Gene Therapy Developments for Pompe Disease

Abstract

Pompe disease is an inherited neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). The most severe form is infantile-onset Pompe disease, presenting shortly after birth with symptoms of cardiomyopathy, respiratory failure and skeletal muscle weakness. Late-onset Pompe disease is characterized by a slower disease progression, primarily affecting skeletal muscles. Despite recent advancements in enzyme replacement therapy management several limitations remain using this therapeutic approach, including risks of immunogenicity complications, inability to penetrate CNS tissue, and the need for life-long therapy. The next wave of promising single therapy interventions involves gene therapies, which are entering into a clinical translational stage. Both adeno-associated virus (AAV) vectors and lentiviral vector (LV)-mediated hematopoietic stem and progenitor (HSPC) gene therapy have the potential to provide effective therapy for this multisystemic disorder. Optimization of viral vector designs, providing tissue-specific expression and GAA protein modifications to enhance secretion and uptake has resulted in improved preclinical efficacy and safety data. In this review, we highlight gene therapy developments, in particular, AAV and LV HSPC-mediated gene therapy technologies, to potentially address all components of the neuromuscular associated Pompe disease pathology.

Keywords: Pompe disease; adeno-associated viral vector; enzyme replacement therapy; hematopoietic stem cell; lentiviral vectors.

Figure 1

Anticipated pharmacokinetics of enzyme replacement therapy (ERT) using recombinant human GAA (rhGAA) protein and single intervention gene therapy. Left: ERT requires intermittent bolus infusions of doses of rhGAA protein to reach above the critical threshold. It has been reported that above 30% GAA enzyme activity present in unaffected individuals is the critical threshold [1]. Peak plasma rhGAA protein levels are present directly after infusion, subsequently taken up by muscles, and degraded over time. Right: Gene therapy applied as a single intervention therapy for curative potential. After transduction of cells of interest, continuous production of therapeutic transgene product provides sustained levels in transduced cells and/or secreted levels in plasma for cross-correction in key tissues. Application of gene therapy may impact the bioavailability of therapeutic enzymes to enhance uptake and correction in key tissues compared to ERT as shown by Costa-Verdera and colleagues [28]. Horizontal dotted line represents the critical threshold to prevent Pompe disease phenotype.

Figure 2

Overview schema of Pompe disease gene therapy modalities. Left panel represents the process of autologous ex vivo lentiviral gene therapy. Briefly, CD34+ cells are isolated in a closed manufacturing system from mobilized peripheral blood. These isolated cells are transduced with a lentiviral vector containing the functional gene of interest, and the genetically modified cells are infused back into a patient who has typically been conditioned with alkylating agents, such as busulfan, to create space in the bone marrow. Long-term repopulating stem cells engraft into the bone marrow niche and repopulate the hematopoietic system with cells capable of secreting functional enzyme, leading to uptake and cross-correction of affected peripheral tissues. The conditioning agent, busulfan not only makes space in the bone marrow for CD34+ to permanently engraft, but also enables the microglia in the CNS to be exchanged for gene-modified microglia derived from the infused cells. The right panel depicts in vivo AAV gene therapy approaches. Utilization of different AAV vector capsid proteins and routes of administration are used to target distinct tissue niches. Transduced cells secrete functional enzyme locally, or systemically depending on targeted tissue. ICV = intracerebroventricular, IT = intrathecal, IV = intravenous, IM = intramuscular, IP = intraperitoneal.

Figure 3

Summary of GAA protein modifications employed in ERT and gene therapy. A redrawn and modified model for maturation of GAA protein, previously reported by Moreland et al. [13]. Modifications to GAA transgene or protein described in literature were subject to changing the N-terminus of the GAA protein to improve secretion and uptake in the key tissues affected in Pompe disease without affecting proteolytic processing steps to the mature GAA protein. Signal peptides have been modified to improve secretion, and tags have been incorporated to enhance uptake in skeletal muscles or cellular delivery to cytoplasm to degrade glycogen more effectively. For enzyme replacement therapy enhanced glycosylation or chaperone has been investigated as well. GT = gene therapy, ERT = enzyme replacement therapy, Fab = antigen-binding fragment. scFv = single-chain variable fragment, hAAT = human alpha-1-antitrypsin.

Figure 4

GAA protein expression in resting microglia in the brain of LV HSPC gene-therapy treated Gaa^−/− mice. Genetically modified Gaa^−/− lineage negative bone marrow cells transduced with a lentiviral vector with codon-optimized GAA, driven by the spleen focus forming virus (SFFV) promoter were infused in to Gaa^−/− mice [29] after busulfex conditioning. Brains were harvested at six months after infusion of genetically modified cells in Gaa^−/− mice, and post saline perfusion fixed in 4% formaldehyde for 24 h, and subsequently processed for immunofluorescence and immunohistochemical staining for GAA protein. Top panels: Representative anti-GAA (green) and Iba1 (red) immunofluorescence staining shows GAA colocalization in engrafted microglia-like cells in the hippocampal region. DAPI is shown in blue. Bottom panels: representative images of immunohistochemical staining for GAA. White arrows indicate microglia cells colocalizing with GAA signal.