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

Figure 2. Uptake of tobrhGAA in human fibroblast cell lines. Human fibroblast (GM4912, GM1935, and GM3329) cell lines from infantile or adult Pompe
disease were exposed to equivalent amounts 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.

Figure 3. Uptake of tobrhGAA in a human Pompe disease myoblast cell line. A human Pompe disease myoblast cell line was exposed to equivalent
amounts of a tobrhGAA seed lysate or rhGAA for 48 h and assayed for GAA and NAG (mean ± standard deviation)
Abbreviations: GAA: Acid a-glucosidase; NAG: Neutral a-glucosidase; tobrhGAA: Tobacco seeds expressing human acid a-glucosidase; rhGAA:
Recombinant human acid a-glucosidase.

stability, and IC for inhibitors (acarbose, castanospermine, an increase in GAA activity in tissues from 8% to 23% of
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deoxynojirimycin, miglitol, and voglibose). Data showed that the wild-type mice tissues (heart, skeletal muscle, liver, and
tobrhGAA was comparable to placental GAA and superior diaphragm) versus treated GAA KO mice (mean ± SD; with
to alglucosidase alfa and other rhGAAs (Table 1). 93,97-107,121,122 the statistical significance indicated) (Table 2).
3.9. Short-term treatment 3.10. Long-term treatment (203 days)
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Forelimb grip strength was measured using a GMS in GAA We treated two groups of GAA KO mice (n = 3) 3 times a
KO mice (exon 6 ) after three oral administrations with week with either a 1× or 3× dose (25 or 75 μg of tobrhGAA,
neo 109
a lysate of tobrhGAA seeds (each dose containing 75 μg of respectively) orally and measured their vertical hang-time
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tobrhGAA) every other day up to day 7. Wild-type mice over 203 days. Both treatment groups exhibited a steady,
(n = 3) averaged 245 ± 21 lbs (SEM) grip at release. Mock- significant improvement in vertical hang-time versus the
treated GAA KO mice (n = 3) averaged 92 ± 3 lbs grip at mock-treated GAA KO mice. Interestingly, the hang-

release (3 – 5 attempts/mouse), and tobrhGAA lysate- time of mice in the 1× group gradually increased until it
treated GAA KO mice (n = 3) averaged 105 ± 3 lbs grip at was equal to that of the 3× dose group by day 92, which
release (P ≤ 0.024 treated vs. mock-treated), which was a 14% suggests that the lower dose can be effective for long-term
improvement in strength. At day 7, mice were sacrificed, and treatment. Both treatment groups showed a 25% – 35%
the tissues were assayed for GAA/NAG and compared to increase versus the mock-treated wild-type mice at day
the mock-treated GAA KO and wild-type mice. We found 82, but by day 203, both groups were almost equal (89%

Volume 4 Issue 1 (2025) 7 doi: 10.36922/gpd.1760