Table 1.

Comparison of the Currently Used Cited In-House and Good Laboratory Practice Amenability Assays

Assay	Lukas et al.34,35	Benjamin et al.10(GLP-HEK Assay)	Oommen et al.36	Lenders et al.37(HEK293T GLA Knockout Assay)	Lenders et al.37(Patient-Derived Urinary Cells)
Cells	HEK293H	HEK293	HEK293H (GripTite 293 MSR)	HEK293T	fibroblast-like primary immortalized cell line
Duration (days)	2.5	5	5	2	2
DGJ (μM)	20	10	10	10 (20)	10 + 20
Expression plasmid	pcDNA3.1/V5-His6	pcDNA6	pcDNA6/V5-His	pcDNA3.1	not required
Transfection efficiency	quantitative western blots	qPCR	commercial SEAP	commercial luciferase	not required
Amenability criteria	1.5-fold over baseline or >5% compared to untreated value	≥1.2-fold over baseline + absolute increase of ≥3% wild-type activity
Pros	high-throughput screening				no overexpression model
	no patient samples required				patient-specific mutation in appropriate genetic background
	identification of amenability in mutations with high residual activity			identification of amenability in mutations with high and low residual activity
		DGJ wash-out (2 h)		DGJ wash-out
				no endogenous AGAL activity	assessment of potential Gb3 depletion
				no heterodimerization between wild-type and mutant AGAL
Cons	overexpression model				requires patients’ urine
	high background due to endogenous AGAL activity				immortalization process
	heterodimerization between wild-type and mutant AGAL				time consuming
	no NAGA inhibition mentioned		overexpression of mutant AGAL might affect SEAP trafficking and secretion	overexpression of mutant AGAL might affect luciferase trafficking and secretion	expensive
	no DGJ wash-out mentioned		no DGJ wash-out mentioned

AGAL, α-galactosidase A; DGJ, 1-deoxygalactonojirimycin; Gb₃, globotriaosylceramide; HEK, human embryonic kidney; NAGA, α-galactosidase B; SEAP, secreted embryonic alkaline phosphatase.