Muscle‐directed gene therapy corrects Pompe disease and uncovers species‐specific GAA immunogenicity
2021; Springer Nature; Volume: 14; Issue: 1 Linguagem: Inglês
10.15252/emmm.202113968
ISSN1757-4684
AutoresMichelle Eggers, Charles H. Vannoy, Jianyong Huang, Pravinkumar Purushothaman, Jacqueline Brassard, Carlos Fonck, Hui Meng, Mariah J. Prom, Michael W. Lawlor, Justine J. Cunningham, Chanchal Sadhu, Fulvio Mavilio,
Tópico(s)RNA regulation and disease
ResumoArticle1 December 2021Open Access Source DataTransparent process Muscle-directed gene therapy corrects Pompe disease and uncovers species-specific GAA immunogenicity Michelle Eggers Corresponding Author Michelle Eggers [email protected] orcid.org/0000-0001-5387-2118 Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Charles H Vannoy Charles H Vannoy orcid.org/0000-0003-3617-8081 Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Jianyong Huang Jianyong Huang Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Pravinkumar Purushothaman Pravinkumar Purushothaman Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Jacqueline Brassard Jacqueline Brassard orcid.org/0000-0002-3305-3163 Jacqueline Brassard Toxicologic Pathology Consulting, Tustin, CA, USA Search for more papers by this author Carlos Fonck Carlos Fonck Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Hui Meng Hui Meng Department of Pathology and Neuroscience Research Center, Medical College of Wisconsin, Milwaukee, WI, USA Search for more papers by this author Mariah J Prom Mariah J Prom Department of Pathology and Neuroscience Research Center, Medical College of Wisconsin, Milwaukee, WI, USA Search for more papers by this author Michael W Lawlor Michael W Lawlor Department of Pathology and Neuroscience Research Center, Medical College of Wisconsin, Milwaukee, WI, USA Search for more papers by this author Justine Cunningham Justine Cunningham Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Chanchal Sadhu Chanchal Sadhu Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Fulvio Mavilio Corresponding Author Fulvio Mavilio [email protected] orcid.org/0000-0003-0459-4320 Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Michelle Eggers Corresponding Author Michelle Eggers [email protected] orcid.org/0000-0001-5387-2118 Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Charles H Vannoy Charles H Vannoy orcid.org/0000-0003-3617-8081 Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Jianyong Huang Jianyong Huang Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Pravinkumar Purushothaman Pravinkumar Purushothaman Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Jacqueline Brassard Jacqueline Brassard orcid.org/0000-0002-3305-3163 Jacqueline Brassard Toxicologic Pathology Consulting, Tustin, CA, USA Search for more papers by this author Carlos Fonck Carlos Fonck Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Hui Meng Hui Meng Department of Pathology and Neuroscience Research Center, Medical College of Wisconsin, Milwaukee, WI, USA Search for more papers by this author Mariah J Prom Mariah J Prom Department of Pathology and Neuroscience Research Center, Medical College of Wisconsin, Milwaukee, WI, USA Search for more papers by this author Michael W Lawlor Michael W Lawlor Department of Pathology and Neuroscience Research Center, Medical College of Wisconsin, Milwaukee, WI, USA Search for more papers by this author Justine Cunningham Justine Cunningham Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Chanchal Sadhu Chanchal Sadhu Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Search for more papers by this author Fulvio Mavilio Corresponding Author Fulvio Mavilio [email protected] orcid.org/0000-0003-0459-4320 Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Author Information Michelle Eggers *,1, Charles H Vannoy1, Jianyong Huang1, Pravinkumar Purushothaman1, Jacqueline Brassard2, Carlos Fonck1, Hui Meng3, Mariah J Prom3, Michael W Lawlor3, Justine Cunningham1,†, Chanchal Sadhu1 and Fulvio Mavilio *,1,4 1Nonclinical, Pharmacology/Toxicology, Audentes Therapeutics, San Francisco, CA, USA 2Jacqueline Brassard Toxicologic Pathology Consulting, Tustin, CA, USA 3Department of Pathology and Neuroscience Research Center, Medical College of Wisconsin, Milwaukee, WI, USA 4Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy †Present address: Sana Biotechnology, South San Francisco, CA, USA *Corresponding author. Tel: +415 851 3437; E-mail: [email protected] *Corresponding author. Tel: +39 059 2055390; E-mail: [email protected] EMBO Mol Med (2021)e13968https://doi.org/10.15252/emmm.202113968 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pompe disease is a severe disorder caused by loss of acid α-glucosidase (GAA), leading to glycogen accumulation in tissues and neuromuscular and cardiac dysfunction. Enzyme replacement therapy is the only available treatment. AT845 is an adeno-associated viral vector designed to express human GAA specifically in skeletal muscle and heart. Systemic administration of AT845 in Gaa−/− mice led to a dose-dependent increase in GAA activity, glycogen clearance in muscles and heart, and functional improvement. AT845 was tolerated in cynomolgus macaques at low doses, while high doses caused anti-GAA immune response, inflammation, and cardiac abnormalities resulting in unscheduled euthanasia of two animals. Conversely, a vector expressing the macaque GAA caused no detectable pathology, indicating that the toxicity observed with AT845 was an anti-GAA xenogeneic immune response. Western blot analysis showed abnormal processing of human GAA in cynomolgus muscle, adding to the species-specific effects of enzyme expression. Overall, these studies show that AAV-mediated GAA delivery to muscle is efficacious in Gaa−/− mice and highlight limitations in predicting the toxicity of AAV vectors encoding human proteins in non-human species. SYNOPSIS Muscle-restricted expression of GAA via systemic AAV gene therapy clears glycogen accumulation and improves function in a mouse model of Pompe disease. Xenogeneic immune responses in non-human primates highlight limitations of animal studies to assess toxicity of vectors encoding human proteins. AT845 is an adeno-associated viral vector designed to express human GAA specifically in skeletal muscle and heart. Systemic administration of AT845 in Gaa−/− mice led to a dose-dependent increase in GAA expression and activity, glycogen clearance in skeletal and cardiac muscles, and functional improvement. In non-human primates, AT845 caused a dose-dependent increase of GAA activity in skeletal muscle and heart. Our results suggest that adverse reactions observed in non-human primates with human GAA were due to an anti-GAA xenogeneic immune response rather than GAA overexpression. The paper explained Problem The current treatment for Pompe disease, enzyme replacement therapy, is limited by the short half-life of the enzyme, insufficient uptake by muscle and heart tissues, and immunogenicity. In addition, immunogenicity of the recombinant enzyme represents a formidable challenge in CRIM-negative patients, for whom therapeutic efficacy is limited even at higher doses and with more intense administration schedules. Gene therapy holds the promise of an efficacious treatment for metabolic diseases such as Pompe disease. Results We developed a systemic gene therapy, AT845, designed to reconstitute human GAA synthesis and activity directly in skeletal muscle and heart. Systemic administration of AT845 led to a robust, dose-dependent increase in GAA activity at therapeutic, supraphysiological levels in both mice and non-human primates, resulting in glycogen clearance and functional improvement in a mouse model of Pompe disease. Toxicity observed at high doses in non-human primates was due to an anti-GAA xenogeneic immune response. Impact Overall, our results show that AT845 leads to a robust, dose-dependent increase in GAA activity at therapeutic, supraphysiological levels in a Pompe mouse model and non-human primates, resulting in glycogen clearance and functional improvement in the Pompe mouse model. Lack of immune responses and toxicity in non-human primates tolerant to the native enzyme supports progression of AT845 to clinical testing. A clinical trial aimed at assessing safety and efficacy of AT845 in patients with CRIM-positive late-onset Pompe disease (Clinical trial identifier: NCT04174105) is activated in the United States and is currently recruiting patients. Introduction Glycogen storage disease type II (GSD-II, OMIM#232300), or Pompe disease (PD), is an autosomal recessive disorder caused by mutations in the lysosomal enzyme acid α-glucosidase (GAA). GAA deficiency causes lysosomal dysfunction, glycogen accumulation in all tissues, muscle weakness, cardiac and respiratory insufficiency, and neurological abnormalities (van der Ploeg & Reuser, 2008). The clinical manifestations of PD vary from the severe, often lethal infantile-onset form (IOPD), associated with < 1% residual GAA activity, to the less severe, late-onset forms (LOPD), characterized by GAA activity up to 20% of normal levels. The only approved treatment for PD is enzyme replacement therapy (ERT), which consists of systemic administration of recombinant human GAA (Kishnani & Beckemeyer, 2014). ERT can extend lifespan in IOPD and slow down or stabilize progression in LOPD (Kishnani et al, 2007; van der Ploeg et al, 2010; Schoser et al, 2017), but its therapeutic efficacy is limited by the short half-life of the enzyme, insufficient uptake by muscle and heart tissues, and immunogenicity, which elicits a neutralizing antibody response in GAA-null (CRIM-negative) patients (Kishnani & Beckemeyer, 2014). In addition, glycogen accumulation impairs lysosomal function, progressively leading to autophagic buildup in muscle fibers that perturbs intracellular trafficking and further reduces exogenous enzyme uptake by receptor-mediated endocytosis (Fukuda et al, 2006a, 2006b). Gene therapy holds the promise of an efficacious treatment for metabolic diseases (Poletti & Biffi, 2019) and particularly for PD (Colella & Mingozzi, 2019; Salabarria et al, 2020). Gene replacement by local or systemic delivery of adeno-associated viral (AAV) vectors of different serotypes expressing GAA under different promoters showed correction of biochemical and functional parameters in a Gaa−/− knockout (KO) mouse model (Fraites et al, 2002; Mah et al, 2005; Sun et al, 2005, 2008; Elmallah et al, 2014; Falk et al, 2015). A Phase I/II clinical trial supported the safety of local delivery of an rAAV1-CMV-GAA vector to the diaphragm, an approach aimed at ameliorating respiratory insufficiency in affected IOPD patients (Byrne et al, 2014; Smith et al, 2017). A local delivery approach has also been proposed for skeletal muscle, using an AAV9 vector expressing GAA under the control of the muscle-restricted desmin promoter (Corti et al, 2015). However, local AAV delivery has major drawbacks, such as incomplete correction of the disease manifestations due to poor cross-correction among muscle fibers, and elicitation of a neutralizing antibody response (Mah et al, 2010; Elmallah et al, 2014). AAV-mediated delivery to muscle through a systemic route of administration also raised an anti-GAA humoral response in GAA-null mice (Sun et al, 2005, 2008; Falk et al, 2015; Doerfler et al, 2016), although it is unclear whether neutralizing antibodies would affect intra-fiber GAA synthesis and activity if an antibody response occurred in CRIM-negative patients (Colella & Mingozzi, 2019). An alternative gene therapy approach to PD is based on systemic delivery of an AAV vector expressing GAA under the control of a liver-specific promoter (Han et al, 2017; Puzzo et al, 2017). Liver-directed synthesis and secretion of GAA could provide a stable source of active enzyme in the circulation, overcoming the pharmacokinetic limitations of ERT, although its availability would remain limited by tissue uptake and may also be affected by neutralizing antibodies. We developed an AAV8 vector encoding the human GAA protein under the control of a muscle-restricted promoter/enhancer element (AT845), designed to reconstitute human GAA synthesis and activity directly in skeletal muscle and heart. Gaa−/− mice treated with AT845 by systemic administration showed dose-dependent reconstitution of GAA expression and activity, glycogen clearance in skeletal and cardiac muscles, and functional improvements as early as 3 months after treatment. GAA activity in murine muscle reached physiological levels at the lowest dose tested (i.e., 3 × 1013 vector genomes per kilogram of body weight [vg/kg]). A toxicology study conducted in cynomolgus macaque non-human primates (NHPs) in the absence of immunosuppression showed anti-human GAA humoral immune response and elevation of liver enzymes and cardiac biomarkers. Inflammatory infiltrates and functional heart abnormalities were observed at the highest doses tested (> 1014 vg/kg). However, animals treated with a vector expressing cynomolgus macaque GAA showed no sign of toxicity nor immune responses at a comparable dose, indicating that the toxicity observed with AT845 was due to an anti-GAA xenogeneic immune response and not GAA expression per se. Overall, our preclinical data show that AT845 is well tolerated when a species-specific transgene is used and leads to a robust, dose-dependent increase in GAA activity at therapeutic, supraphysiological levels in both mice and NHPs and to glycogen clearance and functional improvement in GAA-deficient mice. Lack of immune responses and toxicity in NHPs tolerant of the native enzyme supports the progression of AT845 to clinical testing in CRIM-positive PD patients. Results Systemic administration of AT845 shows dose-dependent correction of GAA expression, enzymatic activity, and glycogen clearance in GAA-deficient mice AT845 (rAAV8-eMCK-hGAA) is a serotype-8 adeno-associated viral (AAV8) vector that expresses a codon-optimized cDNA of the human acid alpha-glucosidase gene (GAA) under the control of a murine muscle creatine kinase (MCK) promoter/enhancer combination (Fig EV1). We assessed the in vivo efficacy of AT845 by systemic administration in a Gaa KO mouse model of PD (B6;129-Gaatm1Rabn/J, hereinafter referred to as Gaa−/−). In this study, 72 Gaa−/− mice (nine males + nine females per group) were treated by a single intravenous (IV) injection at 10–11 weeks of age with either vehicle (Ringer's lactate solution) or AT845 at doses of 3 × 1013 vg/kg (low dose), 1 × 1014 vg/kg (mid-dose), or 3 × 1014 vg/kg (high dose). Mice were followed for 12 weeks after dosing. Additionally, 18 (nine males and nine females) 10- to 11-week-old wild-type mice (Gaa+/+ littermates) were treated with the vehicle as an additional control cohort. Click here to expand this figure. Figure EV1. Structure of the rAAV8-eMCK-hGAA and rAAV8-eMCK-cyno-GAA vectors Scale is approximate. MCK enhancer: murine MCK enhancer element; MCK promoter: murine MCK promoter; Cynomolgus GAA: cynomolgus acid alpha-glucosidase cDNA; Human GAA: human acid alpha-glucosidase cDNA; SV40 PolyA: SV40 late polyadenylation signal sequence. Download figure Download PowerPoint To evaluate the transduction efficiency of AT845 in the cardiac and skeletal muscles, we measured AAV vector genome copy numbers (VCN) per diploid genome (vg/dg) by a real-time PCR test in multiple tissues collected from animals euthanized at 12 weeks post-treatment. We observed a dose-dependent increase in VCN in all tested muscle samples with the highest values in the heart (25.2 ± 12.7 vg/dg) and quadriceps (15.1 ± 10.6 vg/dg) at the highest dose (Fig 1A), which translated into a dose-dependent increase in human GAA protein levels in the same tissues (Fig 1B). GAA enzymatic activity in heart, quadriceps, and diaphragm also increased in a dose-dependent fashion in treated animals when compared to the same tissues from vehicle-treated Gaa−/− mice (Fig 1C). GAA activity levels in AT845-treated Gaa−/− mice exceeded endogenous levels measured in vehicle-treated wild-type littermates in the heart and quadriceps at doses ≥ 3 × 1013 vg/kg and in the diaphragm at doses ≥ 1 × 1014 vg/kg. Figure 1. AAV-mediated gene transfer of an engineered GAA transgene to Gaa−/− mice exhibits dose-dependent transduction efficiency, increases in GAA activity, and clearance of accumulated glycogen in cardiac and skeletal muscle A–D. 10- to 11-week-old mice were treated with vehicle control or AT845 at the vector doses indicated and followed for 12 weeks (N = 7 or 8 per cohort). (A) Vector copy number, (B) GAA levels, (C) GAA activity, and (D) glycogen content in cardiac and skeletal muscles. (C, D) Statistical analysis: two-way ANOVA, Dunnett's test. Data information: Data are presented as box-and-whisker plots with Tukey's whiskers that show minimum, median, and maximum values. Asterisks (*) indicate significant differences compared with untreated Gaa−/− mice (black) or wild-type mice (red). *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. See also Appendix Table S1. Source data are available online for this figure. Source Data for Figure 1 [emmm202113968-sup-0002-SDataFig1.xls] Download figure Download PowerPoint Increases in GAA activity correlated with a significant, dose-dependent reduction in glycogen accumulation in the heart, quadriceps, and diaphragm by 12 weeks after dosing (Fig 1D). The lowest dose of AT845 (3 × 1013 vg/kg) resulted in partial but significant glycogen reduction in the heart, diaphragm, and quadriceps muscles isolated from treated mice when compared to vehicle-treated Gaa−/− mice, while the mid-dose of 1 × 1014 vg/kg resulted in robust reduction of glycogen accumulation in the cardiac and skeletal muscles, with values approaching those of wild-type littermates (Appendix Table S1). At the highest dose of 3 × 1014 vg/kg, AT845 treatment normalized intracellular glycogen levels in the cardiac and skeletal muscles: Most heart samples tested with a minimal residual disease assay resulted in glycogen levels that were below the lower limit of quantitation (LLOQ, Appendix Table S1). Overall, these results indicate that AT845-driven synthesis of physiological or supraphysiological levels of GAA reverts glycogen accumulation in the muscles of GAA-deficient mice at doses ≥ 1014 vg/kg. Administration of AT845 improves skeletal muscle pathology and function in Gaa−/− mice On histological examination, quadriceps muscles from vehicle-treated Gaa−/− mice exhibited minimal-to-mild myofiber vacuolation and minimal-to-mild degenerative and active regenerative changes after hematoxylin and eosin (H&E) staining (Fig 2, top panels). Periodic acid–Schiff (PAS) staining demonstrated abundant, densely stained pink granules, consistent with aggregated glycogen (Fig 2, bottom panels). AT845-treated Gaa−/−mice showed dose-dependent reduction in glycogen accumulation in the quadriceps muscles, accompanied by a lack of or marked decrease in the vacuolar, degenerative, and active regenerative changes observed in vehicle-treated animal muscles (histopathology results are described below). Reduction in glycogen staining was partial at the lower dose and virtually complete at mid- and high doses (Fig 2), in accordance with the reduction in glycogen content measured by the biochemical assay (Fig 1D). Figure 2. AAV-mediated transfer of an engineered GAA transgene improves histopathology and exhibits a reduction in glycogen content in Gaa−/− mice Representative images of H&E (top) and PAS (bottom) staining of the quadriceps in wild-type and Gaa−/− mice untreated or treated with escalating doses of AT845 (3 × 1013, 1 × 1014, and 3 × 1014 vg/kg). Scale bar, 80 µm (see also Appendix Table S1). Download figure Download PowerPoint Gaa−/− mice treated with AT845 at all doses displayed normal weight gain over the time course of the in-life observation period that was comparable to vehicle-treated wild-type littermates (Fig 3A and B). There were no treatment-related differences in mean body weight values. The treated groups had comparable mean body weight values as the wild-type and Gaa−/− vehicle control groups, although some fluctuations were noted in all treated male groups that reached statistical significance. However, treated mean values were within 10% of wild-type controls, and there were no appreciable differences at the end of the 12-week observation period. It should be noted that vehicle-treated Gaa−/− mice also showed an almost normal growth curve, which likely reflects the moderate overall pathology of this animal model. To assess muscle function, we tested the ability of each mouse to grip onto an inverted wire screen for a 60-s period at multiple time points throughout the in-life portion of the study, including predosing and 6 and 12 weeks after AT845 administration. This test evaluates both isotonic and isometric muscle strength and sensory motor coordination, since it requires mice to reflexively respond to a change in the surface orientation on which they are placed. On average, wild-type littermates were able to successfully grip onto the inverted screen for the entire 60-s testing time throughout the observation period, while vehicle-treated Gaa−/− mice showed a progressive decrease in their grip response that was more apparent in males than in females (Fig 3C and D). By Week 12, Gaa−/− mice failed to grip onto the wire for periods longer than ˜3 s. On the contrary, Gaa−/− mice treated with AT845 showed a dose-dependent improvement in grip response that was apparent as early as 6 weeks after dosing and further improved by Week 12. Low-dose- and mid-dose-treated Gaa−/− mice showed partial recovery, resulting in ˜50% and ˜75% of the mice, respectively, improving their grip response. Interestingly, the response was more robust in female than in male animals at low and mid-doses (Fig 3C and D), while both male and female mice treated at the high dose showed complete recovery in grip response and were almost indistinguishable from wild-type littermates at Week 12 (Fig 3C and D). Additional analyses of VCN, GAA, and glycogen data did not provide an explanation for the better performance of females in grip response at the low and mid-dose. Together, these data demonstrate that administration of AT845 leads to a dose-dependent reduction in glycogen accumulation in skeletal muscle, normalization of histological parameters, and preservation of muscle strength in Gaa−/− mice. Figure 3. Dose-dependent functional correction in Gaa−/− mice A–D. (A, B) Body weight curves and (C, D) grip response—ability to grip onto an inverted wire screen for a 60-s period—in wild-type and Gaa−/− mice untreated or treated with escalating doses of AT845 (3 × 1013, 1 × 1014, and 3 × 1014 vg/kg). All groups had n = 9 at all time points, except the 6-week time point (females) and the 12-week time point (males and females) for the Gaa−/− mice, which had n = 8 per group. Female mice dosed with 3 × 1013, 1 × 1014, and 3 × 1014 vg/kg had a 22, 11, and 0% failure rate at Week 12, respectively; in comparison, the female control WT mice had a 22% failure rate and the control Gaa−/− mice had a 38% failure. Male mice dosed with 3 × 1013, 1 × 1014, and 3 × 1014 vg/kg had a 56, 33, and 11% failure rate at Week 12, respectively; in comparison, the male control WT mice had a 11% failure rate and the control Gaa−/− mice had a 100% failure. Bars represent median. Data information: Data show a dose-dependent improvement in grip response following treatment with AT845. Statistical analysis: two-way ANOVA, Dunnett's test. Asterisks (*) indicate significant differences compared with untreated Gaa−/− mice at each respective time point. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. Source data are available online for this figure. Source Data for Figure 3 [emmm202113968-sup-0003-SDataFig3.xls] Download figure Download PowerPoint Mouse histopathology Intravenous injection of AT845 to Pompe mice at doses of 0 (vehicle), 3 × 1013, 1 × 1014, or 3 × 1014 vg/kg resulted in test article-related microscopic findings in the heart, skeletal muscles, and liver. Two Gaa−/− mice in the vehicle group died during the study. In the remaining vehicle-treated GAA−/− mice (eight males and eight females), H&E-stained sections of formalin-fixed skeletal muscle were characterized by the presence of numerous cytoplasmic vacuoles in myofibers that correlated with numerous, densely stained periodic acid–Schiff (PAS)-positive cytoplasmic granules in frozen skeletal muscle. Additionally, there was mild-to-moderate myofiber degeneration and regeneration in H&E-stained sections that were consistent with ongoing myofiber injury and repair. Relative to Pompe vehicle control mice, Pompe AT845-treated mice (nine males and nine females per cohort) had lower heart weights at 3 × 1014 vg/kg and dose-dependent reduction or absence of cardiac myofiber vacuolation at ≥ 3 × 1013 vg/kg. Additionally, AT845-treated Pompe mice had dose-dependent decreased incidence and severity or absence of myofiber vacuolation at ≥ 3 × 1013 vg/kg in skeletal muscle myofibers in H&E-stained sections and in myofiber cytoplasmic glycogen accumulations in PAS-stained sections (Fig 2). These microscopic observations were in agreement with the decrease in glycogen content measured by the biochemical assay (Fig 1D). Systemic delivery of AT845 leads to robust GAA synthesis and activity in skeletal and heart muscle of NHPs To investigate the tolerability, potential toxicity, and safety pharmacology of AT845, we dosed juvenile cynomolgus NHPs at escalating doses via a single IV injection. A total of 22 NHPs with low anti-AAV8-neutralizing antibody titers (< 80 for controls, < 20 for AT845 dosed animals) were enrolled in the study. Six NHPs (three males and three females) per dose cohort were administered AT845 at doses of 6 × 1013 vg/kg (low dose), 2 × 1014 vg/kg (mid-dose), or 5 × 1014 vg/kg (high dose). Given that wild-type NHPs physiologically express GAA in all tissues, we used four control NHPs (two males and two females) injected with vehicle (Ringer's lactate solution with 0.01% poloxamer 188) as a reference for endogenous GAA activity. All NHPs were followed for 12 weeks. Biodistribution of AT845 across treatment cohorts was assessed by measuring VCN in heart, quadriceps, diaphragm, liver, brain, spinal cord, sciatic nerve, kidney, lung, testes, and ovaries (Fig 4A and Appendix Fig S1). At the high dose of 5 × 1014 vg/kg, VCN was highest in the liver (998 ± 324 vg/dg) and lowest in the heart (12.6 ± 6.4 vg/dg), diaphragm (5.9 ± 2.6 vg/dg), and quadriceps (5.8 ± 4.3 vg/dg). VCN decreased in all tissues at decreasing doses, with heart, diaphragm, and quadriceps exhibiting 3.0 ± 1.0, 1.5 ± 0.6 and 1.5 ± 1.0 vg/dg at the lowest dose, respectively. Accordingly, GAA activity exhibited a dose-dependent increase in the cardiac and skeletal muscle acquired from NHPs treated with AT845 compared with vehicle-treated animals (Fig 4B and Appendix Fig S1). More specifically, GAA activity in NHPs treated at the highest dose was 60- to 100-fold higher than values observed in vehicle-treated animals in all tissues analyzed, which included the heart (P < 0.001), quadriceps (P < 0.001), and diaphragm (P < 0.01). GAA activity was detected at background endogenous levels in liver, central nervous system (CNS), and reproductive systems at all doses, confirming the tight restriction in gene expression provided by the MCK enhancer/promoter elements (Fig 4B and Appendix Fig S1). Furthermore, quantitative analysis of mRNA expression from the endogenous cynomolgus GAA gene and the AT845-carried human GAA transgene by RNA sequencing showed a dose-dependent increase in human GAA mRNA in heart and quadriceps, and levels below endogenous GAA mRNA in liver and spinal cord (Fig EV2). At the highest dose, AT845-derived GAA mRNA accumulated at 93- and 16-fold higher levels than endogenous GAA mRNA in the heart and quadriceps, respectively. Figure 4. Dose-dependent increase in transduction level and GAA activity in NHPs A, B. Equal numbers of male and female cynomolgus monkeys were treated with vehicle control (n = 4) or escalating doses AT845 (n = 6) and followed for approximately 12 weeks. (A) Vector copy number and (B) GAA activity in heart, quadriceps, triceps, diaphragm, and liver. Statistical analysis: mixed-effects analysis with Dunnett's test. Data information: Data are presented as box-and-whisker plots with Tukey whiskers that show minimum,
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