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

provides an effective, safe, and affordable treatment option 2.5. Biochemical/enzyme kinetic analyses
as a plant-made pharmaceutical (PMP). 82,87-89,94 We compared a lysate of tobrhGAA seeds to rhGAA (R&D
2. Methods Systems) and mature placental human GAA for specific
activity using 4-MU-Glyc at pH 4.0, maltose and glycogen,
2.1. Animal approval optimal pH, inhibitors (acarbose, castanospermine,
A total of 24 wild-type mice and 34 GAA KO mice were used deoxynojirimycin, miglitol, and voglibose) and heat
in this experiment. The Institutional Biosafety Committee stability 93,97-107 according to standard enzyme kinetic
ratified the project at biosafety level BSL1 containment, methods.
and this study was approved by the Institutional Animal 2.6. Uptake of tobrhGAA by PD human myoblast,
Care and Use Committee at Rutgers, The State University fibroblast, and lymphoid cell lines
of New Jersey (number: PROTO201800168) and at NYU
School of Medicine protocol number 131109, New York, Human lymphoid (GM6314, GM13793, and GM14450) or
NY, USA. fibroblast (GM4912, GM1935, and GM3329) cell lines from
patients with infantile or adult PD were maintained in 15%
2.2. Hydroponic growth of tobacco plants fetal bovine serum, RPMI 1640, or DMEM supplemented
Transgenic tobacco plant #3 expressing human GAA in with glutamine, penicillin, and streptomycin at 37°C in a 5%
the seeds was grown indoors using a hydroponic system CO environment. Cells were plated at a density of 0.3 – 0.4
2
6
(Active Aqua Grow Flow Kit, Hydrobuilder, Inc., USA) × 10 /well in a 6-well plate in 1.5 mL of media 24 h before the
with deionized water, thereby eliminating all water and addition of varying amounts of tobrhGAA or other GAA
formulations. Cells were harvested after various hours of
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soil contaminants. Seed pods were harvested and dried exposure, washed with PBS, lysed by adding 0.5 mL of 0.01
in a freeze dryer, followed by separation from the husk M sodium phosphate (pH 7.5), frozen and thawed 3 times,
by passage through a standard food mesh strainer. To and centrifuged at 13,000 rpm for 5 min. The lysate was
eliminate environmental contamination, seeds were used for human GAA and NAG assay, as described above.
ground for 10 min in a porcelain pestle and mortar until A human PD myoblast cell line (homozygous for IVS1
fine powder was obtained, which was then placed in a UV c.-32 – 13T>G) and normal skeletal muscle cells were
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germicidal incubator for 30 min for sterilization. plated at a density of 0.3 – 0.4 × 10 /well in a 6-well plate
6
2.3. Simulated stomach and small intestinal containing PromoCell skeletal muscle growth media. Cells
environments were exposed to a lysate of tobrhGAA for 48 h and assayed.
Mock-treated GAA, normal myoblast cells, and cells
To mimic the stomach and small intestine environment, treated with equivalent amounts of rhGAA were used as the
we exposed 100 mg of whole or milled tobrhGAA seeds controls (R&D Systems #8329-GH-025). Simultaneously,
to physiologic conditions and times in the stomach with we measured cell proliferation using the Cayman MTT cell
pepsin followed by the conditions and times in the small proliferation assay (#10009365).
intestine trypsin/chymotrypsin. 86,95 Samples were added to
300 μg/mL pepsin at pH 4.0 for 60 min, the pH was adjusted 2.7. Short-term studies in GAA KO mice
to 6.5, and then trypsin at 800 μg/mL and chymotrypsin at We used PD KO mice with exon 6 disruption, wild-
neo
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700 μg/mL were added for 60 min at 37°C, followed by a type BALB/c or 129/J, or mock-treated PD KO mice
GAA assay. with PBS. PD KO mice (4 – 6 months old) were orally
administered a lysate from 300 mg (approximately 75 μg
2.4. Enzyme assay
of tobrhGAA) of transgenic seeds mixed 3:1 with apple
Ground seeds or tissues were resuspended in 10 juice every other day up to day 7 as a safe, non-invasive
mM sodium phosphate (pH 7.5), frozen and thawed oral administration technique, and the grip strength was
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3 times, and centrifuged at 13,000 ×g for 10 min. The measured using a grip-strength meter (GSM) (Columbus
supernatants were assayed for GAA for 18 h using the Inst., OH, USA). On day 7, the mice were sacrificed, and
artificial substrate 4-methylumbelliferyl-a-D-glucoside the tissues were assayed for GAA and NAG and compared
(4-MU-Glyc, 1 mg/mL) at pH 4.0 (0.5 M sodium acetate) to the wild-type and mock (PBS)-treated GAA KO mice.
and neutral alpha-glucosidase (NAG) at pH 7.5 (0.5 M
sodium phosphate) as an internal control. Fluorescence 2.8. Long-term treatment by oral gavage in GAA KO
was determined using a fluorometer with an excitation mice
wavelength of −360 nm and an emission wavelength of We treated two age-matched groups of GAA KO mice
−460 nm (Sequoia-Turner) as previously described. 96 (exon 6 ) (n = 3) 3 times a week with either a 1× or 3×
neo

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