying therapy for XLMTM. Resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) is an investigational adeno-associated virus (AAV8)-mediated gene replacement therapy designed to deliver MTM1 to skeletal muscle cells and achieve long-term correction of XLMTM-related muscle pathology. The clinical trial ASPIRO (NCT03199469) investigating resamirigene bilparvovec in XLMTM is currently paused while the risk:benefit balance associated with this gene therapy is further investigated. METHODS: Muscle biopsies were taken before treatment and 24 and 48 weeks after treatment from ten boys with XLMTM in a clinical trial of resamirigene bilparvovec (ASPIRO; NCT03199469). Comprehensive histopathological analysis was performed. FINDINGS: Baseline biopsies uniformly showed findings characteristic of XLMTM, including small myofibres, increased internal or central nucleation, and central aggregates of organelles. Biopsies taken at 24 weeks post-treatment showed marked improvement of organelle localisation, without apparent increases in myofibre size in most participants. Biopsies taken at 48 weeks, however, did show statistically significant increases in myofibre size in all nine biopsies evaluated at this timepoint. Histopathological endpoints that did not demonstrate statistically significant changes with treatment included the degree of internal/central nucleation, numbers of triad structures, fibre type distributions, and numbers of satellite cells. Limited (predominantly mild) treatment-associated inflammatory changes were seen in biopsy specimens from five participants. INTERPRETATION: Muscle biopsies from individuals with XLMTM treated with resamirigene bilparvovec display statistically significant improvement in organelle localisation and myofibre size during a period of substantial improvements in muscle strength and respiratory function. This study identifies valuable histological endpoints for tracking treatment-related gains with resamirigene bilparvovec, as well as endpoints that did not show strong correlation with clinical improvement in this human study. FUNDING: Astellas Gene Therapies (formerly Audentes Therapeutics, Inc.).

Safety and efficacy of gene replacement therapy for X-linked myotubular myopathy (ASPIRO): a multinational, open-label, dose-escalation trial.

BACKGROUND: X-linked myotubular myopathy is a rare, life-threatening, congenital muscle disease observed mostly in males, which is caused by mutations in MTM1. No therapies are approved for this disease. We aimed to assess the safety and efficacy of resamirigene bilparvovec, which is an adeno-associated viral vector serotype 8 delivering human MTM1. METHODS: ASPIRO is an open-label, dose-escalation trial at seven academic medical centres in Canada, France, Germany, and the USA. We included boys younger than 5 years with X-linked myotubular myopathy who required mechanical ventilator support. The trial was initially in two parts. Part 1 was planned as a safety and dose-escalation phase in which participants were randomly allocated (2:1) to either the first dose level (1·3 × 1014 vector genomes [vg]/kg bodyweight) of resamirigene bilparvovec or delayed treatment, then, for later participants, to either a higher dose (3·5 × 1014 vg/kg bodyweight) of resamirigene bilparvovec or delayed treatment. Part 2 was intended to confirm the dose selected in part 1. Resamirigene bilparvovec was administered as a single intravenous infusion. An untreated control group comprised boys who participated in a run-in study (INCEPTUS; NCT02704273) or those in the delayed treatment cohort who did not receive any dose. The primary efficacy outcome was the change from baseline to week 24 in hours of daily ventilator support. After three unexpected deaths, dosing at the higher dose was stopped and the two-part feature of the study design was eliminated. Because of changes to the study design during its implementation, analyses were done on an as-treated basis and are deemed exploratory. All treated and control participants were included in the safety analysis. The trial is registered with ClinicalTrials.gov, NCT03199469. Outcomes are reported as of Feb 28, 2022. ASPIRO is currently paused while deaths in dosed participants are investigated. FINDINGS: Between Aug 3, 2017 and June 1, 2021, 30 participants were screened for eligibility, of whom 26 were enrolled; six were allocated to the lower dose, 13 to the higher dose, and seven to delayed treatment. Of the seven children whose treatment was delayed, four later received the higher dose (n=17 total in the higher dose cohort), one received the lower dose (n=7 total in the lower dose cohort), and two received no dose and joined the control group (n=14 total, including 12 children from INCEPTUS). Median age at dosing or enrolment was 12·1 months (IQR 10·0-30·9; range 9·5-49·7) in the lower dose cohort, 31·1 months (16·0-64·7; 6·8-72·7) in the higher dose cohort, and 18·7 months (10·1-31·5; 5·9-39·3) in the control cohort. Median follow-up was 46·1 months (IQR 41·0-49·5; range 2·1-54·7) for lower dose participants, 27·6 months (24·6-29·1; 3·4-41·0) for higher dose participants, and 28·3 months (9·7-46·9; 5·7-32·7) for control participants. At week 24, lower dose participants had an estimated 77·7 percentage point (95% CI 40·22 to 115·24) greater reduction in least squares mean hours per day of ventilator support from baseline versus controls (p=0·0002), and higher dose participants had a 22·8 percentage point (6·15 to 39·37) greater reduction from baseline versus controls (p=0·0077). One participant in the lower dose cohort and three in the higher dose cohort died; at the time of death, all children had cholestatic liver failure following gene therapy (immediate causes of death were sepsis; hepatopathy, severe immune dysfunction, and pseudomonal sepsis; gastrointestinal haemorrhage; and septic shock). Three individuals in the control group died (haemorrhage presumed related to hepatic peliosis; aspiration pneumonia; and cardiopulmonary failure). INTERPRETATION: Most children with X-linked myotubular myopathy who received MTM1 gene replacement therapy had important improvements in ventilator dependence and motor function, with more than half of dosed participants achieving ventilator independence and some attaining the ability to walk independently. Investigations into the risk for underlying hepatobiliary disease in X-linked myotubular myopathy, and the need for monitoring of liver function before gene replacement therapy, are ongoing. FUNDING: Astellas Gene Therapies.

High-throughput transcriptome analyses from ASPIRO, a phase 1/2/3 study of gene replacement therapy for X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a severe congenital disease characterized by profound muscle weakness, respiratory failure, and early death. No approved therapy for XLMTM is currently available. Adeno-associated virus (AAV)-mediated gene replacement therapy has shown promise as an investigational therapeutic strategy. We aimed to characterize the transcriptomic changes in muscle biopsies of individuals with XLMTM who received resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) in the ASPIRO clinical trial and to identify potential biomarkers that correlate with therapeutic outcome. We leveraged RNA-sequencing data from the muscle biopsies of 15 study participants and applied differential expression analysis, gene co-expression analysis, and machine learning to characterize the transcriptomic changes at baseline (pre-dose) and at 24 and 48 weeks after resamirigene bilparvovec dosing. As expected, MTM1 expression levels were significantly increased after dosing (p < 0.0001). Differential expression analysis identified upregulated genes after dosing that were enriched in several pathways, including lipid metabolism and inflammatory response pathways, and downregulated genes were enriched in cell-cell adhesion and muscle development pathways. Genes involved in inflammatory and immune pathways were differentially expressed between participants exhibiting ventilator support reduction of either greater or less than 6 h/day after gene therapy compared to pre-dosing. Co-expression analysis identified similarly regulated genes, which were grouped into modules. Finally, the machine learning model identified five genes, including MTM1, as potential RNA biomarkers to monitor the progress of AAV gene replacement therapy. These findings further extend our understanding of AAV-mediated gene therapy in individuals with XLMTM at the transcriptomic level.

Carbon-carbon bond cleavage in activation of the prodrug nabumetone.

Carbon-carbon bond cleavage reactions are catalyzed by, among others, lanosterol 14-demethylase (CYP51), cholesterol side-chain cleavage enzyme (CYP11), sterol 17ß-lyase (CYP17), and aromatase (CYP19). Because of the high substrate specificities of these enzymes and the complex nature of their substrates, these reactions have been difficult to characterize. A CYP1A2-catalyzed carbon-carbon bond cleavage reaction is required for conversion of the prodrug nabumetone to its active form, 6-methoxy-2-naphthylacetic acid (6-MNA). Despite worldwide use of nabumetone as an anti-inflammatory agent, the mechanism of its carbon-carbon bond cleavage reaction remains obscure. With the help of authentic synthetic standards, we report here that the reaction involves 3-hydroxylation, carbon-carbon cleavage to the aldehyde, and oxidation of the aldehyde to the acid, all catalyzed by CYP1A2 or, less effectively, by other P450 enzymes. The data indicate that the carbon-carbon bond cleavage is mediated by the ferric peroxo anion rather than the ferryl species in the P450 catalytic cycle. CYP1A2 also catalyzes O-demethylation and alcohol to ketone transformations of nabumetone and its analogs.

Role of cysteine residues in heme binding to human heme oxygenase-2 elucidated by two-dimensional NMR spectroscopy.

Human heme oxygenases 1 and 2 (HO-1 and HO-2) degrade heme in the presence of oxygen and NADPH-cytochrome P450 reductase, producing ferrous iron, CO, and biliverdin. HO-1 is an inducible enzyme, but HO-2 is constitutively expressed in selected tissues and is involved in signaling and regulatory processes. HO-2 has three cysteine residues that have been proposed to modulate the affinity for heme, whereas HO-1 has none. Here we use site-specific mutagenesis and two-dimensional NMR of l-[3-(13)C]cysteine-labeled proteins to determine the redox state of the individual cysteines in HO-2 and assess their roles in binding of heme. The results indicate that in the apoprotein, Cys(282) and Cys(265) are in the oxidized state, probably in an intramolecular disulfide bond. The addition of a reducing agent converts them to the reduced, free thiol state. Two-dimensional NMR of site-specific mutants reveals that the redox state of Cys(265) and Cys(282) varies with the presence or absence of other Cys residues, indicating that the microenvironments of the Cys residues are mutually interdependent. Cys(265) appears to be in a relatively hydrophilic, oxidizable environment compared with Cys(127) and Cys(282). Chemical shift data indicate that none of the cysteines stably coordinates to the heme iron atom. In the oxidized state of the apoprotein, heme is bound 2.5-fold more tightly than in the reduced state. This small difference in heme affinity between the oxidized and reduced states of the protein is much less than previously reported, suggesting that it is not a significant factor in the physiological regulation of cellular heme levels.

The role of P4-P3' residues flanking Arg336 in facilitating activated protein C-catalyzed cleavage and inactivation of factor VIIIa.

INTRODUCTION: Activated protein C (APC) inactivates factor VIIIa (FVIIIa) through cleavages at Arg336 in the A1 subunit and Arg562 in the A2 subunit. Proteolysis at Arg336 occurs 25-fold faster than at Arg562. Replacing residues flanking Arg336 en bloc with the corresponding residues surrounding Arg562 markedly reduced the rate of cleavage at Arg336, indicating a role for these residues in the catalysis mechanism. MATERIALS AND METHODS: To assess the contributions of individual P4-P3' residues flanking the Arg336 site to cleavage efficiency, point mutations were made based upon those flanking Arg562 of FVIIIa (Pro333Val, Gln334Asp, Leu335Gln, Met337Gly, Lys338Asn, Asn339Gln) and selected residues flanking Arg506 of FVa (Leu335Arg, and Lys338Ile). APC-catalyzed inactivation of the FVIII variants and cleavage of FVIIIa subunits were monitored by FXa generation assays and Western blotting. RESULTS: Specific activity values of the variants were 60-135% of the wild type (WT) value. APC-catalyzed rates of cleavage at Arg336 remained similar to WT for the Pro333Val and Lys338Ile variants and was modestly increased for the Asn339Gln variant; while rates were reduced ~2-3-fold for the Gln334Asp, Leu335Gln, Leu335Arg, and Lys338Asn variants, and 5-fold for the Met337Gly variant. Rates for cofactor inactivation paralleled cleavage at the A1 site. APC slowly cleaves Arg372 in FVIII, a site responsible for procofactor activation. Using FVIII as substrate for APC, the Met337Gly variant yielded significantly greater activation compared with WT FVIII. CONCLUSIONS: These results show that individual P4-P3' residues surrounding Arg336 are in general more favorable to cleavage than those surrounding the Arg562 site.

Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface.

Factor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. The charged residues Glu272, Asp519, Glu665, and Glu1984 appear buried at the interface of the A2 domain with either the A1 or A3 domain, and thus may impact protein stability. To determine the effects of these residues on procofactor/cofactor stability, these residues were individually replaced with either Ala or Val, and stable BHK cell lines expressing the B-domainless proteins were prepared. Specific activity and thrombin generation parameters for 7 of the 8 variants were more than 80% the wild-type value. Factor VIII activity at 52 degrees C to 60 degrees C and the decay of factor VIIIa activity after thrombin activation were monitored. Six of the 7 variants showing wild-type-like activity demonstrated enhanced stability, with the Glu1984Val variant showing a 2-fold increase in thermostability and an approximately 4- to 8-fold increase in stability of factor VIIIa. These results indicate that replacement of buried charged residues is an effective alternative to covalent modification in increasing factor VIII (VIIIa) stability.

Residues surrounding Arg336 and Arg562 contribute to the disparate rates of proteolysis of factor VIIIa catalyzed by activated protein C.

Activated Protein C (APC) inactivates factor VIIIa by cleavage at Arg(336) and Arg(562) within the A1 and A2 subunits, respectively, with reaction at the former site occurring at a rate approximately 25-fold faster than the latter. Recombinant factor VIII variants possessing mutations within the P4-P3' sequences were used to determine the contributions of these residues to the disparate cleavage rates at the two P1 sites. Specific activity values for 336(P4-P3')562, 336(P4-P2)562, and 336(P1'-P3')562 mutants, where indicated residues surrounding the Arg(336) site were replaced with those surrounding Arg(562), were similar to wild type (WT) factor VIII; whereas 562(P4-P3')336 and 562(P4-P2)336 mutants showed specific activity values <1% the WT value. Inactivation rates for the 336 site mutants were reduced approximately 6-11-fold compared with WT factor VIIIa, and approached values attributed to cleavage at Arg(562). Cleavage rates at Arg(336) were reduced approximately 100-fold for 336(P4-P3')562, and approximately 9-16-fold for 336(P4-P2)562 and 336(P1'-P3')562 mutants. Inhibition kinetics revealed similar affinities of APC for WT factor VIIIa and 336(P4-P3')562 variant. Alternatively, the 562(P4-P3')336 variant showed a modest increase in cleavage rate ( approximately 4-fold) at Arg(562) compared with WT, whereas these rates were increased by approximately 27- and 6-fold for 562(P4-P3')336 and 562(P4-P2)336, respectively, using the factor VIII procofactor form as substrate. Thus the P4-P3' residues surrounding Arg(336) and Arg(562) make significant contributions to proteolysis rates at each site, apparently independent of binding affinity. Efficient cleavage at Arg(336) by APC is attributed to favorable P4-P3' residues at this site, whereas cleavage at Arg(562) can be accelerated following replacement with more optimal P4-P3' residues.

pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+.

Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5-6.75 (Kd approximately 4 nM), whereas a Kd approximately 20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 microM total Cu2+) on maximal activity regenerated was negligible at pH 6.25-8.0. However, this level of Cu2+ increased the inter-chain affinity by approximately 5-fold at pH 7.25. This effect resulted from an approximately 1.5-fold increased association rate constant (k(on)) and an approximately 3-fold reduced dissociation rate constant (k(off)). High affinity (Kd=5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.

Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa.

APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate approximately 25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated approximately 9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated approximately 2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a K(m) value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than K(i) values (approximately 9-18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341-345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.