Rare Disease Report

Gene Therapy for Pompe Disease Probed

AUGUST 19, 2015
RDR Staff
Research published in Molecular Therapy by a team of investigators from Japan suggests that the exploration of lentiviral gene transfer as a complimentary therapeutic modality may be key to unlocking a new era of Pompe disease-related breakthroughs.1

Sato et al. generated late-onset Pompe disease-specific induced pluripotent stem cells (iPSC) (derived from a late-onset Pompe disease patient) and differentiated them into cardiomyocytes. The differentiated cardiomyocytes demonstrated clear markers of Pompe disease (e.g., glycogen accumulation and lysosomal enlargement). Subsequently, study authors discovered that lentiviral gene replacement therapy mitigated the cardiomyocyte-related Pompe manifestations in a statistically significant manner.1 

Lentiviral technology is a cutting-edge form of gene therapy in which a transplanted repair gene is introduced into the cell as a `passenger’ transported by a virus which has been stripped of its ability to replicate but - owing to its infective properties and unique ability to penetrate both dividing and non-dividing cells - represents 1 of the most efficient methods of gene delivery currently available.2

Pompe Disease Pathophysiology

Pompe disease is a hereditary lysosomal storage disorder which impacts approximately 1 out of every 40,000 live births. The etiology of this rare, usually-fatal form of muscular dystrophy is rooted in roughly 300 distinct genetic mutations in the acid alpha-glucosidase (GAA) gene, which expresses an enzyme of the same name. The GAA enzyme is an essential facilitator of the glycogen metabolic process. Glycogen is a stored form of sugar. Via interaction with the GAA enzyme in the lysosome, glycogen is converted into glucose. The body uses glucose to fuel muscles. Lysosomes are cytoplasmic organelles which serve as intracellular `waste management centers,’ eliminating debris and breaking down multiple substances into manageable components that are subsequently utilized or disposed of via other cellular processes.3

In the case of Pompe disease, the GAA enzyme is either nonexistent or underrepresented. When this critical protein is missing, lysosomal glycogen builds up throughout the body, resulting, in a spectrum of debilitations which generally culminates in loss of life.3

Age-of-onset ranges widely. Severity is also variable and dependent on degree of GAA enzyme deficiency. Early-onset Pompe is characterized by complete or near complete GAA enzyme deficiency. The typical early-onset Pompe disease patient will die due to severe cardiac muscle deterioration before his or her first birthday. Late-onset Pompe disease, a partial GAA deficiency, generally manifests between the ages of 10 and 60. Late-onset Pompe is also deadly (following a longer period of disease progression of up to several years, during which progressive muscle weakness begins to inhibit respiration, culminating in eventual mortality as a result of respiratory failure).3

Toward A New Pompe Therapy Paradigm?

Since 2006, when the first enzyme replacement therapy (ERT) - alglucosidase alfa - gained US Food and Drug Administration (FDA) approval for early-onset Pompe (approval for an almost biologically identical form of alglucosidase alfa for late-onset Pompe followed in 2010), ERT has represented the-state-of-the-art of Pompe disease treatment. There is no cure for Pompe disease but ERT has been demonstrated to increase skeletal muscle strength, walking distance, respiratory function and survival rates.3

The aforementioned clinical responses, however, are markedly variable. In addition, many Pompe patients become resistant to ERT therapy as a result of neutralizing antibody formation and autophagic buildup. For such cases, “There are few available treatment modalities at this point and novel therapeutic strategy is warranted,” asserted Sato and colleagues. The Japanese research team presented data which positioned lentiviral gene therapy as a potential alternative treatment modality to supplement ERT and other standard clinical interventions.1

Investigators evaluated 3 iPSC clones from 1 late-onset Pompe disease patient and 1 clone from 1 healthy control subject. Several pluripotency markers were assessed via reverse transcription polymerase chain reaction. Both Pompe disease and control iPSC were shown to have similar characteristics; in both, almost all of the analyzed pluripotency markers were expressed equally. Pluripotency is defined as the ability of individual cells to initiate all lineages of the mature human organism. A pluripotent cell is, in essence, a blank slate. Pluripotent stem cells can become any type of tissue in the body, with the exception of placental tissue.4  

Genetic analysis of Pompe disease iPSC demonstrated compound heterozygote mutations which corresponded to late-onset Pompe disease. Electron microscopy was employed to compare glycogen accumulation in the lysosomes of Pompe disease iPSC with control. Pompe disease iPSC were shown to have weak GAA enzyme expression and increased glycogen content compared to control iPSC. Citing the aforementioned evidence, investigators concluded that “Pompe disease iPSC have disease-specific characteristics both pathologically and biochemically.”1 (Figure 1)

Figure 1

Disease-specific change of Pompe disease iPSCs. (a) Mutation analysis shows compound heterozygote mutation (c.796 C>A and c.1316 T>A). (b) Electron microscopy of iPS cell lines (Pompe-1, Pompe2, Pompe3, and control). Arrow is demonstrating accumulated glycogen. Upper scale bar, 5 µm; lower scale bar, 1 µm. (c) GAA enzyme assay of iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). Data were expressed as means ± SEM. (d) Glycogen assay of iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). Data were expressed as means ± SEM. Figure from Sato et al1 who have made the results available at Molecular Therapeutics through a creative commons license.
In the next stage of the investigation, researchers differentiated iPSC into cardiomyocytes. Robust differentiation was observed in both healthy control and Pompe disease iPSC. This was important because various concerns exist among clinicians regarding disease phenotype maintenance following stem cell differentiation. In both Pompe Disease and control iPSC, beating cardiomyocyte tissue was observed at around 10 days following differentiation. (see video clip here of beating cells)

Characteristics of both Pompe Disease and control cardiomyocytes were found to be similar. However, late-onset Pompe pathology was noted in the former. A glycogen accumulation that study authors characterized as “massive” was observed in cardiomyocytes derived from Pompe disease iPSCs; said abnormality was not observed in control.  

In the final phase of the study, a lentivirus delivery mechanism was employed to introduce a replacement GAA gene to Pompe disease iPSC (at multiplicities of infection of 0, 10, 50, and 100). (Figure 2) Findings indicated a dose-dependent increase in GAA enzyme activity in every iPSC-derived cardiomyocyte. In addition, GAA gene transduction (via lentiviral vector) in the highest multiplicity of infection was associated with a statistically significant decrease in glycogen levels (P <  .01). An effect on disease hallmarks such as glycogen accumulation and lysosomal enlargement was clearly evident: “Lentiviral GAA transfer ameliorates pathological and biochemical abnormality seen in patient-specific iPSC,” wrote researchers. Moreover, researchers found that the efficacy of lentiviral gene transfer is maintained after cardiac differentiation and cellular pathology is improved via GAA rescue.1

Figure 2

GAA enzyme assay of transfected iPS cell lines (Control, Pompe1, Pompe2, and Pompe3). Transfection is conducted at the multiplicity of infection of 0, 10, 50, and 100. Data were expressed as means ± SEM. Table from Sato et al1 who have made the results available at Molecular Therapeutics through a creative commons license.


  1. Sato Y, Kobayashi H, Higuchi T, et al. Disease modeling and lentiviral gene transfer in patient-specific induced pluripotent stem cells from late-onset Pompe disease patient. Molecular Therapy — Methods & Clinical Development 2015. http://www.nature.com/articles/mtm201523 Published online July 8, 2015. Accessed online August 18, 2015.
  2. DeTore JM. bluebird bio: transforming the lives of patients with severe genetic and rare diseases. Presented orally at the Jefferies 2015 Global Healthcare Conference, New York, NY. June 1, 2015. http://wsw.com/webcast/jeff88/blue/index.aspx Audio recording and presentation slides accessed on-line on August 18, 2015.
  3. RDR Staff. Exercise and Pompe Disease. Rare Disease Report 2015. http://www.raredr.com/publications/Rare-Disease-Report/2015/october-2015/fitness-pompe Published Online August 13, 2015. Accessed online August 18, 2015.
  4. Wray J, Kalkan T, Smith AG. The ground state of pluripotency. Biochem Soc Trans 2010;38(4):1027-1032.

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