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Gene & Protein in Disease Oral-ERT in PD knockout mice with tobrhGAA

gram in dry conditions, and a GC/MS spectral profile was 3.5. Ames mutagenicity test
generated for future comparisons (data not shown). The Ames mutagenicity test was performed using E. coli
3.2. Long-term stability DH5a and DH5a/pUC19 with various amounts of
tobrhGAA seed lysate. We found no additional bacterial
We compared tobrhGAA in seeds stored for 9 and 15 years or plasmid revertant CFUs generated with the tobrhGAA
at room temperature versus freshly harvested seeds. lysate by exposure to >10 bacteria. 113-117
4
We found there was <15% loss in GAA activity, which
indicated extreme stability (old: 0.20 μg tobrhGAA/g seeds 3.6. In vitro studies in PD fibroblast and lymphoid
vs. fresh: 0.25 μg tobrhGAA/g seeds). cell lines
3.3. Endotoxin levels We evaluated tobrhGAA uptake in fibroblast and lymphoid
cells from infantile, juvenile, and adult-onset patients with
The endotoxin levels were measured in an extract of PD and compared it to human placenta GAA and rhGAA.
tobrhGAA#3 seeds using a LAL endotoxin kit and were Various equivalent concentrations and time points (2, 24, and
found to be <0.25 EU/mL endotoxin or ~25 pg/mL. 48 h) exhibited similar uptake and increases in GAA (mean
These values are lower than the estimate of 300 mg/kg for ± standard error of the mean [SEM]) (Figure 1 and 2). All
oral endotoxin toxicity in mammals and humans. 118-120 treatments were significant at P ≤ 0.05.
Moreover, we found no viable anaerobic or aerobic bacteria
(data not shown). 3.7. Uptake of tobrhGAA in a human PD myoblast
cell line
3.4. Effect of the stomach and small intestinal A human PD myoblast cell line was exposed to equivalent
108
environments
amounts of tobrhGAA seed lysate for 48 h and assayed.
To mimic the stomach and small intestine environments, Mock-treated GAA and normal skeletal muscle cells as
we exposed tobrhGAA to the physiologic conditions and well as cells treated with rhGAA were used as controls.
times of pepsin (stomach) and trypsin/chymotrypsin We found that tobrhGAA increased GAA to 24% – 35%
(small intestine). 86,95 Lysosomal GAA is stable at low of normal (mean ± SD) (Figure 3). All treatments were
pH. None of the enzymes had any effect on tobrhGAA significant at P ≤ 0.05. Simultaneously, we measured cell
activity, thus demonstrating that the conditions in the proliferation using the Cayman MTT assay and found
digestive tract probably do not affect tobrhGAA (data that both tobrhGAA doses slightly reduced cell growth by
not shown). We repeated the conditions with 100 mg of <10% (data not shown).
whole or milled seeds. Interestingly, more than double
the amount of tobrhGAA in the whole and milled seeds 3.8. Biochemical/enzyme kinetic analyses
(600 μg/g seeds) was lysed, which suggests that the acidic We compared the enzyme kinetics for a lysate of tobrhGAA
environment of the stomach more thoroughly disrupts versus placental human placental GAA and rhGAA, as
and releases tobrhGAA from the whole or milled seed well as the limited published data for alglucosidase alfa or
(data not shown). other rhGAAs 99-107 for K , V , optimal pH, thermal heat
m max

Figure 1. Uptake of tobrhGAA in human lymphoid cell lines. Human lymphoid cell lines from infantile or adult Pompe disease (GM6314, GM13793, and
GM14450) were exposed to equivalent amounts of mock, tobrhGAA, placental human GAA, or a rhGAA. Cells were harvested after 2, 24, and 48 h and
assayed for GAA and NAG (mean ± standard deviation)
Abbreviations: GAA: Acid a-glucosidase; NAG: Neutral a-glucosidase; h: Hours; tobrhGAA: Tobacco seeds expressing human acid a-glucosidase;
rhGAA: Recombinant human acid a-glucosidase.

Volume 4 Issue 1 (2025) 6 doi: 10.36922/gpd.1760