Cell type-selective targeted delivery of a recombinant lysosomal enzyme for enzyme therapies
Authors: Andrew D. Baik, Philip Calafati, Xiaoli Zhang, Nina A. Aaron, Antonia Mehra, Sven Moller-Tank, Lawrence Miloscio, Maria Praggastis, Nicholas Giovannone, Cheryl Pan, Yajun Tang, Susannah Bridges, Alejo Mujica, Peter Barbounis, Jean Yanolatos, Nicholas Gale, Ning Li, Christos A. Kyratsous, Christopher J. Schoenherr, Andrew J. Murphy, Aris N. Economides, Katherine D. Cygnar
Journal: Molecular Therapy (2021)

Lysosomal diseases are a class of genetic disorders predominantly caused by loss of lysosomal hydrolases, leading to lysosomal and cellular dysfunction. Enzyme replacement therapy (ERT), where recombinant enzyme is given intravenously, internalized by cells, and trafficked to the lysosome, has been applied to treat several lysosomal diseases. However, current ERT regimens do not correct disease phenotypes in all affected organs because the biodistribution of enzyme uptake does not match that of the affected cells that require the enzyme. We present here targeted ERT, an approach that utilizes antibody-enzyme fusion proteins to target the enzyme to specific cell types. The antibody moiety recognizes transmembrane proteins involved in lysosomal trafficking and that are also preferentially expressed in those cells most affected in disease. Using Pompe disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of PD mice both as a protein replacement therapeutic and as a gene therapy. Lysosomal diseases are a class of genetic disorders predominantly caused by loss of lysosomal hydrolases, leading to lysosomal and cellular dysfunction. Enzyme replacement therapy (ERT), where recombinant enzyme is given intravenously, internalized by cells, and trafficked to the lysosome, has been applied to treat several lysosomal diseases. However, current ERT regimens do not correct disease phenotypes in all affected organs because the biodistribution of enzyme uptake does not match that of the affected cells that require the enzyme. We present here targeted ERT, an approach that utilizes antibody-enzyme fusion proteins to target the enzyme to specific cell types. The antibody moiety recognizes transmembrane proteins involved in lysosomal trafficking and that are also preferentially expressed in those cells most affected in disease. Using Pompe disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of PD mice both as a protein replacement therapeutic and as a gene therapy. IntroductionPompe disease (PD), or glycogen storage disease type II, is a monogenic, lysosomal disease caused by a deficiency in the activity of the enzyme lysosomal acid alpha-glucosidase (GAA). GAA deficiency results in an accumulation of its substrate, glycogen, in the lysosomes of cells in tissues including skeletal and cardiac muscle. This aberrant accumulation of glycogen in myofibers results in progressive damage of muscle tissue, with symptoms that can include cardiomegaly, mild to profound muscle weakness, and ultimately death due to cardiac or respiratory failure1Hirschhorn R. Reuser A. Glycogen Storage Disease Type II: Acid Alpha-glucosidase (acid maltase) Deficiency.in: The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2000: 3389-3420Google Scholar.As is the case for several other lysosomal diseases, PD is currently treated by enzyme replacement therapy (ERT). For PD, recombinant human (rh) GAA is delivered by intravenous infusion into patients every other week. While ERT has been very successful in treating the cardiac manifestations of PD, skeletal muscle and the central nervous system (CNS) remain minimally treated by ERT.2Prater S.N. Banugaria S.G. DeArmey S.M. Botha E.G. Stege E.M. Case L.E. Jones H.N. Phornphutkul C. Wang R.Y. Young S.P. Kishnani P.S. The emerging phenotype of long-term survivors with infantile Pompe disease.Genet. Med. 2012; 14: 800-810Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 3Chien Y.-H. Lee N.-C. Chen C.-A. Tsai F.-J. Tsai W.-H. Shieh J.-Y. Huang H.-J. Hsu W.-C. Tsai T.-H. Hwu W.-L. Long-Term Prognosis of Patients with Infantile-Onset Pompe Disease Diagnosed by Newborn Screening and Treated since Birth.J. Pediat. 2015; 166 (e1-2): 985-991Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 4DeRuisseau L.R. Fuller D.D. Qiu K. DeRuisseau K.C. Donnelly Jr., W.H. Mah C. Reier P.J. Byrne B.J. Neural deficits contribute to respiratory insufficiency in Pompe disease.Proc. Natl. Acad. Sci. USA. 2009; 106: 9419-9424Crossref PubMed Scopus (136) Google ScholarInadequate delivery of rhGAA to skeletal muscle has been suggested as the main reason ERT fails to treat this organ system.5Koeberl D.D. Luo X. Sun B. McVie-Wylie A. Dai J. Li S. Banugaria S.G. Chen Y.T. Bali D.S. Enhanced efficacy of enzyme replacement therapy in Pompe disease through mannose-6-phosphate receptor expression in skeletal muscle.Mol. Genet. Metab. 2011; 103: 107-112Crossref PubMed Scopus (57) Google Scholar The primary mechanism by which rhGAA reaches lysosomes is through uptake by the cation-independent mannose 6-phosphate (M6P) receptor (CI-MPR), which binds M6P on rhGAA. However, CI-MPR expression in skeletal muscle is very low, and rhGAA is poorly mannose 6-phosphorylated.6McVie-Wylie A.J. Lee K.L. Qiu H. Jin X. Do H. Gotschall R. Thurberg B.L. Rogers C. Raben N. O'Callaghan M. et al.Biochemical and pharmacological characterization of different recombinant acid alpha-glucosidase preparations evaluated for the treatment of Pompe disease.Mol. Genet. Metab. 2008; 94: 448-455Crossref PubMed Scopus (74) Google Scholar,7Do H.V. Khanna R. Gotschall R. Challenges in treating Pompe disease: an industry perspective.Ann. Transl. Med. 2019; 7: 291Crossref PubMed Google Scholar In addition, CI-MPR may be misdirected into autophagosomes in affected cells, rather than lysosomes,8Fukuda T. Ahearn M. Roberts A. Mattaliano R.J. Zaal K. Ralston E. Plotz P.H. Raben N. Autophagy and mistargeting of therapeutic enzyme in skeletal muscle in Pompe disease.Mol. Ther. 2006; 14: 831-839Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar while a large amount of the drug is also taken up by the liver (an organ that does not have primary pathology in PD). To overcome these problems, rhGAA is given at a high and frequent dosing (20–40 mg/kg once every 2 weeks); nonetheless, difficulties in treating skeletal muscle persist. Strategies to modify rhGAA to increase delivery to muscle by manipulating the affinity of GAA to CI-MPR, such as increasing the M6P content of GAA9Zhu Y. Jiang J.-L. Gumlaw N.K. Zhang J. Bercury S.D. Ziegler R.J. Lee K. Kudo M. Canfield W.M. Edmunds T. et al.Glycoengineered acid alpha-glucosidase with improved efficacy at correcting the metabolic aberrations and motor function deficits in a mouse model of Pompe disease.Mol. Ther. 2009; 17: 954-963Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar or using the IGF2 binding sites on CI-MPR10Maga J.A. Zhou J. Kambampati R. Peng S. Wang X. Bohnsack R.N. Thomm A. Golata S. Tom P. Dahms N.M. et al.Glycosylation-independent lysosomal targeting of acid α-glucosidase enhances muscle glycogen clearance in pompe mice.J. Biol. Chem. 2013; 288: 1428-1438Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar have been explored in the clinic. Still, these methods fail to address the underlying inherent limitations of using CI-MPR as the mechanism for uptake, i.e., CI-MPR's low level of muscle expression and its largely endosomal rather than surface localization in skeletal muscle, which, combined with high expression in the liver, result in very poor uptake by muscle.11Raben N. Danon M. Gilbert A.L. Dwivedi S. Collins B. Thurberg B.L. Mattaliano R.J. Nagaraju K. Plotz P.H. Enzyme replacement therapy in the mouse model of Pompe disease.Mol. Genet. Metab. 2003; 80: 159-169Crossref PubMed Scopus (176) Google Scholar,12Coutinho M.F. Prata M.J. Alves S. Mannose-6-phosphate pathway: a review on its role in lysosomal function and dysfunction.Mol. Genet. Metab. 2012; 105: 542-550Crossref PubMed Scopus (142) Google ScholarAdeno-associated virus (AAV)-based liver depot gene therapies have shown promise to allow for continuous-dosing ERT by infecting hepatocytes in vivo to produce and secrete GAA.13Puzzo F. Colella P. Biferi M.G. Bali D. Paulk N.K. Vidal P. Collaud F. Simon-Sola M. Charles S. Hardet R. et al.Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid α-glucosidase.Sci. Transl. Med. 2017; 9: eaam6375Crossref PubMed Scopus (66) Google Scholar,14Sun B. Zhang H. Franco L.M. Young S.P. Schneider A. Bird A. Amalfitano A. Chen Y.T. Koeberl D.D. Efficacy of an adeno-associated virus 8-pseudotyped vector in glycogen storage disease type II.Mol. Ther. 2005; 11: 57-65Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar Additionally, liver depot gene therapy of GAA has been shown to tolerize GAA knockout (KO) mice to GAA and mitigate the antibody immune response to GAA in the serum.15Franco L.M. Sun B. Yang X. Bird A. Zhang H. Schneider A. Brown T. Young S.P. Clay T.M. Amalfitano A. et al.Evasion of immune responses to introduced human acid alpha-glucosidase by liver-restricted expression in glycogen storage disease type II.Mol. Ther. 2005; 12: 876-884Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar,16Han S.-o. Ronzitti G. Arnson B. Leborgne C. Li S. Mingozzi F. Koeberl D. Low-Dose Liver-Targeted Gene Therapy for Pompe Disease Enhances Therapeutic Efficacy of Ert Via Immune Tolerance Induction.Mol. Ther. Methods Clin. Dev. 2017; 4: 126-136Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar However, while the immune response was dampened, correction of skeletal muscle glycogen accumulation was only achieved at very high serum levels of GAA,17Ziegler R.J. Bercury S.D. Fidler J. Zhao M.A. Foley J. Taksir T.V. Ryan S. Hodges B.L. Scheule R.K. Shihabuddin L.S. Cheng S.H. Ability of adeno-associated virus serotype 8-mediated hepatic expression of acid alpha-glucosidase to correct the biochemical and motor function deficits of presymptomatic and symptomatic Pompe mice.Hum. Gene Ther. 2008; 19: 609-621Crossref PubMed Scopus (47) Google Scholar likely a reflection of the continued reliance on CI-MPR for uptake by skeletal muscle. It was also unclear what proportion of hepatically secreted GAA is mannose 6-phosphorylated, as overexpression of GAA in other cell systems has been shown to adversely affect M6P content of GAA.6McVie-Wylie A.J. Lee K.L. Qiu H. Jin X. Do H. Gotschall R. Thurberg B.L. Rogers C. Raben N. O'Callaghan M. et al.Biochemical and pharmacological characterization of different recombinant acid alpha-glucosidase preparations evaluated for the treatment of Pompe disease.Mol. Genet. Metab. 2008; 94: 448-455Crossref PubMed Scopus (74) Google Scholar,18Zhu Y. Li X. Kyazike J. Zhou Q. Thurberg B.L. Raben N. Mattaliano R.J. Cheng S.H. Conjugation of mannose 6-phosphate-containing oligosaccharides to acid alpha-glucosidase improves the clearance of glycogen in pompe mice.J. Biol. Chem. 2004; 279: 50336-50341Abstract Full Text Full Text PDF PubMed Scopus (64) Google ScholarTherefore, the current state of the art is one of only incremental progress despite continued attempts at improving ERT. Using PD as an example, we present here targeted ERT, a new technology that addresses the delivery and biodistribution limitations of ERT by employing antibody-enzyme fusion proteins as the drug and testing them in PD mice. In targeted ERT, the antibody moiety targets the enzyme, GAA, to the affected cells by binding to transmembrane proteins that are both trafficked to the lysosome and preferentially expressed on the cell type of interest, thereby achieving superior clearance of the substrate—glycogen—compared to non-targeted GAA. We demonstrate the superior efficacy of this technology by comparing purified targeted GAA protein to GAA alone. We also adapt targeted ERT to gene therapy, using AAV-mediated gene delivery to the liver, in order to provide long-term efficacy while also alleviating the need for repeated dosing. This positions targeted ERT as a potential modality that can be adopted in emerging gene therapy-based treatments for lysosomal diseases (LDs).ResultsAntibody:GAA fusion proteins display enzymatic activity equivalent to GAAIn order to address the delivery limitations associated with rhGAA ERT—i.e., loss of drug to the liver and poor delivery to skeletal muscle—we chose to bypass CI-MPR-mediated delivery of GAA and explore antibody-enzyme fusion proteins as a means to target the enzyme to the affected cells and tissues. These fusion proteins have to display the following properties:1Retain enzymatic function.2Bind to effector proteins that traffic to the lysosome, most likely transmembrane or cell surface proteins that are also preferentially expressed in the cells most affected in disease, while displaying minimal or no expression in non-affected cells and acting independently of CI-MPR.In order to identify proteins with the desired properties specifically for GAA ERT, we screened an internal database of gene expression profiles and defined a set of transmembrane and cell surface proteins that are expressed in skeletal muscles, and which are minimally expressed in the liver. We then looked among them for those with monoclonal antibodies readily available. Two of these proteins, CD63 (Figures S1A, S1B, and Supplemental Methods) and ITGA7,19Uhlén M. Fagerberg L. Hallström B.M. Lindskog C. Oksvold P. Mardinoglu A. Sivertsson Å. Kampf C. Sjöstedt E. Asplund A. et al.Proteomics. Tissue-based map of the human proteome.Science. 2015; 347: 1260419Crossref PubMed Scopus (6810) Google Scholar met all three criteria. Furthermore, CD63 was already known to traffic between the cell surface and lysosomes,20Sincock P.M. Mayrhofer G. Ashman L.K. Localization of the transmembrane 4 superfamily (TM4SF) member PETA-3 (CD151) in normal human tissues: comparison with CD9, CD63, and alpha5beta1 integrin.J. Histochem. Cytochem. 1997; 45: 515-525Crossref PubMed Scopus (170) Google Scholar,21Pols M.S. Klumperman J. Trafficking and function of the tetraspanin CD63.Exp. Cell Res. 2009; 315: 1584-1592Crossref PubMed Scopus (446) Google Scholar suggesting that it could be used as a benchmark to compare other potential effectors.Starting with CD63, we engineered antibody:GAA fusions where the C terminus of full-length immunoglobulin G (IgG)4 antibody or a single-chain fragment variable (scFv) was fused to the N terminus of amino acids 70–952 of GAA with a glycine-serine linker (Figure S1C), expressed, and purified. The enzymatic activity of the IgG:GAA or scFv:GAA purified fusion proteins was found to be comparable to alglucosidase alfa (recombinant GAA). Three different anti-CD63 variable domains were used, one being derived from immunized wild-type mice (denoted as α-hCD631), one derived from immunized mice that produce human antibodies (denoted as α-hCD632),22Murphy A.J. Macdonald L.E. Stevens S. Karow M. Dore A.T. Pobursky K. Huang T.T. Poueymirou W.T. Esau L. Meola M. et al.Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice.Proc. Natl. Acad. Sci. USA. 2014; 111: 5153-5158Crossref PubMed Scopus (240) Google Scholar and finally an anti-mouse CD63 variable domain derived from rat (denoted as α-mCD63). The binding dissociation constant (KD) between the two human antibody variable domains to the extracellular loop 2 of hCD63 was found to be in the nanomolar to subnanomolar range (Figure S1E). The KD of the anti-hCD632 antibody was also measured when formatted into IgG:GAA and scFv:GAA formats (diagrams in Figure S1C), and the binding affinity to hCD63 remained in the nanomolar range.Internalization of antibody:GAA fusions is an antibody-dependent, CI-MPR-independent processTo verify the antibody-mediated properties of the antibody:GAA fusions, the α-hCD631 IgG:GAA fusion was incubated with HEK293 cells overnight to allow for intracellular uptake. Dose-dependent cellular uptake was observed, while a non-binding antibody:GAA control showed no uptake in these cells. Furthermore, α-hCD631 IgG:GAA did not internalize in HEK293 cells that are deficient for hCD63. These results demonstrated that α-hCD631 IgG:GAA internalized specifically through the effector (CD63) recognized by the antibody domain of the fusion protein (Figure 1A). α-hCD631 IgG:GAA was also assayed for internalization in human primary myoblasts (Figure 1B). Uptake saturated at ∼50 nM and was not inhibited by the presence of 5 mM M6P, a competitive inhibitor for CI-MPR binding. The ability of 5 mM M6P to inhibit CI-MPR-mediated uptake of GAA was verified in the same cell line. A glycan analysis (Table S1 and Supplemental methods) of α-hCD631 IgG:GAA showed that the glycan structures were mainly terminally sialylated and mannosylated, and terminal M6P was not detected on any N-glycan sites of GAA.To explore whether the properties observed with α-hCD63:GAA are shared when other effectors are utilized, we tested an α-ITGA7 IgG:GAA fusion protein. This targets mouse integrin alpha-7, a surface protein preferentially expressed in muscle cells.23Song W.K. Wang W. Sato H. Bielser D.A. Kaufman S.J. Expression of alpha 7 integrin cytoplasmic domains during skeletal muscle development: alternate forms, conformational change, and homologies with serine/threonine kinases and tyrosine phosphatases.J. Cell Sci. 1993; 106: 1139-1152Crossref PubMed Google Scholar Purified α-ITGA7 IgG:GAA protein was incubated with C2C12 mouse myoblasts overnight and compared to an α-mCD63 IgG:GAA. Both constructs were internalized in C2C12 myoblasts independently of M6P, as 5 mM M6P did not decrease uptake (Figure 1C). However, α-mCD63 IgG:GAA exhibited higher internalization than α-ITGA7:GAA.Antibody:GAA fusions undergo normal intracellular processing of GAATo determine whether the precursor GAA on the α-hCD63 IgG:GAA fusions retains the intracellular trafficking and proteolytic processing events to form mature lysosomal forms of GAA,24Moreland R.J. Jin X. Zhang X.K. Decker R.W. Albee K.L. Lee K.L. Cauthron R.D. Brewer K. Edmunds T. Canfield W.M. Lysosomal acid alpha-glucosidase consists of four different peptides processed from a single chain precursor.J. Biol. Chem. 2005; 280: 6780-6791Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar especially given that the GAA fusion preparations lacked the canonical M6P residues for trafficking via CI-MPR, we performed a pulse-chase experiment of α-hCD631 IgG:GAA on a Pompe patient fibroblast line that lacks the lysosomal GAA forms. Within 24 h of internalization, accumulation of the 76-kDa lysosomal form of GAA was observed (Figure 1D). The half-life of the lysosomal GAA matched the long intracellular half-life of other rhGAA preparations,10Maga J.A. Zhou J. Kambampati R. Peng S. Wang X. Bohnsack R.N. Thomm A. Golata S. Tom P. Dahms N.M. et al.Glycosylation-independent lysosomal targeting of acid α-glucosidase enhances muscle glycogen clearance in pompe mice.J. Biol. Chem. 2013; 288: 1428-1438Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar while the antibody portion of the fusion quickly degraded over 3 days. Cathepsin inhibitors known to abolish the processing of GAA to the lysosomal form prevented full processing of the IgG:GAA to the lysosomal 76-kDa form (Figure S2),25Wisselaar H.A. Kroos M.A. Hermans M.M. van Beeumen J. Reuser A.J. Structural and functional changes of lysosomal acid alpha-glucosidase during intracellular transport and maturation.J. Biol. Chem. 1993; 268: 2223-2231Abstract Full Text PDF PubMed Google Scholar further indicating that the α-hCD631 IgG:GAA fusion protein is converted in the lysosome into mature GAA.scFv:GAA fusions also mediate uptake and processing of GAAWe then tested whether an scFv:GAA format was able to achieve similar uptake and processing as an IgG:GAA format, as the smaller transgene size would allow for packaging into recombinant AAV genomes for gene therapy. α-hCD632 scFv:GAA or alglucosidase alfa was incubated with HEK293 cells overnight, and the scFv:GAA format, like alglucosidase alfa, also accumulated the 76-kDa form of lysosomal GAA by western blot in HEK cells in a dose-dependent fashion (Figure 2A). We also observed that internalized α-hCD632 scFv:GAA co-localized with LAMP2, a lysosomal marker, similarly to internalized alglucosidase alfa (Figure 2B). Additionally, a glycan analysis of the purified α-hCD632 scFv:GAA protein demonstrated primarily sialylated and mannosylated N-glycan structures with no detectable terminal M6P residues, similar to the α-hCD631 IgG:GAA protein (Table S1).Figure 2An α-hCD63 scFv:GAA fusion protein delivers GAA to lysosomesShow full caption(A) α-hCD632 scFv:GAA and alglucosidase alfa are both processed to lysosomal forms after internalization. HEK293 were incubated with either α-hCD632 scFv:GAA or alglucosidase alfa overnight, and cell lysates were probed for GAA uptake and processing with an anti-GAA antibody (green) and loading control anti-GAPDH (red). A dose-dependent accumulation of the lysosomal GAA form is observed in either GAA molecule. (B) α-hCD632 scFv:GAA colocalizes with lysosomes after internalization. HEK293 cells were incubated with either α-hCD632 scFv:GAA or alglucosidase alfa, cells were fixed 4 h after internalization, and immunofluorescence was performed for GAA (green), nuclei, and Lamp2. Scale bars, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Targeted GAA has superior in vivo efficacy in PD mice versus GAA alone when administered as a protein ERTThe relative efficacy of targeting GAA using α-hCD63 compared to the standard of care, alglucosidase alfa, was tested in a mouse model of PD. Given that these fusion proteins recognize human but not mouse CD63, we first engineered a PD mouse model, Gaa−/−;Cd63hu/hu, where Gaa was replaced by LacZ and the protein-coding region of the Cd63 locus was replaced with its human counterpart (Figures S3A and S3B). Gaa−/−;Cd63hu/hu mice displayed levels of glycogen storage equivalent to a previous Gaa6neo/6neo mouse model (Figure S3C).26Raben N. Nagaraju K. Lee E. Kessler P. Byrne B. Lee L. LaMarca M. King C. Ward J. Sauer B. Plotz P. Targeted disruption of the acid alpha-glucosidase gene in mice causes an illness with critical features of both infantile and adult human glycogen storage disease type II.J. Biol. Chem. 1998; 273: 19086-19092Abstract Full Text Full Text PDF PubMed Scopus (220) Google ScholarAlglucosidase alfa or α-hCD632 scFv:GAA was then dosed intravenously weekly for 4 weeks in 2- to 3-month-old PD mice. Mice were sacrificed 1 week after the last dose. Alglucosidase alfa was given at a dose of 5 mg/kg or 20 mg/kg, and α-hCD632 scFv:GAA were given at the respective equimolar doses of 6.2 mg/kg or 25 mg/kg, respectively. A non-depleting anti-CD4 was administered intraperitoneally 1 day prior to each weekly injection to all groups to mitigate anaphylactic and anti-drug reactions to the test molecules.27Sun B. Banugaria S.G. Prater S.N. Patel T.T. Fredrickson K. Ringler D.J. de Fougerolles A. Rosenberg A.S. Waldmann H. Kishnani P.S. Non-depleting anti-CD4 monoclonal antibody induces immune tolerance to ERT in a murine model of Pompe disease.Mol. Genet. Metab. Rep. 2014; 1: 446-450Crossref PubMed Scopus (11) Google ScholarGlycogen levels were quantified in muscle tissue lysates of the sacrificed animals (Figure 3A). In both low- and high-dose groups, α-hCD632 scFv:GAA removed more glycogen compared to alglucosidase alfa (p < 0.01 in all tissues measured between equivalent molar doses). Furthermore, there was a dose-dependent decrease of glycogen, with 25 mg/kg of α-hCD632 scFv:GAA clearing more glycogen than the 6.2 mg/kg dose (p < 0.01).Figure 3α-hCD63 scFv:GAA outperforms alglucosidase alfa in glycogen removal on a molar basisShow full caption(A) α-hCD632 scFv:GAA removed more glycogen at equivalent molar doses to alglucosidase alfa in skeletal muscle. 2- to 3-month old PD mice were dosed weekly with either α-hCD632 scFv:GAA or alglucosidase alfa, and muscle glycogen was assayed 1 week after the last dose. n = 4–8 per muscle (n = 8 mice in each treatment group). Error bars represent means ± SD. ∗p < 0.01 between groups. (B) Quadriceps show lower LAMP1 staining after treatment with α-hCD632 scFv:GAA. (C) LAMP1-stained quadriceps were quantified for areal staining. Error bars represent means ± SD. ∗p < 0.01 between groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Quadriceps from untreated PD mice showed diffuse staining of LAMP1 lysosomes that was present throughout the muscle fibers rather than restricted mainly to the periphery of the fiber as in wild-type controls (Gaa+/+;Cd63hu/hu) (Figure 3B). Treatment with α-hCD632 scFv:GAA reduced lysosomal staining throughout the muscle fibers at the higher 25 mg/kg dose.AAV-mediated liver depot gene therapy of α-hCD63 scFv:GAA clears muscle glycogen to wild-type levelsIn order to achieve long-term expression and sustained delivery of targeted GAA proteins, we utilized AAV-mediated liver depot gene therapy in our PD mouse model. AAV2/8 viruses encoding α-hCD631 scFv:GAA or GAA cDNAs driven by a liver-specific promoter were injected by tail vein into 2- to 3-month-old PD mice at 1e10 or 1e11 viral genomes (vg)/mouse. Serum levels of GAA proteins were higher in the AAV α-hCD631 scFv:GAA groups than in the AAV GAA-treated groups at equivalent doses (Figure 4A), possibly because of inefficient secretion mediated by the native GAA signal peptide.13Puzzo F. Colella P. Biferi M.G. Bali D. Paulk N.K. Vidal P. Collaud F. Simon-Sola M. Charles S. Hardet R. et al.Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid α-glucosidase.Sci. Transl. Med. 2017; 9: eaam6375Crossref PubMed Scopus (66) Google Scholar AAV-infected PD mice were sacrificed after 3 months, and tissues were assayed for glycogen. Qualitatively, clearance of glycogen to wild-type levels was observed in periodic acid-Schiff (PAS)-stained sections of quadriceps after treatment of 1e11 vg AAV α-hCD631 scFv:GAA but not in the 1e11 vg AAV GAA-treated mice (Figure 4B). Glycogen levels were quantified in muscle tissue lysates (Figure 4C) and in the 1e11 vg AAV α-hCD631:GAA group were reduced to near wild-type levels in heart and all skeletal muscles tested, whereas 1e11 vg AAV GAA was not as efficacious, clearing ∼50% of the glycogen in muscles other than heart. Both soleus (type I myofiber) and extensor digitorum longus (type II myofiber) muscles were cleared of glycogen, with measurements comparable to wild-type levels, in the 1e11 vg AAV α-hCD631 scFv:GAA-treated group.Figure 4Treatment of PD mice with AAV α-hCD631 scFv:GAA restores glycogen to wild-type levels in key musclesShow full caption(A) Serum levels of 1e10 vg/mouse and 1e11 vg/mouse of each AAV were quantified by western blot and are higher in the AAV α-hCD631 scFv:GAA groups at the same dose. (B) PAS-H stain for glycogen of 1e11 vg-treated quadricep muscle sections 3 months after AAV infection show uniform removal of glycogen in the α-hCD631 scFv:GAA group. (C) α-hCD631 scFv:GAA-treated mice have lower glycogen levels in skeletal muscle than GAA-treated mice at equivalent doses. Normalization of muscle glycogen to wild-type levels is seen at the 1e11 vg/mouse dose of α-hCD631 scFv:GAA. Glycogen in muscle tissue lysates was assayed 3 months after AAV administration. Error bars represent means ± SD. ∗∗p < 0.01 between 1e10 vg/mouse groups. ∗p < 0.05 between 1e11 vg/mouse groups. n = 2 for wild type, n = 4 for treatment groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Human GAA RNA levels were measured in lysates of heart, quadriceps, and liver 3 months after infection, and expression was predominant in the liver for all constructs in a viral dose-dependent fashion (Figure S4A). Serum titers against GAA performed 3 months after infection revealed that only the 1e10 vg AAV GAA group, with a mean titer of 1.18e6, was significantly higher compared to control (p = 0.019) (Figure S4B). The 1e11 vg AAV GAA, 1e10 vg AAV α-hCD631 scFv:GAA, and 1e11 vg AAV α-hCD631 scFv:GAA groups had titers in the 104–105 range, but these did not reach statistical significance versus control mice. These results agree with previous studies showing there is a serum level threshold that must be reached in order to induce tolerance to GAA.16Han S.-o. Ronzitti G. Arnson B. Leborgne C. Li S. Mingozzi F. Koeberl D. Low-Dose Liver-Targeted Gene Therapy for Pompe Disease Enhances Therapeutic Efficacy of Ert Via Immune Tolerance Induction.Mol. Ther. Methods Clin. Dev. 2017; 4: 126-136Abstract Full Text Full Text PDF PubMed Scopus (43) Google ScholarMuscles from mice treated with AAV α-hCD631 scFv:GAA showed fewer lysosomes and reduced autophagyLysosomal and autophagosomal accumulation is a hallmark of PD, and effective treatments require their normalization. To visualize lysosomes, sections of quadriceps from the AAV-treated mice and controls were also stained with an anti-LAMP1 antibody and imaged in both confocal (Figure 5A) and wide-field (Figure 5B) microscopy. Quadriceps from PD mice showed a large expansion of lysosomes (Figure 5C), with lysosomes present throughout the muscle fibers rather than restricted mainly to the periphery of the fiber as in wild-type controls (Gaa+/+;Cd63hu/hu) (Figure 5A). Treatment with AAV α-hCD63 scFv:GAA improved lysosomal localization (Figure 5A) and significantly reduced the total lysosomal area (Figures 5B and 5C). In contrast, treatment with AAV GAA showed a trend toward reduced lysosomal area, but the di