Oral Delivery of Acid Alpha Glucosidase Epitopes Expressed in Plant Chloroplasts Suppresses Antibody Formation in Treatment of Pompe Mice

Deficiency of acid alpha glucosidase (GAA) causes Pompe disease in which the patients systemically accumulate lysosomal glycogen in muscles and nervous systems, often resulting in infant mortality. Although enzyme replacement therapy (ERT) is effective in treating patients with Pompe disease, formation of antibodies against rhGAA complicates treatment. In this report, we investigated induction of tolerance by oral administration of GAA expressed in chloroplasts. Because full-length GAA could not be expressed, N-terminal 410-amino acids of GAA (as determined by T-cell epitope mapping) were fused with the transmucosal carrier CTB. Tobacco transplastomic lines expressing CTB-GAA were generated through site-specific integration of transgenes into the chloroplast genome. Homoplasmic lines were confirmed by Southern blot analysis. Despite low-level expression of CTB-GAA in chloroplasts, yellow or albino phenotype of transplastomic lines was observed due to binding of GAA to a chloroplast protein that has homology to mannose-6 phosphate receptor. Oral administration of the plant-made CTB-GAA fusion protein even at 330-fold lower dose (1.5 μg) significantly suppressed immunoglobulin formation against GAA in Pompe mice injected with 500 μg rhGAA per dose, with several-fold lower titre of GAA-specific IgG1 and IgG2a. Lyophilization increased CTB-GAA concentration by 30-fold (up to 190 μg per g of freeze-dried leaf material), facilitating long-term storage at room temperature and higher dosage in future investigations. This study provides the first evidence that oral delivery of plant cells is effective in reducing antibody responses in ERT for lysosomal storage disorders facilitating further advances in clinical investigations using plant cell culture system or in vitro propagation

Targeted approaches to induce immune tolerance for Pompe disease therapy

Enzyme and gene replacement strategies have developed into viable therapeutic approaches for the treatment of Pompe disease (acid α-glucosidase (GAA) deficiency). Unfortunately, the introduction of GAA and viral vectors encoding the enzyme can lead to detrimental immune responses that attenuate treatment benefits and can impact patient safety. Preclinical and clinical experience in addressing humoral responses toward enzyme and gene therapy for Pompe disease have provided greater understanding of the immunological consequences of the provided therapy. B- and T-cell modulation has been shown to be effective in preventing infusion-associated reactions during enzyme replacement therapy in patients and has shown similar success in the context of gene therapy. Additional techniques to induce humoral tolerance for Pompe disease have been the targeted expression or delivery of GAA to discrete cell types or tissues such as the gut-associated lymphoid tissues, red blood cells, hematopoietic stem cells, and the liver. Research into overcoming preexisting immunity through immunomodulation and gene transfer are becoming increasingly important to achieve long-term efficacy. This review highlights the advances in therapies as well as the improved understanding of the molecular mechanisms involved in the humoral immune response with emphasis on methods employed to overcome responses associated with enzyme and gene therapies for Pompe disease

Immune Responses and Hypercoagulation in ERT for Pompe Disease Are Mutation and rhGAA Dose Dependent

<div><p>Enzyme replacement therapy (ERT) with recombinant human acid-α-glucosidase (rhGAA) is the only FDA approved therapy for Pompe disease. Without ERT, severely affected individuals (early onset) succumb to the disease within 2 years of life. A spectrum of disease severity and progression exists depending upon the type of mutation in the GAA gene (<i>GAA</i>), which in turn determines the amount of defective protein produced and its enzymatic activity. A large percent of the early onset patients are also cross reactive immunological material negative (CRIM-) and develop high titer immune responses to ERT with rhGAA. New insights from our studies in pre-clinical murine models reveal that the type of <i>Gaa</i> mutation has a profound effect on the immune responses mounted against ERT and the associated toxicities, including activation of clotting factors and disseminated intravascular coagulation (DIC). Additionally, the mouse strain affects outcomes, suggesting the influence of additional genetic components or modifiers. High doses of rhGAA (20 mg/kg) are currently required to achieve therapeutic benefit. Our studies indicate that lower enzyme doses reduce the antibody responses to rhGAA, reduce the incidence of immune toxicity and avoid ERT-associated anaphylaxis. Therefore, development of rhGAA with increased efficacy is warranted to limit immunotoxicities.</p></div

Antibody responses to varying doses of rhGAA in null mutation (n = 6) or P545L mutant mice (n = 5).

<p><b>A</b>) Anti-rhGAA IgG1 in 1 mg/kg rhGAA injected GAA-/- 129SVE mice tested weekly <b>B</b>) Anti-rhGAA IgG1 in 5 mg/kg rhGAA injected GAA-/- 129SVE mice <b>C</b>) Anti-rhGAA IgG1 in 20 mg/kg rhGAA injected GAA-/- 129SVE mice <b>D</b>) Anti-rhGAA IgG1 response in 20 mg/kg rhGAA injected P545L mice <b>E</b>) Anti-rhGAA IgG2a in 1 mg/kg rhGAA injected GAA-/- 129SVE mice <b>F</b>) Anti-rhGAA IgG2a in 5 mg/kg rhGAA injected GAA-/- 129SVE mice <b>G</b>) Anti-rhGAA IgG2a in 20 mg/kg rhGAA injected GAA-/- 129SVE mice <b>H</b>) Anti-rhGAA IgG2a in 20 mg/kg rhGAA injected P545L mice. Arrows indicate fold decrease over corresponding 20 mg/kg cohort time point. p<0.05 *, p<0.005 **, p<0.0005 ***, ns = not significant.</p

Vital signs measured by pulse oxymetry, prior to and 5 minutes after the 8^th rhGAA IV injection in wt BALB/c and wt 129SVJ mice (n = 5).

<p><b>A</b>) Percentage oxygen saturation <b>B</b>) Heart rate <b>C</b>) Breath distention <b>D</b>) Pulse distention, p<0.05 *, ns = not significant, (inj; injection).</p

Immune Responses and Hypercoagulation in ERT for Pompe Disease Are Mutation and rhGAA Dose Dependent - Figure 6

<p><b>A</b>) Reduction of clotting times measured in activated partial thromboplastin time (aPTT) in GAA-/- 129SVE and P545L mice. <b>B</b>) D-Dimer levels in GAA-/-129SVE naïve and rhGAA injected mice. Changes in hemorheologic values from complete blood counts in 129SVE GAA-/- mice and P545L naive and 20 mg/kg rhGAA injected mice (5 min post rhGAA injection). <b>C</b>) Platelet count, <b>D</b>) Mean platelet volume, <b>E</b>) Platelet width distribution, p<0.05 *, p<0.005 **, p<0.0005 ***, ns = not significant.</p

Representative examples of hematoxylin and eosin (H&E) staining of paraffin embedded sections of liver, kidney and heart (n = 3) in A-C) naïve GAA-/- 129SVE mice and D-F) 20 mg/kg rhGAA IV injected GAA-/- 129SVE indicating residual RBC, G-I) 20 mg/kg rhGAA IV injected P545L mice.

<p>Representative examples of hematoxylin and eosin (H&E) staining of paraffin embedded sections of liver, kidney and heart (n = 3) in A-C) naïve GAA-/- 129SVE mice and D-F) 20 mg/kg rhGAA IV injected GAA-/- 129SVE indicating residual RBC, G-I) 20 mg/kg rhGAA IV injected P545L mice.</p

Comparison of pulse oxymetry measurements of vital signs prior to and post the 4^th rhGAA (1 mg/kg, 5 mg/kg or 20 mg/kg) ERT IV injection in GAA-/- 129SVE (n = 6) and P545L mice (20 mg/kg rhGAA; n = 5).

<p>A) oxygen saturation B) heart rate C) pulse distention D) breath distention, E) Time taken for the formation of a platelet plug post-injury (tail-snip) prior to and post-rhGAA IV administration. Changes in hemorheologic values from complete blood counts in 129SVE GAA-/- mice and P545L naive and 20 mg/kg rhGAA injected mice (5 min post rhGAA injection). F) Hematocrit, G) Hemoglobin, p<0.05 *, p<0.005 **, p<0.0005 ***, ns = not significant.</p

Temperature measurements prior to and post rhGAA IV injections in A-C) GAA-/-129SVE mice (n = 6) receiving 1 mg/kg, 5 mg/kg or 20 mg/kg doses of rhGAA IV D) GAA-/-129SVE mice injected with PBS E) P545L mice (n = 5) receiving 20 mg/kg of rhGAA IV, F) Experimental timeline indicating GAA-/- 129SVE mice or P545L C57BL/6 x 129SVE mice injected with 1 mg/kg, 5 mg/kg or 20 mg/kg doses of rhGAA G) Survival curve.

<p>Temperature measurements prior to and post rhGAA IV injections in A-C) GAA-/-129SVE mice (n = 6) receiving 1 mg/kg, 5 mg/kg or 20 mg/kg doses of rhGAA IV D) GAA-/-129SVE mice injected with PBS E) P545L mice (n = 5) receiving 20 mg/kg of rhGAA IV, F) Experimental timeline indicating GAA-/- 129SVE mice or P545L C57BL/6 x 129SVE mice injected with 1 mg/kg, 5 mg/kg or 20 mg/kg doses of rhGAA G) Survival curve.</p