The interplay of psychosis and non-compliance with fatal outcome in an adult with MSUD.

Significant progress has been achieved in enhancing early outcomes for individuals with maple syrup urine disease (MSUD), a rare metabolic disorder that leads to the accumulation of branched-chain amino acids leucine, isoleucine, and valine, where leucine is known as the primary neurotoxic metabolite. Newborn screening is helpful in early diagnosis and implementation of dietary treatment, thus reducing neurological deterioration and complications in young children. However, patients face the life-long challenge of maintaining metabolic control through adherence to a strict low-leucine diet to avoid long-term consequences of chronic hyperleucinemia, which include cognitive deficits, mood disorders, and movement disorders. This case report exemplifies the complex involvement of MSUD in adult survivors. Despite presenting early in life, the patient thrived until the onset of psychiatric symptoms. The subject of this case is a 25-year-old woman with MSUD, who remained in her usual state of health until presentation to the emergency department (ED) with psychosis and altered mental status. However, due to a lack of medical records and poor communication, there was a delay in considering MSUD as a primary cause of her psychiatric symptoms. Although a genetics consultation was later arranged and efforts were made to decrease plasma leucine to the therapeutic range, these interventions proved inadequate in halting her deterioration in health. Her condition worsened within 72 h, culminating in her untimely death. This case emphasizes the comorbidity of psychiatric involvement in MSUD, which contributes to metabolic decompensation that can lead to cerebral edema and death. This case also highlights the pressing need for enhanced strategies for the acute management and long-term care of MSUD patients with psychiatric involvement, particularly in scenarios where mental disturbance could lead to noncompliance.

Interim analyses of a first-in-human phase 1/2 mRNA trial for propionic acidaemia.

Propionic acidaemia is a rare disorder caused by defects in the propionyl-coenzyme A carboxylase α or ß (PCCA or PCCB) subunits that leads to an accumulation of toxic metabolites and to recurrent, life-threatening metabolic decompensation events. Here we report interim analyses of a first-in-human, phase 1/2, open-label, dose-optimization study and an extension study evaluating the safety and efficacy of mRNA-3927, a dual mRNA therapy encoding PCCA and PCCB. As of 31 May 2023, 16 participants were enrolled across 5 dose cohorts. Twelve of the 16 participants completed the dose-optimization study and enrolled in the extension study. A total of 346 intravenous doses of mRNA-3927 were administered over a total of 15.69 person-years of treatment. No dose-limiting toxicities occurred. Treatment-emergent adverse events were reported in 15 out of the 16 (93.8%) participants. Preliminary analysis suggests an increase in the exposure to mRNA-3927 with dose escalation, and a 70% reduction in the risk of metabolic decompensation events among 8 participants who reported them in the 12-month pretreatment period.

Critical sample collection delayed? Urine organic acid analysis can still save the day! A new case of HMG-CoA synthase deficiency.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (mHS) deficiency is an autosomal recessive disorder of ketone body synthesis caused by biallelic pathogenic variants in HMGCS2. Clinical symptoms are precipitated by prolonged fasting and/or intercurrent illness with onset before the first year of life. Clinically, patients may present with hypo-/ non-ketotic hypoglycemia, metabolic acidosis, hyperammonemia, lethargy, hepatomegaly, and encephalopathy. During periods of decompensation, elevations of 4-hydroxy-6-methyl-2-pyrone (4-HMP), several hydroxylated hexanoic and hexenoic acid species, and medium-chain dicarboxylic acids in the absence of significant ketonuria may be observed in the urine organic acid profile. Abnormalities may also be observed in plasma which includes elevated acetylcarnitine (C2) and 3-hydroxybutyryl/3-hydroxyisobutyryl (C4-OH) carnitine. We report a patient who presented to the ED at 13 months of age with an undetectable point-of-care blood glucose level. Continuous infusion of dextrose-containing intravenous (IV) fluids were required to correct the hypoglycemia and routine chemistries were notable for an anion gap metabolic acidosis, transaminasemia, and elevated creatine kinase and lactate dehydrogenase. Urine and blood ketones were undetectable. Qualitative assessment of urine organic acids collected ∼46 and âˆ¼ 99 h post-admission were significant for mild elevations of 4-HMP and hydroxy-hexanoic and hydroxy-hexenoic acid species with a notable absence of ketones. Previously, biochemical abnormalities in urine have been shown to normalize in as few as 27 h after treatment giving providers a narrow window with which to obtain a critical sample. Direct communication of laboratory findings to the ordering provider guided the molecular testing and assisted in results interpretation to confirm the molecular diagnosis. Our case emphasizes the importance of collecting samples for biochemical analysis even if the critical period has been missed and acute metabolic decompensation seems to be resolved, as residual abnormalities observed in our patient greatly narrowed the differential diagnosis.

Gene therapy for glycogen storage diseases.

Glycogen storage disorders (GSDs) are inherited disorders of metabolism resulting from the deficiency of individual enzymes involved in the synthesis, transport, and degradation of glycogen. This literature review summarizes the development of gene therapy for the GSDs. The abnormal accumulation of glycogen and deficiency of glucose production in GSDs lead to unique symptoms based upon the enzyme step and tissues involved, such as liver and kidney involvement associated with severe hypoglycemia during fasting and the risk of long-term complications including hepatic adenoma/carcinoma and end stage kidney disease in GSD Ia from glucose-6-phosphatase deficiency, and cardiac/skeletal/smooth muscle involvement associated with myopathy +/- cardiomyopathy and the risk for cardiorespiratory failure in Pompe disease. These symptoms are present to a variable degree in animal models for the GSDs, which have been utilized to evaluate new therapies including gene therapy and genome editing. Gene therapy for Pompe disease and GSD Ia has progressed to Phase I and Phase III clinical trials, respectively, and are evaluating the safety and bioactivity of adeno-associated virus vectors. Clinical research to understand the natural history and progression of the GSDs provides invaluable outcome measures that serve as endpoints to evaluate benefits in clinical trials. While promising, gene therapy and genome editing face challenges with regard to clinical implementation, including immune responses and toxicities that have been revealed during clinical trials of gene therapy that are underway. Gene therapy for the glycogen storage diseases is under development, addressing an unmet need for specific, stable therapy for these conditions.

Corrigendum to "Elevated amyloid beta peptides and total tau in cerebrospinal fluid in individuals with Creatine transporter deficiency".

[This corrects the article DOI: 10.1016/j.ymgmr.2023.101001.].

Elevated amyloid beta peptides and total tau in cerebrospinal fluid in individuals with Creatine transporter deficiency.

Background: Creatine transporter deficiency (CTD) is a rare X-linked disorder of creatine transport caused by pathogenic variants in SLC6A8 (Xq28). The disorder is marked by developmental delay, especially speech delay. The biomarkers Aß40, Aß42 and total tau are abnormal in Alzheimer disease (AD), a common neurodegenerative disorder pathologically characterized by Aß peptide containing amyloid plaques and tau neurofibrillary tangles. Although CTD results in neuronal energy deficiency, the pathological processes underlying the CTD phenotype are not fully characterized. Methods: Cerebral spinal fluid (CSF) was collected as an optional part of a natural history study of CTD. Aß40, Aß42 and total tau levels were quantified in CSF from individuals with CTD and from age-appropriate comparison samples. Neuro3-Plex enzyme-linked immunoassay was performed on a Quanterix SR-X instrument. The Vineland Adaptive Behavior Scale, 3rd Edition was used to determine an overall Adaptive Behavior Composite (ABC) standard score. Results: CSF from 12 individuals with CTD and 23 age appropriate non-CTD comparison samples were analyzed. We found that levels of total tau [t(32) = 4.05, p = 0.0003], Aß40 [t(31) = 6.11, p < 0.0001], and Aß42 [t(32) = 3.20, p = 0.003] were elevated in the participants with CTD relative to the comparison group. Additionally, except for one individual that we considered an outlier, all three biomarkers correlated inversely with the adaptive behavior score (total tau: ρ = -0.60 [-0.88, 0.005]; Aß40: ρ = -0.67 [-0.91, -0.12]; Aß42: ρ = -0.62 [-0.89, -0.02]). Conclusion: We describe here the novel finding of elevated protein biomarkers in the CSF of individuals with CTD. Aß40, Aß42 and total tau are markedly elevated in individuals with CTD compared to comparison samples, and increased levels of these biomarkers inversely correlated with ABC scores. We hypothesize that elevated CSF levels of Aß40 and Aß42 are due to cellular energy deficiency. Elevated CSF total tau levels may indicate ongoing neuronal damage. The observed inverse correlation of Vineland ABC scores with increased biomarker levels needs to be confirmed in a larger CTD cohort; however, our observation of increased Aß40, Aß42 and total tau levels in CSF from individuals with CTD may provide insight into pathological mechanisms contributing to the CTD phenotype and may prove useful as supportive data in future therapeutic trials.

Screening data from 19 patients with late-onset Pompe disease for a phase I clinical trial of AAV8 vector-mediated gene therapy.

Late-onset Pompe disease (LOPD) is a multisystem disorder with significant myopathy. The standard treatment is enzyme replacement therapy (ERT), a therapy that is lifesaving, yet with limitations. Clinical trials have emerged for other potential treatment options, including adeno-associated virus (AAV) gene therapy. We present clinical parameters and AAV antibody titers for 19 individuals with LOPD undergoing screening for a Phase I clinical trial with an AAV serotype 8 vector targeting hepatic transduction (AAV2/8-LSPhGAA). Reported clinical parameters included GAA genotype, assessments of muscle function, upright and supine spirometry, anti-recombinant human GAA antibody titers, and biomarkers. Variability in measured parameters and phenotypes of screened individuals was evident. Eligibility criteria required that all participants have six-minute walk test (6MWT) and upright forced vital capacity (FVC) below the expected range for normal individuals, and were stably treated with ERT for >2 years. All participants had Pompe disease diagnosed by enzyme deficiency, and all had the common c.-32-13T>G LOPD pathogenic variant. Screening identified 14 patients (74%) with no or minimal detectable neutralizing antibodies against AAV8 (titer ≤1:5). 6MWT distance varied significantly (percent of expected distance ranging from 24% to 91% with an average of 60 and standard deviation of 21). Upright FVC percent predicted ranged from 35% predicted to 91% predicted with an average of 66 and standard deviation of 18. None of the participants had significantly elevated alanine transaminase, which has been associated with LOPD and could complicate screening for hepatitis related to AAV gene therapy. We review the parameters considered in screening for eligibility for a clinical trial of AAV8 vector-mediated gene therapy.

Clinical, biochemical and molecular characterization of 12 patients with pyruvate carboxylase deficiency treated with triheptanoin.

Pyruvate carboxylase (PC) deficiency is a rare autosomal recessive mitochondrial neurometabolic disorder of energy deficit resulting in high morbidity and mortality, with limited therapeutic options. The PC homotetramer has a critical role in gluconeogenesis, anaplerosis, neurotransmitter synthesis, and lipogenesis. The main biochemical and clinical findings in PC deficiency (PCD) include lactic acidosis, ketonuria, failure to thrive, and neurological dysfunction. Use of the anaplerotic agent triheptanoin on a limited number of individuals with PCD has had mixed results. We expand on the potential utility of triheptanoin in PCD by examining the clinical, biochemical, molecular, and health-related quality-of-life (HRQoL) findings in a cohort of 12 individuals with PCD (eight with Type A and two each with Types B and C) treated with triheptanoin ranging for 6 days to about 7 years. The main endpoints were changes in blood lactate and HRQoL scores, but collection of useful data was limited to about half of subjects. An overall trend of lactate reduction with time on triheptanoin was noted, but with significant variability among subjects and only one subject reaching close to statistical significance for this endpoint. Parent reported HRQoL assessments with treatment showed mixed results, with some subjects showing no change, some improvement, and some worsening of overall scores. Subjects with buried amino acids in the pyruvate carboxyltransferase domain of PC that undergo destabilizing replacements may be more likely to respond (with lactate reduction or HRQoL improvement) to triheptanoin compared to those with replacements that disrupt tetramerization or subunit-subunit interface contacts. The reason for this difference is unclear and requires further validation. We observed significant variability but an overall trend of lactate reduction with time on triheptanoin and mixed parent reported outcome changes by HRQoL assessments for subjects with PCD on long-term triheptanoin. The mixed results noted with triheptanoin therapy in this study could be due to endpoint data limitation, variability of disease severity between subjects, limitation of the parent reported HRQoL tool, or subject genotype variability. Alternative designed trials and more study subjects with PCD will be needed to validate important observations from this work.

Clenbuterol Treatment Is Safe and Associated With Slowed Disease Progression in a Small Open-Label Trial in Patients With Amyotrophic Lateral Sclerosis.

OBJECTIVE: Clenbuterol, a beta-agonist, has plausible mechanisms for treating amyotrophic lateral sclerosis (ALS). In this highly inclusive open-label trial (NCT04245709), we aimed to study the safety and efficacy of clenbuterol in patients with ALS. METHODS: All participants received clenbuterol starting at 40 µg daily and increased to 80 µg twice daily. Outcomes included safety, tolerability, ALS Functional Rating Score (ALSFRS-R) progression, forced vital capacity (FVC) progression, and myometry. ALSFRS-R and FVC slopes measured during treatment were compared with slopes before treatment (calculated by assuming ALSFRS-R was 48 and FVC was 100% at ALS onset). RESULTS: The 25 participants had a mean age of 59, mean disease duration of 43 months, ALSFRS-R score at enrollment 34, and FVC at enrollment 77%. Forty-eight percent were female, 68% were taking riluzole, and none were taking edaravone. Two participants experienced severe adverse events, neither related to the study. Twenty-four participants experienced adverse events, most commonly tremors/jitters, cramps/spasms, insomnia, and stiffness/spasticity. Fourteen participants withdrew early from the trial, 13 due to adverse events. Patients who withdrew early were significantly older and more likely to be male. Per-protocol and intention-to-treat analyses showed meaningfully slower ALSFRS-R and FVC progression during treatment. Hand grip dynamometry and myometry changes were highly variable between participants; most declined slowly, but some showed improvements. CONCLUSIONS: Clenbuterol was safe but less tolerable at the doses we selected compared with an earlier Italian case series. Consistent with that series, our study suggested benefits on ALS progression. However, the latter result should be interpreted with caution as our study is limited by small sample size, large drop out, lack of randomization, and blinding and placebo controls. A larger, more traditional trial now seems warranted.

Genome editing using Staphylococcus aureus Cas9 in a canine model of glycogen storage disease Ia.

Glycogen storage disease type Ia (GSD Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), associated with life-threatening hypoglycemia and long-term complications, including hepatocellular carcinoma formation. Gene replacement therapy fails to stably reverse G6Pase deficiency. We attempted genome editing using two adeno-associated virus vectors, one that expressed Staphylococcus aureus Cas9 protein and a second containing a donor transgene encoding G6Pase, in a dog model for GSD Ia. We demonstrated donor transgene integration in the liver of three adult-treated dogs accompanied by stable G6Pase expression and correction of hypoglycemia during fasting. Two puppies with GSD Ia were treated by genome editing that achieved donor transgene integration in the liver. Integration frequency ranged from 0.5% to 1% for all dogs. In adult-treated dogs, anti-SaCas9 antibodies were detected before genome editing, reflecting prior exposure to S. aureus. Nuclease activity was low, as reflected by a low percentage of indel formation at the predicted site of SaCas9 cutting that indicated double-stranded breaks followed by non-homologous end-joining. Thus, genome editing can integrate a therapeutic transgene in the liver of a large animal model, either early or later in life, and further development is warranted to provide a more stable treatment for GSD Ia.

Successful AAV8 readministration: Suppression of capsid-specific neutralizing antibodies by a combination treatment of bortezomib and CD20 mAb in a mouse model of Pompe disease.

BACKGROUND: A major challenge to adeno-associated virus (AAV)-mediated gene therapy is the presence of anti-AAV capsid neutralizing antibodies (NAbs), which can block viral vector transduction even at very low titers. In the present study, we examined the ability of a combination immunosuppression (IS) treatment with bortezomib and a mouse-specific CD20 monoclonal antibody to suppress anti-AAV NAbs and enable readministration of AAV vectors of the same capsid in mice. METHODS: An AAV8 vector (AAV8-CB-hGAA) that ubiquitously expresses human α-glucosidase was used for initial gene therapy and a second AAV8 vector (AAV8-LSP-hSEAP) that contains a liver-specific promoter to express human secreted embryonic alkaline phosphatase (hSEAP) was used for AAV readministration. Plasma samples were used for determination of anti-AAV8 NAb titers. Cells isolated from whole blood, spleen, and bone marrow were analyzed for B-cell depletion by flow cytometry. The efficiency of AAV readministration was determined by the secretion of hSEAP in blood. RESULTS: In näive mice, an 8-week IS treatment along with AAV8-CB-hGAA injection effectively depleted CD19+ B220+ B cells from blood, spleen, and bone marrow and prevented the formation of anti-AAV8 NAbs. Following administration of AAV8-LSP-hSEAP, increasing levels of hSEAP were detected in blood for up to 6 weeks, indicating successful AAV readministration. In mice pre-immunized with AAV8-CB-hGAA, comparison of IS treatment for 8, 12, 16, and 20 weeks revealed that the 16-week IS treatment demonstrated the highest plasma hSEAP level following AAV8-LSP-hSEAP readministration. CONCLUSIONS: Our data suggest that this combination treatment is an effective IS approach that will allow retreatment of patients with AAV-mediated gene therapy. A combination IS treatment with bortezomib and a mouse-specific CD20 monoclonal antibody effectively suppressed anti-AAV NAbs in naïve mice and in mice with pre-existing antibodies, allowing successful readministration of the same AAV capsid vector.

Moving away from one disease at a time: Screening, trial design, and regulatory implications of novel platform technologies.

Most rare diseases are caused by single-gene mutations, and as such, lend themselves to a host of new gene-targeted therapies and technologies including antisense oligonucleotides, phosphomorpholinos, small interfering RNAs, and a variety of gene delivery and gene editing systems. Early successes are encouraging, however, given the substantial number of distinct rare diseases, the ability to scale these successes will be unsustainable without new development efficiencies. Herein, we discuss the need for genomic newborn screening to match pace with the growing development of targeted therapeutics and ability to rapidly develop individualized therapies for rare variants. We offer approaches to move beyond conventional "one disease at a time" preclinical and clinical drug development and discuss planned regulatory innovations that are necessary to speed therapy delivery to individuals in need. These proposals leverage the shared properties of platform classes of therapeutics and innovative trial designs including master and platform protocols to better serve patients and accelerate drug development. Ultimately, there are risks to these novel approaches; however, we believe that close partnership and transparency between health authorities, patients, researchers, and drug developers present the path forward to overcome these challenges and deliver on the promise of gene-targeted therapies for rare diseases.

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.

Expanding allelic and phenotypic spectrum of ZC4H2-related disorder: A novel hypomorphic variant and high prevalence of tethered cord.

ZC4H2 (MIM# 300897) is a nuclear factor involved in various cellular processes including proliferation and differentiation of neural stem cells, ventral spinal patterning and osteogenic and myogenic processes. Pathogenic variants in ZC4H2 have been associated with Wieacker-Wolff syndrome (MIM# 314580), an X-linked neurodevelopmental disorder characterized by arthrogryposis, development delay, hypotonia, feeding difficulties, poor growth, skeletal abnormalities, and dysmorphic features. Zebrafish zc4h2 null mutants recapitulated the human phenotype, showed complete loss of vsx2 expression in brain, and exhibited abnormal swimming and balance problems. Here we report 7 new patients (four males and three females) with ZC4H2-related disorder from six unrelated families. Four of the 6 ZC4H2 variants are novel: three missense variants, designated as c.142T>A (p.Tyr48Asn), c.558G>A (p.Met186Ile) and c.602C>T (p.Pro201Leu), and a nonsense variant, c.618C>A (p.Cys206*). Two variants were previously reported : a nonsense variant c.199C>T (p.Arg67*) and a splice site variant (c.225+5G>A). Five patients were on the severe spectrum of clinical findings, two of whom had early death. The male patient harboring the p.Met186Ile variant and the female patient that carries the p.Pro201Leu variant have a relatively mild phenotype. Of note, 4/7 patients had a tethered cord that required a surgical repair. We also demonstrate and discuss previously under-recognized phenotypic features including sleep apnea, arrhythmia, hypoglycemia, and unexpected early death. To study the effect of the missense variants, we performed microinjection of human ZC4H2 wild-type or variant mRNAs into zc4h2 null mutant zebrafish embryos. The p.Met186Ile mRNA variant was able to partially rescue vsx2 expression while p.Tyr48Asn and p.Pro201Leu mRNA variants were not. However, swimming and balance problems could not be rescued by any of these variants. These results suggest that the p.Met186Ile is a hypomorphic allele. Our work expands the genotypes and phenotypes associated with ZC4H2-related disorder and demonstrates that the zebrafish system is a reliable method to determine the pathogenicity of ZC4H2 variants.

Itavastatin and resveratrol increase triosephosphate isomerase protein in a newly identified variant of TPI deficiency.

Triosephosphate isomerase (TPI) deficiency (TPI Df) is an untreatable glycolytic enzymopathy that results in hemolytic anemia, progressive muscular impairment and irreversible brain damage. Although there is a 'common' mutation (TPIE105D), other pathogenic mutations have been described. We identified patients who were compound heterozygous for a newly described mutation, TPIQ181P, and the common TPIE105D mutation. Intriguingly, these patients lacked neuropathy or cognitive impairment. We then initiated biochemical and structural studies of TPIQ181P. Surprisingly, we found that purified TPIQ181P protein had markedly impaired catalytic properties whereas crystallographic studies demonstrated that the TPIQ181P mutation resulted in a highly disordered catalytic lid. We propose that genetic complementation occurs between the two alleles, one with little activity (TPIQ181P) and one with low stability (TPIE105D). Consistent with this, TPIQ181P/E105D fibroblasts exhibit a significant reduction in the TPI protein. These data suggest that impaired stability, and not catalytic activity, is a better predictor of TPI Df severity. Lastly, we tested two recently discovered chemical modulators of mutant TPI stability, itavastatin and resveratrol, and observed a significant increase in TPI in TPIQ181P/E105D patient cells.

Minimum Effective Dose to Achieve Biochemical Correction with Adeno-Associated Virus Vector-Mediated Gene Therapy in Mice with Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder caused by deficiency of acid α-glucosidase (GAA), resulting in skeletal muscle weakness and cardiomyopathy. Muscle weakness progresses despite currently available therapy, which has prompted the development of gene therapy with adeno-associated virus (AAV) type 2 vectors cross-packaged as AAV8 (2/8). Preclinical studies of gene therapy demonstrated that the minimum effective dose (MED) for biochemical correction with AAV2/8-LSPhGAA was ∼2 × 1011 vector genomes (vg)/kg body weight. The current study examined the transduction of AAV2/8-LSPeGFP vector in adult GAA-KO mice with Pompe disease, and correlated that degree of transduction with the biochemical correction achieved by the same dose of AAV2/8-LSPhGAA. The MED was found to be ∼2 × 1011 vg/kg, with all hepatocytes variably transducing at this dose. At this dose, liver GAA significantly increased, while liver glycogen significantly decreased. The 2 × 1011 vg/kg dose was sufficient to significantly decrease diaphragm glycogen. However, the heart, diaphragm, and quadriceps all required a fourfold higher dose to achieve correction of GAA deficiency in association with significant clearance of stored glycogen, which correlated with increased serum GAA activity. These data indicate that AAV2/8-LSPeGFP transduced all hepatocytes when the 2 × 1011 vg/kg dose was administered, which correlated with partial biochemical correction from the equivalent dose of AAV2/8-LSPhGAA. Altogether, these data support the conclusion that substantial transduction of the liver is required to achieve biochemical correction from AAV2/8-LSPhGAA.

Efficacious Androgen Hormone Administration in Combination with Adeno-Associated Virus Vector-Mediated Gene Therapy in Female Mice with Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder caused by deficiency of acid α-glucosidase (GAA), resulting in skeletal muscle weakness and cardiomyopathy that progresses despite currently available therapy in some patients. The development of gene therapy with adeno-associated virus (AAV) vectors revealed a sex-dependent decrease in efficacy in female mice with Pompe disease. This study evaluated the effect of testosterone on gene therapy with an AAV2/8 vector containing a liver-specific promoter to drive expression of GAA (AAV2/8-LSPhGAA) in female GAA-knockout (KO) mice that were implanted with pellets containing testosterone propionate before vector administration. Six weeks after treatment, neuromuscular function and muscle strength were improved as demonstrated by increased Rotarod and wirehang latency for female mice treated with testosterone and vector, in comparison with vector alone. Biochemical correction improved after the addition of testosterone as demonstrated by increased GAA activity and decreased glycogen content in the skeletal muscles of female mice treated with testosterone and vector, in comparison with vector alone. An alternative androgen, oxandrolone, was evaluated similarly to reveal increased GAA in the diaphragm and extensor digitorum longus of female GAA-KO mice after oxandrolone administration; however, glycogen content was unchanged by oxandrolone treatment. The efficacy of androgen hormone treatment in females correlated with increased mannose-6-phosphate receptor in skeletal muscle. These data confirmed the benefits of brief treatment with an androgen hormone in mice with Pompe disease during gene therapy.

Three-dimensional tissue-engineered human skeletal muscle model of Pompe disease.

In Pompe disease, the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) causes skeletal and cardiac muscle weakness, respiratory failure, and premature death. While enzyme replacement therapy using recombinant human GAA (rhGAA) can significantly improve patient outcomes, detailed disease mechanisms and incomplete therapeutic effects require further studies. Here we report a three-dimensional primary human skeletal muscle ("myobundle") model of infantile-onset Pompe disease (IOPD) that recapitulates hallmark pathological features including reduced GAA enzyme activity, elevated glycogen content and lysosome abundance, and increased sensitivity of muscle contractile function to metabolic stress. In vitro treatment of IOPD myobundles with rhGAA or adeno-associated virus (AAV)-mediated hGAA expression yields increased GAA activity and robust glycogen clearance, but no improvements in stress-induced functional deficits. We also apply RNA sequencing analysis to the quadriceps of untreated and AAV-treated GAA-/- mice and wild-type controls to establish a Pompe disease-specific transcriptional signature and reveal novel disease pathways. The mouse-derived signature is enriched in the transcriptomic profile of IOPD vs. healthy myobundles and partially reversed by in vitro rhGAA treatment, further confirming the utility of the human myobundle model for studies of Pompe disease and therapy.